Developmental Biology

Early neural development involves a combination of planar signals from the vertebrate organiser and vertical signals from its derived structures, the prechordal plate and notochord. However, the relative contribution of each structure to neural development is not clear. Here, we isolate anterior tissues from the primitive streak at successively later stages of development, to identify the extent of patterning that can occur prior to, during, and after the formation of the organiser and its later derivatives. Our results show that acquisition of neural identity occurs gradually and that exposure to planar signals from the developing node is necessary for neural plate specification. We also show that planar node-derived signals are required for AP patterning in isolated anterior tissues and give evidence that early neural tissue is of anterior character which subsequently becomes caudalised by signals (in part) from the developing node. However, anterior neural identity is lost without long-term contact with vertical signals from the axial mesendoderm. These results reveal a previously unappreciated level of autonomy in anterior neural development in the absence of node derived tissues. Summary statementCulture of isolated anterior tissues from the chick embryo reveal the roles of planar and vertical organiser signals for neural specification and anteroposterior patterning and maintenance.

BackgroundThe trigeminal ganglion (TG) is a structure of the peripheral nervous system, composed of neuronal and non-neuronal cell types, that integrates sensory input from the face and jaw. The developing TG is derived from two embryonic cell populations: neural crest and cranial placode. Both populations play critical roles in TG development and must interact to coordinate changes in gene expression that regulate specification, differentiation, and organization. However, the molecular characteristics of the heterogeneous cell populations within the developing TG remain poorly defined. ResultsWe performed single-cell RNA-sequencing (scRNA-seq) on TG from developing chick embryos at HH17. Our high-resolution dataset (14 clusters, [~]87000 cells) provides insight into cellular diversity within the developing TG. As expected, we identified placode-derived neurons as well as neural crest cells prior to neuronal differentiation. In addition to classic markers, we identified novel transcripts with unknown roles in TG development, including several long non-coding RNAs (lncRNAs). ConclusionsWe generated a single-cell atlas of the developing chick trigeminal ganglion during early axonogenesis and defined the transcriptomic states of its diverse cell populations. Our results provide a useful resource for better understanding the cell populations contributing to TG development and gene expression that drives cell identity and differentiation.

Differentiated cells can return to a progenitor-like state in response to injury via the evolutionarily conserved cellular program called paligenosis. Paligenosis proceeds by three stages: 1) autophagy/autodegradation of differentiated cell architecture, 2) metaplasia/progenitor gene induction, 3) TOR complex 1 (TORC1)-dependent cell cycle re-entry. Using multiple injury and reverse-lineage-tracing approaches in the Drosophila gut, we show that mature polyploid enterocytes dedifferentiate into diploid progenitors in response to epithelial injury. Several key findings suggest a role for paligenosis. Shortly after injury, enterocytes dramatically increased autophagic flux (stage 1); additionally, pharmacological and genetic inhibition of autophagy blocked progenitor recruitment. Rapamycin also blocked recruitment, indicating that TORC1 is required (stage 3). Finally, RNAi knockdown of ifrd1, an evolutionarily conserved protein required for paligenosis, blocked progenitor recruitment. Thus, replenishment of diploid progenitors from differentiated polyploid cells may occur by paligenosis. The Drosophila gut may offer a versatile system for dissecting the mechanisms of this evolutionarily conserved pathway. Impact StatementMature, polyploid Drosophila enterocytes may dedifferentiate to a stem cell-like, diploid state via the paligenosis cell regeneration program, adding to evidence that paligenosis is a fundamental cellular process and highlighting Drosophila gut as a potential model system for its study.

Neural crest (NC) cells are dynamic embryonic stem cells that undergo an epithelial-to-mesenchymal transition (EMT) and alter their cell states from tightly adherent to migratory and invasive during early development. While EMT transcriptional programs are well characterized, how cytoskeletal architecture is developmentally patterned across EMT states remains poorly understood. Here, we present a spatial and temporal atlas of - and {beta}-tubulin isotype gene expression during NC EMT in the chick embryo. Single cell RNA-sequencing reveals diversity in tubulin isotype gene expression from ubiquitous (TUBA1A, TUBA1B) to cell type specific (TUBAL3, TUBB4B). In addition, we identified novel enrichment of several tubulin isotypes in NC and NC-associated clusters (TUBB3, TUBA3E, TUBG1). Using fluorescent in situ hybridization chain reaction (HCR), we focus on NC EMT and migration states to validate and spatially resolve these expression patterns. Additional characterization in differentiated cells reveals tubulin gene expression in specific neuronal and myogenic populations. We further identify expression of the microtubule motor genes KIF11 and DYNC1LI1 within neural tube and NC populations, suggesting coordinated regulation of microtubule composition and cargo transport capacity. Together, these data establish that vertebrate NC EMT is accompanied by systematic reprogramming of tubulin gene expression and provide a developmental resource for investigating cytoskeletal control of cell state transitions. SUMMARY STATEMENTThis study defines when and where distinct tubulin genes are expressed during neural crest epithelial-to-mesenchymal transition in the chicken embryo providing a resource for understanding cytoskeletal organization across embryonic cell state changes.

Hox genes have been broadly implicated in nervous system development, but the molecular and genetic mechanisms that act downstream of Hox factors remain to be identified. The MAB-5 antennapedia-like Hox transcription factor is both necessary and sufficient to cause posterior migration of the Q neuroblast descendants in Caenorhabditis elegans. In response to MAB-5, the left-side QL descendants QL.a and QL.ap undergo a three-stage migration process, with each stage characterized by a posterior lamellipodial protrusion followed by cell body migration. The QL.ap cell differentiates into the PQR neuron posterior to the anus. Previous studies showed that the MAB-5-regulated gene efn-4/Ephrin was required for the third and final stage of QL.ap migration, with efn-4 mutation resulting in placement of PQR immediately anterior to the anus. This subtle and previously-undescribed phenotype opens the possibility that other known neuronal development genes could be involved. In this work, we screened known signaling mutants for third-stage PQR migration defects. We found that mutations in SAX-3/Robo signaling, UNC-6/Netrin signaling, and heparan sulfate proteoglycans (HSPGs) all displayed third-stage PQR migration defects. The effects in single mutants were weak compared to efn-4, and double mutant analysis revealed lack of genetic synergy, consistent with all of these molecules converging on a common pathway. This genetic analysis is consistent with physical interaction studies in vitro from another group that suggest that these molecules form connected communities of interacting extracellular domains, raising the possibility that they are all components of a large extracellular signaling complex required for posterior QL.ap migration. In this model, we envision that MAB-5/Hox drives EFN-4/Ephrin expression in QL.ap, which then seeds the formation of an extracellular signaling complex containing SAX-3/Robo signaling, UNC-6/Netrin signaling, and HSPGs that drives posterior lamellipodial formation and posterior migration.

Nervous systems display extensive diversity in structure and organization, yet a broadly conserved set of signaling pathway components and transcription factors is consistently associated with early neurogenesis in many animal lineages. Determining how these conserved markers map onto the spatiotemporal organization of neurogenic territories across phylogenetically informative but underrepresented lineages, particularly within Spiralia, is critical for refining inferences about the evolutionary origins and diversification of nervous systems. Chaetognaths, a spiralian lineage frequently recovered close to Gnathifera, have a compact and centralized nervous system but lack detailed molecular descriptions of early neural development. Here, we generate an expression-based developmental map of early neurogenesis in the chaetognath Spadella cephaloptera by combining nuclear-staining-based anatomical staging with spatiotemporal analyses of conserved developmental genes associated with early neurogenesis and axial patterning from gastrulation through early post-embryonic stages. Sce-soxB1-like1 and Sce-neuroD expressions mark a lateral neuroectodermal territory during gastrulation. Notably, Sce-neuroD is activated early in a broad ectodermal domain and is expressed within mitotically active neuroectodermal cells, consistent with early deployment in proliferative neurogenic territories. Sce-soxB1 and Sce-soxB2 show delayed and more spatially restricted expression relative to Sce-soxB1-like1, suggesting a paralog-specific partitioning of SoxB deployment during chaetognath neurogenesis. Sce-bmp2/4 and Sce-chd exhibit reciprocal dorsoventral expression during gastrulation that coincides with early neurogenic territory formation, before transitioning to more localized expression later in development. Sce-nk6 and Sce-hb9 reveal early ventral regionalization of the developing ventral nerve center (VNC), with Sce-hb9 occupying a subset of a broader Sce-nk6 domain, in line with conserved ventral subtype-associated regionalization. Sce-th (tyrosine hydroxylase) is detected in a small bilateral subset of hatchling VNC cells, while Sce-dbh (dopamine beta-hydroxylase) is first detected only in early juveniles in the anterior VNC and head domains, suggesting stage-dependent and region-specific deployment of catecholamine-pathway components. Together, these expression-based datasets provide a comparative reference for early neurogenesis in chaetognaths and a framework for assessing conserved and lineage-specific features of early neurogenic patterning across Spiralia.

Adult hydrozoan cnidarians undergo extensive tissue turnover, generating neural cell types including nematocytes (stinging cells) and gland cells from interstitial stem cells (i-cells) expressing stemness proteins such as Piwi and Nanos. The contribution of i-cells during embryogenesis, however, has been unclear. Here we address the origin of neural cells during development of the Clytia hemisphaerica planula larva. Marker gene in situ hybridisation revealed that Piwi/Nanos1-expressing cells within the early gastrula presumptive endoderm generate a substantial pool of nematoblasts, a few of which migrate and differentiate in the planula ectoderm. Some neurogenic and neuronal markers, however, showed a markedly distinct expression profile, developing within a basal layer of the aboral/lateral ectoderm during gastrulation. Embryo bisection and lineage tracing experiments confirmed that sensory neurons and secretory cell types derive from gastrula ectoderm, while nematocytes and at least some ganglionic neurons derive from i-cells. Knockdown and inhibitor treatments revealed steps in neuron and nematocyte development regulated by Wnt-{beta}-catenin. We conclude that two distinct neurogenesis pathways operate during Clytia embryogenesis, one involving aboral ectoderm delamination, and one generating mainly nematocytes from i-cell-like precursors. Summary statementDuring embryogenesis in the hydrozoan Clytia neural cell types derive both from Piwi/Nanos expressing "i-cells" and from ectodermal delamination during gastrulation.

Neural tube closure is a critical developmental process, essential to the proper formation of the vertebrate nervous system. This process starts with the invagination of neural plate cells. Its borders then converge, leading to the closure of the neural tube, propagating like a zipper. Afterwards, cell intercalation and proliferation allow the tube to elongate. Neural tube closure involves thousands of cells in vertebrates. However, the closest invertebrates to vertebrates, the tunicates, such as Ciona, close a hollow dorsal neural tube with fewer than 20 neural cells. This minimal model makes it easier to study the mechanisms of this intricated process. In Ciona, the transcription factor Lmx1 is expressed in the most dorsal cells of the developing neural tube, like its vertebrate orthologs. In vertebrates, Lmx1 paralogs are involved in neural tube patterning. However, no function related to morphogenesis has been uncovered. Here, we explore Ciona Lmx1 roles during neural tube closure. Lmx1 Knockdown leads to slight but significant defects in neural tube closure. The overexpression of a repressive Lmx1 variant prevents the proper intercalation of the dorsal neural tube cells, impeding the anterior progression of the zipper. Furthermore, studies of Lmx1 regulatory sequences indicate that Pax3/7, ZicL, and Nodal signaling may directly regulate its transcription. These transcription factors are present at the vertebrate neural plate border, suggesting that Lmx1 regulation is conserved across chordates. It raises the possibility of an unrecognized role for Lmx1 during vertebrate neural tube morphogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/709676v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@f409b1org.highwire.dtl.DTLVardef@1a88180org.highwire.dtl.DTLVardef@1ce2a89org.highwire.dtl.DTLVardef@4aba89_HPS_FORMAT_FIGEXP M_FIG C_FIG

Bone morphogenetic proteins (BMPs) play an important role in dorsal spinal cord patterning. Their presence in the roof plate of the midbrain indicates a role in its development. We examined whether the BMP signaling contributes to dorsal midbrain size expansion in chick embryos by missexpressing pathway activators and inhibitors. Overactivation of BMP4 did not affect midbrain development, whereas GDF7 reduced midbrain growth. In contrast, expression of a truncated dominant-negative BMP receptor type 1b or the extracellular inhibitor Chordin had no detectable effect. Ectopic expression of SMAD6, the intracellular inhibitor of the BMP/ TGF-{beta} pathway, significantly reduced midbrain size, which correlated with decreased proliferation rates of SMAD6-overexpressing cells. In some cases, SMAD6 also disrupted MTN axon trajectory. These results indicate an important role for SMAD-dependent signaling pathways in early dorsal midbrain growth.

The integration of growth and patterning in developing tissues is a complex process involving both intrinsic and extrinsic cues. The Hippo pathway, a conserved regulator of organ size, controls growth and patterning in Drosophila, including the development of the eye-antennal imaginal disc into adult structures. Defective Proventriculus (Dve), a SATB1/2 ortholog and K-50 type transcription factor regulates dorsal-ventral (DV) patterning during Drosophila eye development. Dve works with Wingless (Wg) to suppress eye development and promote head cuticle fate, thereby influencing the positioning of eyes and interocular distance. Our study investigates the role of the Hippo effector Yorkie (Yki), and dve in coordinating growth and patterning during eye development, specifically focusing on the regulation of the head cuticle domain. Here we show that Hippo signaling, mediated by Yki, regulates the size of the head cuticle domain and morphogenetic furrow (MF) progression, and that Dve suppresses Yki activity in the dorsal head region. Furthermore, Yki regulates several DV patterning genes like pnr, wg and mirr, to coordinate eye and head development. Mutations in mammalian orthologs of yki, dve, pnr and wg are associated with facial dysmorphia and several developmental disorders. Our studies thus reveal new genetic mechanisms by which growth and patterning are coordinated for head and eye development across species.

Spinal motor nerves are an integral component of the nervous system whose development requires the coordination of many diverse cell types, including motor neurons, glia, and muscle. Although several molecular mechanisms guiding these interactions are known, many remain to be uncovered. Extracellular matrix (ECM) proteins also play a critical role in motor nerve assembly, yet their functions are less understood compared to classical pathfinding and guidance cues. Here, we identify a role for tenascin-n (tnn), an ECM glycoprotein, in spinal motor nerve development in zebrafish. Using in situ hybridization and immunohistochemistry, we show that tnn/Tnn is expressed and localized along vertical myosepta and the border of the ventral neural tube during spinal motor nerve development. To assess its function, we generated a CRISPR/Cas9 mutant allele, tnnuva96, and performed in vivo imaging and morphological analysis throughout motor nerve development. Loss of tnn leads to a subtle and transient increase in ectopic motor axon exit and aberrant motor axon branching in the zebrafish trunk. Our findings reveal a previously unrecognized role for tnn in spinal motor nerve assembly and expand our understanding of the diverse molecular contributors to spinal motor nerve development and morphogenesis.

The Meckels cartilage (MC) is a fundamental component of mandibular development across vertebrates. In mammals, MC is transient and functions primarily as an early template for mandibular ossification, whereas other vertebrates, including zebrafish, retain MC within the mandible throughout life. Despite its importance, the requirements for MC in sustaining mandibular growth and how signaling pathways implicated in MC development contribute to this process remain unclear. Here, we investigated the dosage-dependent roles of BMP antagonists during zebrafish MC development using mutant alleles of grem1a, nog2, and nog3. Compound mutant adults exhibited fully penetrant mandibular truncation. MC shortening emerged after early larval stages, indicating a requirement for BMP antagonism to sustain cartilage growth. Chondrocyte number remained unchanged as phenotypes developed, but mutants displayed disorganized cartilage morphology and increased chondrocyte volume. Molecular analyses revealed reduced col2a1a domains and expanded ihha and col10a1a expression, consistent with ectopic hypertrophic-like differentiation. Constitutive activation of BMP receptor signaling in chondrocytes recapitulated these phenotypes. Although osteogenesis appeared unaffected by 14 dpf, loss of a tnn skeletal mesenchyme population was observed. Together, these findings demonstrate that BMP antagonists sustain MC growth by regulating chondrocyte differentiation and cartilage organization to support mandibular growth in non-mammalian vertebrates. Summary StatementThis study leverages zebrafish to define the cellular and molecular mechanisms by which BMP antagonism sustains mandibular growth.

Light and transmission electron microscopy were used to identify changes in ultrastructure of the olfactory pit of larval zebrafish (Danio rerio) that occur as a result of age and altered environmental light levels. Larvae were reared under control/cyclic light or constant light condition until 4, 8, and 15 days postfertilization (dpf). The larval olfactory pit consisted of an epithelium that varies from simple to pseudostratified to stratified and contained three types of receptor cells: ciliated, microvillar and ciliated crypt. A variety of non-receptor cells were also identified: kinociliate non-sensory supporting cells, vesicular supporting cells, basal cells and an occasional intruder, such as a neutrophil or a lymphocyte. Microvilli projecting from microvillus receptor, kinociliate, and vesicular supporting cells were single, forked, or doubly forked. Junctional complexes were evident between a variety of cells including adjacent epidermal cells, an epidermal cell and a kinociliate cell, a kinociliate and a vesicular supporting cell, and two vesicular supporting cells. Desmosomes were also observed between adjacent cell types. With age, the olfactory epithelium thinned and vesicle number varied. In larvae reared in constant light, mitotic figures were evident, microvillar receptor cells were absent, and, at 4 dpf, some ultrastructural components were similar to those observed in 8 dpf control animals, suggesting precocious development. These findings suggest that constant light rearing alters the timing of receptor replacement, supporting previous work showing that rearing light levels impact sensory system growth and development.

BackgroundDuring development, axons are organized into bundles, a process known as axonal fasciculation. The zebrafish lateral line nerve has been used as a model to study axonal fasciculation; however, the underlying mechanisms are not yet fully understood. Although Fgf3 and Fgf10a are well known to regulate the migration of the lateral line primordium along which the lateral line nerve projects, their roles in the organization of the lateral line nerve itself have not been clarified. Resultsfgf3,10a double mutants exhibited lateral line axonal defasciculation accompanied by an increased number of Schwann cells. Live imaging revealed a marked increase in Schwann cell proliferation and demonstrated that newly divided Schwann cells migrate along axons and infiltrate interaxonal spaces, thereby expanding these spaces and disrupting axonal fasciculation. Pharmacological manipulations further implicated a contribution of Nrg1-ErbB signaling to this phenotype. ConclusionsOur findings suggest that Fgf3 and Fgf10a are required to restrict Schwann cell proliferation and infiltration, thereby ensuring axonal fasciculation during lateral line development.

Cardiac neural crest cells (CNCCs) contribute to key cardiac structures during embryonic development. Disruption of CNCC patterning or function can lead to congenital heart defects. Here, we investigate whether hemodynamic perturbation alters CNCC behavior in chick embryos. We use the left atrial ligation model to modify intracardiac blood flow in the early common-atrium, common-ventricle heart and track retrovirally labelled CNCCs for lineage tracing and single-cell transcriptomic analysis. Results revealed a significant reduction of CNCC derivatives in major cardiac regions, including the pharyngeal arch arteries and myocardium, in flow-perturbed embryos compared with controls. Notably, despite reduced CNCC numbers in the PAAs, their relative proportion increased, suggesting retention within the PAAs and delayed differentiation. Transcriptional analysis shows the expression of CNCC post-migratory markers (HAND1, FOXC2, GATA6, and TBX2) were consistently downregulated at 4, 24, and 48 hours after LAL. Together, these findings indicate that hemodynamic perturbation impairs CNCC migration and differentiation while preserving their capacity to contribute to mature cardiac structures.

Regeneration in Drosophila has primarily been studied in imaginal discs, which are organized into distinct compartments defined by strict lineage boundaries. While cells typically do not cross these borders, regeneration can bridge them to restore the original organ specification, which is determined by Hox genes. Although differences in Hox gene expression are known to segregate cell populations, the role of such differences as a potential barrier to regeneration remains unclear. We investigated this by analyzing two experimental settings: the analia (derived from the Hox-compound genital disc) and haltere discs with mutations in Ultrabithorax regulatory regions (bithorax or postbithorax). Our findings demonstrate that Hox gene differences are not an absolute impediment to regeneration across segments or compartments. However, we observed occasional regenerative limits, which were non-specifically enhanced in the postbithorax background.

Microtubules are essential components of the cytoskeleton that support epithelial organization, polarity, and tissue morphogenesis. They are composed of - and {beta}-tubulin heterodimers, each encoded by distinct genes that generate closely related but functionally distinct isotypes. Although several tubulin isotypes have been implicated in ocular development and disease, how isotype diversity is organized during corneal morphogenesis remains poorly defined. Herein, we use the developing chick embryo as a model system to investigate the conservation and spatiotemporal localization of tubulin isotypes during corneal development. Through comparative amino acid sequence analysis, we show that chick and human - and {beta}-tubulin isotypes are highly conserved at structural and catalytic domains, with divergence concentrated in C-terminal regions associated with post-translational modifications. To relate these molecular features to tissue-level organization, we performed a longitudinal immunohistochemical analysis of five tubulin isotypes across key stages of corneal development. We identify distinct and dynamic patterns of isotype enrichment along apico-basal and central-peripheral axes within the cornea, as well as isotype-specific redistribution during epithelial maturation and corneal endothelial differentiation. Notably, TUBA5/TUBA4A exhibits tightly regulated localization, including enrichment at the leading edge of migratory corneal stromal progenitor cells and within the maturing corneal endothelium. Together, these data establish the chick embryo as a conserved and tractable model for studying tubulin isotype diversity in the cornea, and more broadly across other tissues, and to provide a developmental resource linking tubulin sequence identity to spatially defined microtubule organization during epithelial morphogenesis. SUMMARY STATEMENTThis study defines when and where distinct tubulin proteins are deployed during corneal development, providing a resource for understanding cytoskeletal organization in the developing eye.

Mitochondria undergo significant structural and functional changes during human pre-implantation embryogenesis, yet the transcriptional activity of both nuclear-encoded mitochondria-associated genes and mitochondrially transcribed genes across this developmental window remains poorly characterized. While mitochondria are established as the primary energy source for the early embryo, emerging evidence suggests they may also influence lineage specification through epigenetic regulation and metabolite availability. To investigate this, we reanalyzed two publicly available human single-cell RNA sequencing datasets filtered for mitochondria-associated genes using the MitoCarta 3.0 reference database, with separate analyses conducted on the nuclear-encoded and mitochondrially transcribed subsets. The first dataset spanned individual blastomeres from the oocyte through blastocyst stage, and the second compared trophectoderm and inner cell mass cells isolated from blastocysts. Mitochondria-associated gene expression was sufficient to cluster human blastomeres by developmental stage, with morula and blastocyst stage cells forming well-defined clusters. Mitochondrially transcribed genes were found to be the primary drivers of clustering in earlier developmental stages, while nuclear-encoded mitochondria-associated genes drove clustering at the blastocyst stage. A pronounced shift in the expression of both gene sets was identified at the transition from the 4-cell to the 8-cell stage, with 115 unique differentially expressed genes identified across the two stages immediately following this transition, compared to only 5 across the two prior stages. The timing of this transcriptional upregulation, preceding the known onset of oxidative phosphorylation at approximately the 32-cell stage, suggests a mitochondrial role in early embryogenesis beyond energy production. Analysis of trophectoderm and inner cell mass cells showed that mitochondrial gene expression profiles partially distinguished these two lineages, consistent with known differences in mitochondrial activity between them. These findings suggest that both nuclear-encoded and mitochondrially transcribed gene expression is upregulated prior to the first lineage specification event in the human embryo, potentially contributing to epigenetic regulation and cell fate determination through altered metabolite availability. A limitation of this study is its reliance on transcriptomic data alone; future work incorporating functional metabolite measurements will be needed to establish causality. Nonetheless, these data reframe mitochondria as active participants in early human developmental programming rather than passive energy suppliers.

The evolution of visual systems has compelled numerous investigations of developmental processes underlying eye patterning across Bilateria. It is well-established that homologs of the transcription factor Pax6 play a highly conserved role in eye fate specification and are at the top of the retinal determination gene network (RDGN) hierarchy. In insects, the two Pax6 homologs eyeless (ey) and twin of eyeless (toy) are required for the development of the two visual systems broadly found within the phylum (i.e., median and lateral eyes). Curiously, Pax6 homologs do not appear to maintain this function in well-studied chelicerate models, with emphasis on spiders, a lineage of arachnids with great diversity of eye form and acuity. It was recently proposed that the gene Pax2 (shaven; sv) may have subsumed the role of eye fate specification in chelicerates, a hypothesis predicated upon the observation that one of two spider Pax2 copies is strongly expressed in the developing lateral eyes during embryogenesis. However, no functional data are available for any Pax homologs across Chelicerata. We examined the incidence of Pax family genes across Chelicerata, as well as interrogated the expression and function of Pax2 and Pax6 homologs in the daddy-longlegs Phalangium opilio, an arachnid recently discovered to bear a highly plesiomorphic arrangement of visual systems. Here, we show that ey and toy are expressed early in the developing head lobes of P. opilio, whereas sv is not expressed until well after stages when downstream RDGN members (eyes absent and sine oculis) are already activated. Gene silencing of ey, toy, and sv individually had no discernible effect on eye development. By contrast, double knockdown of ey and toy resulted in an array of median eye defects, spanning loss of some cells of the eye to total loss of the median eyes. Gene expression assays also showed that depletion of the two Pax6 copies resulted in failure of the vestigial median and vestigial lateral eyes. These data are consistent with a conserved role for Pax6 homologs in patterning both visual systems and all three eye pairs in the daddy-longlegs. Our results comprise the first functional data for Pax6 genes in any chelicerate and suggest that heterochronic shifts in expression, rather than changes in function, underlie the atypical dynamics of Pax genes in derived arachnid groups such as spiders.

ABSTRACT/SUMMARYDevelopment of the visual system is dependent upon precise regulation of cell fate specification. In the mammalian retina, a single pool of multipotent progenitor cells becomes competent to produce the seven major retinal cell classes in distinct but overlapping windows. MicroRNAs (miRNAs) have been implicated in controlling retinal progenitor competence and risk for retinal disease, but the specific contribution of individual miRNAs and how they may be regulated is still unclear. Here we characterize a deeply conserved gene regulatory unit that includes the miRNA, miR-9-2, and a retinal-disease-associated enhancer that controls its expression. Loss of miR-9-2, one of three mammalian miR-9 paralogs, delays the emergence of late-born retinal cell classes and leads to misspecification of Muller glial cells to a hybrid neuronal-glial fate. Further, we identify transcription factors and gene regulatory networks directly controlled by miR-9-2 during retinal development. Lastly, we provide evidence of a negative feedback loop through which miR-9-2 regulates itself. Altogether, this study provides insight into mechanisms that regulate the timing of retinal progenitor competence and glial cell identity, and how this gene regulatory unit may contribute to retinal disease. HIGHLIGHTSO_LIA functionally conserved, disease-associated enhancer regulates miR9-2 expression in human and mouse retina. C_LIO_LImiR9-2 regulates key transcription factors in progenitor cells and glia. C_LIO_LImiR9-2 controls the timing of retinal cell class specification. C_LIO_LIRegulation of miR9-2 is required to establish and maintain proper glial cell identity. C_LI