The ability to image blood flow in early-stage avian embryos has significant applications in developmental biology, drug and vaccine testing, as well as determining sex differentiation. In this project, a recently developed laser speckle contrast imaging (LSCI) system was used to non-invasively image extraembryonic blood vessels and used these images to attempt early sex identification of chick embryos. Specifically, blood vessels images were captured from 1,251 living chicken embryos between day three and day four of incubation. Then, deep neural network (DNN) models were applied to evaluate whether it is possible to differentiate sex based on vascular patterns. Using ResNetBiT and YOLOv5s-cls models, our results indicate that sex differentiation from extraembryonic blood vessel images was not achievable with sufficiently high accuracy or statistical significance for practical use. Specifically, ResNetBiT had a five-fold cross-validated average accuracy of 56% ± 4% (p-value of 0.28 across cross-validation folds) at day 3 and 57% ± 3% (p-value of 0.07 across cross-validation folds) at day 4. YOLOv5s-cls had a five-fold cross-validated average accuracy of 55% ± 2% (p-value of 0.13 across folds) at day 3 and 57% ± 3% (p-value of 0.10 across folds) at day 4. Our findings suggest that under the current experimental conditions and modeling approaches, per-egg evaluation did not produce sufficiently accurate or statistically robust results for early sex classification.

While the enteric nervous system (ENS) of jawed vertebrates is largely derived from the vagal neural crest, lamprey are jawless vertebrates that lack the vagal neural crest, yet possess enteric neurons derived from late-migrating Schwann cell precursors. To illuminate homologies between the ENS of jawed and jawless vertebrates, here we examine the diversity and distribution of neuronal subtypes within the intestine of the sea lamprey during late embryonic and ammocete stages. In addition to previously described 5-HT-immunoreactive serotonergic neurons, we identified NOS+ and VIP+ neurons, consistent with motor neuron identity. Moreover, the presence of Calbindin+ neurons was suggestive of sensory IPANs. Quantification of neural numbers by subtype across the length of the intestine revealed significant, albeit subtle differences in distribution of neuronal markers at different axial levels, suggesting that the complex organizational features of the ENS may have emerged much earlier in the vertebrate lineage than previously appreciated.

Cranial neural crest cells (NCCs) undergo dynamic processes during embryonic development, including delamination from the neural tube by epithelial-to-mesenchymal transition (EMT), migration to the periphery, condensation via mesenchymal-to-epithelial transition (MET) and differentiation into structures like the trigeminal ganglion. Here, we identify and characterize the function of a core set of miRNAs involved in these transitions during the formation of the trigeminal ganglion in the chick embryo. We further identify putative targets of miRNAs involved in neural crest EMT and MET. Notably, introducing MET-involved miRNAs into trunk NCCs endows these cells with the ability to condense and differentiate into neurons in vivo in a manner reminiscent of cranial rather than trunk NCCs. Our findings shed light on the intricate regulatory networks governing NCC behavior, positioning miRNAs as key regulatory elements required for migratory transitions and axial level specific differentiation capabilities.

The neural crest is a highly migratory and multipotent cell population that contributes to many defining features of vertebrates. As a uniquely vertebrate cell type, the neural crest is an excellent model for studying cell lineage and diversification during embryonic development because of its multipotency, motility, and capacity to form a plethora of derivatives. Neural crest cells migrate extensively throughout the body and contribute to many of the defining features of vertebrate embryos, including the craniofacial skeleton, most of the peripheral nervous system and pigmentation of the skin. What guides their migration and subsequent formation of discrete structures? Interactions between neural crest cells and their environment, including other cell types like placode cells, play a major role in guiding their migration and condensation into numerous derivatives. In this review, we discuss aspects of neural crest induction, migration and axial level differences, highlighting what is currently known regarding molecular cues that govern their formation, migratory behavior, and differentiation as they reach their final destinations. We particularly focus on formation of cranial sensory ganglia. New technologies are playing an important role in furthering our understanding of the molecular mechanisms underlying neural crest migration and what leads to cessation of their movement and onset of differentiation.

The ability to image blood flow in early-stage avian embryos has significant applications in developmental biology, drug and vaccine testing, as well as determining sex differentiation. In this project, we used our recently developed laser speckle contrast imaging (LSCI) system to non-invasively image extraembryonic blood vessels and used these images to attempt early sex identification of chick embryos. Specifically, we captured images of blood vessels from 1,251 living chicken embryos between day three and day four of incubation. We then applied deep neural network (DNN) models to evaluate whether it is possible to differentiate sex based on vascular patterns. Using ResNetBiT and YOLOv5 models, our results indicate that sex differentiation from extraembryonic blood vessel images was not achievable with sufficiently high accuracy or statistical significance for practical use. Specifically, ResNetBiT had a five-fold cross-validated average accuracy of 59%±5% (fold-wise p-value, p ≤ 0.3) at day 3 and 61%±3% (fold-wise, p ≤ 0.04) at day 4. YOLOv5 had a five-fold cross-validated average accuracy of 55%±3% (fold-wise, p ≤ 0.3) at day 3 and 53%±3% (fold-wise, p ≤ 0.5) at day 4. Our findings suggest that using vascular pattern imaging alone is inconclusive for reliable early sex identification in chicken embryos. ### Competing Interest Statement The authors have declared no competing interest.

Human fertility is suboptimal in part by error-prone divisions during early cleavage stages, which frequently result in chromosomal aneuploidy. Most human pre-implantation embryos are mosaics of euploid and aneuploid cells, yet those with a low proportion of aneuploid cells can develop to term at rates similar to fully euploid embryos. How embryos manage aneuploidy during early development remains poorly understood – yet this knowledge is crucial for improving fertility outcomes and reducing developmental defects. To investigate these mechanisms, we established a new mouse model of chromosome mosaicism to trace the fate of aneuploid cells during pre-implantation development. We previously used the Mps1 inhibitor reversine to induce aneuploidy. Here, we demonstrate that the more specific Mps1 inhibitor AZ3146 similarly disrupts chromosome segregation but supports higher developmental potential than reversine. AZ3146-treated embryos transiently upregulate hypoxia-inducible factor-1A (HIF1A) without triggering Trp53 activation. Given that pre-implantation embryos develop in a hypoxic environment in vivo, we further explored the role of oxygen tension. Hypoxia exposure in vitro reduced DNA damage in response to Mps1 inhibition and increased the proportion of euploid cells in mosaic epiblast. Conversely, HIF1A inhibition decreased the proportion of aneuploid cells. Together, these findings uncover a role for hypoxia signaling in modulating the response to chromosomal errors and suggest new strategies to improve the developmental potential of mosaic human embryos.

Neural crest induction begins at the neural plate border and involves the intricate interplay of signaling and transcriptional events. In this review, we examine the literature on neural crest induction, focusing primarily on the chick model due to the extended time during which the induction process occurs. While it is well-established that induction initiates during mid-gastrulation, evidence from tissue recombination and transcriptomic analyses suggests that the process continues until neural tube closure. Along the body axis, distinct neural crest populations with varying developmental potentials emerge in a rostral to caudal progression. Testing axial level differences has revealed axial level specific subcircuits that influence region-specific neural crest cell fate decision, though what leads to axial level specification remains unknown.

During vertebrate development, the heart primarily arises from mesoderm, with crucial contributions from cardiac neural crest (CdNC) cells that migrate to the heart and form a variety of cardiovascular derivatives. Here, by integrating bulk and single cell RNA-seq with ATAC-seq, we identify a gene regulatory subcircuit specific to migratory cardiac crest cells composed of key transcription factors egr1, sox9a, tfap2a, and ets1. Notably, we show that cells expressing the canonical neural crest gene sox10 are essential for proper cardiac regeneration in adult zebrafish. Furthermore, expression of all transcription factors from the migratory cardiac crest gene subcircuit are reactivated after injury at the wound edge. Together, our results uncover a developmental gene regulatory network that is important for CdNC fate determination, with key factors of the program reexpressed during regeneration.

The evolution of vertebrates from protochordate ancestors marked the beginning of the gradual transition to predatory lifestyles. Enabled by the acquisition of multipotent neural crest and cranial placode cell populations, vertebrates developed an elaborate peripheral nervous system, equipped with paired sense organs, which aided in adaptive behaviors and ultimately, successful colonization of diverse environmental niches. Underpinning the enduring success of vertebrates is the highly adaptable nature of the peripheral nervous system, which is enabled by the exceptional malleability of the neural crest and placode developmental programs. Here, we explore the embryonic origins of the vertebrate senses from the neural crest and cranial placodes and discuss the evolutionary trajectory of the senses in the context of adaptation to novel environments.

The cardiac neural crest is critical for the normal development of the heart, as its surgical ablation in the chick recapitulates common human congenital heart defects such as 'Common Arterial Trunk' and 'Double Outlet Right Ventricle' (DORV). While left-right asymmetry is known to be important for heart development, little is known about potential asymmetric differences between right and left cardiac neural folds with respect to heart development. Here, through surgical ablation of either left or right cardiac neural crest, we find that right ablation results in more varied and more severe heart defects. Embryos with Common Arterial Trunk and with missing arteries occurred in right-ablated embryos but were not observed in left-ablated embryos; moreover, embryos with DORV and with misalignment of the arteries were more prevalent following right versus left cardiac crest ablation. In addition, survival of right-ablated embryos was lower than left-ablated embryos. Together, these data raise the intriguing possibility that there may be differences in left versus right cardiac neural crest during heart development.

The inner ear is an intricate structure that houses six sensory organs responsible for both hearing and balance. The development of the inner ear begins with the formation of the otic placode, a transient ectodermal thickening that emerges early during embryonic development. The otic placode undergoes a series of morphological changes, from thickening to invagination and then pinching off from the ectoderm to form the otic vesicle, which further differentiates into the specialized structures of the inner ear. These developmental processes require a coordinated interplay between intrinsic transcription factors and extrinsic signaling molecules, which regulate the patterning, proliferation, and differentiation of the inner ear components. In this review, we focus on the role of Sonic hedgehog (Shh) signaling in the development of the mammalian inner ear. We explore how Shh signaling is involved at multiple stages of inner ear development, including the patterning of the otic vesicle and the differentiation of specific cell types within the cochlea. Additionally, we discuss the mechanisms by which Shh signaling integrates with other signaling pathways and transcription factors to ensure the proper development and function of the inner ear. Understanding the molecular basis of these processes provides valuable insights into inner ear development and its disorders.

The endostyle is an endodermal organ unique to nonvertebrate chordates except for lamprey larvae, where it serves as forerunner to the adult thyroid. Here, we examine whether the acquisition of neural crest in the vertebrate lineage played a role in the elaboration of the endostyle. CM-DiI lineage tracing reveals a neural crest contribution to the endostyle, and CRISPR-Cas9 mutagenesis of key neural crest genes causes endostyle defects including formation of a single rather than bilobed structure. RNA sequencing reveals gene profiles characteristic of embryonic neural crest cells and Schwann cell precursors in the developing endostyle. Contrasting with the prevailing view that the endostyle is an endoderm-derived organ, we propose that the acquisition of the neural crest played a critical step in promoting thyroid evolution from chordate endostyle.

The hindbrain is a life-sustaining brain region. In one model, a common neural progenitor generates all brain regions. Here our studies of mouse embryos and human pluripotent stem cells (hPSCs) support a different model: two parallel brain progenitors emerge simultaneously during gastrulation, anterior neural ectoderm (forebrain/midbrain progenitor) and posterior neural ectoderm (hindbrain progenitor). Not only are they lineage-committed to respectively form forebrain/midbrain vs. hindbrain in vitro, but they also have diverging chromatin landscapes foreshadowing future forebrain/midbrain vs. hindbrain identities. Leveraging these differences, we differentiated hPSCs into hindbrain rhombomere 5/6-specific motor neurons, hitherto difficult to generate in vitro. We postulate the brain is a composite organ emanating from two lineage-restricted progenitors; these dual progenitors may be evolutionarily conserved across 550 million years from hemichordates to mammals.

The vertebrate enteric nervous system (ENS) is derived from vagal neural crest cells, which enter the foregut as progenitors that migrate from rostral to caudal to populate the entire length of the gut. Here, we show that transcription factors sox11a and sox11b, zebrafish orthologs of the human SOX11 gene, are highly expressed in neural crest cells transitioning from progenitors to differentiating neuronal subtypes. Accordingly, CRISPR-Cas9 depletion shows that loss of sox11 paralogs reduces the number of neurons that express the inhibitory motor neuron marker adcyap1b without affecting cell proliferation or death. Transcription factor footprinting analysis of open chromatin regions identified by ATAC-seq reveals Sox11 binding sites in the adcyap1b enhancer. Furthermore, mutational analysis shows these binding sites are required for mediating enhancer-driven reporter expression. Taken together, our results demonstrate an important and previously unrecognized role for sox11a and sox11b in neuronal subtype specification in the developing zebrafish ENS.

Neural crest cells give rise to the neurons of the enteric nervous system (ENS) that innervate the gastrointestinal (GI) tract to regulate gut motility. The immense size and distinct subregions of the gut present a challenge to understanding the spatial organization and sequential differentiation of different neuronal subtypes. Here, we profile enteric neurons (ENs) and progenitors at single-cell resolution during zebrafish embryonic and larval development to provide a near-complete picture of transcriptional changes that accompany the emergence of ENS neurons throughout the GI tract. Multiplex spatial RNA transcript analysis identifies the temporal order and distinct localization patterns of neuronal subtypes along the length of the gut. Finally, we show that functional perturbation of select transcription factors Ebf1a, Gata3, and Satb2 alters the cell fate choice, respectively, of inhibitory, excitatory, and serotonergic neuronal subtypes in the developing ENS.

The myriad regenerative abilities across the animal kingdom have fascinated us for centuries. Recent advances in developmental, molecular, and cellular biology have allowed us to unearth a surprising diversity of mechanisms through which these processes occur. Developing an all-encompassing theory of animal regeneration has thus proved a complex endeavor. In this chapter, we frame the evolution and loss of animal regeneration within the broad developmental constraints that may physiologically inhibit regenerative ability across animal phylogeny. We then examine the mouse as a model of regeneration loss, specifically the experimental systems of the digit tip and heart. We discuss the digit tip and heart as a positionally-limited system of regeneration and a temporally-limited system of regeneration, respectively. We delve into the physiological processes involved in both forms of regeneration, and how each phase of the healing and regenerative process may be affected by various molecular signals, systemic changes, or microenvironmental cues. Lastly, we also discuss the various approaches and interventions used to induce or improve the regenerative response in both contexts, and the implications they have for our understanding regenerative ability more broadly.

The neural crest is an embryonic stem cell population unique to vertebrates1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3-8. Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine β-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates.

In amniote limbs, Fibroblast Growth Factor 10 (FGF10) is essential for limb development, but whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established Fgf10 mutant lines in the newt Pleurodeles waltl which has amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs fail to develop and downregulate FGF target genes. Despite these developmental defects, Fgf10 mutants were able to regenerate normal hindlimbs rather than recapitulating the embryonic phenotype. Together, our results demonstrate an important role for FGF10 in hindlimb formation, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.

While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, by using chick embryos, we show that the microRNA (miR)-203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. By employing cell-specific electroporations for either miR-203 sponging or genomic editing using CRISPR/Cas9, we elucidated that neural crest cells serve as the source, while placode cells serve as the site of action for miR-203 in trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses an miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.

Imaging blood vessels in early-stage avian embryos has a wide range of practical applications for developmental biology studies, drug and vaccine testing, and early sex determination. Optical imaging such as brightfield transmission imaging offers a compelling solution due to its safe non-ionizing radiation, and operational benefits. However, it comes with challenges such as eggshell opacity and light scattering. To address these, we have revisited an approach based on laser speckle contrast imaging (LSCI) and demonstrated a high quality, comprehensive and non-invasive visualization of blood vessels in few-days-old chicken eggs, with blood vessel as small as 100 µm in diameter (with LSCI profile full-width-at-half-maximum of 275 µm). We present its non-invasive use for monitoring blood flow, measuring the embryo’s heartbeat, and determining the embryo’s developmental stages using machine learning with 85% accuracy from stage HH15 to HH22. This method can potentially be used for non-invasive longitudinal studies of cardiovascular development and angiogenesis, as well as egg screening for the poultry industry. ### Competing Interest Statement The authors have declared no competing interest.