Isolation and Preparation of Embryonic Zebrafish Retinal Cells for Single-Cell RNA Sequencing.

Recent technological advances in single-cell RNA sequencing (scRNA-Seq) have enabled scientists to answer novel questions in biology with unparalleled precision. Indeed, in the field of ocular development and regeneration, scRNA-Seq studies have resulted in a number of exciting discoveries that have begun to revolutionize the way we think about these processes. Despite the widespread success of scRNA-Seq, many scientists are wary to perform scRNA-Seq experiments due to the uncertainty of obtaining high-quality viable cell populations that are necessary for the generation of usable data that enable rigorous computational analyses. Here, we describe methodology to reproducibility generate high-quality single-cell suspensions from embryonic zebrafish eyes. These single-cell suspensions served as inputs to the 10× Genomics v3.1 system and yielded high-quality scRNA-Seq data in proof-of-principle studies. In describing methodology to quantitatively assess cell yields, cell viability, and other critical quality control parameters, this protocol can serve as a useful starting point for others in designing their scRNA-Seq experiments in the zebrafish eye and in other developing or regenerating tissues in zebrafish or other model systems.

Nuclei Isolation from Ocular Tissues of the Embryonic Chicken for Single-Nucleus Profiling.

The generation of quality data from a single-nucleus profiling experiment requires nuclei to be isolated from tissues in a gentle and efficient manner. Nuclei isolation must be carefully optimized across tissue types to preserve nuclear architecture, prevent nucleic acid degradation, and remove unwanted contaminants. Here, we present an optimized workflow for generating a single-nucleus suspension from ocular tissues of the embryonic chicken that is compatible with various downstream workflows. The described protocol enables the rapid isolation of a high yield of aggregate-free nuclei from the embryonic chicken eye without compromising nucleic acid integrity, and the nuclei suspension is compatible with single-nucleus RNA and ATAC sequencing. We detail several stopping points, either via cryopreservation or fixation, to enhance workflow adaptability. Further, we provide a guide through multiple QC points and demonstrate proof-of-principle using two commercially available kits. Finally, we demonstrate that existing in silico genotyping methods can be adopted to computationally derive biological replicates from a single pool of chicken nuclei, greatly reducing the cost of biological replication and allowing researchers to consider sex as a variable during analysis. Together, this tutorial represents a cost-effective, simple, and effective approach to single-nucleus profiling of embryonic chicken eye tissues and is likely to be easily modified to be compatible with similar tissue types.

Acquisition and Analysis Methods for Hi-C Data from Medaka Early Embryos.

The three-dimensional (3D) structure of the genome undergoes dynamic changes during the developmental process. While Hi-C is a technique that enables the acquisition of genome 3D structure data across various species and cell types, existing Hi-C analysis programs may face challenges in detecting and comparing structures effectively depending on the characteristics of the genome or cell type. Here, we describe a method for acquiring Hi-C data from medaka early embryos and quantifying the structural changes during the developmental process.

The Drosophila tracheal terminal cell as a model for branching morphogenesis.

The terminal cells of the Drosophila larval tracheal system are perhaps the simplest delivery networks, providing an analogue for mammalian vascular growth and function in a system with many fewer components. These cells are a prime example of single-cell morphogenesis, branching significantly over time to adapt to the needs of the growing tissue they supply. While the genetic mechanisms governing local branching decisions have been studied extensively, an understanding of the emergence of a global network architecture is still lacking. Mapping out the full network architecture of populations of terminal cells at different developmental times of Drosophila larvae, we find that cell growth follows scaling laws relating the total edge length, supply area, and branch density. Using time-lapse imaging of individual terminal cells, we identify that the cells grow in three ways: by extending branches, by the side budding of new branches, and by internally growing existing branches. A generative model based on these modes of growth recapitulates statistical properties of the terminal cell network data. These results suggest that the scaling laws arise from the coupled contributions of branching and internal growth. This study establishes the terminal cell as a uniquely tractable model system for further studies of transportation and distribution networks.

What Makes Us Human: Insights from the Evolution and Development of the Human Neocortex.

"What makes us human?" is a central question of many research fields, notably anthropology. In this review, we focus on the development of the human neocortex, the part of the brain with a key role in cognition, to gain neurobiological insight toward answering this question. We first discuss cortical stem and progenitor cells and human-specific genes that affect their behavior. We thus aim to understand the molecular foundation of the expansion of the neocortex that occurred in the course of human evolution, as this expansion is generally thought to provide a basis for our unique cognitive abilities. We then review the emerging evidence pointing to differences in the development of the neocortex between present-day humans and Neanderthals, our closest relatives. Finally, we discuss human-specific genes that have been implicated in neuronal circuitry and offer a perspective for future studies addressing the question of what makes us human.

A survey of water chemistry used in zebrafish facilities and their effects on early zebrafish development.

Background: There are a variety of published standard methods and water chemistry recommendations for zebrafish ( Danio rerio) husbandry, but empirical evidence for their justification is often lacking, as is information on some variables that have important biological effects on fish. Importantly, these different recommendations could contribute to variability in results and fish welfare between or within institutions. Methods: Here we document the current range of water chemistry used by various research institutions around the world and report initial findings on their effects on the development and growth of zebrafish. Over 40 institutes responded to a survey that revealed a large variation in water chemistry used for zebrafish husbandry including differences in the set-points and acceptable ranges for temperature, pH and conductivity. In subsequent experiments, zebrafish ( D. rerio, WIK) embryos/larvae exposed to a large range of salt concentrations (50µM to 10mM Na + or 30 - 2500 µS/cm) and CO 2 levels (400 - 8,000 µatm). Results: Larvae exposed to the lowest salt concentration (5 µM Na + or < 30µS/cm) had a slower response to touch and their swim bladders were not inflated. Larvae exposed to 5-100 µM Na + were 5 % shorter in total body length than those exposed to higher salt concentrations (>100 µM Na +). Zebrafish embryo/larvae exposed to intermediate pCO 2 values (~2000 µatm) were 1 to 3.5% longer than those exposed to either ambient (400 µatm) or higher (4000 µatm) pCO 2, but pCO 2 did not affect developmental endpoints up to 4 dpf. Conclusions: Overall, we highlight the magnitude of variation in water chemistry used within zebrafish research and provide some empirical evidence to show that not all of these water conditions might be optimal for developing zebrafish and reproducibility of research, although further research is necessary to determine longer-term effects of water chemistry on older larvae, juveniles and adults.

Foetal cerebral blood redistribution in idiopathic polyhydramnios and the impact on perinatal outcomes.

BACKGROUND: Idiopathic polyhydramnios is associated with adverse neonatal outcome. The aim of this study was to examine the value of the middle cerebral artery pulsatility index (MCA-PI) in predicting adverse perinatal outcome in pregnancies affected by idiopathic polyhydramnios. METHODS: A retrospective study was performed during 2013-2022 at a tertiary-care university affiliated hospital. The study included singleton pregnancies with idiopathic polyhydramnios. Obstetrical and perinatal outcomes were compared between women with MCA-PI <10th percentile and women with MCA-P ≥ 10th percentile. A composite adverse perinatal outcome was defined as an Apgar score <7 at 5 minutes, umbilical artery pH <7.15, emergent caesarean delivery (CD) or operative delivery due to foetal distress, neonatal intensive care admission or foetal death. RESULTS: Among 45,459 pregnancies, 128 (0.3%) had idiopathic polyhydramnios; 27 exhibited MCA-PI <10th percentile. Among the latter, compared to pregnancies with MCA-PI ≥10th percentile group, the rates were higher of emergent CD (25.9% vs. 7.9%, p = 0.017) and adverse perinatal outcomes (37.0% vs. 15.8%, p = 0.020). These differences persisted in a subgroup analysis of women with moderate or severe polyhydramnios. In the MCA-PI <10th percentile group, the median MCA-PI and cerebroplacental ratio were lower: 0.9 vs. 1.7, p < 0.001 and 0.7 vs. 2.1, p < 0.001, respectively. Receiver operating characteristic curve analysis indicated a significant association of MCA-PI with emergent CD/operative delivery for foetal distress (area under curve = 0.672, p = 0.031); the sensitivity was 46.7% and specificity 82.3%. Cerebroplacental ratio values were not associated with adverse perinatal outcomes. CONCLUSIONS: Idiopathic polyhydramnios might be associated with foetal cerebral blood flow redistribution, potentially contributing to an increased risk of adverse neonatal outcomes. Prospective studies are required to establish the role of foetal Doppler studies in the antenatal surveillance of idiopathic polyhydramnios, and to determine whether evidence of abnormal MCA-PI serves as a reliable predictor of perinatal outcomes, potentially necessitating labour induction.

Polyhydramnios is a condition in pregnancy characterized by an excessive amount of amniotic fluid, and in many cases, the cause remains unidentified, referred to as idiopathic polyhydramnios. This study aimed to determine whether changes in blood flow to the fetus's brain, specifically in the middle cerebral artery (MCA), could predict adverse outcomes during delivery. To assess this, the researchers used Doppler ultrasound to measure the pulsatility index (PI) of the MCA, which reflects the blood flow resistance in the artery. The study compared two groups of pregnancies with idiopathic polyhydramnios­those with reduced MCA blood flow (below the 10th percentile) and those with normal blood flow. It was observed that pregnancies with reduced MCA-PI were more likely to experience complications, such as emergency cesarean deliveries due to fetal distress and the need for neonatal intensive care unit admissions. These findings suggest that altered blood flow in the fetal brain might indicate increased risks of complications during delivery. The results support the idea that monitoring MCA blood flow in pregnancies complicated by polyhydramnios could offer valuable insights for early intervention and management. However, further research is necessary to confirm whether MCA Doppler assessments can reliably predict which pregnancies are at higher risk and whether such monitoring should guide clinical decisions like early induction of labor.

Linkage between Fuz and Gpr161 genes regulates sonic hedgehog signaling during mouse neural tube development.

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing the primary cilium, the cell's antenna, which acts as a signaling hub. Fuz, an effector of planar cell polarity signaling, regulates Shh signaling by facilitating cilia formation, and the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of the genes encoding these proteins is similar; however, their functional relationship has not been previously explored. This study identified the genetic and biochemical linkage between Fuz and Gpr161 in mouse neural tube development. Fuz was found to be genetically epistatic to Gpr161 with respect to regulation of Shh signaling in mouse neural tube development. The Fuz protein biochemically interacts with Gpr161, and Fuz regulates Gpr161-mediated ciliary localization, a process that might utilize ß-arrestin 2. Our study characterizes a previously unappreciated Gpr161-Fuz axis that regulates Shh signaling during mouse neural tube development.

Actin-driven nanotopography promotes stable integrin adhesion formation in developing tissue.

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

Calcium wave dynamics in the embryonic mouse gut mesenchyme: impact on smooth muscle differentiation.

Intestinal smooth muscle differentiation is a complex physico-biological process involving several different pathways. Here, we investigate the properties of Ca2+ waves in the developing intestinal mesenchyme using GCamp6f expressing mouse embryos and investigate their relationship with smooth muscle differentiation. We find that Ca2+ waves are absent in the pre-differentiation mesenchyme and start propagating immediately following α-SMA expression. Ca2+ waves are abrogated by CaV1.2 and gap-junction blockers, but are independent of the Rho pathway. The myosine light-chain kinase inhibitor ML-7 strongly disorganized or abolished Ca2+ waves, showing that perturbation of the contractile machinery at the myosine level also affected the upstream Ca2+ handling chain. Inhibiting Ca2+ waves and contractility with CaV1.2 blockers did not perturb circular smooth muscle differentiation at early stages. At later stages, CaV1.2 blockers abolished intestinal elongation and differentiation of the longitudinal smooth muscle, leading instead to the emergence of KIT-expressing interstitial cells of Cajal at the gut periphery. CaV1.2 blockers also drove apoptosis of already differentiated, CaV1.2-expressing smooth muscle and enteric neural cells. We provide fundamental new data on Ca2+ waves in the developing murine gut and their relation to myogenesis in this organ.

Maternal diet during pregnancy and adaptive changes in the maternal and fetal pancreas have implications for future metabolic health.

Fetal and neonatal development is a critical period for the establishment of the future metabolic health and disease risk of an individual. Both maternal undernutrition and overnutrition can result in abnormal fetal organ development resulting in inappropriate birth size, child and adult obesity, and increased risk of Type 2 diabetes and cardiovascular diseases. Inappropriate adaptive changes to the maternal pancreas, placental function, and the development of the fetal pancreas in response to nutritional stress during pregnancy are major contributors to a risk trajectory in the offspring. This interconnected maternal-placental-fetal metabolic axis is driven by endocrine signals in response to the availability of nutritional metabolites and can result in cellular stress and premature aging in fetal tissues and the inappropriate expression of key genes involved in metabolic control as a result of long-lasting epigenetic changes. Such changes result is insufficient pancreatic beta-cell mass and function, reduced insulin sensitivity in target tissues such as liver and white adipose and altered development of hypothalamic satiety centres and in basal glucocorticoid levels. Whilst interventions in the obese mother such as dieting and increased exercise, or treatment with insulin or metformin in mothers who develop gestational diabetes, can improve metabolic control and reduce the risk of a large-for-gestational age infant, their effectiveness in changing the adverse metabolic trajectory in the child is as yet unclear.

Variation in the thermal plasticity of avian embryos is produced by the developmental environment, not genes.

Limited evidence suggests that variation in phenotypic plasticity within populations may arise largely from environmental sources, thereby constraining its evolvability. This is of concern for temperature-sensitive metabolism in the face of climate change. We quantified the relative influence of the developmental environment versus genes on the metabolic plasticity of avian embryos to temperature. We partially cross-fostered 602 house sparrow eggs (Passer domesticus), measured the heart rate plasticity of these embryos to egg temperature and partitioned variance in plasticity. We found that the foster (incubation) environment was the sole meaningful source of variance in embryonic plasticity (not genes, pre-laying effects or ambient conditions). In contrast to heart rate plasticity, offspring growth was influenced by the foster environment, genes/pre-laying parental effects and ambient conditions. Although embryonic plasticity to temperature varied in this population, these results suggest that it is unlikely to evolve quickly. Nevertheless, the expression of this plasticity may be able to shift between generations in response to changes in the developmental environment. Whether the multidimensional plasticity of heart rate to both current temperature and the developmental environment is itself an adaptive, evolved trait allowing avian embryos to optimize their metabolic plasticity to their current environment remains to be tested.

A role for organ level dynamics in morphogenesis of the C. elegans hermaphrodite distal tip cell.

The morphology of cells in vivo can arise from a variety of mechanisms. In the Caenorhabditis elegans hermaphrodite gonad, the distal tip cell (DTC) elaborates into a complex plexus over a relatively short developmental time period, but the mechanisms underlying this change in cell morphology are not well defined. We correlated the time of DTC elaboration with the L4-to-adult molt, but ruled out a relevant heterochronic pathway as a cue for DTC elaboration. Instead, we found that the timing of gonad elongation and aspects of underlying germline flux influence DTC elaboration. We propose a 'hitch and tow' aspect of organ-level dynamics that contributes to cellular morphogenesis, whereby germline flux drags the flexible DTC cell cortex away from its stationary cell body. More broadly, we speculate that this mechanism may contribute to cell shape changes in other contexts with implications for development and disease.

EZH2 specifically regulates ISL1 during embryonic urinary tract formation.

Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10- 24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D' = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 × 10- 20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2-/- knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2-/- ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.

Alternative splicing dynamically regulates common carp embryogenesis under thermal stress.

BACKGROUND: Thermal stress is a major environmental factor affecting fish development and survival. Common carp (Cyprinus carpio) are susceptible to heat stress in their embryonic and larval phases, but the thermal stress response of alternative splicing during common carp embryogenesis remains poorly understood. RESULTS: Using RNA-seq data from eight developmental stages and four temperatures, we constructed a comprehensive profile of alternative splicing (AS) during the embryogenesis of common carp, and found that AS genes and events are widely distributed among all stages. A total of 5,835 developmental stage-specific AS (SAS) genes, 21,368 temperature-specific differentially expressed genes (TDEGs), and 2,652 temperature-specific differentially AS (TDAS) genes were identified. Hub TDAS genes in each developmental stage, such as taf2, hnrnpa1, and drg2, were identified through protein-protein interaction (PPI) network analysis. The early developmental stages may be more sensitive to temperature, with thermal stress leading to a massive increase in the number of expressed transcripts, TDEGs, and TDAS genes in the morula stage, followed by the gastrula stage. GO and KEGG analyses showed that from the morula stage to the neurula stage, TDAS genes were more involved in intracellular transport, protein modification, and localization processes, while from the optic vesicle stage to one day post-hatching, they participated more in biosynthetic processes. Further subgenomic analysis revealed that the number of AS genes and events in subgenome B was generally higher than that in subgenome A, and the homologous AS genes were significantly enriched in basic life activity pathways, such as mTOR signaling pathway, p53 signaling pathway, and MAPK signaling pathway. Additionally, lncRNAs can play a regulatory role in the response to thermal stress by targeting AS genes such as lmnl3, affecting biological processes such as apoptosis and axon guidance. CONCLUSIONS: In short, thermal stress can affect alternative splicing regulation during common carp embryogenesis at multiple levels. Our work complemented some gaps in the study of alternative splicing at both levels of embryogenesis and thermal stress in C. carpio and contributed to the comprehension of environmental adaptation formation in polyploid fishes during embryogenesis.

Placental extracellular vesicles promote cardiomyocyte maturation and fetal heart development.

Congenital heart defects are leading causes of neonatal mortality and are often associated with placental abnormalities, but mechanisms linking placenta and heart development are poorly understood. Herein, we investigated a potential signaling network connecting the placenta and nascent heart in mice. We found that fetal hearts exposed to media conditioned by placental tissue or differentiated wild-type trophoblast stem (TS) cells, but not undifferentiated TS cells, showed increased heart rate and epicardial cell outgrowth. This effect was not observed when hearts were exposed to media from TS cells lacking OVO-Like 2, a transcription factor required for trophoblast differentiation and placental development. Trophoblasts released abundant extracellular vesicles into media, and these vesicles were sufficient to mediate cardio-promoting effects. Our findings provide a potential mechanism whereby the placenta communicates with the fetal heart to promote cardiac morphogenesis, and offers insight into the link between poor placentation and a higher incidence of heart defects.

SLMAP3 is essential for neurulation through mechanisms involving cytoskeletal elements, ABP, and PCP.

SLMAP3 is a tail-anchored membrane protein that targets subcellular organelles and is believed to regulate Hippo signaling. The global loss of SLMAP3 causes late embryonic lethality in mice, with some embryos exhibiting neural tube defects such as craniorachischisis. We show here that SLMAP3 -/- embryos display reduced length and increased width of neural plates, signifying arrested convergent extension. The expression of planar cell polarity (PCP) components Dvl2/3 and the activity of the downstream targets ROCK2, cofilin, and JNK1/2 were dysregulated in SLMAP3 -/- E12.5 brains. Furthermore, the cytoskeletal proteins (γ-tubulin, actin, and nestin) and apical components (PKCζ and ZO-1) were mislocalized in neural tubes of SLMAP3 -/- embryos, with a subsequent decrease in colocalization of PCP proteins (Fzd6 and pDvl2). However, no changes in PCP or cytoskeleton proteins were found in cultured neuroepithelial cells depleted of SLMAP3, suggesting an essential requirement for SLMAP3 for these processes in vivo for neurulation. The loss of SLMAP3 had no impact on Hippo signaling in SLMAP3 -/- embryos, brains, and neural tubes. Proteomic analysis revealed SLMAP3 in an interactome with cytoskeletal components, including nestin, tropomyosin 4, intermediate filaments, plectin, the PCP protein SCRIB, and STRIPAK members in embryonic brains. These results reveal a crucial role of SLMAP3 in neural tube development by regulating the cytoskeleton organization and PCP pathway.

VEGF-dependent testicular vascularisation involves MEK1/2 signalling and the essential angiogenesis factors, SOX7 and SOX17.

BACKGROUND: Abnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4-eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor. RESULTS: RNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes, the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5. In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane. CONCLUSIONS: Together, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis.

Gα13 controls pharyngeal endoderm convergence by regulating E-cadherin expression and RhoA activation.

Pharyngeal endoderm cells undergo convergence and extension (C&E), which is essential for endoderm pouch formation and craniofacial development. Our previous work implicates Gα13/RhoA-mediated signaling in regulating this process, but the underlying mechanisms remain unclear. Here, we have used endoderm-specific transgenic and Gα13 mutant zebrafish to demonstrate that Gα13 plays a crucial role in pharyngeal endoderm C&E by regulating RhoA activation and E-cadherin expression. We showed that during C&E, endodermal cells gradually establish stable cell-cell contacts, acquire apical-basal polarity and undergo actomyosin-driven apical constriction, which are processes that require Gα13. Additionally, we found that Gα13-deficient embryos exhibit reduced E-cadherin expression, partially contributing to endoderm C&E defects. Notably, interfering with RhoA function disrupts spatial actomyosin activation without affecting E-cadherin expression. Collectively, our findings identify crucial cellular processes for pharyngeal endoderm C&E and reveal that Gα13 controls this through two independent pathways - modulating RhoA activation and regulating E-cadherin expression - thus unveiling intricate mechanisms governing pharyngeal endoderm morphogenesis.

Stretch triggers microtubule stabilization and MARCKS-dependent membrane incorporation in the shaft of embryonic axons.

Neurons have a unique polarized nature that must adapt to environmental changes throughout their lifespan. During embryonic development, axon elongation is led by the growth cone,1 culminating in the formation of a presynaptic terminal. After synapses are formed, axons elongate in a growth cone-independent manner to accompany body growth while maintaining their ultrastructure and function.2,3,4,5,6 To further understand mechanical strains on the axon shaft, we developed a computer-controlled stretchable microfluidic platform compatible with multi-omics and live imaging. Our data show that sensory embryonic dorsal root ganglia (DRGs) neurons have high plasticity, with axon shaft microtubules decreasing polymerization rates, aligning with the direction of tension, and undergoing stabilization. Moreover, in embryonic DRGs, stretch triggers yes-associated protein (YAP) nuclear translocation, supporting its participation in the regulatory network that enables tension-driven axon growth. Other than cytoskeleton remodeling, stretch prompted MARCKS-dependent formation of plasmalemmal precursor vesicles (PPVs), resulting in new membrane incorporation throughout the axon shaft. In contrast, adolescent DRGs showed a less robust adaptation, with axonal microtubules being less responsive to stretch. Also, while adolescent DRGs were still amenable to strain-induced PPV formation at higher stretch rates, new membrane incorporation in the axon shaft failed to occur. In summary, we developed a new resource to study the biology of axon stretch growth. By unraveling cytoskeleton adaptation and membrane remodeling in the axon shaft of stretched neurons, we are moving forward in understanding axon growth.