Open questions

An interactive platform for navigating embryonic cell morphologies and contacts over development is lacking.

p. 11: 'A well-designed platform that allows interactive navigation of cell morphologies and contacts over development is required in the future.'
Cao et al. 2020. Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation

Are tardigrade trunk ganglia homologous to arthropod segmental ganglia (same position relative to segments, development and gene expression) or convergent?

pp271-272: "Future studies on the structure, development, and pattern of gene expression of the tardigrade nervous system will help resolve the issue of their putative homology with arthropod ganglia."
Mayer & Whitington 2009. Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda

Are the oscillating pair-rule genes linked into a single circuit, is it sufficient to oscillate, what is its topology, and how far has it diverged between arthropod lineages?

p. 5: 'it is not yet clear whether the oscillating genes are linked into a single circuit, whether this circuit is sufficient to generate oscillations, what the topology of this circuit is likely to be, nor indeed the extent to which it may have diverged in different lineages'
Clark et al. 2019. Arthropod segmentation

Are the primordial germ cells of H. exemplaris specified by induction, by inherited germ plasm, or by a mix of both, and which modes occur across Tardigrada?

p. 52: "It remains an open question what modes of PGC specification exist in the tardigrade phylum." … "Future studies are needed to clarify the mode of PGC specification in this system."
Heikes et al. 2023. The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris

Can automated lineaging be extended through the last round of embryonic divisions, when cells migrate actively and nuclei pack closely (C. elegans beyond the 350-cell stage)?

p. 707: 'Extending automated lineaging to include the last round of embryonic cell division ... remains a challenging goal because of the active cell migrations and close packing of nuclei in the late embryo.'
Murray et al. 2008. Automated analysis of embryonic gene expression with cellular resolution in C. elegans

Can intracellular determinants of the division axis (e.g. PAR gradients) be combined with cell mechanics into a true self-organisation model of early embryo arrangement?

p. 1810: "Intracellular events that determine the cells' division axis, e.g., PAR protein gradients (23), need to be included to arrive at a true self-organization model that integrates both mechanical and biochemical cues."
Fickentscher et al. 2013. Mechanical cues in the early embryogenesis of Caenorhabditis elegans

Can label-free holotomography follow H. exemplaris embryogenesis and organogenesis live at cellular resolution through the eggshell?

p. 9: "future research could employ this technique for detailed time-lapse monitoring of embryogenesis, organogenesis, and cellular dynamics"
Hong et al. 2025. A Non-Invasive, Label-Free Method for Examining Tardigrade Anatomy Using Holotomography

Do embryological and molecular data (segment-polarity and Hox expression in the head) confirm that the three tardigrade brain lobes come from three segments homologous to the arthropod proto-, deuto- and tritocerebrum?

p188 right col.: "To get confirmation on the morphological data, more embryological and molecular investigations are needed."
Persson et al. 2014. Brain anatomy of the marine tardigrade Actinarctus doryphorus (Arthrotardigrada)

Do heterotardigrades share the H. exemplaris pattern in which left and right ventromedian muscle attachment sites meet at the midline only in some segments, and is it ancestral for Tardigrada?

PDF p 13: "Whether or not the pattern revealed in H. dujardini and M. tardigradum exists among heterotardigrade species has not been reported"
Smith & Jockusch 2014. The metameric pattern of Hypsibius dujardini(Eutardigrada) and its relationship to that of other panarthropods

Do tardigrade adults and embryos survive subzero temperatures by the same mechanisms, and can tardigrades tolerate intracellular freezing?

pp. 302, 307 (PDF pp 304, 309): "It is not known yet whether adult tardigrades and embryos use similar mechanisms to survive subzero temperatures." … "It is still not known if tardigrades tolerate intracellular freezing."
Hengherr S, Schill RO 2018. Environmental Adaptations: Cryobiosis. In: Schill RO (ed.) Water Bears: The Biology of Tardi

Do tardigrade embryos have any extra-embryonic tissue, or cells that do not contribute to the final animal, or was it lost with miniaturisation?

p. 2: "studies explicitly focusing on this topic would be required in order to obtain information about the presence or absence of dorsal and ventral extra-embryonic tissues in tardigrades, including more detailed cell lineage tracings using four-dimensional microscopy to identify cells that do not directly contribute to the final morphology of the animal"; p. 11: "Search for (rudiments of) extra-embryonic tissues in tardigrades to clarify their existence or potential loss"
Treffkorn et al. 2022. Review of extra-embryonic tissues in the closest arthropod relatives, onychophorans and tardigrades

Do the dorsal and ventral extra-embryonic cells of onychophorans (and arthropods) contribute cells to the embryo and adult, and are they homologous across Panarthropoda?

p. 11: "Tracing of individual cell lineages in extra-embryonic tissues of arthropods and onychophorans to clarify their ontogenetic fate"; p. 9: "it is also unclear whether the dorsal extra-embryonic tissues of onychophorans and arthropods are homologous."
Treffkorn et al. 2022. Review of extra-embryonic tissues in the closest arthropod relatives, onychophorans and tardigrades

Do the unusual chromosome and nuclear features of early H. exemplaris embryos occur in other tardigrades and in other desiccation-tolerant animals such as rotifers?

PDF p 9: "It is not known if the unusual features we observed in embryos exist in tardigrades more generally, or in just this species"
Papell et al. 2026. Chromosomes remain individualized through interphase in embryos of the tardigrade Hypsibius exemplaris

Do transverse commissures form, even transiently, in the trunk ganglia of tardigrade embryos?

p. 33: "Whether segmental commissures are transiently formed during embryonic development is unknown."
Zantke et al. 2008. Three-dimensional reconstruction of the central nervous system of Macrobiotus hufelandi (Eutardigrada, Parachela): implications for the phylogenetic position of Tardigrada

Do yolk granules limit microtubule asters as steric obstacles or by affecting microtubule growth molecularly?

p. 679: "Whether yolk granules act as steric obstacles or affect MT growth by molecular means remains an important open question (Dogterom and Yurke, 1997; Wuhr et al., 2010)."
Pierre et al. 2016. Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos

Does an arthrotardigrade with primary, secondary and tertiary clavae (e.g. Neoarctus primigenius) have additional brain lobes or innervation that would count as further cephalised segments?

p188 left col.: "This could be supported by an investigation on the brain structure of tardigrades possessing primary, secondary and tertiary clavae, such as Neoarctus primigenius"
Persson et al. 2014. Brain anatomy of the marine tardigrade Actinarctus doryphorus (Arthrotardigrada)

Does the C. elegans AB blastomere have a higher cortical tension than P1 (as the 2-cell interface curvature suggests)?

p. 6: 'whether AB has stronger surface tension than P1 needs further experimental verification, such as fluorescent imaging on cytoskeleton density or direct measurement on surface tension'
Kuang et al. 2022. Computable early Caenorhabditis elegans embryo with a phase field model

Does the thickness of the gonad set the shape (aspect ratio) of the egg across nematode species?

p. 4445: "In future studies, it would be of interest to investigate the correlation between the thickness of the gonad and the shape of the eggs by examining various nematode species (Goldstein, 2001; Schulze and Schierenberg, 2011)."
Yamamoto & Kimura 2017. An asymmetric attraction model for the diversity and robustness of cell arrangement in nematodes

How are the attractive forces between blastomeres regulated when par-3 is knocked down, where their strength becomes highly variable?

p. 4444: "Therefore, an understanding of the mechanism underlying the regulation of attractive forces under par-3 (RNAi) conditions is needed in order to develop a model that accounts for this condition."
Yamamoto & Kimura 2017. An asymmetric attraction model for the diversity and robustness of cell arrangement in nematodes

How are the cell movements that sort embryonic regions into an invariant arrangement regulated, and how much genetic information does sorting several hundred cells require?

p. 260: "How cell migrations that contribute to establishing developmental form are regulated is unknown." … "It is an intriguing question how complex the genetic information needs to be to sort 558 cells in the C. elegans embryo."
Schnabel et al. 1997. Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of dev

How do species with post-embryonic segmentation coordinate segment patterning with the moult cycle, given that ecdysone-related genes have segmentation roles in some embryos?

Box 2 (p. 9): 'Another interesting question is how species that undergo post-embryonic segmentation coordinate segment patterning with the moult cycle. Ecdysone-related genes play segmentation roles in some embryos'
Clark et al. 2019. Arthropod segmentation

How do the brain and trunk ganglia of H. exemplaris coordinate the legs into gaits, switch between gaits and single-limb movements, and does this circuitry share an ancestral plan with insects?

pp. 4, 14: "How the integration of these movement modes produces cohesive whole-body behavior remains unknown." … "the neural mechanisms that coordinate limb movements to produce gaits, or facilitate transitions between gaits, are unknown"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

How do the inner cavities (blastocoel) of the early C. elegans embryo form and change, and what are they for?

p. 11: 'However, systematic characterization of such cavities is still lacking.'
Cao et al. 2020. Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation

How does pad-1 control P2 division orientation in C. elegans, and is the pad-1 RNAi 8-cell misarrangement caused by the misoriented P2 division?

p. 15: 'more in vivo experiments are needed to further explore how it functions in P2 division and whether the morphogenetic chaos in the RNAi experiment is attributed to the erroneous P2 division.'
Kuang et al. 2022. Computable early Caenorhabditis elegans embryo with a phase field model

How is the competition between bulk (cytoplasmic, length-dependent) and cortical microtubule pulling forces titrated in different cells and species?

p. 679: "How this competition may be titrated in different systems remains an important question to address experimentally (Minc and Piel, 2012; Mitchison et al., 2012)."
Pierre et al. 2016. Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos

How many neural progenitor cells build the brain in tardigrade embryos (T. stephaniae; H. exemplaris), and when do they delaminate relative to the ganglion progenitors?

p. 1353: "Although we could not determine the number of cells forming the brain"
Hejnol & Schnabel 2005. The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations

How much contact area and contact duration does contact-dependent signalling (Notch, Wnt) need between embryonic cells?

p. 7: 'the actual sensitivity of intercellular signaling including both contact area and contact duration is still poorly understood.'
Cao et al. 2020. Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation

In H. exemplaris embryos, does FGF signal from segmental ectoderm to the endomesoderm during pouch formation, and does BMP signalling pattern the dorsal–ventral axis of the lateral ectoderm?

bioRxiv p. 2 (abstract): "possible FGF signaling between ectoderm and endomesoderm, and possible roles of BMP signaling in dorsal-ventral patterning of lateral ectoderm"
Heikes et al. 2024. FGF and BMP pathway genes are expressed in segmentally iterated domains during segmentation of germ layers in a tardigrade

Is hairy part of the arthropod segmentation clock (it is unclear in Tribolium, though hairy knockdown truncates Dermestes), or is its role masked by its paralogue deadpan?

p. 4: 'First, whether hairy is involved in the Tribolium segmentation clock or not remains unclear.'
Clark et al. 2019. Arthropod segmentation

Is independent evolution of alleles common among diploid clonal parthenogens such as H. exemplaris, and are such lineages evolutionary dead ends or do they occasionally reproduce non-clonally?

pp. 2, 10: "Whether independent evolution of alleles is a widespread phenomenon among diploid clonal parthenogens remains an open question in the field." … "Whether these species are doomed in the long run, represent rare survivors against the usual fate of clonal reproduction"
Coke et al. 2026. Modified meiosis in the tardigrade Hypsibius exemplaris maintains heterozygosity across the genome

Is the absence of hth/exd proximal identity and dac intermediate identity in tardigrade legs derived, or the ancestral state of Panarthropoda?

p. 10: "However, it is less clear whether the absence of proximal identity specified by hth/exd and intermediate identity specified by dac represents a derived state of H. exemplaris and other tardigrades or the ancestral state of Panarthropoda."
Game & Smith 2025. An Sp6-9 ortholog is expressed in developing legs in the tardigrade Hypsibius exemplaris

Is the caterpillar midgut pushed forward by the terminal segments or pulled by the head and thorax during crawling, and does gut movement actively contribute to locomotion?

p. 661: "It is not clear if the gut is being pushed forward by the terminal segments or being pulled forward by the head and thorax (Simon et al., 2010b)."
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

Is the segmentation clock period set explicitly by the concentrations of timing factors such as Caudal, or is the slowing of the clock an inherently transient step in its switch from oscillating to non-oscillating?

p. 6: 'Currently, it is unknown whether the period of the clock is indeed explicitly determined by the concentrations of particular timing factors ... or whether the slowing of the segmentation clock is an inherently transient phenomenon'
Clark et al. 2019. Arthropod segmentation

Is the tardigrade head one segment (Mayer 2013a, Smith 2016) or at least two to three (Persson 2012/2014; two pairs of stylet glands), and which frame should the model's segment numbering use?

p. 1 (abstract): "it is unclear whether the tardigrade head and its enclosed brain comprises one, or several segments, or a non-segmental structure"
Mayer et al. 2013. Neural markers reveal a one-segmented head in tardigrades (water bears)

Is there any significant difference in the general principles of embryogenesis between an invariant-lineage embryo (C. elegans) and embryos with variable lineages?

p. 264, Conclusion: "A key question arising now is whether there is any significant difference in the general principles of embryogenesis between C. elegans and other organisms."
Schnabel et al. 1997. Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of dev

To what extent is the onychophoran nervous system segmented at the level of neuron identity (e.g. segmentally repeated engrailed-expressing motoneurons) rather than gross anatomy?

p272 left col.: "Future studies using various neural and molecular markers will show to what extent the onychophoran nervous system is segmented."
Mayer & Whitington 2009. Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda

To which head segment does each onychophoran post-oral commissure (#1–#5) belong, given that their somata lie in both the jaw (deutocerebral) and slime-papilla segments?

p. 8: "The anteriorly shifted position of somata makes it difficult to assign each post-oral commissure to a particular head segment."
Martin & Mayer 2015. Insights into the segmental identity of post-oral commissures and pharyngeal nerves in Onychophora based on retrograde fills

Was Lim1 expressed broadly or in regional domains in the legs of the panarthropod ancestor, given broad expression in H. exemplaris and tip-only expression in E. kanangrensis?

PDF p 11: "whether Lim1 ancestrally has an expression pattern that was broad or more regionalized remains unknown"
Mapalo et al. 2024. Expression of distal limb patterning genes in Hypsibius exemplaris indicate regionalization and suggest distal identity of tardigrade legs

Were Wnt1, Wnt6, Wnt7, Wnt8 and Wnt10 lost in the ancestor of all tardigrades or only within the lineage leading to H. exemplaris and R. varieornatus?

p. 9: "whether these genes were lost in an ancestor of all tardigrades, or within crown group Tardigrada is unclear"
Chavarria et al. 2021. Extensive loss of Wnt genes in Tardigrada

What activates He-Dll in the lateral ectoderm where H. exemplaris legs form: does He-Sp6-9A activate or maintain it, and which Wnt ligand (Wnt4 from endomesoderm?) replaces wingless?

p. 10: "Functional studies are required to precisely determine the upstream regulators of He-Dll expression in H. exemplaris."; p. 2: "it is unclear whether the upstream mechanisms that activate Dll expression during appendage development are conserved in this clade."
Game & Smith 2025. An Sp6-9 ortholog is expressed in developing legs in the tardigrade Hypsibius exemplaris

What do 3D spatial transcriptomes of earlier Drosophila embryonic stages show about tissue developmental trajectories, given that most organogenesis happens before the late stages profiled?

p. 1280–1281 (Limitations): 'it is difficult to construct tissue development trajectories with only late-stage embryo and larva samples ... We will include more stages of embryo data'
Wang et al. 2022. High-resolution 3D spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae

What do engrailed, Pax3/7, Wnt4, Wnt16B, hedgehog and other homeobox genes do in H. exemplaris segmentation, and does Engrailed act before segments are visible?

p. 15: "The precise developmental roles of Wnt4, Wnt16B, and other Wnt genes need to be clarified" … "other commonly conserved components of the segment polarity network, such as hh, need to be investigated in tardigrades"
Chavarria et al. 2021. Extensive loss of Wnt genes in Tardigrada

What do the actin fibres in onychophoran extra-embryonic tissue do during germ-band closure?

p. 8: "Since the function of these actin fibres is currently unknown, future studies should focus on identifying the processes involved in the germ band closure of onychophorans"
Treffkorn et al. 2022. Review of extra-embryonic tissues in the closest arthropod relatives, onychophorans and tardigrades

What do the opsins that are enriched in H. exemplaris eggs (rhabdomeric and related lineages) do during embryogenesis?

PDF p 8: "further work would be required to determine the exact function of these new tardigrade egg rhabdomeric opsins"
Fleming et al. 2021. New Tardigrade Opsins and Differential Expression Analyses Show Ontogenic Variation in Light Perception

What do the tardigrade-specific genes expressed in development do (e.g. the grey60 module at limb-bud and pharynx stages, suggested to act in ecdysone synthesis and storage-cell formation), and how do cells vary along developmental trajectories?

p. 11 of 15: "The identification of the specific genes influencing life activities during developmental stages, along with the roles of TSGs, is still incomplete. Further in‐depth analyses and experiments, such as single‐cell transcriptomics ... while in situ hybridization experiments can provide insights into the spatial expression"
Li et al. 2024. Conserved and specific gene expression patterns in the embryonic development of tardigrades. Evol Dev 26

What mechanisms reduce nuclear growth at later preimplantation stages, so that daughter nuclei downscale without halving?

p. 8: 'This suggests that nuclear growth is reduced at later developmental stages (Fig. S12). Future work is required to uncover the specific biological mechanisms controlling developmental-stage-dependent nuclear growth.'
Nunley et al. 2024. Nuclear instance segmentation and tracking for preimplantation mouse embryos

What roles do He-hth, He-exd3 and He-Dll play in patterning the proximodistal axis of H. exemplaris legs (proximal and distal identity, segmental leg identity with Hox genes, or other roles)?

p. 6: "Functional data for He-hth, He-exd3 and He-Dll are required to determine the precise roles of these genes in regulating the development of PD axes in Tardigrada."
Game & Smith 2020. Loss of intermediate regions of perpendicular body axes contributed to miniaturization of tardigrades

What was the ancestral arthropod clock-and-wavefront system (clock network topology, timing-factor wavefronts from a retracting SAZ, and their interaction), and how did segmentation networks diversify into simultaneous and double-segment modes?

p. 12: 'We anticipate that future investigation will centre on two contrasting but inter-related tasks. First, better resolving the nature of the ancestral arthropod clock-and-wavefront system ... Second, reconstructing how arthropod segmentation networks have diversified over time' and 'The most pressing next step is to collect good-quality multiplexed expression data from a variety of arthropod species'
Clark et al. 2019. Arthropod segmentation

What, if anything, does He-Sp6-9B do in embryogenesis given its diffuse expression, and what were the consequences of losing Sp5 in tardigrades?

p. 7: "It is unclear what functions, if any, He-Sp6-9B could be playing during embryogenesis, given its broad and diffuse expression."; "The consequences of the loss of Sp5 in tardigrades remain unclear."
Game & Smith 2025. An Sp6-9 ortholog is expressed in developing legs in the tardigrade Hypsibius exemplaris

When between the eight-cell stage and 12 hpl does vasa mRNA become enriched in the PGC lineage, and is Vasa protein localised earlier?

p. 47: "it remains an open question exactly when enrichment for vasa begins in a subset of cells"
Heikes et al. 2023. The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris

When do mesodermal cells become distinct from endoderm in the endomesodermal pouches of H. exemplaris?

PDF p 3: "It is unclear precisely when mesodermal cells differentiate in H. exemplaris."
Smith et al. 2024. Developmental and genomic insight into the origin of the tardigrade body plan

When, how often and by what regulatory change (new read-out enhancers or new clock genes) did double-segment (pair-rule) periodicity evolve in insects and centipedes?

p. 8: 'it is not clear exactly when or how many times pair-rule patterning evolved in either of the centipede or insect lineages' ... 'it will be informative to find out how the gene expression and enhancer logic of pair-rule species compares with their closest segmental relatives'
Clark et al. 2019. Arthropod segmentation

Where and how has self-organisation been selected over determinism in the evolution of cleavage patterns?

p. 679: "More quantitative studies of the generic designs that regulate cleavage may unravel where and how self-organization has been selected over determinism in evolution."
Pierre et al. 2016. Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos

Which Paired-box orthologue(s) does the cross-reactive antibody detect in the developing brain and ganglia of H. exemplaris (Gabriel & Goldstein 2007)?

p. 334, Fig. 4 legend: "However, it is unclear which Paired box ortholog(s) were actually detected in this study, because the cross-reactive antibody used detects several distinct Paired box orthologs in euarthropods (Davis et al., 2005)."
Smith & Goldstein 2017. Segmentation in Tardigrada and diversification of segmental patterns in Panarthropoda

Which gene-regulatory changes account for the loss of terminal segment addition in the tardigrade lineage, given that caudal is still expressed posteriorly?

p. 338, section 6: "this approach might provide valuable insight into the gene regulatory changes that account for the loss of terminal segment addition in the tardigrade lineage."
Smith & Goldstein 2017. Segmentation in Tardigrada and diversification of segmental patterns in Panarthropoda

Why do embryos rotate inside the eggshell during cleavage and gastrulation, and why in different directions from similar starting orientations?

p. 248: "We do not know currently what causes the rotation of the embryos."; p. 256: "An appealing question to ask is why the embryos rotate at all and why in different directions from an apparently similar initial orientation."
Schnabel et al. 1997. Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of dev

Why does the pha-4::HIS-24::DsRed strain (RW10007) express brightly in the non-pharyngeal sister lineages ABalpp, ABarap and MSxp, unlike the mCherry pha-4 strains, and is this biologically relevant?

p. 706: 'Whereas determining the cause and biological relevance of this observation will require additional experiments, our ability to detect the differences in pattern demonstrates the utility of lineage analysis'
Murray et al. 2008. Automated analysis of embryonic gene expression with cellular resolution in C. elegans

Are tardigrade pigment-cup eyes homologous to arthropod median ocelli, to onychophoran eyes, or to other bilaterian eyes, and how diverse are photoreceptors across Tardigrada?

pp. 401, 406: "the significance of these eyes for the evolution of arthropod visual systems is unclear yet" … "future studies may reveal a larger structural diversity of photoreceptors in the Tardigrada with traits usable for phylogenetic interpretations"
Greven 2007. Comments on the eyes of tardigrades

Are tardigrades the sister group of arthropods, of onychophorans plus arthropods, or closer to nematodes and other cycloneuralians?

p. 12: "the position of tardigrades, and the issue of panarthropod phylogeny in general, remains an open question"
Gross & Mayer 2015. Neural development in the tardigrade Hypsibius dujardini based on anti-acetylated α-tubulin immunolabeling

Can a neuromechanical simulation replay an animal's pose-estimated kinematics in real time to infer contacts and drive closed-loop experimental perturbations?

p. 2361: "NeuroMechFly could be used during experiments to replay an animal's kinematics as captured by pose estimation methods, enabling the real-time inference of dynamic variables such as contacts"
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

Can the principles of whole-brain organisation found in C. elegans (spatial neighbourhoods, core versus variable circuit, layered modular map) inform other nervous systems?

p. 105 (PDF p. 1): 'Thus, it remains to be determined whether lessons about whole-brain organization in C. elegans can inform questions and approaches for other systems.'
Brittin et al. 2021. A multi-scale brain map derived from whole-brain volumetric reconstructions

Can whole-body connectomics and cellular-resolution transcriptomics be integrated for an entire animal across developmental stages, and do registrations hold for less stereotyped animals?

PDF p. 16 (Cell 184:4833): 'With our new resource, this can now be extended to integrate connectomics and transcriptomics for an entire animal and for different developmental stages.'
Vergara et al. 2021. Whole-body integration of gene expression and single-cell morphology

Do multidendritic body-wall sensory neurons (MDC) or stretch receptor organs have a role in caterpillar locomotion?

p. 665: "There is no direct evidence that the MDC has a role in locomotion"
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

Do protostome (including tardigrade) neuropsin/opsin-5 homologues work as UV-sensitive receptors or as retinal photoisomerases?

p. 2387: "it would be interesting to know whether the protostome neuropsin/opsin-5 homologs also have a function as UV-sensitive GPCRs or rather act as retinal photoisomerases"
Hering & Mayer 2014. Analysis of the opsin repertoire in the tardigrade Hypsibius dujardini provides insights into the evolution of opsin genes in panarthropoda

Do the chelicerate-like brain features of onychophorans (arcuate body, heterolateral mushroom body lobes, protocerebral glomeruli) reflect common descent or convergence, given that molecular phylogenies place Onychophora outside Euarthropoda?

p194 left col.: "Are the features described here sufficient to propose that the Onychophora might be considered a highly divergent branch of the chelicerates ...? At present, this issue remains unresolved."
Strausfeld et al. 2006. The organization and evolutionary implications of neuropils and their neurons in the brain of the onychophoran Euperipatoides rowelli

Does H. exemplaris have a subpharyngeal ganglion linking brain and first trunk ganglia (reported by TEM and Persson 2012/2014, not found by Zantke 2008 or Mayer 2013a)?

p. 1353: "Although the early origin of the suboesophageal ganglion remains unclear, it seems likely that it is formed by an outgrowth of the brain"
Hejnol & Schnabel 2005. The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations

Does the bifurcating onychophoran optic nerve (to arcuate body and to mushroom body) mean the single onychophoran eye corresponds to both the median and the lateral eyes of spiders?

p191 left col.: "Might this bifurcation of the retinal representation to central neuropils suggest that the single eye of the velvet worm corresponds to the median and primary lateral eyes of araneans?"
Strausfeld et al. 2006. The organization and evolutionary implications of neuropils and their neurons in the brain of the onychophoran Euperipatoides rowelli

Does the onychophoran optic tract supply one or two visual neuropils?

p. 583: "Another yet unresolved issue concerns the number of visual neuropils supplied by the onychophoran eyes."
Martin et al. 2017. The nervous and visual systems of onychophorans and tardigrades: learning about arthropod evolution from their closest relatives

How are light signals transduced in the eyes of H. exemplaris, integrated in the brain and turned into phototaxis?

p. 13: "the pathways by which light signals are transduced, integrated, and transformed into behavioral outputs like phototaxis remain unknown"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

How is the reproducible neighbourhood organisation of the C. elegans nerve ring orchestrated during development (pioneer neurites, guidance molecules, nested mechanisms)?

p. 109 (PDF p. 6): 'How the neighbourhood organization is developmentally orchestrated remains an open question.'
Brittin et al. 2021. A multi-scale brain map derived from whole-brain volumetric reconstructions

How large is the variable part of the C. elegans nerve-ring connectome, beyond the conserved core?

p. 109 (PDF p. 6): 'Although determining the extent of the variable circuit is challenging due to the technical limitations of synaptic scoring and will therefore require multiple further connectomes, it is noteworthy that conserved synapses—like most variable ones—are constrained by the same contactome.'
Brittin et al. 2021. A multi-scale brain map derived from whole-brain volumetric reconstructions

How many degrees of freedom do the Drosophila head–pedicel and pedicel–funiculus antennal joints have?

Methods (PDF p. 11): "We gave the remaining joints (that is, head–pedicel and pedicel–funiculus) all three rotational DoFs because the real number of DoFs in these antennal joints remains unknown."
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

How many neurons does H. exemplaris have, and is the number fixed?

p. 3: "the exact number of neurons in any tardigrade life-history stage remains unknown"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

How widespread are the opsin duplications found in single tardigrade genera: is the Echiniscus ciliary-opsin duplication shared by Echiniscoidea or Heterotardigrada, are the Batillipes duplications shared by Arthrotardigrada, and what do the independently duplicated arthropsins do?

PDF p 4: "Further studies will help elucidate whether this duplication is specific to the genus, or to Heterotardigrada or Echiniscoidea as a group." … "Further study of the independent duplications of arthropsin within both Tardigrada and Daphnia could advance our understanding of the function of this recently discovered homolog." … "Whether the duplications in Batillipes are shared among other members of the Arthrotardigrada is unclear"
Fleming et al. 2021. New Tardigrade Opsins and Differential Expression Analyses Show Ontogenic Variation in Light Perception

Is Er-c-opsin transcribed from both strands in E. rowelli (as the sense-probe signal suggests), and if so what does the antisense transcript do?

p920 left col.: "The putative role of the Er-c-opsin antisense transcript in E. rowelli is unknown, but it might be involved in a self-regulation of Er-c-opsin expression"
Beckmann et al. 2015. Spectral sensitivity in Onychophora (velvet worms) revealed by electroretinograms, phototactic behaviour and opsin gene expression

Is direct visual input from median ocelli (or their homologues) to the central body part of the euarthropod ground pattern, as in onychophorans and chelicerates?

p242 right col.: "Further studies should clarify whether this feature belongs to the ground pattern of Euarthropoda since there is evidence that neuropils of the nauplius eyes ... in malacostracan crustaceans are connected with a cluster of cells that also innervate the central body"
Mayer 2006. Structure and development of onychophoran eyes: what is the ancestral visual organ in arthropods?

Is the arthropod labrum a derived arthropod structure, or do onychophorans or tardigrades have a homologous structure?

p. 579: "it is still unclear whether the labrum represents a derived feature of arthropods or whether homologous structures occur in other animal groups"
Martin et al. 2017. The nervous and visual systems of onychophorans and tardigrades: learning about arthropod evolution from their closest relatives

To what extent do behavioural differences between individual worms come from differences in connectivity, and how much does activity or experience reinforce connections?

p. 68 (PDF p. 6): 'Reinforcement of connectivity by circuit activity and hence experience has been demonstrated but little studied so far. The extent to which differences in the behaviour of individual worms is due to differences in connectivity remains to be explored.'
Cook et al. 2019. Whole-animal connectomes of both Caenorhabditis elegans sexes

Were the ventral nerve cords of the last common ancestor of Panarthropoda and of Ecdysozoa paired or unpaired, and medullary or ganglionated?

p. 15: "the ancestral condition of the ventral nerve cords within Ecdysozoa (paired versus unpaired) remains ambiguous and requires further investigation"
Martin et al. 2017. Assessing segmental versus non-segmental features in the ventral nervous system of onychophorans (velvet worms)

What are the electron-dense pigment granules of the H. exemplaris eye pigment cell made of?

bioRxiv p. 10, lines 296–297: "whose biochemical" … "composition remains unknown"
Dutta et al. 2026. Extensive opsin gene expansion and non-cerebral origin of the minimalist eye in a model tardigrade

What becomes of the third, laterally oriented mushroom-body peduncle of the 6-dpf Platynereis worm in later stages?

PDF p. 11 (Cell 184:4828): 'A third prominent peduncle of unknown fate is oriented laterally and perpendicular to the dorsal and ventral peduncles'
Vergara et al. 2021. Whole-body integration of gene expression and single-cell morphology

What do comparative connectomes within and across species reveal about the origin of similarities and differences in nervous-system structure and behaviour?

p. 260 (PDF p. 4), Discussion: 'Comparative connectomics is needed to reveal the origin of similarities and differences in structure and behaviour within and across species.'
Witvliet et al. 2021. Connectomes across development reveal principles of brain maturation

What do the mushroom bodies, 'olfactory' glomeruli and giant neurons of the onychophoran brain do, and how many giant neurons are there and where do they project (E. rowelli)?

p. 9: "The function of the onychophoran mushroom bodies is unknown."
Schumann et al. 2016. Immunolocalization of Arthropsin in the Onychophoran Euperipatoides rowelli (Peripatopsidae)

What do the three PDF neuropeptides of H. exemplaris do, including their signalling to storage cells, and what are the ventral extracerebral PDF-immunoreactive cells?

pp. 11–12: "the identity, detailed morphology and potential function of the ventral extracerebral PDF-ir cells remain to be clarified" … "The functional role of these differences between the three peptides as well as their different localization in the head (figure 8a–c) is unclear." … "The role of PDF/PDFR signalling in storage cells is unclear"
Dutta et al. 2025. Pigment-dispersing factor neuropeptides act as multifunctional hormones and modulators in tardigrades

What does c-opsin do in onychophoran optic ganglion cells and protocerebral neurons: non-visual, circadian photoreception?

p920 left col.: "Although the function of the onychophoran c-opsin protein is unknown, it might be involved in non-visual, extraocular photoreception associated with circadian clock mechanisms"
Beckmann et al. 2015. Spectral sensitivity in Onychophora (velvet worms) revealed by electroretinograms, phototactic behaviour and opsin gene expression

What neuron types, transmitters and synaptic connections make up the H. exemplaris nervous system?

p. 3: "neuronal identities and types have not been established, their connectome has yet to be reconstructed"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

Where does the onychophoran heart nerve originate, and is it connected to the brain?

p. 570: "its origin and the question of whether or not it is associated with the brain remain to be clarified"
Martin et al. 2017. The nervous and visual systems of onychophorans and tardigrades: learning about arthropod evolution from their closest relatives

Which cell types keep proliferating after hatching in different tardigrade species, and is the absence of proliferation in epidermis and nervous system shared across clades?

PDF p 7: "it would be valuable to reevaluate the proliferative capacities of different cell types in a range of tardigrade species using contemporary molecular techniques like EdU incorporation"
Quiroga-Artigas & Moriel-Carretero 2024. Storage cell proliferation during somatic growth establishes that tardigrades are not eutelic organisms

Which of the C. elegans connections that differ between the hermaphrodite and male reconstructions are true sex differences rather than individual variability or reconstruction error?

PDF p. 27 (Extended Data legend): 'Whether the edges highlighted here are true sex differences needs to be tested by methodology that allows many animals to be examined.'
Cook et al. 2019. Whole-animal connectomes of both Caenorhabditis elegans sexes

Which opsins does each H. exemplaris eye photoreceptor express, and what are the spectral sensitivities of He-c-opsin-1 and -2?

PDF p 8: "Further analysis of the Tardigrada would be required to positive confirm the lack of dual expression within the eye."
Fleming et al. 2021. New Tardigrade Opsins and Differential Expression Analyses Show Ontogenic Variation in Light Perception

Why do the four trunk ganglia differ in size (narrower first and fourth), and is a differentiated first and fourth ganglion ancestral for Tardigrada?

PDF p 12: "The functional significance, if any, for different sizes among ganglia remains unclear."
Smith & Jockusch 2014. The metameric pattern of Hypsibius dujardini(Eutardigrada) and its relationship to that of other panarthropods

Would measured tendons and muscles of the Drosophila musculoskeletal system, and connectome-constrained motor circuits, improve the interface between neural controllers and the simulated body?

p. 2361: "careful measurements and analyses of the Drosophila musculoskeletal system (that is, tendons and muscles) could improve the interface between neural network controllers and the biomechanical embodiment"
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

By what mechanism do Drosophila legs release adhesion at lift-off?

p. 2355: "Although liftoff mechanisms are known for some insects36,37, they are not known for D. melanogaster."
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

Did the tardigrade stylet apparatus evolve from the ancestral panarthropod frontal appendages, from a pair of legs, or from no appendage at all?

p. 338, section 6: "did the frontal appendages give rise to the stylet apparatus of tardigrades (Ou et al., 2012), or is the stylet apparatus derived from a pair of legs (Nielsen, 2001)? ... possibly the stylet apparatus has a non-appendicular origin, which may become apparent via studies of canonical appendage patterning genes in tardigrades."
Smith & Goldstein 2017. Segmentation in Tardigrada and diversification of segmental patterns in Panarthropoda

Has long-term laboratory culture changed H. exemplaris behaviour, such as the locomotor differences seen against Ramazzottius?

p. 20: "The differences between H. exemplaris and Ramazzottius could potentially arise from effects of long-term culturing in the lab; further work will be required to investigate this possibility."
Anderson et al. 2024. Comparative analysis of tardigrade locomotion across life stage, species, and disulfiram treatment

How do walking kinematics vary across tardigrade species and over a wider range of speeds, and does the stance-duration model of interleg coordination hold across panarthropods?

bioRxiv p. 11: "Future work should expand upon both of these parameters, both by surveying walking dynamics across Tardigrada"
Nirody et al. 2021. Tardigrades exhibit robust interlimb coordination across walking speeds and terrains

How does disulfiram disrupt locomotion in H. exemplaris, and is it by blocking octopamine synthesis through tyramine β-hydroxylase?

p. 20: "While the mechanism by which disulfiram disrupts locomotion in tardigrades remains unknown"
Anderson et al. 2024. Comparative analysis of tardigrade locomotion across life stage, species, and disulfiram treatment

How does locomotion vary across caterpillar species, given it has not been studied widely or described systematically?

p. 657: "Despite the environmental and agricultural importance of caterpillars, their locomotion has not been studied widely or described systematically."
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

How many muscles enter each leg of H. exemplaris under one explicit counting rule (fibres vs fused muscles; include lateral-group fibres or not)? Reported: Hypsibius sp. 14/15/14/6 (dorsal+ventral), H. exemplaris 14/12/11/10 (sweep summary), other eutardigrades 10–14 in legs I–III and 4–6 in leg IV.

PDF p 2: "Whether these and other discrepancies are attributable to species differences or perhaps result from different techniques is unclear."
Gross & Mayer 2019. Cellular morphology of leg musculature in the water bear Hypsibius exemplaris (Tardigrada) unravels serial homologies

Is caterpillar crawling driven by a central pattern generator in the intact animal, given CPGs are confirmed only in fictive preparations?

p. 666: "CPGs in Manduca sexta have only been confirmed in fictive preparations"; p. 667: "direct evidence in intact animals is lacking"
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

What is the mechanism of inching locomotion in caterpillars?

p. 656: "The mechanism of inching has not been investigated in detail, but crawling is now well understood"
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

What is the source of idiothetic cues for path integration in flies; are they ascending leg proprioceptive and tactile signals?

p. 2357: "The source of these idiothetic cues for path integration is unknown but may, in principle, be derived from ascending leg proprioceptive and tactile signals."
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

Why do late fifth-instar (wandering) Manduca caterpillars not take extra thoracic steps if those steps serve to probe the substrate?

p. 662: "It is not clear why extra steps are not seen in these larvae if their function is indeed to examine the substrate."
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

Can spatial transcriptome reconstructions be guided by the actual anatomy, by integrating imaging of each cryosection with the Stereo-seq data from the same slice, so that reconstructed tissues match real boundaries?

p. 1280 (Limitations): 'dissimilarities remain between 3D reconstructed tissue models and their actual locations and boundaries ... In the future, this can be achieved by integrating imaging information of the cryosection slice and Stereo-seq data from the same slice.'
Wang et al. 2022. High-resolution 3D spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae

Do Dsup proteins of other eutardigrades, including H. exemplaris, protect DNA in vivo as R. varieornatus Dsup does, and what do heterotardigrades without Dsup use instead?

pp. 1819, 1826: "it is unclear whether other eutardigrade species’ Dsup proteins have the same protective abilities" … "even within those lineages it is unclear to what extent Dsup plays a role in IR"
Clark-Hachtel et al. 2024. The tardigrade Hypsibius exemplaris dramatically upregulates DNA repair pathway genes in response to ionizing radiation

Do tardigrades tolerate desiccation and radiation with the same molecular mechanisms?

p. 325 (PDF p 327): "the extent to which tolerance to desiccation is related to tolerance to radiation, and relies on the same molecular mechanisms, is still unclear"
Jönsson KI, Levine EB, Wojcik A, Haghdoost S, Harms-Ringdahl M 2018. Environmental Adaptations: Radiation Tolerance. In:

Do the abundant heat-soluble proteins of Echiniscus testudo (EtAHS) contribute to its desiccation tolerance?

PDF p 9: "Functional experiments would be required as a future work to fully confirm the contribution of EtAHS in the desiccation tolerance of heterotardigrada."
Murai et al. 2021. Multiomics study of a heterotardigrade, Echinisicus testudo, suggests the possibility of convergent evolution of abundant heat-soluble proteins in Tardigrada

Does time spent in anhydrobiosis extend the life span of H. exemplaris (the 'Sleeping Beauty' hypothesis), and how does drying affect later reproduction?

p. 275 (PDF p 278): "Very little is known about the effects of anhydrobiosis on life cycle and longevity achieved thereby."
Schill RO, Hengherr S 2018. Environmental Adaptations: Desiccation Tolerance. In: Schill RO (ed.) Water Bears: The Biolo

How does H. exemplaris repair widespread DNA damage without producing chromosome translocations and fusions?

PDF pp 2 (abstract), 3: "How tardigrades can repair widespread DNA damage without producing frequent, large-scale chromosome structural abnormalities, like chromosome translocations and fusions, is unknown." … "It is unknown how H. exemplaris endures or fixes DNA damage from these extreme environments without extensively impacting the genome."
Papell et al. 2026. Chromosomes remain individualized through interphase in embryos of the tardigrade Hypsibius exemplaris

How much do DNA protection (Dsup), DNA repair, protection of proteins against carbonylation and low cell proliferation each contribute to the radiation tolerance of H. exemplaris and other tardigrades?

p. 11: "Further studies are needed to evaluate the relative importance of eutely vs. other mechanisms (e.g., DNA repair system, protein protection, apoptosis avoidance) in radiation tolerance of tardigrades."
Beltrán-Pardo et al. 2015. Tolerance to Gamma Radiation in the Tardigrade Hypsibius dujardini from Embryo to Adult Correlate Inversely with Cellular Proliferation

In which tissues are the CAHS paralogues and other tardigrade-specific protectants expressed during anhydrobiosis, and do CAHS subfamilies have different roles?

pp. 1, 7: "little is known about the actual tissue specificity or the actual workings of these tardigrade-specific IUPs within tardigrades during anhydrobiosis" … "future studies should analyze in more detail the differences in CAHS subfamilies and their tissue specificity"
Tanaka, Aoki & Arakawa 2023. In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging

Which genes beyond the known protectants contribute to anhydrobiosis in H. exemplaris: induced cytochrome P450s, solute carriers and apolipoproteins, calcium–calmodulin signalling, or proteins rich in low-complexity and disordered regions?

pp. 14, 22: "The contributions to anhydrobiosis of additional genes identified as up-regulated (including cytochrome P450, several solute carrier families, and apolipoproteins) are unknown." … "but it is still unclear how this is related to anhydrobiosis"
Yoshida et al. 2017. Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus

Why are Cas9 double-strand breaks in H. exemplaris mostly rejoined without indels, and does Dsup protect the broken ends?

p. 201: "Although the mechanisms underlying the preferred no-indel end-joining remain unclear"
Kumagai et al. 2022. Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades

Can H. exemplaris be stably transformed, edited heritably and screened by forward genetics?

pp. 186, 192: "Perhaps the methods most needed now for diverse kinds of further studies are transgenesis and CRISPR-mediated DNA editing." … "To date, forward genetic screening using mutagenesis has not been reported."
Goldstein 2022. Tardigrades and their emergence as model organisms

Can reference-based age estimation be adapted to data other than genome-wide expression, such as genome-wide binding data?

p. 974: 'Another avenue for improvement is to adapt this approach to data other than genome-wide gene expression, such as genome-wide binding data.'
Bulteau & Francesconi 2022. Real age prediction from the transcriptome with RAPToR

How can the local ordering and orientation of small adjacent contigs in Hi-C (3D-DNA) assemblies be resolved, given that Hi-C gives long-range but weak short-range information?

p. 95: 'the current approach is not perfect for local ordering of small adjacent contigs. This might be circumvented by more sophisticated analysis of Hi-C data. Additional data (such as long or paired-end reads) could also improve the results.'
Dudchenko et al. 2017. De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds

Which promoter lengths, genetic cargo and cofactors give reliable, cell-type-specific transgene expression in H. exemplaris, and are tardigrade regulatory sequences unusually compact?

p. 5: "The choice of genetic cargo and necessary cofactors is an open question." … "it is unclear whether this promoter length is optimal, or if regulatory sequences in tardigrades are uniquely compact"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

Why did unedited G0 siblings of DIPA-CRISPR-injected R. varieornatus mothers show the same growth retardation as frameshift mutants?

p. 13: "It remains unclear why the unedited siblings exhibited similar growth retardation."
Kondo et al. 2024. Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR

Are the large 'one-cell' glands of arthrotardigrades homologous to insect oenocytes, and are they involved in moulting?

p189 left col.: "the conspicuous 'one-cell' glands ... could be an important morphological character if they were proven to be homologues to oenocytes of insects ... hence it could have merit to conduct research on these cells in the future."
Persson et al. 2014. Brain anatomy of the marine tardigrade Actinarctus doryphorus (Arthrotardigrada)

Can the rare males that appear in thelytokous tardigrade strains (Milnesium sp.) fertilise eggs, and what triggers their appearance?

p. 232 (PDF p 237): "It is unknown whether these males can actually function in sexual reproduction"
Altiero T, Suzuki AC, Rebecchi L 2018. Reproduction, Development and Life Cycles. In: Schill RO (ed.) Water Bears: The B

Can transcriptome staging (RAPToR) be extended to stage samples on multiple branching trajectories rather than one developmental or ageing trajectory?

p. 974: 'RAPToR can currently only stage on one developmental or ageing trajectory, so a future improvement will be to provide RAPToR with the ability to stage on multiple branching trajectories.'
Bulteau & Francesconi 2022. Real age prediction from the transcriptome with RAPToR

How do cell size, cell number and fat stores change with feeding, starvation and DNA damage, and how do they set body size in H. exemplaris?

PDF p 15: "Future studies examining cellular dimensions, numbers, and fat depots under various conditions will be crucial"
Quiroga-Artigas et al. 2026. Compromised DNA replication in gut cells underlies sensitivity to genotoxic stress in the tardigrade Hypsibius exemplaris

How is pseudostratified epithelial organisation acquired and maintained under growth and morphogenetic deformation?

p. 303: 'How this structure is acquired and maintained under growth and morphogenetic deformation is still largely unknown.'
Runser et al. 2024. SimuCell3D: three-dimensional simulation of tissue mechanics with cell polarization

How long has H. exemplaris reproduced asexually, and is its heterozygosity still rising or at equilibrium?

pp. 8, 10: "it remains an open question whether heterozygosity in this spe" … "Future work to compare heterozygosity levels between tardigrade lineages may shed additional light on the question of how long this and other tardigrade species have been reproducing asexually."
Coke et al. 2026. Modified meiosis in the tardigrade Hypsibius exemplaris maintains heterozygosity across the genome

Do the properties of jammed deformable-particle packings depend on surface roughness (vertex spheres versus smooth sphero-polyhedra)?

p. 9911: 'It will be interesting to determine whether any of the properties of jammed packings of deformable particles depend on the surface roughness.'
Wang et al. 2021. The structural, vibrational, and mechanical properties of jammed packings of deformable particles in three dimensions

How can shape-resolving multicellular simulations be scaled to thousands of cells at acceptable cost?

p. 9: 'there is still a great need from the field to keep increasing the cell number and decreasing the computational cost. For example, the early Drosophila embryogenesis involves thousands of cells'
Kuang et al. 2023. MorphoSim: an efficient and scalable phase-field framework for accurately simulating multicellular morphologies

How do cell stiffness and surface tension shape the dynamics of engineered multicellular aggregates, and how can parameters be chosen to optimise a target function?

p. 8: '(1) How do biological parameters like cell stiffness and surface tension affect the dynamics? ... and (2) How to choose the parameter combination to optimize a specific function?'
Kuang et al. 2023. MorphoSim: an efficient and scalable phase-field framework for accurately simulating multicellular morphologies

How should one choose a cell-mechanics model (particle, phase field, deformable mesh) for a given biological system with enough accuracy at low cost?

p. 18: 'How to choose a model for a specific biophysical scene with enough accuracy and low cost becomes a practical problem and merits further study'
Kuang et al. 2022. Computable early Caenorhabditis elegans embryo with a phase field model

What forces drive interkinetic nuclear migration in pseudostratified epithelia?

p. 304: 'processes such as interkinetic nuclear migration in pseudostratified epithelia could be simulated at unprecedented resolution to address open questions regarding the driving forces.'
Runser et al. 2024. SimuCell3D: three-dimensional simulation of tissue mechanics with cell polarization

What links quartic eigenmodes of the dynamical matrix to the non-trivial power-law scaling of shear modulus with pressure in jammed deformable particles?

p. 9911: 'We encourage future studies to understand the link between quartic eigenmodes of the dynamical matrix and the non-trivial power-law scaling of ⟨G(P)⟩.'
Wang et al. 2021. The structural, vibrational, and mechanical properties of jammed packings of deformable particles in three dimensions

Are onychophoran nephridia homologous to annelid nephridia, given their different innervation (from somata in the nerve cords, via a medioventrolateral loop)?

p1205 left col.: "The different innervation patterns and the absence of specific correspondences in the nephridial anatomy and nephridiogenesis ... challenge the homology hypothesis of nephridial organs in Annelida and Panarthropoda ... However, further comparative studies are necessary to clarify this issue."
Mayer & Harzsch 2008. Distribution of serotonin in the trunk of Metaperipatus blainvillei (Onychophora, Peripatopsidae): implications for the evolution of the nervous system in Arthropoda

Can temporal information (video-level deep networks or temporal filtering) improve markerless pose estimation beyond frame-by-frame detection, especially where occlusion is common?

p. 1288: "While temporal information could indeed be beneficial in certain contexts, challenges remain to using end-to-end-trained deep architectures for video data to extract postures ... As this is an active area of research, we believe this situation is likely to change"
Mathis et al. 2018. DeepLabCut: markerless pose estimation of user-defined body parts with deep learning

Do tardigrades use peroxisomes, given their losses of peroxisome biogenesis genes, and are peroxisomes used only during recovery from cryptobiosis?

PDF p 14: "Overall, it remains unclear if and how tardigrades utilize peroxisomes."
Kamilari et al. 2019. Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages

Do the six disk-shaped structures at the 30 h mouth become the six peribuccal lobes, and why is the mouth closed at 45 h?

p. 570: "Exactly how this happens is unclear"
Gross et al. 2017. External morphogenesis of the tardigrade Hypsibius dujardini as revealed by scanning electron microscopy

Does H. exemplaris really lack a nanos ortholog, or was it missed by BLAST?

p. 51: "because a tardigrade homolog of nanos does not exist, or because the published sequence databases are incomplete"
Heikes et al. 2023. The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris

Does the heterotardigrade Echiniscoides sigismundi really lack all p53-family genes?

PDF p 10: "We state the latter with caution and acknowledge that this finding needs further investigation."
Kamilari et al. 2019. Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages

How can Lagrangian surface parameterisation be extended to tubes that branch, fuse or separate?

p. 1986: 'Extending to higher-order networks of tubes and shapes that fuse or separate poses future challenges.'
Mitchell & Cislo 2023. TubULAR: tracking in toto deformations of dynamic tissues via constrained maps

How can rare, scattered cell types (e.g. intestinal stem and enteroendocrine cells) be captured in spatial transcriptomes, for instance by integrating scRNA-seq from the same line?

p. 1280 (Limitations): 'capture of rare cell types with Stereo-seq remains challenging ... we will obtain scRNA-seq data of embryos and larval tissues and integrate them with Stereo-seq data of the same fly line'
Wang et al. 2022. High-resolution 3D spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae

What is the transient developmental function of onychophoran ventral organs in the trunk?

p269 left col.: "Their rapid increase in size and degeneration by apoptosis suggest that the ventral organs play a transient, albeit an important role in onychophoran development. This function remains unclear, except in the antennal segment"
Mayer & Whitington 2009. Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda

Which of the several dicer genes of tardigrades act in development, RNAi and antiviral defence, and how does their expression vary across species?

p. 12: "it is unclear which dicers may also be involved in antiviral defense" … "it is unclear how expression may vary across the other tardigrade species we investigated" … "Future research could focus on characterizing tardigrade small RNAs across various"
Weinberg et al. 2025. Lineage-specific expansions of the Dicer gene family in tardigrades

Which population is the male Hypsibius cf. dujardini in Altiero et al. 2018 Fig. 8.4b, and do males ever occur in H. exemplaris?

p. 556: "It would be useful to be able to both maintain clonal organisms and cross-fertilize organisms as needed, but only parthenogenetic females have been recognized in our cultures to date."
Gabriel et al. 2007. The tardigrade Hypsibius dujardini, a new model for studying the evolution of development

What is the mechanism of inching locomotion in caterpillars?

p. 656: "The mechanism of inching has not been investigated in detail, but crawling is now well understood"
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

How does locomotion vary across caterpillar species, given it has not been studied widely or described systematically?

p. 657: "Despite the environmental and agricultural importance of caterpillars, their locomotion has not been studied widely or described systematically."
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

Is the caterpillar midgut pushed forward by the terminal segments or pulled by the head and thorax during crawling, and does gut movement actively contribute to locomotion?

p. 661: "It is not clear if the gut is being pushed forward by the terminal segments or being pulled forward by the head and thorax (Simon et al., 2010b)."
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

Why do late fifth-instar (wandering) Manduca caterpillars not take extra thoracic steps if those steps serve to probe the substrate?

p. 662: "It is not clear why extra steps are not seen in these larvae if their function is indeed to examine the substrate."
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

Is caterpillar crawling driven by a central pattern generator in the intact animal, given CPGs are confirmed only in fictive preparations?

p. 666: "CPGs in Manduca sexta have only been confirmed in fictive preparations"; p. 667: "direct evidence in intact animals is lacking"
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

Do multidendritic body-wall sensory neurons (MDC) or stretch receptor organs have a role in caterpillar locomotion?

p. 665: "There is no direct evidence that the MDC has a role in locomotion"
van Griethuijsen & Trimmer 2014. Locomotion in caterpillars

Is hairy part of the arthropod segmentation clock (it is unclear in Tribolium, though hairy knockdown truncates Dermestes), or is its role masked by its paralogue deadpan?

p. 4: 'First, whether hairy is involved in the Tribolium segmentation clock or not remains unclear.'
Clark et al. 2019. Arthropod segmentation

Are the oscillating pair-rule genes linked into a single circuit, is it sufficient to oscillate, what is its topology, and how far has it diverged between arthropod lineages?

p. 5: 'it is not yet clear whether the oscillating genes are linked into a single circuit, whether this circuit is sufficient to generate oscillations, what the topology of this circuit is likely to be, nor indeed the extent to which it may have diverged in different lineages'
Clark et al. 2019. Arthropod segmentation

Is the segmentation clock period set explicitly by the concentrations of timing factors such as Caudal, or is the slowing of the clock an inherently transient step in its switch from oscillating to non-oscillating?

p. 6: 'Currently, it is unknown whether the period of the clock is indeed explicitly determined by the concentrations of particular timing factors ... or whether the slowing of the segmentation clock is an inherently transient phenomenon'
Clark et al. 2019. Arthropod segmentation

When, how often and by what regulatory change (new read-out enhancers or new clock genes) did double-segment (pair-rule) periodicity evolve in insects and centipedes?

p. 8: 'it is not clear exactly when or how many times pair-rule patterning evolved in either of the centipede or insect lineages' ... 'it will be informative to find out how the gene expression and enhancer logic of pair-rule species compares with their closest segmental relatives'
Clark et al. 2019. Arthropod segmentation

How do species with post-embryonic segmentation coordinate segment patterning with the moult cycle, given that ecdysone-related genes have segmentation roles in some embryos?

Box 2 (p. 9): 'Another interesting question is how species that undergo post-embryonic segmentation coordinate segment patterning with the moult cycle. Ecdysone-related genes play segmentation roles in some embryos'
Clark et al. 2019. Arthropod segmentation

What was the ancestral arthropod clock-and-wavefront system (clock network topology, timing-factor wavefronts from a retracting SAZ, and their interaction), and how did segmentation networks diversify into simultaneous and double-segment modes?

p. 12: 'We anticipate that future investigation will centre on two contrasting but inter-related tasks. First, better resolving the nature of the ancestral arthropod clock-and-wavefront system ... Second, reconstructing how arthropod segmentation networks have diversified over time' and 'The most pressing next step is to collect good-quality multiplexed expression data from a variety of arthropod species'
Clark et al. 2019. Arthropod segmentation

How many neurons does H. exemplaris have, and is the number fixed?

p. 3: "the exact number of neurons in any tardigrade life-history stage remains unknown"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

What neuron types, transmitters and synaptic connections make up the H. exemplaris nervous system?

p. 3: "neuronal identities and types have not been established, their connectome has yet to be reconstructed"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

How are light signals transduced in the eyes of H. exemplaris, integrated in the brain and turned into phototaxis?

p. 13: "the pathways by which light signals are transduced, integrated, and transformed into behavioral outputs like phototaxis remain unknown"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

How do the brain and trunk ganglia of H. exemplaris coordinate the legs into gaits, switch between gaits and single-limb movements, and does this circuitry share an ancestral plan with insects?

pp. 4, 14: "How the integration of these movement modes produces cohesive whole-body behavior remains unknown." … "the neural mechanisms that coordinate limb movements to produce gaits, or facilitate transitions between gaits, are unknown"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

Which promoter lengths, genetic cargo and cofactors give reliable, cell-type-specific transgene expression in H. exemplaris, and are tardigrade regulatory sequences unusually compact?

p. 5: "The choice of genetic cargo and necessary cofactors is an open question." … "it is unclear whether this promoter length is optimal, or if regulatory sequences in tardigrades are uniquely compact"
Lyons & Kato 2025. The tardigrade as an emerging model organism for systems neuroscience

By what mechanism do Drosophila legs release adhesion at lift-off?

p. 2355: "Although liftoff mechanisms are known for some insects36,37, they are not known for D. melanogaster."
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

What is the source of idiothetic cues for path integration in flies; are they ascending leg proprioceptive and tactile signals?

p. 2357: "The source of these idiothetic cues for path integration is unknown but may, in principle, be derived from ascending leg proprioceptive and tactile signals."
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

How many degrees of freedom do the Drosophila head–pedicel and pedicel–funiculus antennal joints have?

Methods (PDF p. 11): "We gave the remaining joints (that is, head–pedicel and pedicel–funiculus) all three rotational DoFs because the real number of DoFs in these antennal joints remains unknown."
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

Would measured tendons and muscles of the Drosophila musculoskeletal system, and connectome-constrained motor circuits, improve the interface between neural controllers and the simulated body?

p. 2361: "careful measurements and analyses of the Drosophila musculoskeletal system (that is, tendons and muscles) could improve the interface between neural network controllers and the biomechanical embodiment"
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

Can a neuromechanical simulation replay an animal's pose-estimated kinematics in real time to infer contacts and drive closed-loop experimental perturbations?

p. 2361: "NeuroMechFly could be used during experiments to replay an animal's kinematics as captured by pose estimation methods, enabling the real-time inference of dynamic variables such as contacts"
Wang-Chen et al. 2024. NeuroMechFly v2: simulating embodied sensorimotor control in adult Drosophila

When between the eight-cell stage and 12 hpl does vasa mRNA become enriched in the PGC lineage, and is Vasa protein localised earlier?

p. 47: "it remains an open question exactly when enrichment for vasa begins in a subset of cells"
Heikes et al. 2023. The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris

Does H. exemplaris really lack a nanos ortholog, or was it missed by BLAST?

p. 51: "because a tardigrade homolog of nanos does not exist, or because the published sequence databases are incomplete"
Heikes et al. 2023. The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris

Are the primordial germ cells of H. exemplaris specified by induction, by inherited germ plasm, or by a mix of both, and which modes occur across Tardigrada?

p. 52: "It remains an open question what modes of PGC specification exist in the tardigrade phylum." … "Future studies are needed to clarify the mode of PGC specification in this system."
Heikes et al. 2023. The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris

Does the onychophoran optic tract supply one or two visual neuropils?

p. 583: "Another yet unresolved issue concerns the number of visual neuropils supplied by the onychophoran eyes."
Martin et al. 2017. The nervous and visual systems of onychophorans and tardigrades: learning about arthropod evolution from their closest relatives

Is the arthropod labrum a derived arthropod structure, or do onychophorans or tardigrades have a homologous structure?

p. 579: "it is still unclear whether the labrum represents a derived feature of arthropods or whether homologous structures occur in other animal groups"
Martin et al. 2017. The nervous and visual systems of onychophorans and tardigrades: learning about arthropod evolution from their closest relatives

Where does the onychophoran heart nerve originate, and is it connected to the brain?

p. 570: "its origin and the question of whether or not it is associated with the brain remain to be clarified"
Martin et al. 2017. The nervous and visual systems of onychophorans and tardigrades: learning about arthropod evolution from their closest relatives

How widespread are the opsin duplications found in single tardigrade genera: is the Echiniscus ciliary-opsin duplication shared by Echiniscoidea or Heterotardigrada, are the Batillipes duplications shared by Arthrotardigrada, and what do the independently duplicated arthropsins do?

PDF p 4: "Further studies will help elucidate whether this duplication is specific to the genus, or to Heterotardigrada or Echiniscoidea as a group." … "Further study of the independent duplications of arthropsin within both Tardigrada and Daphnia could advance our understanding of the function of this recently discovered homolog." … "Whether the duplications in Batillipes are shared among other members of the Arthrotardigrada is unclear"
Fleming et al. 2021. New Tardigrade Opsins and Differential Expression Analyses Show Ontogenic Variation in Light Perception

What do the opsins that are enriched in H. exemplaris eggs (rhabdomeric and related lineages) do during embryogenesis?

PDF p 8: "further work would be required to determine the exact function of these new tardigrade egg rhabdomeric opsins"
Fleming et al. 2021. New Tardigrade Opsins and Differential Expression Analyses Show Ontogenic Variation in Light Perception

Which opsins does each H. exemplaris eye photoreceptor express, and what are the spectral sensitivities of He-c-opsin-1 and -2?

PDF p 8: "Further analysis of the Tardigrada would be required to positive confirm the lack of dual expression within the eye."
Fleming et al. 2021. New Tardigrade Opsins and Differential Expression Analyses Show Ontogenic Variation in Light Perception

What activates He-Dll in the lateral ectoderm where H. exemplaris legs form: does He-Sp6-9A activate or maintain it, and which Wnt ligand (Wnt4 from endomesoderm?) replaces wingless?

p. 10: "Functional studies are required to precisely determine the upstream regulators of He-Dll expression in H. exemplaris."; p. 2: "it is unclear whether the upstream mechanisms that activate Dll expression during appendage development are conserved in this clade."
Game & Smith 2025. An Sp6-9 ortholog is expressed in developing legs in the tardigrade Hypsibius exemplaris

What, if anything, does He-Sp6-9B do in embryogenesis given its diffuse expression, and what were the consequences of losing Sp5 in tardigrades?

p. 7: "It is unclear what functions, if any, He-Sp6-9B could be playing during embryogenesis, given its broad and diffuse expression."; "The consequences of the loss of Sp5 in tardigrades remain unclear."
Game & Smith 2025. An Sp6-9 ortholog is expressed in developing legs in the tardigrade Hypsibius exemplaris

Is the absence of hth/exd proximal identity and dac intermediate identity in tardigrade legs derived, or the ancestral state of Panarthropoda?

p. 10: "However, it is less clear whether the absence of proximal identity specified by hth/exd and intermediate identity specified by dac represents a derived state of H. exemplaris and other tardigrades or the ancestral state of Panarthropoda."
Game & Smith 2025. An Sp6-9 ortholog is expressed in developing legs in the tardigrade Hypsibius exemplaris

Which Paired-box orthologue(s) does the cross-reactive antibody detect in the developing brain and ganglia of H. exemplaris (Gabriel & Goldstein 2007)?

p. 334, Fig. 4 legend: "However, it is unclear which Paired box ortholog(s) were actually detected in this study, because the cross-reactive antibody used detects several distinct Paired box orthologs in euarthropods (Davis et al., 2005)."
Smith & Goldstein 2017. Segmentation in Tardigrada and diversification of segmental patterns in Panarthropoda

Did the tardigrade stylet apparatus evolve from the ancestral panarthropod frontal appendages, from a pair of legs, or from no appendage at all?

p. 338, section 6: "did the frontal appendages give rise to the stylet apparatus of tardigrades (Ou et al., 2012), or is the stylet apparatus derived from a pair of legs (Nielsen, 2001)? ... possibly the stylet apparatus has a non-appendicular origin, which may become apparent via studies of canonical appendage patterning genes in tardigrades."
Smith & Goldstein 2017. Segmentation in Tardigrada and diversification of segmental patterns in Panarthropoda

Which gene-regulatory changes account for the loss of terminal segment addition in the tardigrade lineage, given that caudal is still expressed posteriorly?

p. 338, section 6: "this approach might provide valuable insight into the gene regulatory changes that account for the loss of terminal segment addition in the tardigrade lineage."
Smith & Goldstein 2017. Segmentation in Tardigrada and diversification of segmental patterns in Panarthropoda

Do tardigrade embryos have any extra-embryonic tissue, or cells that do not contribute to the final animal, or was it lost with miniaturisation?

p. 2: "studies explicitly focusing on this topic would be required in order to obtain information about the presence or absence of dorsal and ventral extra-embryonic tissues in tardigrades, including more detailed cell lineage tracings using four-dimensional microscopy to identify cells that do not directly contribute to the final morphology of the animal"; p. 11: "Search for (rudiments of) extra-embryonic tissues in tardigrades to clarify their existence or potential loss"
Treffkorn et al. 2022. Review of extra-embryonic tissues in the closest arthropod relatives, onychophorans and tardigrades

Do the dorsal and ventral extra-embryonic cells of onychophorans (and arthropods) contribute cells to the embryo and adult, and are they homologous across Panarthropoda?

p. 11: "Tracing of individual cell lineages in extra-embryonic tissues of arthropods and onychophorans to clarify their ontogenetic fate"; p. 9: "it is also unclear whether the dorsal extra-embryonic tissues of onychophorans and arthropods are homologous."
Treffkorn et al. 2022. Review of extra-embryonic tissues in the closest arthropod relatives, onychophorans and tardigrades

What do the actin fibres in onychophoran extra-embryonic tissue do during germ-band closure?

p. 8: "Since the function of these actin fibres is currently unknown, future studies should focus on identifying the processes involved in the germ band closure of onychophorans"
Treffkorn et al. 2022. Review of extra-embryonic tissues in the closest arthropod relatives, onychophorans and tardigrades

How are the cell movements that sort embryonic regions into an invariant arrangement regulated, and how much genetic information does sorting several hundred cells require?

p. 260: "How cell migrations that contribute to establishing developmental form are regulated is unknown." … "It is an intriguing question how complex the genetic information needs to be to sort 558 cells in the C. elegans embryo."
Schnabel et al. 1997. Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of dev

Why do embryos rotate inside the eggshell during cleavage and gastrulation, and why in different directions from similar starting orientations?

p. 248: "We do not know currently what causes the rotation of the embryos."; p. 256: "An appealing question to ask is why the embryos rotate at all and why in different directions from an apparently similar initial orientation."
Schnabel et al. 1997. Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of dev

Is there any significant difference in the general principles of embryogenesis between an invariant-lineage embryo (C. elegans) and embryos with variable lineages?

p. 264, Conclusion: "A key question arising now is whether there is any significant difference in the general principles of embryogenesis between C. elegans and other organisms."
Schnabel et al. 1997. Assessing normal embryogenesis in Caenorhabditis elegans using a 4D microscope: variability of dev

Do yolk granules limit microtubule asters as steric obstacles or by affecting microtubule growth molecularly?

p. 679: "Whether yolk granules act as steric obstacles or affect MT growth by molecular means remains an important open question (Dogterom and Yurke, 1997; Wuhr et al., 2010)."
Pierre et al. 2016. Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos

How is the competition between bulk (cytoplasmic, length-dependent) and cortical microtubule pulling forces titrated in different cells and species?

p. 679: "How this competition may be titrated in different systems remains an important question to address experimentally (Minc and Piel, 2012; Mitchison et al., 2012)."
Pierre et al. 2016. Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos

Where and how has self-organisation been selected over determinism in the evolution of cleavage patterns?

p. 679: "More quantitative studies of the generic designs that regulate cleavage may unravel where and how self-organization has been selected over determinism in evolution."
Pierre et al. 2016. Generic Theoretical Models to Predict Division Patterns of Cleaving Embryos

To what extent is the onychophoran nervous system segmented at the level of neuron identity (e.g. segmentally repeated engrailed-expressing motoneurons) rather than gross anatomy?

p272 left col.: "Future studies using various neural and molecular markers will show to what extent the onychophoran nervous system is segmented."
Mayer & Whitington 2009. Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda

Are tardigrade trunk ganglia homologous to arthropod segmental ganglia (same position relative to segments, development and gene expression) or convergent?

pp271-272: "Future studies on the structure, development, and pattern of gene expression of the tardigrade nervous system will help resolve the issue of their putative homology with arthropod ganglia."
Mayer & Whitington 2009. Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda

What is the transient developmental function of onychophoran ventral organs in the trunk?

p269 left col.: "Their rapid increase in size and degeneration by apoptosis suggest that the ventral organs play a transient, albeit an important role in onychophoran development. This function remains unclear, except in the antennal segment"
Mayer & Whitington 2009. Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda

Does an arthrotardigrade with primary, secondary and tertiary clavae (e.g. Neoarctus primigenius) have additional brain lobes or innervation that would count as further cephalised segments?

p188 left col.: "This could be supported by an investigation on the brain structure of tardigrades possessing primary, secondary and tertiary clavae, such as Neoarctus primigenius"
Persson et al. 2014. Brain anatomy of the marine tardigrade Actinarctus doryphorus (Arthrotardigrada)

Do embryological and molecular data (segment-polarity and Hox expression in the head) confirm that the three tardigrade brain lobes come from three segments homologous to the arthropod proto-, deuto- and tritocerebrum?

p188 right col.: "To get confirmation on the morphological data, more embryological and molecular investigations are needed."
Persson et al. 2014. Brain anatomy of the marine tardigrade Actinarctus doryphorus (Arthrotardigrada)

Are the large 'one-cell' glands of arthrotardigrades homologous to insect oenocytes, and are they involved in moulting?

p189 left col.: "the conspicuous 'one-cell' glands ... could be an important morphological character if they were proven to be homologues to oenocytes of insects ... hence it could have merit to conduct research on these cells in the future."
Persson et al. 2014. Brain anatomy of the marine tardigrade Actinarctus doryphorus (Arthrotardigrada)

Can the principles of whole-brain organisation found in C. elegans (spatial neighbourhoods, core versus variable circuit, layered modular map) inform other nervous systems?

p. 105 (PDF p. 1): 'Thus, it remains to be determined whether lessons about whole-brain organization in C. elegans can inform questions and approaches for other systems.'
Brittin et al. 2021. A multi-scale brain map derived from whole-brain volumetric reconstructions

How is the reproducible neighbourhood organisation of the C. elegans nerve ring orchestrated during development (pioneer neurites, guidance molecules, nested mechanisms)?

p. 109 (PDF p. 6): 'How the neighbourhood organization is developmentally orchestrated remains an open question.'
Brittin et al. 2021. A multi-scale brain map derived from whole-brain volumetric reconstructions

How large is the variable part of the C. elegans nerve-ring connectome, beyond the conserved core?

p. 109 (PDF p. 6): 'Although determining the extent of the variable circuit is challenging due to the technical limitations of synaptic scoring and will therefore require multiple further connectomes, it is noteworthy that conserved synapses—like most variable ones—are constrained by the same contactome.'
Brittin et al. 2021. A multi-scale brain map derived from whole-brain volumetric reconstructions

Can spatial transcriptome reconstructions be guided by the actual anatomy, by integrating imaging of each cryosection with the Stereo-seq data from the same slice, so that reconstructed tissues match real boundaries?

p. 1280 (Limitations): 'dissimilarities remain between 3D reconstructed tissue models and their actual locations and boundaries ... In the future, this can be achieved by integrating imaging information of the cryosection slice and Stereo-seq data from the same slice.'
Wang et al. 2022. High-resolution 3D spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae

How can rare, scattered cell types (e.g. intestinal stem and enteroendocrine cells) be captured in spatial transcriptomes, for instance by integrating scRNA-seq from the same line?

p. 1280 (Limitations): 'capture of rare cell types with Stereo-seq remains challenging ... we will obtain scRNA-seq data of embryos and larval tissues and integrate them with Stereo-seq data of the same fly line'
Wang et al. 2022. High-resolution 3D spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae

What do 3D spatial transcriptomes of earlier Drosophila embryonic stages show about tissue developmental trajectories, given that most organogenesis happens before the late stages profiled?

p. 1280–1281 (Limitations): 'it is difficult to construct tissue development trajectories with only late-stage embryo and larva samples ... We will include more stages of embryo data'
Wang et al. 2022. High-resolution 3D spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae

Does the C. elegans AB blastomere have a higher cortical tension than P1 (as the 2-cell interface curvature suggests)?

p. 6: 'whether AB has stronger surface tension than P1 needs further experimental verification, such as fluorescent imaging on cytoskeleton density or direct measurement on surface tension'
Kuang et al. 2022. Computable early Caenorhabditis elegans embryo with a phase field model

How does pad-1 control P2 division orientation in C. elegans, and is the pad-1 RNAi 8-cell misarrangement caused by the misoriented P2 division?

p. 15: 'more in vivo experiments are needed to further explore how it functions in P2 division and whether the morphogenetic chaos in the RNAi experiment is attributed to the erroneous P2 division.'
Kuang et al. 2022. Computable early Caenorhabditis elegans embryo with a phase field model

How should one choose a cell-mechanics model (particle, phase field, deformable mesh) for a given biological system with enough accuracy at low cost?

p. 18: 'How to choose a model for a specific biophysical scene with enough accuracy and low cost becomes a practical problem and merits further study'
Kuang et al. 2022. Computable early Caenorhabditis elegans embryo with a phase field model

How much contact area and contact duration does contact-dependent signalling (Notch, Wnt) need between embryonic cells?

p. 7: 'the actual sensitivity of intercellular signaling including both contact area and contact duration is still poorly understood.'
Cao et al. 2020. Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation

How do the inner cavities (blastocoel) of the early C. elegans embryo form and change, and what are they for?

p. 11: 'However, systematic characterization of such cavities is still lacking.'
Cao et al. 2020. Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation

An interactive platform for navigating embryonic cell morphologies and contacts over development is lacking.

p. 11: 'A well-designed platform that allows interactive navigation of cell morphologies and contacts over development is required in the future.'
Cao et al. 2020. Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation

Does H. exemplaris have a subpharyngeal ganglion linking brain and first trunk ganglia (reported by TEM and Persson 2012/2014, not found by Zantke 2008 or Mayer 2013a)?

p. 1353: "Although the early origin of the suboesophageal ganglion remains unclear, it seems likely that it is formed by an outgrowth of the brain"
Hejnol & Schnabel 2005. The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations

How many neural progenitor cells build the brain in tardigrade embryos (T. stephaniae; H. exemplaris), and when do they delaminate relative to the ganglion progenitors?

p. 1353: "Although we could not determine the number of cells forming the brain"
Hejnol & Schnabel 2005. The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations

Is the tardigrade head one segment (Mayer 2013a, Smith 2016) or at least two to three (Persson 2012/2014; two pairs of stylet glands), and which frame should the model's segment numbering use?

p. 1 (abstract): "it is unclear whether the tardigrade head and its enclosed brain comprises one, or several segments, or a non-segmental structure"
Mayer et al. 2013. Neural markers reveal a one-segmented head in tardigrades (water bears)

What do engrailed, Pax3/7, Wnt4, Wnt16B, hedgehog and other homeobox genes do in H. exemplaris segmentation, and does Engrailed act before segments are visible?

p. 15: "The precise developmental roles of Wnt4, Wnt16B, and other Wnt genes need to be clarified" … "other commonly conserved components of the segment polarity network, such as hh, need to be investigated in tardigrades"
Chavarria et al. 2021. Extensive loss of Wnt genes in Tardigrada

Were Wnt1, Wnt6, Wnt7, Wnt8 and Wnt10 lost in the ancestor of all tardigrades or only within the lineage leading to H. exemplaris and R. varieornatus?

p. 9: "whether these genes were lost in an ancestor of all tardigrades, or within crown group Tardigrada is unclear"
Chavarria et al. 2021. Extensive loss of Wnt genes in Tardigrada

Has long-term laboratory culture changed H. exemplaris behaviour, such as the locomotor differences seen against Ramazzottius?

p. 20: "The differences between H. exemplaris and Ramazzottius could potentially arise from effects of long-term culturing in the lab; further work will be required to investigate this possibility."
Anderson et al. 2024. Comparative analysis of tardigrade locomotion across life stage, species, and disulfiram treatment

How does disulfiram disrupt locomotion in H. exemplaris, and is it by blocking octopamine synthesis through tyramine β-hydroxylase?

p. 20: "While the mechanism by which disulfiram disrupts locomotion in tardigrades remains unknown"
Anderson et al. 2024. Comparative analysis of tardigrade locomotion across life stage, species, and disulfiram treatment

How does H. exemplaris repair widespread DNA damage without producing chromosome translocations and fusions?

PDF pp 2 (abstract), 3: "How tardigrades can repair widespread DNA damage without producing frequent, large-scale chromosome structural abnormalities, like chromosome translocations and fusions, is unknown." … "It is unknown how H. exemplaris endures or fixes DNA damage from these extreme environments without extensively impacting the genome."
Papell et al. 2026. Chromosomes remain individualized through interphase in embryos of the tardigrade Hypsibius exemplaris

Do the unusual chromosome and nuclear features of early H. exemplaris embryos occur in other tardigrades and in other desiccation-tolerant animals such as rotifers?

PDF p 9: "It is not known if the unusual features we observed in embryos exist in tardigrades more generally, or in just this species"
Papell et al. 2026. Chromosomes remain individualized through interphase in embryos of the tardigrade Hypsibius exemplaris

Do tardigrades use peroxisomes, given their losses of peroxisome biogenesis genes, and are peroxisomes used only during recovery from cryptobiosis?

PDF p 14: "Overall, it remains unclear if and how tardigrades utilize peroxisomes."
Kamilari et al. 2019. Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages

Does the heterotardigrade Echiniscoides sigismundi really lack all p53-family genes?

PDF p 10: "We state the latter with caution and acknowledge that this finding needs further investigation."
Kamilari et al. 2019. Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages

How long has H. exemplaris reproduced asexually, and is its heterozygosity still rising or at equilibrium?

pp. 8, 10: "it remains an open question whether heterozygosity in this spe" … "Future work to compare heterozygosity levels between tardigrade lineages may shed additional light on the question of how long this and other tardigrade species have been reproducing asexually."
Coke et al. 2026. Modified meiosis in the tardigrade Hypsibius exemplaris maintains heterozygosity across the genome

Is independent evolution of alleles common among diploid clonal parthenogens such as H. exemplaris, and are such lineages evolutionary dead ends or do they occasionally reproduce non-clonally?

pp. 2, 10: "Whether independent evolution of alleles is a widespread phenomenon among diploid clonal parthenogens remains an open question in the field." … "Whether these species are doomed in the long run, represent rare survivors against the usual fate of clonal reproduction"
Coke et al. 2026. Modified meiosis in the tardigrade Hypsibius exemplaris maintains heterozygosity across the genome

Why do the four trunk ganglia differ in size (narrower first and fourth), and is a differentiated first and fourth ganglion ancestral for Tardigrada?

PDF p 12: "The functional significance, if any, for different sizes among ganglia remains unclear."
Smith & Jockusch 2014. The metameric pattern of Hypsibius dujardini(Eutardigrada) and its relationship to that of other panarthropods

Do heterotardigrades share the H. exemplaris pattern in which left and right ventromedian muscle attachment sites meet at the midline only in some segments, and is it ancestral for Tardigrada?

PDF p 13: "Whether or not the pattern revealed in H. dujardini and M. tardigradum exists among heterotardigrade species has not been reported"
Smith & Jockusch 2014. The metameric pattern of Hypsibius dujardini(Eutardigrada) and its relationship to that of other panarthropods

Can intracellular determinants of the division axis (e.g. PAR gradients) be combined with cell mechanics into a true self-organisation model of early embryo arrangement?

p. 1810: "Intracellular events that determine the cells' division axis, e.g., PAR protein gradients (23), need to be included to arrive at a true self-organization model that integrates both mechanical and biochemical cues."
Fickentscher et al. 2013. Mechanical cues in the early embryogenesis of Caenorhabditis elegans

Does the thickness of the gonad set the shape (aspect ratio) of the egg across nematode species?

p. 4445: "In future studies, it would be of interest to investigate the correlation between the thickness of the gonad and the shape of the eggs by examining various nematode species (Goldstein, 2001; Schulze and Schierenberg, 2011)."
Yamamoto & Kimura 2017. An asymmetric attraction model for the diversity and robustness of cell arrangement in nematodes

How are the attractive forces between blastomeres regulated when par-3 is knocked down, where their strength becomes highly variable?

p. 4444: "Therefore, an understanding of the mechanism underlying the regulation of attractive forces under par-3 (RNAi) conditions is needed in order to develop a model that accounts for this condition."
Yamamoto & Kimura 2017. An asymmetric attraction model for the diversity and robustness of cell arrangement in nematodes

Is direct visual input from median ocelli (or their homologues) to the central body part of the euarthropod ground pattern, as in onychophorans and chelicerates?

p242 right col.: "Further studies should clarify whether this feature belongs to the ground pattern of Euarthropoda since there is evidence that neuropils of the nauplius eyes ... in malacostracan crustaceans are connected with a cluster of cells that also innervate the central body"
Mayer 2006. Structure and development of onychophoran eyes: what is the ancestral visual organ in arthropods?

Does the bifurcating onychophoran optic nerve (to arcuate body and to mushroom body) mean the single onychophoran eye corresponds to both the median and the lateral eyes of spiders?

p191 left col.: "Might this bifurcation of the retinal representation to central neuropils suggest that the single eye of the velvet worm corresponds to the median and primary lateral eyes of araneans?"
Strausfeld et al. 2006. The organization and evolutionary implications of neuropils and their neurons in the brain of the onychophoran Euperipatoides rowelli

Do the chelicerate-like brain features of onychophorans (arcuate body, heterolateral mushroom body lobes, protocerebral glomeruli) reflect common descent or convergence, given that molecular phylogenies place Onychophora outside Euarthropoda?

p194 left col.: "Are the features described here sufficient to propose that the Onychophora might be considered a highly divergent branch of the chelicerates ...? At present, this issue remains unresolved."
Strausfeld et al. 2006. The organization and evolutionary implications of neuropils and their neurons in the brain of the onychophoran Euperipatoides rowelli

Is Er-c-opsin transcribed from both strands in E. rowelli (as the sense-probe signal suggests), and if so what does the antisense transcript do?

p920 left col.: "The putative role of the Er-c-opsin antisense transcript in E. rowelli is unknown, but it might be involved in a self-regulation of Er-c-opsin expression"
Beckmann et al. 2015. Spectral sensitivity in Onychophora (velvet worms) revealed by electroretinograms, phototactic behaviour and opsin gene expression

What does c-opsin do in onychophoran optic ganglion cells and protocerebral neurons: non-visual, circadian photoreception?

p920 left col.: "Although the function of the onychophoran c-opsin protein is unknown, it might be involved in non-visual, extraocular photoreception associated with circadian clock mechanisms"
Beckmann et al. 2015. Spectral sensitivity in Onychophora (velvet worms) revealed by electroretinograms, phototactic behaviour and opsin gene expression

Can automated lineaging be extended through the last round of embryonic divisions, when cells migrate actively and nuclei pack closely (C. elegans beyond the 350-cell stage)?

p. 707: 'Extending automated lineaging to include the last round of embryonic cell division ... remains a challenging goal because of the active cell migrations and close packing of nuclei in the late embryo.'
Murray et al. 2008. Automated analysis of embryonic gene expression with cellular resolution in C. elegans

Why does the pha-4::HIS-24::DsRed strain (RW10007) express brightly in the non-pharyngeal sister lineages ABalpp, ABarap and MSxp, unlike the mCherry pha-4 strains, and is this biologically relevant?

p. 706: 'Whereas determining the cause and biological relevance of this observation will require additional experiments, our ability to detect the differences in pattern demonstrates the utility of lineage analysis'
Murray et al. 2008. Automated analysis of embryonic gene expression with cellular resolution in C. elegans

What becomes of the third, laterally oriented mushroom-body peduncle of the 6-dpf Platynereis worm in later stages?

PDF p. 11 (Cell 184:4828): 'A third prominent peduncle of unknown fate is oriented laterally and perpendicular to the dorsal and ventral peduncles'
Vergara et al. 2021. Whole-body integration of gene expression and single-cell morphology

Can whole-body connectomics and cellular-resolution transcriptomics be integrated for an entire animal across developmental stages, and do registrations hold for less stereotyped animals?

PDF p. 16 (Cell 184:4833): 'With our new resource, this can now be extended to integrate connectomics and transcriptomics for an entire animal and for different developmental stages.'
Vergara et al. 2021. Whole-body integration of gene expression and single-cell morphology

Which of the C. elegans connections that differ between the hermaphrodite and male reconstructions are true sex differences rather than individual variability or reconstruction error?

PDF p. 27 (Extended Data legend): 'Whether the edges highlighted here are true sex differences needs to be tested by methodology that allows many animals to be examined.'
Cook et al. 2019. Whole-animal connectomes of both Caenorhabditis elegans sexes

To what extent do behavioural differences between individual worms come from differences in connectivity, and how much does activity or experience reinforce connections?

p. 68 (PDF p. 6): 'Reinforcement of connectivity by circuit activity and hence experience has been demonstrated but little studied so far. The extent to which differences in the behaviour of individual worms is due to differences in connectivity remains to be explored.'
Cook et al. 2019. Whole-animal connectomes of both Caenorhabditis elegans sexes

Can transcriptome staging (RAPToR) be extended to stage samples on multiple branching trajectories rather than one developmental or ageing trajectory?

p. 974: 'RAPToR can currently only stage on one developmental or ageing trajectory, so a future improvement will be to provide RAPToR with the ability to stage on multiple branching trajectories.'
Bulteau & Francesconi 2022. Real age prediction from the transcriptome with RAPToR

Can reference-based age estimation be adapted to data other than genome-wide expression, such as genome-wide binding data?

p. 974: 'Another avenue for improvement is to adapt this approach to data other than genome-wide gene expression, such as genome-wide binding data.'
Bulteau & Francesconi 2022. Real age prediction from the transcriptome with RAPToR

What forces drive interkinetic nuclear migration in pseudostratified epithelia?

p. 304: 'processes such as interkinetic nuclear migration in pseudostratified epithelia could be simulated at unprecedented resolution to address open questions regarding the driving forces.'
Runser et al. 2024. SimuCell3D: three-dimensional simulation of tissue mechanics with cell polarization

How is pseudostratified epithelial organisation acquired and maintained under growth and morphogenetic deformation?

p. 303: 'How this structure is acquired and maintained under growth and morphogenetic deformation is still largely unknown.'
Runser et al. 2024. SimuCell3D: three-dimensional simulation of tissue mechanics with cell polarization

Do the properties of jammed deformable-particle packings depend on surface roughness (vertex spheres versus smooth sphero-polyhedra)?

p. 9911: 'It will be interesting to determine whether any of the properties of jammed packings of deformable particles depend on the surface roughness.'
Wang et al. 2021. The structural, vibrational, and mechanical properties of jammed packings of deformable particles in three dimensions

What links quartic eigenmodes of the dynamical matrix to the non-trivial power-law scaling of shear modulus with pressure in jammed deformable particles?

p. 9911: 'We encourage future studies to understand the link between quartic eigenmodes of the dynamical matrix and the non-trivial power-law scaling of ⟨G(P)⟩.'
Wang et al. 2021. The structural, vibrational, and mechanical properties of jammed packings of deformable particles in three dimensions

How can shape-resolving multicellular simulations be scaled to thousands of cells at acceptable cost?

p. 9: 'there is still a great need from the field to keep increasing the cell number and decreasing the computational cost. For example, the early Drosophila embryogenesis involves thousands of cells'
Kuang et al. 2023. MorphoSim: an efficient and scalable phase-field framework for accurately simulating multicellular morphologies

How do cell stiffness and surface tension shape the dynamics of engineered multicellular aggregates, and how can parameters be chosen to optimise a target function?

p. 8: '(1) How do biological parameters like cell stiffness and surface tension affect the dynamics? ... and (2) How to choose the parameter combination to optimize a specific function?'
Kuang et al. 2023. MorphoSim: an efficient and scalable phase-field framework for accurately simulating multicellular morphologies

How many muscles enter each leg of H. exemplaris under one explicit counting rule (fibres vs fused muscles; include lateral-group fibres or not)? Reported: Hypsibius sp. 14/15/14/6 (dorsal+ventral), H. exemplaris 14/12/11/10 (sweep summary), other eutardigrades 10–14 in legs I–III and 4–6 in leg IV.

PDF p 2: "Whether these and other discrepancies are attributable to species differences or perhaps result from different techniques is unclear."
Gross & Mayer 2019. Cellular morphology of leg musculature in the water bear Hypsibius exemplaris (Tardigrada) unravels serial homologies

Which population is the male Hypsibius cf. dujardini in Altiero et al. 2018 Fig. 8.4b, and do males ever occur in H. exemplaris?

p. 556: "It would be useful to be able to both maintain clonal organisms and cross-fertilize organisms as needed, but only parthenogenetic females have been recognized in our cultures to date."
Gabriel et al. 2007. The tardigrade Hypsibius dujardini, a new model for studying the evolution of development

Do the six disk-shaped structures at the 30 h mouth become the six peribuccal lobes, and why is the mouth closed at 45 h?

p. 570: "Exactly how this happens is unclear"
Gross et al. 2017. External morphogenesis of the tardigrade Hypsibius dujardini as revealed by scanning electron microscopy

In H. exemplaris embryos, does FGF signal from segmental ectoderm to the endomesoderm during pouch formation, and does BMP signalling pattern the dorsal–ventral axis of the lateral ectoderm?

bioRxiv p. 2 (abstract): "possible FGF signaling between ectoderm and endomesoderm, and possible roles of BMP signaling in dorsal-ventral patterning of lateral ectoderm"
Heikes et al. 2024. FGF and BMP pathway genes are expressed in segmentally iterated domains during segmentation of germ layers in a tardigrade

When do mesodermal cells become distinct from endoderm in the endomesodermal pouches of H. exemplaris?

PDF p 3: "It is unclear precisely when mesodermal cells differentiate in H. exemplaris."
Smith et al. 2024. Developmental and genomic insight into the origin of the tardigrade body plan

Do transverse commissures form, even transiently, in the trunk ganglia of tardigrade embryos?

p. 33: "Whether segmental commissures are transiently formed during embryonic development is unknown."
Zantke et al. 2008. Three-dimensional reconstruction of the central nervous system of Macrobiotus hufelandi (Eutardigrada, Parachela): implications for the phylogenetic position of Tardigrada

Are tardigrades the sister group of arthropods, of onychophorans plus arthropods, or closer to nematodes and other cycloneuralians?

p. 12: "the position of tardigrades, and the issue of panarthropod phylogeny in general, remains an open question"
Gross & Mayer 2015. Neural development in the tardigrade Hypsibius dujardini based on anti-acetylated α-tubulin immunolabeling

Were the ventral nerve cords of the last common ancestor of Panarthropoda and of Ecdysozoa paired or unpaired, and medullary or ganglionated?

p. 15: "the ancestral condition of the ventral nerve cords within Ecdysozoa (paired versus unpaired) remains ambiguous and requires further investigation"
Martin et al. 2017. Assessing segmental versus non-segmental features in the ventral nervous system of onychophorans (velvet worms)

What do the mushroom bodies, 'olfactory' glomeruli and giant neurons of the onychophoran brain do, and how many giant neurons are there and where do they project (E. rowelli)?

p. 9: "The function of the onychophoran mushroom bodies is unknown."
Schumann et al. 2016. Immunolocalization of Arthropsin in the Onychophoran Euperipatoides rowelli (Peripatopsidae)

To which head segment does each onychophoran post-oral commissure (#1–#5) belong, given that their somata lie in both the jaw (deutocerebral) and slime-papilla segments?

p. 8: "The anteriorly shifted position of somata makes it difficult to assign each post-oral commissure to a particular head segment."
Martin & Mayer 2015. Insights into the segmental identity of post-oral commissures and pharyngeal nerves in Onychophora based on retrograde fills

Are tardigrade pigment-cup eyes homologous to arthropod median ocelli, to onychophoran eyes, or to other bilaterian eyes, and how diverse are photoreceptors across Tardigrada?

pp. 401, 406: "the significance of these eyes for the evolution of arthropod visual systems is unclear yet" … "future studies may reveal a larger structural diversity of photoreceptors in the Tardigrada with traits usable for phylogenetic interpretations"
Greven 2007. Comments on the eyes of tardigrades

Do protostome (including tardigrade) neuropsin/opsin-5 homologues work as UV-sensitive receptors or as retinal photoisomerases?

p. 2387: "it would be interesting to know whether the protostome neuropsin/opsin-5 homologs also have a function as UV-sensitive GPCRs or rather act as retinal photoisomerases"
Hering & Mayer 2014. Analysis of the opsin repertoire in the tardigrade Hypsibius dujardini provides insights into the evolution of opsin genes in panarthropoda

What are the electron-dense pigment granules of the H. exemplaris eye pigment cell made of?

bioRxiv p. 10, lines 296–297: "whose biochemical" … "composition remains unknown"
Dutta et al. 2026. Extensive opsin gene expansion and non-cerebral origin of the minimalist eye in a model tardigrade

What do the three PDF neuropeptides of H. exemplaris do, including their signalling to storage cells, and what are the ventral extracerebral PDF-immunoreactive cells?

pp. 11–12: "the identity, detailed morphology and potential function of the ventral extracerebral PDF-ir cells remain to be clarified" … "The functional role of these differences between the three peptides as well as their different localization in the head (figure 8a–c) is unclear." … "The role of PDF/PDFR signalling in storage cells is unclear"
Dutta et al. 2025. Pigment-dispersing factor neuropeptides act as multifunctional hormones and modulators in tardigrades

How do walking kinematics vary across tardigrade species and over a wider range of speeds, and does the stance-duration model of interleg coordination hold across panarthropods?

bioRxiv p. 11: "Future work should expand upon both of these parameters, both by surveying walking dynamics across Tardigrada"
Nirody et al. 2021. Tardigrades exhibit robust interlimb coordination across walking speeds and terrains

Can H. exemplaris be stably transformed, edited heritably and screened by forward genetics?

pp. 186, 192: "Perhaps the methods most needed now for diverse kinds of further studies are transgenesis and CRISPR-mediated DNA editing." … "To date, forward genetic screening using mutagenesis has not been reported."
Goldstein 2022. Tardigrades and their emergence as model organisms

Why did unedited G0 siblings of DIPA-CRISPR-injected R. varieornatus mothers show the same growth retardation as frameshift mutants?

p. 13: "It remains unclear why the unedited siblings exhibited similar growth retardation."
Kondo et al. 2024. Single-step generation of homozygous knockout/knock-in individuals in an extremotolerant parthenogenetic tardigrade using DIPA-CRISPR

Why are Cas9 double-strand breaks in H. exemplaris mostly rejoined without indels, and does Dsup protect the broken ends?

p. 201: "Although the mechanisms underlying the preferred no-indel end-joining remain unclear"
Kumagai et al. 2022. Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades

How much do DNA protection (Dsup), DNA repair, protection of proteins against carbonylation and low cell proliferation each contribute to the radiation tolerance of H. exemplaris and other tardigrades?

p. 11: "Further studies are needed to evaluate the relative importance of eutely vs. other mechanisms (e.g., DNA repair system, protein protection, apoptosis avoidance) in radiation tolerance of tardigrades."
Beltrán-Pardo et al. 2015. Tolerance to Gamma Radiation in the Tardigrade Hypsibius dujardini from Embryo to Adult Correlate Inversely with Cellular Proliferation

Do Dsup proteins of other eutardigrades, including H. exemplaris, protect DNA in vivo as R. varieornatus Dsup does, and what do heterotardigrades without Dsup use instead?

pp. 1819, 1826: "it is unclear whether other eutardigrade species’ Dsup proteins have the same protective abilities" … "even within those lineages it is unclear to what extent Dsup plays a role in IR"
Clark-Hachtel et al. 2024. The tardigrade Hypsibius exemplaris dramatically upregulates DNA repair pathway genes in response to ionizing radiation

Do tardigrades tolerate desiccation and radiation with the same molecular mechanisms?

p. 325 (PDF p 327): "the extent to which tolerance to desiccation is related to tolerance to radiation, and relies on the same molecular mechanisms, is still unclear"
Jönsson KI, Levine EB, Wojcik A, Haghdoost S, Harms-Ringdahl M 2018. Environmental Adaptations: Radiation Tolerance. In:

Which genes beyond the known protectants contribute to anhydrobiosis in H. exemplaris: induced cytochrome P450s, solute carriers and apolipoproteins, calcium–calmodulin signalling, or proteins rich in low-complexity and disordered regions?

pp. 14, 22: "The contributions to anhydrobiosis of additional genes identified as up-regulated (including cytochrome P450, several solute carrier families, and apolipoproteins) are unknown." … "but it is still unclear how this is related to anhydrobiosis"
Yoshida et al. 2017. Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus

Do the abundant heat-soluble proteins of Echiniscus testudo (EtAHS) contribute to its desiccation tolerance?

PDF p 9: "Functional experiments would be required as a future work to fully confirm the contribution of EtAHS in the desiccation tolerance of heterotardigrada."
Murai et al. 2021. Multiomics study of a heterotardigrade, Echinisicus testudo, suggests the possibility of convergent evolution of abundant heat-soluble proteins in Tardigrada

In which tissues are the CAHS paralogues and other tardigrade-specific protectants expressed during anhydrobiosis, and do CAHS subfamilies have different roles?

pp. 1, 7: "little is known about the actual tissue specificity or the actual workings of these tardigrade-specific IUPs within tardigrades during anhydrobiosis" … "future studies should analyze in more detail the differences in CAHS subfamilies and their tissue specificity"
Tanaka, Aoki & Arakawa 2023. In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging

Which of the several dicer genes of tardigrades act in development, RNAi and antiviral defence, and how does their expression vary across species?

p. 12: "it is unclear which dicers may also be involved in antiviral defense" … "it is unclear how expression may vary across the other tardigrade species we investigated" … "Future research could focus on characterizing tardigrade small RNAs across various"
Weinberg et al. 2025. Lineage-specific expansions of the Dicer gene family in tardigrades

Does time spent in anhydrobiosis extend the life span of H. exemplaris (the 'Sleeping Beauty' hypothesis), and how does drying affect later reproduction?

p. 275 (PDF p 278): "Very little is known about the effects of anhydrobiosis on life cycle and longevity achieved thereby."
Schill RO, Hengherr S 2018. Environmental Adaptations: Desiccation Tolerance. In: Schill RO (ed.) Water Bears: The Biolo

Do tardigrade adults and embryos survive subzero temperatures by the same mechanisms, and can tardigrades tolerate intracellular freezing?

pp. 302, 307 (PDF pp 304, 309): "It is not known yet whether adult tardigrades and embryos use similar mechanisms to survive subzero temperatures." … "It is still not known if tardigrades tolerate intracellular freezing."
Hengherr S, Schill RO 2018. Environmental Adaptations: Cryobiosis. In: Schill RO (ed.) Water Bears: The Biology of Tardi

Can the rare males that appear in thelytokous tardigrade strains (Milnesium sp.) fertilise eggs, and what triggers their appearance?

p. 232 (PDF p 237): "It is unknown whether these males can actually function in sexual reproduction"
Altiero T, Suzuki AC, Rebecchi L 2018. Reproduction, Development and Life Cycles. In: Schill RO (ed.) Water Bears: The B

Which cell types keep proliferating after hatching in different tardigrade species, and is the absence of proliferation in epidermis and nervous system shared across clades?

PDF p 7: "it would be valuable to reevaluate the proliferative capacities of different cell types in a range of tardigrade species using contemporary molecular techniques like EdU incorporation"
Quiroga-Artigas & Moriel-Carretero 2024. Storage cell proliferation during somatic growth establishes that tardigrades are not eutelic organisms

How do cell size, cell number and fat stores change with feeding, starvation and DNA damage, and how do they set body size in H. exemplaris?

PDF p 15: "Future studies examining cellular dimensions, numbers, and fat depots under various conditions will be crucial"
Quiroga-Artigas et al. 2026. Compromised DNA replication in gut cells underlies sensitivity to genotoxic stress in the tardigrade Hypsibius exemplaris

Can label-free holotomography follow H. exemplaris embryogenesis and organogenesis live at cellular resolution through the eggshell?

p. 9: "future research could employ this technique for detailed time-lapse monitoring of embryogenesis, organogenesis, and cellular dynamics"
Hong et al. 2025. A Non-Invasive, Label-Free Method for Examining Tardigrade Anatomy Using Holotomography

Was Lim1 expressed broadly or in regional domains in the legs of the panarthropod ancestor, given broad expression in H. exemplaris and tip-only expression in E. kanangrensis?

PDF p 11: "whether Lim1 ancestrally has an expression pattern that was broad or more regionalized remains unknown"
Mapalo et al. 2024. Expression of distal limb patterning genes in Hypsibius exemplaris indicate regionalization and suggest distal identity of tardigrade legs

What do the tardigrade-specific genes expressed in development do (e.g. the grey60 module at limb-bud and pharynx stages, suggested to act in ecdysone synthesis and storage-cell formation), and how do cells vary along developmental trajectories?

p. 11 of 15: "The identification of the specific genes influencing life activities during developmental stages, along with the roles of TSGs, is still incomplete. Further in‐depth analyses and experiments, such as single‐cell transcriptomics ... while in situ hybridization experiments can provide insights into the spatial expression"
Li et al. 2024. Conserved and specific gene expression patterns in the embryonic development of tardigrades. Evol Dev 26

What roles do He-hth, He-exd3 and He-Dll play in patterning the proximodistal axis of H. exemplaris legs (proximal and distal identity, segmental leg identity with Hox genes, or other roles)?

p. 6: "Functional data for He-hth, He-exd3 and He-Dll are required to determine the precise roles of these genes in regulating the development of PD axes in Tardigrada."
Game & Smith 2020. Loss of intermediate regions of perpendicular body axes contributed to miniaturization of tardigrades

Are onychophoran nephridia homologous to annelid nephridia, given their different innervation (from somata in the nerve cords, via a medioventrolateral loop)?

p1205 left col.: "The different innervation patterns and the absence of specific correspondences in the nephridial anatomy and nephridiogenesis ... challenge the homology hypothesis of nephridial organs in Annelida and Panarthropoda ... However, further comparative studies are necessary to clarify this issue."
Mayer & Harzsch 2008. Distribution of serotonin in the trunk of Metaperipatus blainvillei (Onychophora, Peripatopsidae): implications for the evolution of the nervous system in Arthropoda

What mechanisms reduce nuclear growth at later preimplantation stages, so that daughter nuclei downscale without halving?

p. 8: 'This suggests that nuclear growth is reduced at later developmental stages (Fig. S12). Future work is required to uncover the specific biological mechanisms controlling developmental-stage-dependent nuclear growth.'
Nunley et al. 2024. Nuclear instance segmentation and tracking for preimplantation mouse embryos

How can Lagrangian surface parameterisation be extended to tubes that branch, fuse or separate?

p. 1986: 'Extending to higher-order networks of tubes and shapes that fuse or separate poses future challenges.'
Mitchell & Cislo 2023. TubULAR: tracking in toto deformations of dynamic tissues via constrained maps

What do comparative connectomes within and across species reveal about the origin of similarities and differences in nervous-system structure and behaviour?

p. 260 (PDF p. 4), Discussion: 'Comparative connectomics is needed to reveal the origin of similarities and differences in structure and behaviour within and across species.'
Witvliet et al. 2021. Connectomes across development reveal principles of brain maturation

Can temporal information (video-level deep networks or temporal filtering) improve markerless pose estimation beyond frame-by-frame detection, especially where occlusion is common?

p. 1288: "While temporal information could indeed be beneficial in certain contexts, challenges remain to using end-to-end-trained deep architectures for video data to extract postures ... As this is an active area of research, we believe this situation is likely to change"
Mathis et al. 2018. DeepLabCut: markerless pose estimation of user-defined body parts with deep learning

How can the local ordering and orientation of small adjacent contigs in Hi-C (3D-DNA) assemblies be resolved, given that Hi-C gives long-range but weak short-range information?

p. 95: 'the current approach is not perfect for local ordering of small adjacent contigs. This might be circumvented by more sophisticated analysis of Hi-C data. Additional data (such as long or paired-end reads) could also improve the results.'
Dudchenko et al. 2017. De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds