Developmental Biology

Background: During vertebrate embryogenesis, the isthmic region spans the midbrain hindbrain-boundary of the neural tube and includes an organizer (IsO) that is essential for proper formation of adjacent brain regions, yet the molecular and cellular composition of the isthmic region remains unresolved. Results: We employed combined single-nucleus ATAC-seq and RNA-seq (scMultiome) in 13 and 16 hours-post-fertilization zebrafish embryos to molecularly resolve cell populations in the isthmic region and validated our findings in vivo by RNA fluorescence in situ hybridization. We identified two distinct isthmic cell populations (isthmic midbrain [IsMB] and isthmic hindbrain [IsHB]) that share expression of canonical isthmic genes, but that differ in their expression of midbrain vs hindbrain genes. We also uncovered a previously unrecognized heterogeneity within the IsHB, reflecting a canonical fgf8-expressing population anteriorly (IsO/r0a), and a novel fgf8-negative population posteriorly (r0p). We find that inhibition of Fgf signaling disrupts formation of the isthmic region, leading to loss of isthmic cell populations except a residual population characterized by a mixed neural identity. Conclusions: Using transcriptional and epigenetic characterization, we expand on prior anatomical and genetic analyses of the isthmic region to refine our understanding of its cellular organization and demonstrate that it consists of several subdomains.

Periodic patterns are a frequent motif in biology that occurs across diverse tissues and species. In mammals, pigmentation patterns such as zebra stripes or tiger stripes are well-known examples of periodic patterns; more recently, the myenteric plexus (MP) of the enteric nervous system (ENS), which controls gastrointestinal motility, has been found to exhibit a striped organization in humans and laboratory mice. In domestic cats and other felids, the Transmembrane aminopeptidase Q (Taqpep) gene plays a key role in color pattern establishment during skin development, but its patterning role has not been examined in other tissues. Here, we show that, in laboratory mice, Taqpep is required for normal patterning of developing hair follicles and the MP. Using both sequencing and histologic techniques, we found Taqpep is expressed in mesenchymal cells in embryonic skin and intestine directly adjacent to where periodic patterning occurs. We generated Taqpep mutant mice, which exhibit disrupted epidermal patterning akin to the changes in periodic coat patterning observed in Taqpep mutant cats. The intestine of Taqpep mutants has irregularly periodicity of enteric neuronal stripes, and enteric neurons in Taqpep mutants exhibit disrupted Wnt signaling. This work provides new insight into the mechanism of enteric neuronal patterning and identify Taqpep as a common and conserved mediator of periodic patterning across mammalian tissues and organisms. Author summaryPeriodic patterning is a frequent motif in biology. Examples include pigmentation patterning such as tiger stripes and, as recently identified in both mouse and human, the striped organization of enteric neurons in the myenteric plexus of the intestine. In domestic and wild cats, the Transmembrane aminopeptidase Q (Taqpep) gene is essential for the establishment of periodic patterning. Whether this gene plays a conserved role in periodic patterning across other tissues and species has yet to be explored. We found that Taqpep is expressed in mesenchymal cells in embryonic mouse skin and intestine at key locations and developmental stages to instruct periodic patterning. We next generated Taqpep mutant mice that exhibit disrupted periodic patterns in both developing skin follicles and in enteric neuron organization. Thus, Taqpep is essential in establishing periodic patterning in diverse mammals and tissues.

Zebrafish possess three distinct sources of retinal progenitors that produce identical photoreceptor subtypes throughout life. All photoreceptor progenitors simultaneously express multiple transcription factors specifying different identities, requiring mechanisms to repress alternative fates. Disruption of the tbx2 paralogues, tbx2a or tbx2b, resulted in a cell-fate switch of sws1 cones into rods. Here, we demonstrate that tbx2b was necessary for sws1 cone differentiation during embryogenesis and outgrowth at the retinal margin, but tbx2a was necessary during photoreceptor regeneration. Transgenic overexpression of Tbx2b was not sufficient to drive the sws1 cone fate or sws1 opsin expression. Rather, Tbx2b repressed the synergistic activity of Nrl and Crx at the rhodopsin promoter. Targeting the transcription factor thr{beta}2 on wildtype and tbx2 mutant backgrounds revealed a hierarchy wherein early progenitors have the potential to be respecified from lws cones into sws1 cones or rods. But late progenitors are limited to either the sws1 cone or rod fate. These data support a model in which transcriptional repressors, like tbx2a and tbx2b, orchestrate progression through competency states.

UNC-6/Netrin is a conserved regulator of dorsal-ventral axon and cell migrations. UNC-6 is composed of a Laminin N-terminal domain (LN), three epidermal growth factor repeats (EGF), and a Netrin C terminal domain (NC). Here, we identified missense mutations in distinct UNC-6 domains and assessed their roles in dorsal VD/DD motor axon guidance and ventral AVM axon guidance. A missense mutation in a conserved residue of the LN domain (G289D) resulted in dorsal and ventral axon guidance defects similar to unc-6 null. A distinct missense mutation in the LN domain (S120F) was hypomorphic and strongly perturbed ventral AVM axon guidance with minimal effects on dorsal VD/DD axon guidance, showing that S120F is predominantly required for ventral guidance. Missense mutations altering conserved cysteine residues involved in di-sulfide bonding in the EGF domains were analyzed. EGF1(C321G) caused both ventral and dorsal axon guidance defects albeit weaker than unc-6 null, indicating that EGF1 is required for both. EGF2(C347Y) strongly affected dorsal VD/DD axon guidance similar to unc-6 null, with weaker perturbation of ventral AVM axon guidance. Previous results revealed that EGF3(C410Y) specifically disrupted dorsal axon guidance, a result that we confirmed. Our studies using missense mutations in the endogenous unc-6 locus complement previous structure-function studies using transgenic expression, and identify domains specifically required for ventral AVM guidance (S120Y in the LN domain) and dorsal VD/DD axon guidance (C410Y in EGF3). The crystal structure of UNC-6 indicates conserved N-linked glycosylation at N114 and N128. Mutation of these sites in UNC-6 had no effect on dorsal ventral axon guidance, showing that they do not play a major role. However, the N114 and N128 mutations interacted genetically with unc-40 and unc-5 mutations, indicating that these glycosylation sites indeed have a role in UNC-6 signaling. Our results will inform studies on how these distinct UNC-6 domains interact with guidance receptors (e.g. UNC-40/DCC and UNC-5) and other extracellular molecules to mediate dorsal-ventral axon guidance.

The hallmark of sessile adult ascidians is a vase-like shape with a single oral and atrial siphon. Ciona, however, develops two atrial siphons after metamorphosis, which subsequently fuse into one. The mechanisms underlying this fusion are unknown. This study suggests that Hox2 controls this process. Hox2-knockout animals using Transcription-Activator-Like Effector Nuclease (TALEN) retain two atrial siphons throughout their lives. During normal fusion, epidermal cells between the siphons flatten along the anterior-posterior axis. This cellular flattening does not occur in Hox2-knockout animals, suggesting that the shape change in the epidermal cells produces tension, allowing the atrial siphon openings to converge at the midline for fusion. Hox2-knockout animals lack cupular organs, which are suspected hydrodynamic sensors in the internal epithelium of the fused atrial siphon and on the sperm duct. Among several knockout attempts, atrial siphon fusion was reproduced by only one TALEN pair, suggesting that this phenotype is driven by a mutation having a broader effect than those abolishing protein function. Many ascidians, unlike Ciona, develop a single atrial siphon shortly after metamorphosis. Our findings suggest that a phylogenetically conserved gene, Hox2, establishes this group-specific atrial siphon formation mechanism in Ciona.

The mature kidney contains approximately one million nephrons, and defects arising during nephron development can result in lifelong renal impairment, often culminating in kidney failure and transplantation. Nephric tubule formation requires coordinated epithelial processes, including polarity, adhesion, signaling, and vesicle transport; however, how these processes are integrated during kidney morphogenesis remains unclear. Dynamin binding protein (Dnmbp) is a multi-domain scaffolding protein expressed in human kidneys that is involved in several cellular processes. Using the Xenopus embryonic kidney, we previously demonstrated that Dnmbp is essential for nephrogenesis, yet the mechanisms by which it influences nephron development remain undefined. Here, we identify Dnmbp as a novel interacting partner of the Wnt/planar cell polarity effector Daam1. The interaction between Daam1 and Dnmbp was independently identified in two yeast two-hybrid screens, biochemically verified, and supported by structural modeling predictions of a Daam1-Dnmbp complex. In developing Xenopus laevis kidneys, Dnmbp localized to punctate structures associated with E-cadherin-rich cell-cell contacts. Dnmbp depletion significantly reduced junctional E-cadherin localization in both epithelializing and mature nephric tubules without affecting total E-cadherin levels, indicating a role in E-cadherin recruitment or stabilization at adherens junctions. Furthermore, expression of human DNMBP rescued the junctional defects, confirming the specificity of the loss-of-function phenotype. Together, these findings identify Dnmbp as an essential regulator of kidney development and support a model in which Dnmbp provides a mechanistic link between Wnt/PCP signaling, Cdc42 activation, and adherens junction formation during nephrogenesis.

Zebrafish combines the power of genetics and unparalleled in vivo imaging for investigating the dynamics of vertebrate hematopoietic development. Across species, the transcription factor Runx1 is essential for definitive hematopoiesis. We generated a zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Cre recombinase Runx1 lineage tracing and characterized its activity using the ubi:Switch recombinase-dependent fluorescence reporter, microscopic live imaging and flow cytometry. Tamoxifen treatment beginning at gastrula stage labeled all expected Runx1 lineages in the early embryo, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and nascent hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Runx1 HSPCs colonized the larval caudal hematopoietic tissue and thymus from three to five days of development. Timed tamoxifen induction of Cre activity allowed separation of Runx1 primitive hematopoiesis from definitive HSPC emergence and larval stem cell niche colonization. Flow cytometry of kidney marrow and peripheral blood from adults treated with tamoxifen at gastrula stage revealed Runx1 embryonic hematopoietic cells contributed to adult hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The zebrafish runx1-2A-creERT2 line provides a powerful tool for precise spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis. Key PointsO_LIzebrafish endogenous runx1-2A-creERT2 provides inducible Cre recombinase genetic analysis in all runx1 neuromesodermal and blood lineages C_LIO_LIzebrafish runx1-2A-creERT2 line enables in vivo spatial and temporal analysis of embryonic and adult hematopoiesis C_LI

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K1-10). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG211, the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (~130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

Conserved dorsoventral patterning systems have been proposed as evidence for a common evolutionary origin of centralized nervous systems in Bilateria, yet functional evidence outside vertebrates and arthropods remains limited. Here, we investigated the role of pax6 in the annelid Platynereis dumerilii using a mutant carrying a 61 bp deletion in the paired-domain coding region. Loss of pax6 disrupted ventral neuroectodermal patterning at 34 hpf, causing a shift in nk2.2 expression, narrowing of the nk6 domain, and downregulation of pax3/7, while msx expression remained largely unaffected. These early patterning defects were followed by selective neuronal abnormalities at 48 hpf, including displacement of TrpH-positive serotonergic cells and loss of posterior hb9-positive motoneuron domains. By 6 dpf, additional defects were observed in TrpH, ChAT, VAChT, and nk2.2 expression, accompanied by severe disruption of ventral nerve cord morphology and loss of the characteristic rope-ladder architecture. Together, these findings identify pax6 as a key regulator linking dorsoventral progenitor patterning, neuronal subtype specification, and nervous system morphogenesis in Platynereis. Our results provide functional evidence that the conserved dorsoventral patterning network plays an essential role in annelid ventral nerve cord development and support the view that important components of bilaterian nervous system patterning predate the divergence of major animal lineages.

The caudal fin is an anomaly among vertebrate locomotory appendages: its internal skeleton is as asymmetric as the human hand, but it lacks the Shh-secreting Zone of Polarizing Activity (ZPA) and the Gli3/HoxD/Hand2 programs that govern anterior-posterior patterning in other appendages. As the caudal fin was also the first appendage to evolve, deciphering its alternative patterning program may provide clues to the ancestral state. Pax9 is one of few conserved appendage patterning factors reported to also be active in the caudal fin, specifically in the anterior domain. We report that loss of pax9 function in zebrafish not only disrupts anterior-specific caudal fin anatomy, but also results in a spectacular fusion of the caudal and anal fins along the ventral midline. The dorsal fin is also expanded to a lesser degree, the paired fins not at all. The mutant caudal fin initially forms as an irregularly patterned structure lacking anterior molecular identity, with supernumerary elements spilling out beyond the normal anterior boundary. Unexpectedly, this phenotype is subsequently compounded by neighboring trunk somites erroneously deploying skeletal mesenchyme in the normally finless caudal peduncle region, completing the ectopic skeleton. scRNAseq analysis at caudal fin bud stage indicates that pax9 mutants gain skeletal mesenchyme at the expense of a specialized type of fibroblast involved in the formation of fin fold actinotrichia. Median fin skeletal mesenchyme and fin fold fibroblasts both arise from the sclerotome, a somite compartment that also robustly expresses pax9. We propose that, within the sclerotome, Pax9 pushes progenitors towards fin fold fibroblast fate, limiting how many cells will later be available to make median fin skeleton. Within the fin bud, it drives anterior identity, with the strongest impact on the ZPA-free caudal fin bud. These dual sites of action make Pax9 a uniquely powerful governor of median fin development.

The endoderm is specified at the onset of gastrulation and subsequently undergoes extensive migration before forming an epithelial sheet that gives rise to multiple organs, including the gut and respiratory tracts. Although the gene regulatory network underlying endoderm specification and the later processes that regionalize the gut are increasingly well understood, comparatively little is known about the intervening developmental events. Using single cell transcriptomics, we profiled the zebrafish sox17 lineage, comprising endoderm and dorsal forerunner cells, throughout and immediately after gastrulation. We found that dorsal forerunner cells remain transcriptionally homogeneous while undergoing coordinated temporal changes, associated with ciliogenesis and epithelial organization, during assembly of Kupffers vesicle. In contrast, endoderm cells transition from a migratory to an epithelial transcriptional state while progressively acquiring distinct regional identities. These findings indicate that endoderm regionalization emerges within the context of a broadly shared transcriptional program associated with migration and epithelialization.

Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation. Highlights- FAF2 is required to regulate ER homeostasis in neural progenitor cells - FAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation. - Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.

Tissue patterning is integral to development and regeneration, yet the factors that initiate morphogenetic patterning remain to be explored. Here, using embryonic chicken skin as a model, we show that perturbation of calcium signaling induces de novo feather bud formation in regions that normally do not form feather buds. This is achieved through coordinated changes in calcium dynamics, endogenous bioelectric currents, transcriptional regulation of calcium and potassium channel genes, and morphogen signaling. Different combinations of channel perturbations altered the number, distribution, size, and shape of induced feather buds. Live calcium imaging and extracellular electrophysiological recordings revealed homeostatic regulation, in which initially depressed calcium activity is followed by elevated calcium activity. Inward bioelectric currents emerge as de novo feather buds appear. Potassium channel blockade suppressed calcium dynamics, abolished endogenous currents, and inhibited new bud formation. Canonical feather morphogenesis pathways including Shh and {beta}-catenin are induced in these new buds. Our findings support a model in which developmental bioelectricity contributes to regulating the threshold of feather bud formation. These results identify developmental bioelectricity as an unrecognized regulatory layer of tissue patterning that warrants further study. Bullet points- Calcium signaling perturbation induces de novo feather bud formation in apteric skin - Ion channel perturbations regulate the formation, distribution and shape of new buds across a continuum, depending on channel type(s) and perturbation strength. - Elevated calcium activity and inward bioelectric currents accompany feather bud induction - Developmental bioelectricity represents an unrecognized regulatory layer for morphogenesis

The morphogenesis of the optic cup provides a robust system for studying how two apposed epithelial monolayers with distinct properties fold and stretch in a coordinated manner to form the primordial eye. While much research has been conducted on the temporal dynamics of retinal neuroepithelium invagination, the spatial organization and stretching of the retinal pigment epithelium has received less attention. The fish species Astyanax mexicanus offers a unique model to examine the mechanisms of optic tissue morphogenesis through a comparative lens, as it exhibits natural variation in eye development between its river-dwelling and cave-adapted morphs. Using quantitative 3D imaging of optic cups from both morphs, we found that RPE morphogenesis involves transient, graded, and anisotropic cell stretching that patterns the epithelium during optic cup shaping. Analyses of RPE nuclear spacing and cell morphology showed that tissue stretching gradually increases along the proximo-distal axis, suggesting maximal tension in the elongated distal RPE cells aligned along the optic cup meridians. Furthermore, nuclear volumes and apical surface areas of RPE cells scaled spatially along the same axis, independently of endoreplication. In the cavefish natural mutant, RPE expansion was delayed by over six hours and proximal stretching exhibited altered isotropy, indicative of disrupted temporal coordination and suggesting modified mechanical constraints. These results demonstrate that RPE morphogenesis is a highly heterogeneous process from a spatiotemporal perspective, offering new insights into the study of the biomechanical principles of eye development in vertebrates. Summary statementThis study reveals the emergence of cell morphology gradients within the retinal pigment epithelium during morphogenesis of the eye in two distinct populations of the same species of fish.

A central unresolved question in development biology is how systems of overwhelming complexity arise from relatively few families of growth factors. Compounding this issue, signaling pathways often show signal convergence, where many ligands interact with fewer receptors, which then signal through a single second messenger complex. Here we investigate this question in the context of bone morphogenetic protein (BMP) signaling and its role directing dorsal spinal cord development, focusing on two receptor-regulated (R) Smads, Smad1 and Smad5. Multiple models have been proposed for their mode of action from acting redundantly through combined signal strength, to having distinct activities that drive different fate outcomes. We sought to distinguish between these models by generating CRISPR-edited Smad1 and Smad5 null mouse embryonic stem cell (ESC) lines to dissect the cell fate of activities of individual R-Smads, with a resolution not possible in vivo. Using a directed differentiation protocol for dorsal interneurons (dI), together with bioinformatic analyses, we have defined the roles of the R-Smads at key decision points along the dI specification timeline. Together, these findings support a model in which Smad1 and Smad5 play largely distinct roles in dorsal spinal cord development. While both R-Smads can activate canonical BMP-responsive transcriptional targets, they asymmetrically contribute to cell fate specification. Smad1 plays a restricted role, while Smad5 has a dominant role, regulating dorsal progenitor transcriptional dynamics and reiteratively directing the dorsal-most dI fates.

Most of the visceral organs are anatomically asymmetric across the left-right axis. These asymmetries can be traced to a well-studied molecular cascade leading to left-sided gene expression, including Pitx2, in the mesoderm. Yet how these early differences in gene expression are converted into differential shaping of organs at later stages remains incompletely understood, and for many organs, such as the lung, the question has not even been explored. Meanwhile, the signaling pathways responsible for the morphogenesis of the lung have been intensively studied, but no insight has been reported regarding whether they should differ on the left and right sides. Here we identify Fgf10 as a Pitx2-sensitive signal in the mesenchyme of the developing mouse lung. Fgf10 expression increases as Pitx2 decreases, making the right lung, which lacks Pitx2 expression, grow faster than the left during the budding stage. Modulating Fgf10 dosage in the left mesenchyme is sufficient to alter lung budding asymmetry. At the cellular level, the faster growth of the right lung is established by increased levels of epithelial proliferation, without significant differences in directional migration into the mesenchyme. Conditional genetics further show that Pitx2 acts during the budding stage to establish later branching asymmetry. Thus, Pitx2 converts left-right mesenchymal identity into organ asymmetry by quantitatively tuning Fgf10-dependent epithelial growth during early organogenesis.

The developing spinal cord contains both neural and non-neural tissues that arise within a shared morphogen signaling environment, raising the question of how distinct lineage identities are established and maintained. A striking example of this is the floor plate (FP), a mesoderm-derived, non-neural midline structure that functions as a critical signaling center adjacent to neural progenitor domains. Here, using zebrafish, we show that the pioneer transcription factor foxa2 is expressed in the FP, adjacent p3 neural progenitors, and p3-derived Kolmer-Agduhr" (KA") interneurons. Loss of foxa2 results in a complete loss of canonical FP identity and an expansion of p3 progenitors. Lineage tracing reveals that, in the absence of foxa2, FP cells undergo a fate transformation into neuron-producing p3-like cells, indicating that Foxa2 functions as a lineage barrier to preserve non-neural FP identity. In contrast, within the neural lineage, loss of foxa2 leads to elevated Sonic hedgehog (Shh) pathway activity and impaired KA" differentiation, suggesting that Foxa2 negatively regulates Shh responsiveness. Conversely, Foxa2 overexpression induces ectopic FP and KA" marker expression in a stage-dependent manner. Together, our findings reveal a dual role for Foxa2 in maintaining the non-neural FP lineage while fine-tuning morphogen responsiveness in neighboring neural progenitors during spinal cord development.

Closely related neurons that share core differentiation programs can be difficult to distinguish by gene expression, even when they differ in connectivity and function. Cranial motor neurons (cMNs) exemplify this challenge, forming discrete nuclei that control behaviors as diverse as eye movement, feeding, facial expression, and regulation of visceral organ function. However, the developmental programs that drive cMN target choices and functional specialization have not been comprehensively studied in any vertebrate. Here, we present an integrated single-cell RNA-sequencing atlas of zebrafish cMN development from 18 to 144 hours post-fertilization, combining FACS-purified cMNs with cMNs computationally extracted from published whole-embryo datasets and perform extensive validation by HCR in situ hybridization. We find that each cranial motor nucleus expresses a distinct transcriptional signature, and in many cases, we identify transcriptional correlates to functional subtypes within individual nuclei as determined by retrograde labeling. We find that identity often precedes axon targeting, indicating that cranial motor neuron fate is genetically specified early in development. These distinct identities are shaped by the intersection of shared function, rhombomere origin, and developmental time. A cross-species comparison reveals a largely one-to-one correspondence between zebrafish and mouse cMN nuclei, indicating that these genetic programs are conserved. Together, this atlas provides a nucleus-resolved molecular framework for understanding cranial motor neuron diversification, and for interpreting human cranial dysinnervation disorders.

Neuromuscular contractures arising from neonatal brachial plexus injuries (NBPI) are highly disabling and currently incurable. We previously showed that contractures involve impaired longitudinal growth of denervated muscles, a defect mediated through myostatin (MSTN) signaling, a potent negative regulator of muscle size. However, MSTN-mediated contractures occur independent of canonical signaling pathways, including SMAD 2/3 and AKT/mTOR. Through a mouse model of NBPI, our present study extended these findings by revealing pharmacologic inhibition of JNK signaling, a noncanonical pathway downstream of MSTN, partially rescues contractures without restoring muscle length. Rather, JNK activation upregulates myofiber expression of the target gene Lmna, which encodes the nuclear envelope proteins Lamin A and Lamin C that are vital for nuclear stability, resulting in pervasive myonuclear displacement. These results suggest that other factors contribute to contracture pathology beyond deficits in longitudinal muscle growth. Further, while JNK inhibition does not restore length of denervated muscles, it impedes size and mass of normally innervated neonatal muscles, suggesting a requirement of JNK signaling for neonatal muscle growth. Our collective findings thereby establish new mechanistic insights into the molecular basis of aberrant muscle growth and neuromuscular contracture formation, potentially leading to novel targets for muscle restorative strategies and medical contracture prevention.

The evolution of flippers for wing-powered diving in penguins is a striking example of tetrapod limb specialization. The modern penguin flipper is structurally reinforced by a characteristic dorsoventral flattening of the long bones accompanied by a reduction in distal forelimb musculature, features which emerged convergently in flightless diving birds and aquatic mammals. While an extensive fossil record informs the morphological sequence through which these changes occurred, the evolutionary pressures and developmental mechanisms underlying these modifications are unknown. We find that in avian and mammalian forelimbs, a flattened bone morphology only emerged in aquatic lineages that lost ancestral modes of locomotion, including in flightless diving birds, pinnipeds, and cetaceans. Using penguin embryos as an accessible model for investigating flipper development, we demonstrate that early patterning of forelimb musculoskeletal morphology is similar to that seen in forelimbs of non-aquatic birds. Instead, later modifications of gene expression and cell and tissue behaviors underlie flipper phenotypes. Thus, we find that in the early penguin forelimb, the initial cues that pattern the muscle do not differ from other avian species, however late embryonic changes in proliferation result in dramatic reduction of muscle. Likewise, forelimb bones in penguins initially have similar cross-sectional proportions to those in flighted birds. The shape of these bones is, however, remodeled late in embryonic development through a process that shares molecular hallmarks with bone ridge formation at tendon attachment sites. In these bones, ridge-forming tissue initiates at the ends of the bones (the epiphyses) and extends into tendon-like connective tissue along the lateral edges of the bone, widening the long bones along the anterior-posterior axis and producing a flattened bone. Using spatial transcriptomics and comparative genomic tools we determine that differentially expressed genes between the ridge-forming tissues and long bone cartilage are significantly enriched for signals of selection in the penguin lineage and that these genes may also be convergently evolving in marine mammals. Together, these data show that the evolution of musculoskeletal morphology in the penguin flipper occurred through expansion or novel deployment of molecular programs typically associated with tendon-attachment sites during late embryonic development.