Developmental Dynamics

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.

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.

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.

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.

Zebrafish are widely recognized as a powerful vertebrate model for studying epimorphic regeneration due to their remarkable ability to restore complex tissues. However, regenerative efficiency declines with age, potentially due to alterations in gene regulatory networks and cellular metabolism. In the present study, we investigated the molecular and bioenergetic basis of age-associated regenerative decline by comparing young adult (<1 year) and old adult (>3 years) zebrafish during caudal fin regeneration. To further examine the contribution of mitochondrial function, mitochondrial dysfunction was experimentally induced using rotenone (20 nM), a mitochondrial Complex I inhibitor. Regenerative progression was assessed morphologically at 12hpa, 1dpa, 2dpa, 3dpa, and 7dpa, revealing a pronounced delay in fin regrowth in aged and rotenone-treated fish compared with young controls. Behavioral analysis indicated subtle but non-significant changes across experimental groups. Gene expression analysis using quantitative real-time PCR revealed age- and mitochondria-associated dysregulation of key regenerative gene families involved in developmental patterning, extracellular matrix organization, cellular signaling, and mitochondrial metabolism. Proteomic profiling further identified differential expression of proteins associated with mitochondrial bioenergetics, extracellular matrix remodeling, and signaling pathways required for blastema formation and tissue outgrowth. Ultrastructural examination by transmission electron microscopy revealed pronounced mitochondrial abnormalities, including enlarged mitochondria with fragmented or disrupted cristae, in aged and rotenone-treated regenerating tissues. Collectively, our integrative analysis establishes a mechanistic link between aging, mitochondrial dysfunction, and compromised regenerative capacity in zebrafish. The findings provide broader insights into metabolic constraints underlying age-related decline in regenerative potential in vertebrates.

To achieve a stereotypic lineage, each embryo of Caenorhabditis elegans follows an invariant cell differentiation process arising from a combination of cell polarisation, asymmetric or symmetric divisions, combined with intercellular signalling processes. This pattern of embryonic cell differentiation is driven by regulated segregation of molecules occurring at each cell division, including polarity proteins or cell fate determinants, transcription factors, p-granules and mRNAs. These distribution patterns are coupled with a robust spatio-temporal orchestration of cortical actin dynamics, which also plays a crucial role in these processes. However, compared to other molecular contents, how the actin per se is segregated from the first asymmetric division onward remains poorly understood. This study presents a thorough quantification of the intracellular distribution from the zygote to the 4-cell stage of key actors related to actin polymerisation: two nucleators (a formin and the Arp2/3 complex), a capping protein and E-cadherin. We additionally developed a novel method to assess actin polymerisation capacities from single blastomere extracts. We found that actin-related signatures arise at these early stages and that differential mechanisms of protein segregation and homeostasis occur, depending both on the cell pair and on the protein considered. Notably, if asymmetric divisions correlated with unequal partitioning of actin-related contents in a process linked with embryonic polarity, differences were revealed between AB daughter cells upon their separation. Taken together, these actin-related asymmetric distributions are adding a layer to the complexity of cell fate acquisition mechanisms in the early embryo.

AbstractThe lymphatic system is essential for maintaining fluid homeostasis, lipid transport and supporting immune function. Despite its central role in health and disease, advancements in understanding human lymphatic vasculature has been constrained, in part because primary human LECs are difficult to access and study in disease-relevant contexts. This study describes an efficient and scalable feeder-free method to differentiate human iPSCs into lymphatic endothelial cells (LECs) that are transcriptionally and phenotypically similar to primary fetal LECs. An iPSC-derived LEC system overcomes a drawback of primary cells by enabling precise genetic perturbations, supporting study of lymphatic diseases of interest in a human context. By grounding our approach in in vivo stages of lymphangiogenisis, we describe a staged protocol that recapitulates the key milestones of lymphatic development. We first adapted a published method to differentiate human iPSCs into venous endothelial cells (VECs) and then initiate transdifferentiation of VECs into LECs. Using immunocytochemistry, qPCR, as well as flow cytometry, we demonstrated expression of lymphatic-specific markers in the differentiated population. We further characterized our induced VECs (iVECs) and LECs (iLECs) through bulk RNA sequencing analysis and compared the populations to pseudobulk VEC and LEC transcriptomic datasets generated from human fetal heart endothelia at 12, 13 and 14 weeks of gestation. Through this work, we expanded the repertoire of approaches for accessing LECs, with the goal of accelerating discoveries in lymphatic biology and therapeutics. Abstract summary image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/712968v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a9a406org.highwire.dtl.DTLVardef@4faec6org.highwire.dtl.DTLVardef@15b4e73org.highwire.dtl.DTLVardef@17b9c36_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dauer larvae are a dormant developmental stage in nematodes that is induced by a range of environmental cues. The molecular mechanisms that transduce these cues to regulate dauer entry have been well characterized in Caenorhabditis elegans, whereas those in other nematode species remain unclear. The closest known sibling species of C. elegans, Caenorhabditis inopinata, occupies a distinct ecological niche and shows an extremely low frequency of dauer formation by starvation in laboratory conditions, suggesting that it could serve as a useful comparative model for analyzing dauer-inducing mechanisms. To support such analysis, we generated a fluorescent dauer reporter, Cin-col-183p::mCherry, in C. inopinata based on a previously reported dauer-specific reporter in C. elegans. This reporter showed fluorescence specifically in the pre-dauer and dauer stages, but not in other developmental stages, indicating that it functions as a dauer-specific marker in C. inopinata. Using these marker strains, we compared the responses to high temperature and RNAi-mediated knockdown of insulin/IGF-1 pathway genes (daf-2, age-1, and pdk-1), and found that dauer induction differs mechanistically between C. elegans and C. inopinata. This dauer-specific fluorescent strain will be a useful tool for investigating the diversity of dauer-inducing mechanisms across nematode species. Article SummaryDauer is a dormant developmental stage in nematodes induced by environmental stress. Although its regulation is well studied in Caenorhabditis elegans, the mechanisms in other species remain unclear. Here, we developed a fluorescent dauer reporter, Cin-col-183p::mCherry, in Caenorhabditis inopinata, a close relative of C. elegans. The reporter was specifically expressed in pre-dauer and dauer stages, confirming its usefulness as a dauer marker. Using this strain, we found that responses to high temperature and insulin/IGF-1 pathway gene knockdown differ between C. elegans and C. inopinata. This reporter will help reveal diversity in dauer-inducing mechanisms across nematode species.

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

Bats are the only mammals capable of powered flight and roost head-down. However, the molecular changes shaping bat limbs remain largely unknown. Here, we used comparative functional genomics coupled with mouse-bat sequence swaps to identify key regulatory elements important in bat limb development. We generated and compared bat and mouse forelimb and hindlimb genomic datasets at key wing developmental timepoints, followed by mouse enhancer assays to characterize sequences showing differences between species. We then swapped six mouse enhancer sequences with their corresponding bat sequences, obtaining a variety of bat limb associated phenotypes, including ossification delay, longer digits, thicker skin and symmetrical hindlimb digits. Our work provides a genomic catalog of genes and regulatory elements involved in bat limb development and through extensive characterization in mice shows how changes in regulatory elements lead to small phenotypic changes that together contribute to bat limb development.

ABSTRACT/SUMMARYVascular remodeling within the developing fetus and placenta is essential for supporting the growth and function of emerging tissues and organs. Pericytes (PCs) play a central role in stabilizing and maturing microvascular networks by extending along endothelial cells (ECs) and reinforcing vessel integrity. In the placenta, as in other organs, PC-EC communication is mediated in part by platelet-derived growth factor-BB (PDGF-BB) signaling, which governs PC differentiation, proliferation, migration, and survival, ultimately enabling their recruitment and retention along capillaries. In this study, we identified progressive PC investment along feto-placental capillaries in both murine and human tissues across gestation, supported by morphological and molecular evidence. Placental PCs displayed phenotypic heterogeneity comparable to that observed in the brain and heart, suggesting conserved diversity across organ systems. In addition to characterizing PC dynamics, we examined the expression of recently identified soluble PDGF Receptor-{beta} (sPDGFR{beta}) isoforms. These variants were detected at the protein and transcript levels in mouse and human placentas, as well as in a murine trophoblast-embryonic stem cell (TESC) differentiation model that recapitulates aspects of early placental vascular development. Within this model, sPDGFR{beta} expression was independent of ADAM10 activity and exogenous growth factors during early vessel formation but was markedly upregulated during hypoxia. To assess how elevated sPDGFR{beta} might influence PDGF-BB signaling, we exposed TESCl-derived vascular networks to excess PDGF-BB with or without a sPDGFR{beta} mimetic. PDGF-BB alone reduced full-length PDGFR{beta} levels while increasing receptor phosphorylation, consistent with known ligand-induced regulatory mechanisms. Inclusion of the sPDGFR{beta} mimetic shifted these responses toward baseline, suggesting a potential modulatory or feedback role for soluble receptor variants. Together, these findings demonstrate that PCs are progressively recruited to placental capillaries and exhibit diverse phenotypes during development, and that soluble PDGFR{beta} isoforms may modulate PDGF-BB signaling in a manner sensitive to oxygen tension. Understanding these mechanisms provides insight into the regulation of placental vascular maturation and may inform strategies to improve human health by targeting disorders rooted in impaired placental development.

Postnatal growth of the mandibular condyle requires coordinated expansion of fibrocartilage and production of chondrocytes, yet the cellular populations that organize this process remain incompletely defined. Here we identify a Wnt-responsive fibrocartilage progenitor population that contributes to postnatal mandibular condylar cartilage growth. Using a direct Wnt activity reporter (R26-WntVis), inducible genetic lineage tracing (Axin2CreERT2), and single-cell transcriptomics, we define a Wnt-enriched progenitor-like cluster localized predominantly within the fibrocartilage zone. Lineage tracing demonstrates that Axin2-lineage cells expand laterally within fibrocartilage and generate vertically aligned chondrocytes in the chondrocartilage compartment, indicating bidirectional growth contribution in vivo. Conditional ablation of {beta}-catenin in Axin2-lineage cells results in depletion of the fibrocartilage compartment and premature activation of chondrogenic differentiation programs, whereas constitutive {beta}-catenin activation disrupts compartmental organization without enhancing proliferation. Mechanistically, we identify Foxm1 as a Wnt-associated proliferative mediator enriched in fibrocartilage, and genetic reduction of Foxm1 cooperates with {beta}-catenin deficiency to impair condylar growth. In parallel, {beta}-catenin loss derepresses TGF-{beta}-Smad signaling and enhances chondrogenic differentiation, indicating that canonical Wnt activity coordinates proliferative maintenance while restraining lineage commitment within the same cellular compartment. Together, these findings identify a Wnt-responsive fibrocartilage progenitor system that regulates postnatal mandibular condylar cartilage growth by coupling Foxm1-associated proliferative maintenance with suppression of TGF-{beta}-dependent chondrogenic differentiation during temporomandibular joint development. Graphical abstractWnt-responsive fibrocartilage progenitors coordinate postnatal mandibular condylar cartilage growth through Foxm1-dependent proliferative maintenance and suppression of TGF-{beta}-driven chondrogenic differentiation.

Stem cell technologies have become a vital component of conservation efforts around the globe. Biobanks and pluripotent stem cell lines help to ensure species and their genetic diversity are preserved. These efforts have however, focussed mostly on mammals and birds, and the cryopreservation protocols for embryos and cells were developed decades ago laying the basis for artificial reproductive techniques for species conservation. With over 20% of non-avian reptile species facing extinction, it is imperative to establish protocols for reptiles to ensure species preservation and also to facilitate the establishment of new reptile model organisms to match the standard of mammals. Here, we have generated a cryopreservation method for preserving early gastrulating veiled chameleon embryos as a representative squamate species. To this end, we first developed a tissue culture method for maintaining cells extracted from peri-gastrulation chameleon embryos and then tested different cryopreservation methods altering the concentration of the penetrating cryoprotectant DMSO and assessing the effect of the addition of non-penetrating cryoprotectants Trehalose and Sucrose. We then optimised a protocol for whole embryo vitrification in 20% DMSO with added Trehalose or Sucrose that can easily be adapted for fieldwork. Taken together, our method not only provides a protocol for conservation efforts but also lays the basis for mechanistic studies of early squamate embryo development by enabling cryopreservation of whole embryos in a fieldwork setting, which facilitates their live transport back to a laboratory for functional experiments or molecular analyses.

Binding of Fibroblast growth factor (FGF) to a heparan sulfate proteoglycan (HSPG) is required for paracrine FGF signaling. To improve our understanding of FGF:HSPG association, we developed a method to monitor export of the Drosophila FGF ortholog Branchless (Bnl) in vivo. We detected Bnl on the surface of approximately 10% of Bnl-producing cells, but Bnl on the surface of cells depleted of HS was much reduced. HS depletion also non-autonomously decreased the activity of cytonemes that extend from cells that receive Bnl. These results are consistent with the idea that Bnl export to the cell surface is regulated, that intracellular binding of an HSPG to Bnl in producing cells is essential for export, and that cells that take up Bnl actively participate in its release from producing cells. SummaryLevels of FGF exported to the surface of FGF-expressing cells are dependent on intracellular heparan sulfate proteoglycans.

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