Developmental Dynamics

Peripherin (PRPH) is a class III intermediate filament protein expressed in peripheral nerves and upregulated during axon outgrowth and regeneration. In this study, we developed a transgenic Xenopus laevis line for long-term in vivo visualization of the peripheral nervous system. Deletion and motif analyses identified cis-regulatory regions within the promoter and intron 1 that are important for neuronal expression of the X. laevis prph gene. Stable lines exhibited robust EGFP reporter activity in developing neural primordia in embryos and in the peripheral nerves of tadpoles. Transgenic tadpoles enabled in vivo imaging of peripheral nerves throughout limb development. During larval limb regeneration, we observed modest early nerve entry into the blastema, recapitulating that seen in early limb development. In contrast, post-metamorphic limb blastemas displayed extensive innervation from the early phase of regeneration. Moreover, increased reporter activity in the nerves of the regenerating adult forelimb suggests regeneration-associated regulation of peripheral innervation and its potential role in blastema formation. This transgenic line will serve as a versatile tool for analyzing such large-scale neural remodeling across development, metamorphosis, and regeneration.

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.

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.

Proper forebrain development relies on precise spatial and temporal control of early neural stem cell (NSC) proliferation and later neurogenesis. Brain malformations can arise when these processes are defective. Joubert Syndrome (JS) is a neurodevelopmental disorder that is diagnosed by a mid-hindbrain malformation, but often includes forebrain defects such as microcephaly, which are less understood. One gene recently linked to Joubert Syndrome with microcephaly is Togaram1, which encodes a TOG domain microtubule binding protein shown to affect primary cilia. In the embryonic dorsal forebrain, NSCs have primary cilia on their apical membranes that play a role in regulating proliferation and neurogenesis, but how they do this is not well understood. Here we investigate the role of Togaram1 in mammalian forebrain development using a mouse knockout. We find that Togaram1 is crucial for forebrain size, thickness, and morphology. In particular, knockout forebrains have sporadic indentations of the lateral ventricles, and the neuronal layer is thin with gaps and heterotopias. The dorsal forebrain NSCs have increased proliferation and apoptosis. Finally, the primary cilia of Togaram1 knockout NSCs have abnormal morphology and function. This study begins to elucidate the role of Togaram1 in forebrain morphogenesis and the involvement of NSC primary cilia in forebrain malformations.

The human fallopian tube plays a critical role in reproductive biology, yet the structural organization and immune repertoire of this tissue remain incompletely characterized. Here, we performed an in-depth analysis of human fallopian tube tissue from women of reproductive age across three distinct anatomical regions (isthmus, ampulla, and fimbriae) across the menstrual cycle. Using antibody-based imaging for EPCAM, CD8A, and CD20 together with automated image analysis, the epithelial thickness and spatial distribution of T and B lymphocytes was assessed. No significant differences in epithelial thickness were observed between proliferative and secretory phases within any tubal region. In contrast, significant regional differences were identified, with the epithelium being thickest in the isthmus and thinnest in the ampulla. Both CD8A+ T lymphocytes and CD20+ B lymphocytes were detected throughout the fallopian tube, and a strong correlation between T and B lymphocyte abundance was observed across patients. Spatial analysis further revealed that both lymphocyte populations were preferentially localized within the mucosal compartment adjacent to the lumen. Notably, intraepithelial B lymphocytes were identified throughout the fallopian tube. Together, these findings provide new insight into epithelial organization and immune cell distribution in the human fallopian tube, highlighting the complexity of the tubal immune microenvironment and its potential relevance for reproductive biology.

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.

Congenital heart defects frequently arise from alterations in the elongation of the cardiac outflow tract (OFT). Proper elongation of the OFT depends on the coordinated deployment of progenitor cells from the second heart field (SHF) and on dynamic interactions with the extracellular matrix (ECM). Among ECM components, fibronectin (Fn1) and tenascin-C (TnC) have emerged as key regulators of cardiac morphogenesis. Studies in mouse embryos have shown that mesodermal Fn1 is required to maintain proper TnC localization within SHF cells. To study heart development, mammalian models are challenging to use because of their in utero development. This limitation highlights the need for alternative models with external development, where direct observation is possible; however, in these systems, the cellular organization of the SHF and the dynamics of its ECM environment remain poorly characterized Here, we investigated the cellular and extracellular architecture of SHF cells localized to the dorsal pericardial wall (DPW) during heart development in Xenopus laevis. We show that SHF cells undergo a stage-dependent transition from a predominantly monolayered organization at NF35 to a multilayered structure at NF42. This transition is accompanied by dynamic remodeling of the ECM, characterized by increased expression of Fn1, TnC, and Collagen I (ColI) and by redistribution of ECM components within the DPW. Functional experiments revealed that depletion of Fn1 disrupts cardiac morphogenesis, leading to shortening of the OFT and reduced ventricular size. Moreover, loss of Fn1 decreases TnC and ColI levels and alters the spatial organization of TnC within the DPW, indicating that Fn1 is required for proper ECM assembly within the SHF cells. These findings identify Fn1 as a key regulator of ECM assembly within the DPW and highlight how ECM remodeling contributes to the organization of SHF progenitor cells during OFT elongation. Altogether, we demonstrated that Xenopus laevis is a powerful model for studying ECM-driven mechanisms of cardiac morphogenesis.

In many oviparous animals, egg yolk is the sole source of nutrition until feeding begins, and carbohydrates are present in only small amounts in the yolk. Glucose plays an important role in the developmental processes of various animals. In addition, gluconeogenesis has been reported to occur in the yolk syncytial layer (YSL) of cartilaginous fish and teleosts. In contrast, the role of gluconeogenesis in tetrapods remains unclear. In this study, we used Xenopus tropicalis, an anuran amphibian, which lacks YSL, and therefore provide an opportunity to examine the evolutionary conservation of gluconeogenic mechanisms among vertebrates. In X. tropicalis, liquid chromatography/mass spectrometry revealed that glucose levels increased before liver formation. Subsequent tracer experiments using 13C-labeled metabolic substrates detected gluconeogenesis activity from glycerol and lactate. Expression analyses showed that gluconeogenic genes are expressed in the epidermis and endoderm. Consistently, G0 knockout of fbp1, a key gluconeogenic gene, resulted in a significant reduction in glucose levels, affecting brain development. These findings first demonstrate that gluconeogenesis supports development of X. tropicalis. To the best of our knowledge, gluconeogenesis in developing epidermis has not been reported, highlighting previously unrecognized diversity in tissue-specific metabolism during vertebrate development. Comparative analyses across species will provide further insights into the evolution and functional significance of embryonic gluconeogenesis and nutrient metabolism.

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.

Chondrichthyans (cartilaginous fishes) form the sister group to osteichthyans (bony fishes) and therefore occupy a key phylogenetic position for comparative studies of early vertebrate evolution. Despite their importance, chondrichthyan development remains understudied relative to established model systems such as mouse, chick, and zebrafish, in part because of limited embryo accessibility and the lack of standardized laboratory resources for rearing. Here, we present the epaulette shark Hemiscyllium ocellatum, a small, oviparous shark as a tractable laboratory system for studying shark development. We provide an overview of epaulette shark husbandry requirements and generate a comprehensive micro-computed tomography imaging series spanning embryonic development through hatching. This dataset provides a three-dimensional anatomical atlas of development for a representative chondrichthyan species. By preserving whole embryos in three dimensions, micro-CT imaging enables developmental morphologies to be visualized at high resolution and in near-native anatomical context. Together with the recently published epaulette shark genome, this developmental atlas helps establish the Epaulette shark for comparative anatomical, developmental, and genomic studies.

Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 hours post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression. HighlightsPrdm3 and Prdm16 associate with nucleosomes for regulation of gene expression Gene targets are altered in prdm3 and prdm16 mutant zebrafish Reduced binding is observed in respective mutants

BackgroundPrecise control of DYRK1A dosage is essential for embryonic development, including craniofacial morphogenesis. While LZTS2 is among the most consistently identified DYRK1A-interacting proteins, its roles in embryonic development remain incompletely understood, and its potential contribution to craniofacial development has not been examined. Xenopus laevis was used to test the role of LZTS2 in craniofacial development and its functional relationship with DYRK1A. ResultsLzts2 and Dyrk1a showed overlapping expression during craniofacial development, with both proteins present in developing facial tissues. Knockdown of Lzts2 disrupted craniofacial morphogenesis and reduced expression of the neural crest-associated genes sox9 and pax3. These phenotypes closely resembled those caused by decreasing Dyrk1a function. Sub-phenotypic reductions of Lzts2 and Dyrk1a synergized to produce craniofacial defects, while partial reduction of Lzts2 attenuated aspects of the phenotype caused by Dyrk1a overexpression. Comparative analysis of human phenotypes associated with copy number gains of LZTS2 and DYRK1A revealed striking overlap, consistent with a potential functional interaction between these genes in humans. ConclusionsThese findings identify Lzts2 as a previously unrecognized regulator of craniofacial development and support a functional interaction with Dyrk1a during embryogenesis. Modulating LZTS2 or related regulatory partners may provide a strategy to selectively tune DYRK1A-dependent developmental pathways

Congenital heart defects affect females and males differently. Several congenital heart defects arise during the formation of the heart tube, suggesting that heart tube morphogenesis may differ between females and males. We investigated if the fruit fly Drosophila melanogaster displays sexual dimorphisms in the cellular mechanisms of heart tube formation. Quantitative microscopy revealed no differences between females and males in the migration of cardiac progenitors to form the heart tube. Our results suggest that Drosophila do not display sexual dimorphisms in early cardiac development, and support the omission of sex as an experimental variable when investigating Drosophila heart tube morphogenesis.

Ehmt2 is a key H3K9 methyltransferase that regulates genome silencing and structural integrity during animal development. In addition to this canonical function, Ehmt2 has also been implicated in neural tissues mediating both direct and indirect transcriptional activation, and exon splicing, to facilitate proper neural cell differentiation and survival. Several germline loss-of-function animal models have been developed showing both conserved and divergent phenotypes that range from embryonic lethality to behavioural deficits in adult, fertile animals. Here, we generated the first maternal-zygotic ehmt2 loss of function mutant in zebrafish using CRISPR-Cas9 mutagenesis. An assessment of the pattern of H3K9 methylation in mutant embryos by ChIP-seq indicates that there are aberrant levels of this repressive mark, including reduction in discrete 5 non-coding regions of genes, but with no significant change in the overall pattern distribution of these marks across the genome. Global transcriptome and morphological analyses demonstrated that mutant embryos displayed greater variation in the timing of developmental progression that is, on average, slower compared to controls. Despite this, mutant embryos ultimately survive and are fertile. Through examination of progenitor cell dynamics and gene expression profiles, we found that the delay in embryonic development was associated with longer rates of S-M phases of the progenitor cell cycle in mutants leading to deficits in tissue growth. Finally, our data suggest a robust network of epigenetic regulators can potentially compensate for Ehmt2 loss of function and permit embryonic development and survival in ehmt2 mutant zebrafish. Our work establishes a zebrafish ehmt2 loss of function model that will facilitate examination of the complex and varied roles of Ehmt2 in vertebrate development.

Immunohistochemistry (IHC) is widely used to assess protein expression in corneal tissue, yet staining outcomes are strongly influenced by tissue preparation methods and regional differences within the cornea. This study aimed to systematically compare three preparation techniques including paraffin (wax) embedding, wax embedding with antigen retrieval (wax AR), and cryosectioning for IHC analysis in embryonic day 18 chicken corneal tissue. Markers representing key biological functions were evaluated, including progenitor activity (PAX6, P40), tissue architecture (actin), and immune surveillance (TAP1, CD68), across central and limbal regions. Cryosectioning consistently produced the most specific staining for nuclear and antigen-sensitive markers. PAX6 and P40 exhibited strong, nuclear-localized expression in the corneal epithelium only under cryo conditions, whereas wax-based methods resulted in reduced specificity and irregular signal distribution. TAP1-positive immune cells were detectable in the limbal stroma exclusively in cryosections, highlighting improved antigen preservation. In contrast, actin staining, was best preserved with wax AR, and provided superior structural clarity and expected expression patterns across corneal layers. CD68 showed minimal or inconsistent staining in corneal tissue across all methods despite positive control validation. These findings demonstrate that optimal IHC outcomes in corneal tissue are marker-dependent and influenced by preparation methods and regional tissue context. Cryosectioning is recommended for detecting nuclear and immune-related antigens, while wax AR is preferable for preserving tissue architecture. This study provides a practical framework for improving reproducibility and interpretation of corneal immunostaining in avian models.

Lymphatic vessels are formed during embryonic and postnatal development to facilitate interstitial fluid clearance and immune regulation after birth. Their organ-specific heterogeneity in organisation and function is preceded by heterogenous origins of lymphatic endothelial cells (LECs), the main building blocks of lymphatic vessels. In the dermis, a subset of LECs was reported to arise from blood capillaries, which themselves differentiate, in part, from paraxial mesoderm. However, it is not known whether additional cell lineages contribute to the dermal LEC population. Here, we have combined transcriptomic analyses with genetic lineage tracing and wholemount immunostaining to show that 60% of LECs in the embryonic day (E) 13.5 and E15.5 dermis are derived from a cell lineage that expresses Csf1r, a marker of myeloid cells and their progeny. Csf1r lineage LECs persist in adult dermal lymphatic vasculature and are indispensable for normal lymphatic development, because Prox1 deletion within the Csf1r lineage causes dermal oedema and blood-filled lymphatic vessels. As Csf1r lineage dermal LECs do not themselves express Csf1r and also do not arise from Csf1r-expressing differentiated myeloid cells, our findings imply the existence of a Csf1r-expressing non-LEC precursor population for the majority of dermal LECs and will prompt further work to identify this cell population.

Pericytes are mural cells that provide support to the endothelium of small blood vessels. Pericyte soma are regularly spaced along vessels, and their processes overlap only slightly. Given that vessel patterning is imprecise, we explore the interplay between vessel growth and pericyte recruitment that leads to even pericyte spacing. After recruitment to the zebrafish brain central arteries (CtAs), pericytes undergo rapid expansion, followed by morphological differentiation. Blocking angiogenesis by reducing Gpr124 (Wnt) or Vegf signaling reduces the length of the vessel network and the number of pericytes, preserving spacing, suggesting proportional recruitment of pericytes to cover the network and the territorial nature of pericytes. However, these initial brain pericytes have low proliferation rates. We demonstrate that additional pericytes are recruited firstly through migration of col5a1- and later col1a2-expressing fibroblasts into the brain. These second-wave pericytes retain some fibroblast properties and show elevated col1a2 levels in a model of pericyte loss (notch3 mutants). Our data provide new insights into the developmental timing, expansion, and novel origins of late-arriving brain pericytes during embryogenesis. SUMMARY STATEMENTThis article demonstrates that brain pericytes originate from multiple sources, including fibroblast-derived populations, and how pericyte numbers are adjusted in proportion to vessel development.

Understanding the functions of maternal effect genes during oocyte growth is essential for elucidating the mechanisms of oogenesis and early embryonic development. However, conventional gene knockout and conditional knockout approaches require extensive breeding and are time-consuming. Here, we present a rapid in vitro gene functional analysis system that combines microinjection of mRNA, siRNA and plasmid DNA into mouse secondary follicles with a two-step oocyte growth culture system. Mouse secondary follicles were subjected to microinjection of mCherry mRNA and subsequently cultured for 15 days to produce fully grown oocytes. mCherry fluorescence persisted throughout the oocyte growth period but declined rapidly after fertilization. Despite minor cellular damage occasionally caused by microinjection, injected follicles developed normally and retained developmental competence. To evaluate the efficiency of gene suppression, we introduced siRNA targeting Dnmt3l, which is abundantly expressed during oocyte growth phase. Although Dnmt3l deficiency is known not to affect oocyte growth, we observed that oocyte growth was maintained normally despite a marked reduction in endogenous Dnmt3l mRNA levels in our knockdown model. These results demonstrate that this method enables efficient manipulation of gene expression specifically during oocyte growth while preserving developmental competence, providing a versatile platform for rapid functional screening of maternal effect genes in vitro.

Fertilization is the process in which two specialized cells, the sperm and egg, interact, adhere, and fuse their membranes. This occurs in all sexually reproducing organisms. Several transmembrane and secreted proteins have been shown to be required for fertilization. Genetic mutations can alter these proteins and disrupt fertilization, leading to reduced or no offspring. When fertilization-specific sperm proteins are mutated, sperm production, motility, and activation are unaffected, but the sperm lose the ability to successfully fertilize an egg. In this study, we report on the sperm-specific protein SPE-40/FAM187, which is a single-pass transmembrane protein with an immunoglobulin-like domain. When spe-40 is mutated in C. elegans the animals are severely sub-fertile due to a sperm-specific defect. All the characteristics of the sperm that we have evaluated in the mutant are normal, yet sperm lacking SPE-40 do not fertilize. SPE-40 has orthologs in other species, including humans. Thus, we have established a role for SPE-40/FAM187 in fertilization that suggests it represents a conserved component of the fertilization synapse.