Developmental Dynamics

The Notch signaling pathway is a critical means to regulate cell fate choice in animals. Appropriate regulation of this pathway is also required for human face formation as both loss and gain of function mutations of Notch signaling can cause syndromes with craniofacial abnormalities. Here we examine the consequences of manipulation of Notch signaling in the early mouse embryonic ectoderm by either removing the transcriptional effector Rbpj or expressing a constitutively active form of the Notch1 intracellular domain. Loss of Rbpj resulted in cleft secondary palate but strikingly was also associated with the ectopic mineralization of vibrissa follicles. In contrast, activation of Notch signaling resulted in multiple embryonic defects including a fully penetrant bilateral cleft lip and palate. Further, single cell RNA-seq data indicated a switch from a basal epithelial cell identity towards periderm when Notch signal transduction was elevated. These cell fate changes were accompanied by misregulation of genes and pathways known to impact human and mouse orofacial clefting including Grhl3, Irf6, and Wnt pathway. Together, these findings provide insight into human craniofacial conditions caused by misregulated Notch activity. SUMMARY STATEMENTOur studies demonstrate that Notch signaling in the embryonic ectoderm stimulates periderm cell fate while also repressing transformation of the inner root sheath of whisker follicles into mineralized tissue.

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.

The orientation and distribution of fibrillar collagen are critical determinants of the shape and mechanical properties of bones and organs.1-3 However, how they are spatially organized within tissues is still poorly understood,4-7 as visualizing these collagen architectures remains challenging. Actinotrichia (AT), the spear-shaped fibrillar collagen structures located at the distal tips of fish fins, are easily observable due to their large size and distinctive morphology8-14 and have recently emerged as a model system for studying collagen fiber organization.15-19 In this study, we generated knockout lines for the fish-specific extracellular matrix (ECM) genes actinodin1 and actinodin2 (and1/2), which are lost in tetrapods.12 Loss of these genes dramatically altered the orientation of collagen fibers, thereby inducing changes in fin morphology. In the wild-type fins, AT are orderly arranged beneath the epidermis, forming layers parallel to the fin surface, and their individual fibers radiate distally toward the fin tip. In contrast, double knockout (dKO) of and1/2 results in overall fin reduction accompanied by increased thickness. Examination of the collagen structure distribution revealed the presence of aberrant collagen fibers oriented perpendicular to the fin epidermis. Moreover, the vertically oriented fibers contributed to thickening of the mesenchymal region in which they were distributed. The number of abnormal fibers increased with the severity of and1/2 deficiency, suggesting that collagen fibers in fins inherently tend to align perpendicular to the epidermis when these genes are absent. Furthermore, in tetrapods lacking the and gene family--specifically amphibians, the tetrapod group most closely related to fish20--examination of the developing limb, the organ homologous to paired fins in fish,21 revealed collagen fibers oriented perpendicular to the epidermis. The distribution pattern also resembled that observed in the fin buds of and1/2 dKO fish. Together, these findings highlight collagen patterning alterations as a previously unrecognized factor contributing to the evolutionary divergence between thinned fins and thickened limbs. Moreover, the identification of mutants that dramatically alter collagen fiber orientation is unprecedented, suggesting that analysis of Actinodin (And) function unveil the mechanisms underlying collagen matrix formation.22-28

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.

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.

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.

The mammalian auditory sensory epithelium, the organ of Corti, contains a number of unique cell types, including the inner and outer pillar cells that form the walls of the tunnel of Corti. The limited number of pillar cells and their close physical proximity to other cells within the organ of Corti has limited efforts to transcriptionally characterize their development. To identify potential regulators of pillar cell formation, we isolated cochlear duct epithelial cells at time points between embryonic day 11 and 16 for single cell RNA-sequencing. The resulting data were used to build a developmental trajectory from undifferentiated precursor cells to each of the major cell types in the organ of Corti including inner pillar cells. Bioinformatic analyses including SCENIC, TradeSeq and CellOracle identified the Etv4/5/1 transcription factors as likely regulators of inner pillar cell development. To specifically examine the role of Etv4/5/1 in pillar cell development, conditional triple mutants were generated. Results indicate defects in the formation of inner pillar cells as well as changes in other cochlear cells likely as a result of secondary interactions. Finally, we demonstrate that expression of Etv4/5/1 in pillar cells is dependent on Fgfr3 and identify downstream targets of Fgfr3/Etv signaling in inner pillar cells. These results provide significant insights regarding the specification and early development of inner pillar cells, which will have implications for understanding congenital deficits and potential applications in the development of regenerative strategies.

BackgroundCranial neural crest cells (cNCC) generate craniofacial cartilage, bone, and peripheral neurons and glia, and birth defects arise when the cartilage/neuronal/glial progenitor fail to differentiate. PRDM16 is a transcriptional regulator containing both zinc-finger and SET domains, implicated in craniofacial development and orofacial clefting, but its role in cranial sensory ganglion formation has not been defined. ResultsHere, we demonstrate that prdm16 is required for trigeminal ganglion (TG) assembly and sensory neurogenesis from cranial neural crest lineages. In zebrafish, prdm16 is expressed in TG beginning by 18 hours post fertilization (hpf) and persists through the later developmental stage at 48 hpf. In the prdm16 loss-of-function zebrafish, fewer HuC+ TG neurons are present at 24 hpf and 48 hpf, along with reduced overall ganglion size. Live imaging in Tg(sox10:mRFP; elavl3:GFP) embryos demonstrates similar numbers of sox10+ cNCCs migrating to the TG region and reduced cell numbers and overall smaller size of TG in prdm16-/-. Acetylated {beta}-tubulin immunostaining shows fewer trigeminal axon projections early and an altered projection pattern by 48 hpf. A reduction in a defined sensory neuron population, p2rx3b+ cells displayed a weaker signal and decreased cell number in prdm16-/- TG. Transcriptomic analysis of FACS-isolated sox10+ cranial neural crest cells supported reduced expression of key neurogenic and sensory lineage genes. Finally, in mouse embryos, PRDM16 is expressed in TG neurons, and Prdm16csp1/csp1 embryos exhibited reduced TG volume, area and fewer HuC+ neurons at E18.5. ConclusionTogether, these data identify Prdm16 as a conserved regulator of trigeminal ganglion growth and sensory neuron differentiation, linking PRDM-family chromatin regulators to the development of the peripheral sensory nervous system.

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.

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.

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.

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

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.

Visualizing protein expression dynamics with high temporal resolution is essential for understanding how cells acquire specific fates and functions during development, where key decisions can occur within minutes. Conventional direct fluorescent tagging often fails to capture these rapid changes in protein expression due to the relatively slow fluorophore maturation time. Indirect epitope-based labeling strategies offer a promising alternative, yet only a limited number of these systems have been developed and used in the context of multicellular organisms. Here, we evaluate and combine four epitope-based indirect labeling systems for live-imaging of proteins in C. elegans: the SunTag, Frankenbody, MoonTag and AlfaTag systems. Each system uses a fluorescently labeled high-affinity single-chain antibody or nanobody to recognize short peptide epitopes fused to a protein of interest, enabling immediate visualization of newly synthesized proteins. We demonstrate that all four systems specifically label epitope-tagged endogenous proteins and show no detectable cross-reactivity when used in dual-color combinations, enabling simultaneous visualization of distinct proteins within the same embryo. In addition, we show that the SunTag system offers three major advantages over direct labeling: earlier detection of proteins, enhanced sensitivity through signal amplification (as illustrated by CAM-1) and less impact on the function (as demonstrated for ERM-1). Together, this expanded toolkit of epitope-based labeling systems offers many new opportunities for visualizing rapid protein dynamics and for dissecting how their dynamics drive cell fate decisions during development. SUMMARYThe development of epitope-labeling systems has improved live-imaging quality of proteins. Unfortunately, limited systems exist for multicellular organisms to study protein expression in the context of development. Here, we expand the epitope-labeling toolbox for C. elegans by combining SunTag or Frankenbody with MoonTag or AlfaTag. Our data indicates that these systems simultaneously visualize different endogenous proteins without cross-reactivity. Moreover, the SunTag system shows advantages over direct labeling: earlier detection, enhanced sensitivity through signal amplification and less impact on protein function. This expanded epitope-labeling toolbox in C. elegans provides opportunities for accurate visualization of different proteins that drive cell fate decisions. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/703904v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@449fe0org.highwire.dtl.DTLVardef@15c68cforg.highwire.dtl.DTLVardef@1e51ff8org.highwire.dtl.DTLVardef@196114d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.