The extracellular matrix (ECM) provides biophysical and biochemical cues necessary for cellular migration, differentiation and survival during development. Laminins are major ECM proteins consisting of α, β and γ chains. However, the function of laminin β4, encoded by LAMB4, remains unknown. Using human pluripotent stem cells (hPSCs), we characterize the role of LAMB4 in human peripheral sensory neuron (SN) biology. We found that LAMB4 is expressed during early SN specification, and it is required for SN development and survival. To assess clinical relevance, we examined familial dysautonomia (FD), a genetic disorder specifically affecting peripheral neurons. LAMB4 variants previously identified in individuals with severe FD sharply downregulated LAMB4 expression in SNs. Moreover, restoring a healthy ECM rescued the FD-related developmental phenotypes, suggesting that ECM defects contribute significantly to the etiology of FD. Finally, we showed that LAMB4/laminin β4 interacts with laminin α4 and laminin γ3 to form the previously unreported laminin-443 and is required for actin filament formation in SNs. Together, these results identify LAMB4 as a crucial regulator of SN development and survival with clinical implications.
SRY-box transcription factor 17 (Sox17) is critical for hepato-pancreato-biliary (HPB) development. We identified two distributed enhancers of Sox17, Sox17e-13 and Sox17e-231, within the likely murine Sox17 topologically associating domain. Individual CRISPR-mediated enhancer deletions temporally reduce endodermal Sox17 expression, delaying the transition of bipotent ventral pancreato-biliary progenitor cells to a lineage-unipotent ventral pancreatic fate, reducing pancreas size and increasing gallbladder size. Deleting both enhancers further reduces Sox17 expression and delays conversion of bipotent progenitor cells to ventral pancreatic or biliary fates, reducing both pancreas and gallbladder size. Crossing each enhancer mutation to a Sox17 null allele revealed differential and profound sensitivities of the extrahepatic biliary system to alterations of Sox17 expression, as demonstrated by gallbladder hypoplasia, gallbladder agenesis with a cystic duct, and biliary atresia. These studies indicate that Sox17e-13 and Sox17e-231 additively modulate Sox17 expression, the differentiation of bipotential ventral pancreato-biliary cells to unipotent fates is Sox17 dosage dependent, and that thresholds for Sox17 action during pancreato-biliary formation vary by developmental stage, further defining the role of Sox17 in HPB formation and function.
Multiple signaling pathways and transcription factors (TFs) establish organ domains in the developing gastrointestinal tract. How these are integrated into spatial-temporal networks to regulate organogenesis and how disruptions to those networks lead to congenital syndromes remain poorly understood. Using human pluripotent stem cell cultures and Xenopus embryos, we demonstrate that retinoic acid (RA) from the lateral plate mesoderm directly activates expression of the TF rfx6 in posterior foregut endoderm. Rfx6 subsequently promotes posterior foregut identity while suppressing Wnt-dependent hindgut and Bmp-dependent pharyngeal fates through direct and indirect mechanisms. Rfx6 can directly activate the expression of several key foregut TFs (onecut1 and pdx1) and Wnt antagonists (sfrp2/5) while indirectly restricting expression of Wnt and Bmp ligands. Rfx6 also directly suppresses transcription of the Wnt-dependent hindgut TF cdx2 and the Bmp-dependent pharyngeal TFs nkx2-5 and nkx2-6. Thus, Rfx6 acts at multiple levels to integrate RA, Wnt, and Bmp activity into a network with lineage-promoting TFs to control gut tube patterning. These results provide insight into the molecular basis of Mitchell-Riley Syndrome congenital anomalies, which are caused by RFX6 mutations.
Mechanical forces shape multiscale biological processes across many living systems, ranging from cell proliferation and apoptosis, to tissue folding and pattern formation. In recent years, tissue pressure has emerged as a fundamental regulator of morphogenesis across animal and plant kingdoms. However, a fundamental understanding of the definition and origin of tissue pressure remains lacking. In this Review, we synthesise how tissue pressure is generated from diverse sources, including tissue growth, actomyosin contractility, topological defects and fluid- or extracellular matrix-driven swelling, and how it influences developmental processes such as proliferation, apoptosis, differentiation, tissue folding and pattern formation. We also discuss how homeostatic pressure governs tissue growth and competition, while highlighting the cellular and molecular signalling pathways implicated in compressive stress responses. Finally, drawing on examples from animal, plant, bacterial and organoid systems, we outline emerging experimental and computational approaches to measure and manipulate tissue pressure in vivo, and identify key challenges in dissecting compression-specific mechanosignalling pathways.
Alizarin Red S (AZ) is a dye that is commonly used in histological studies and textiles. We find that AZ is teratogenic to Lytechinus variegatus sea urchin larvae. Here, we demonstrate that embryos exposed to AZ have abnormal skeletal patterning and perturbed migration of skeletogenic mesenchyme cells, along with mild ectodermal abnormalities and reduced neural connectivity. Temporal transcriptomics reveal delayed development and implicate wide-reaching changes in gene expression with AZ treatment. Particle image velocimetry experiments show that AZ treatment perturbs the normal pattern of fluid flow away from the larval mouth and inhibits larval feeding. Finally, we show that AZ promotes a significant elevation of reactive oxygen species (ROS) via catalase inhibition. ROS-mediated effects are expected to be broad, in agreement with the broad effects of AZ detected by transcriptomics. Catalase knockdown and hydrogen peroxide treatment are each sufficient to quantitatively phenocopy AZ-mediated skeletal patterning defects, while catalase overexpression is sufficient to rescue the AZ skeletal phenotype. This study is the first to define the teratogenic consequences of AZ exposure on development, skeletal patterning and biomineralization.
The apical extracellular matrix (aECM) plays crucial roles during morphogenesis and forms the interface through which tissues communicate with the environment. The aECM exhibits complex molecular composition and structure, including multi-layered organization, localized cell-type-specific specializations, three-dimensional elaborations and dynamic temporal remodeling. Yet, until recently, the aECM received relatively little attention compared to the much more well-studied basal ECM (i.e. the basement membrane and stromal matrix). Here, we discuss aECM composition and variation across species, and summarize emerging ideas from a growing community of aECM researchers related to potential mechanisms that control aECM development.
Robust tissue growth control requires long-range communication between the rate of progenitor addition and tissue expansion. However, the regulatory mechanisms that couple these processes are unknown. In zebrafish, notochord morphogenesis is a driver of axis extension through both posterior progenitor addition and anterior vacuolation. To elucidate how progenitor dynamics and vacuole-driven cell expansion interact to elongate the notochord, we generated a mathematical model linking progenitor addition rate to the expansion of cells from anterior-to-posterior to simulate vacuolation rate. Comparing this with empirical measurements, we find that progenitor incorporation together with vacuolation, produces a linear gradient in nearest neighbour distance. We next explored the role of YAP/TAZ in regulating progenitor addition in mutants for YAP/TAZ inhibitor vgll4b. We find that vgll4b expression and YAP activity are enriched in posterior midline progenitors. Loss of vgll4b elevates YAP signaling, enhances progenitor addition, restricts vacuole expansion, and—after a transient buffering phase—compromises A-P axis elongation. These results support a long-range feedback mechanism linking progenitor recruitment to vacuolation, enabling the notochord to balance cellular input with volumetric expansion, thereby maintaining tissue proportions.
Esophageal atresia/tracheoesophageal fistula (EA/TEF) are congenital malformations of the foregut, and we identified two EA/TEF patients with two variants in the BMP/TGFβ repressor SMAD6. We investigated the function of SMAD6 in tracheoesophageal development using two orthogonal approaches in Xenopus embryos, both resulting in foregut malformations including EA/TEF. We then used human Pluripotent Stem Cell-derived foregut epithelium and mesenchyme to explore the separate roles of SMAD6 in these germ layers. CRISPR-mediated disruption of human SMAD6 caused an increase in BMP signaling in foregut epithelium and mesenchyme consistent with its role as a BMP repressor. Loss of SMAD6 caused patterning defects in both tissue types; SMAD6-/- endoderm shows increased expression of distal gut tube markers and SMAD6-/- mesenchyme shows increased expression of ventral and posterior markers including markers of cardiac and liver mesenchyme lineages. Furthermore, SMAD6-/- mesenchyme had decreased ability to form CD31-positive endothelial cells. Our results demonstrate that SMAD6 is required for foregut development and that rare variants in this gene are likely causative for foregut malformations in EA/TEF patients.
The optic nerve develops from the neuroectodermal optic stalk, which undergoes coordinated morphogenesis and gives rise to optic nerve astrocytes that support retinal ganglion cell axons. Here, we define the progression of astrocyte formation from the optic stalk and identify stage-specific functions of the SWI/SNF scaffolding subunits Smarcc1 and Smarcc2. Both factors are co-expressed in retinal pigment epithelium (RPE) and optic stalk progenitors, with Smarcc2 persisting in differentiated RPE and astrocytes. Conditional deletion using Dct-Cre revealed compensatory activity in pigmented lineages, whereas Smarcc1 loss uniquely disrupted optic nerve head morphogenesis, resulting in glial lamina collapse, retinal ganglion cell degeneration and progressive visual decline. Spatial transcriptomics and functional assays show that Smarcc1 enables dorsal optic stalk progenitors to transition from a pigmented, RPE-like state to astrocyte progenitors by repressing pigment gene programs and permitting Pax2 and Sox2 activity. After specification, Smarcc1 is also required for glial lamina assembly and astrocyte migration into the inner retina. These findings demonstrate that Smarcc1-dependent chromatin remodeling coordinates astrocyte specification with optic nerve head morphogenesis to maintain long-term retinal function.
Programmed cell death (apoptosis) during oogenesis is conserved across metazoans and linked to regulation of oocyte number and quality. In oogenic germlines, the removal of developing oocytes by apoptosis ensures that oocytes do not contain DNA damage or multiple nuclei. Beyond this chromatin-quality control assurance role, it was unknown how apoptosis contributes to oocyte quality. We used the nematode Caenorhabditis elegans to study the consequences of loss of apoptosis on oogenesis. Blocking apoptosis reduced fecundity in hermaphrodites at peak fertility and caused germline architectural defects, such as abnormal rachis morphology and perturbed arrangement and distribution of oogenic germline compartments. Our results suggest that the loss of germline apoptosis arises due to lack of sufficient space for, and reduced cytoplasmic flows into, developing oogonia. In support of this idea, oocytes and embryos are abnormally small and exhibit low viability in animals unable to execute apoptosis. These findings suggest that, in addition to preventing ploidy defects during oogenesis, apoptosis contributes to fertility by preserving the homeostatic germline structure required for the fidelity of oogenesis.
Craniosynostosis is a congenital disorder characterized by premature fusion of cranial sutures. Lambdoid synostosis is a rare and severe form of the disease, but its developmental etiology remains poorly understood. Although ectopic cartilage has been associated with lambdoid suture fusion, whether abnormal chondrocyte development is causative has not been directly tested. Here, we have investigated the role of platelet-derived growth factor receptor alpha (PDGFRα) signaling in chondrocyte development and lambdoid suture morphogenesis. Mesoderm-specific expression of an autoactivated Pdgfra allele (Pdgfra+/K) caused premature fusion of the lambdoid and occipitointerparietal sutures, preceded by excessive cartilage expansion during embryogenesis. Spatial transcriptomic analysis identified a PDGFRα-dependent transcriptional program characterized by increased Col2a1, Sox9 and Sfrp2 expression, indicating maintenance of chondrocytes in a proliferative progenitor state. Consistent with this, PDGFRα-activated chondrocytes exhibited increased proliferation. Moreover, expression of Pdgfra+/K in the Col2a1Cre lineage selectively induced occipitointerparietal suture fusion. Together, these findings demonstrate that PDGFRα signaling promotes proliferative cartilage and impairs chondrocyte maturation, identifying dysregulated chondrogenesis as a developmental mechanism underlying lambdoid craniosynostosis.
Ovarian follicles in most species are assumed to develop using a single pathway. However, in Drosophila pupae, lineage tracing 2937 single-cell clones identified three follicle 'waves' that follow distinct programs arrayed anterior to posterior in the germarium and developing ovarioles. 40 primordial germ cells (PGCs) become anterior germline stem cells that produce 'wave 2' follicles throughout adulthood. 100 PGCs posterior to wave 2 develop into 'wave 1.5' follicles that form the earliest laid eggs. Different follicle stem cells sequentially occupy the same two niches timed to provide both waves with specific follicle cells, consistent with programmed differences between waves 2 and 1.5. 'Wave 1' PGCs located even further posteriorly proliferate, form cysts, interact with swarm cells, degenerate, break from the ovary 22-26 h after puparium formation, and release lipid-enriched vacuoles. Some testis germ cells behave similarly. We speculate that wave 1 germ cells contribute directly or indirectly to the pharate adult ecdysone pulse that mediates adult development and sex-specific neural remodeling. Why Drosophila follicle waves generally resemble those clarified recently in mouse pre-adult ovaries merits additional study.
Despite advances in deep-learning and bioimage analysis, manual curation of segmented and tracking data is still required to extract accurate quantitative single cell temporal information from large tissue/embryo movies. However, very few tools address specifically this challenge. We present here EpiCure (Epithelial Curation), a versatile tool designed to streamline and accelerate manual curation of segmentation and tracking in 2D movies of large epithelial tissues. EpiCure uses temporal information and morphometric parameters to automatically identify segmentation and tracking errors and provides user-friendly tools to correct them. It focuses on ergonomics and offers visualization options to help navigate movies covering a large number of cells, speeding up the detection and curation of errors. EpiCure is highly interoperable, supports input from diverse segmentation tools and includes multiple export filters enabling seamless integration with downstream analysis pipelines. Using movies from several animal models, we highlight the importance of curating cell segmentation and tracking for accurate downstream analysis, and how EpiCure helps by extracting single cell dynamics and detecting cellular events in a large dataset.
The primitive endoderm (PE), or hypoblast in humans, is a conserved extra-embryonic lineage essential for mammalian development that fulfills key roles in embryo patterning, such as contributing to the gut endoderm, providing nutrient and gas exchange, and hosting the first wave of hematopoiesis. In this Review, we summarize our current understanding of PE development, focusing on the mouse and, where appropriate, the human context. We examine the molecular mechanisms involved in the segregation of the PE and epiblast lineages during pre-implantation development, exploring how stochastic processes and positional history initiate fate specification, and how robust ratio control of the two lineages is ensured. Furthermore, we evaluate the expanding repertoire of in vitro stem cell models of embryos, categorizing them according to their PE representation and functional utility in studying peri- and post-implantation development.
Teeth serve as a powerful model to dissect epithelial-mesenchymal interaction-mediated organogenesis. While most non-mammalian vertebrates exhibit lifelong tooth renewal (polyphyodonty), mammals have largely lost this capacity and become predominantly diphyodont. Elucidating this evolutionary transition is crucial for understanding developmental constraints on regeneration, yet key mechanisms, particularly how the successional dental lamina persists and how dormant stem cell niches are reactivated, remain largely unresolved. Recent advances in lineage tracing, single-cell sequencing, multi-omics profiling and organoid models have uncovered remarkable cellular diversity and lineage potential during tooth development and homeostasis across vertebrates, reshaping our view of dental stem cell identity and its roles in tissue regeneration. In this Review, we summarize recent progress in tooth development, replacement and molecular regulation, and our current understanding of dental stem cells and organoid models, aiming to delineate a roadmap for translational tooth regeneration.
Anterior-posterior patterning in Drosophila relies on positional information supplied by the graded distributions of three maternally supplied transcription factors originally identified in large-scale mutagenesis screens. Here, I examine the development of embryos in which these three maternal gradients have been flattened, but set at different levels. Following such manipulations, expression patterns at the blastoderm stage are uniform along the anterior-posterior axis, but their specific nature depends on the combination and concentration level of the three systems. Each of the three gradients are then examined in contexts in which the others are flattened but set at different levels. This reveals that each system generates a greater fraction of the wild-type pattern than that deleted when that system is removed by loss-of-function mutations, with the exact pattern depending on the levels to which the two remaining flattened systems are set. These simplified patterns are then used to analyze interactions between the maternal systems and to investigate how the pattern of gap and pair rule genes are modulated by network interactions downstream of the maternal inputs themselves.
RNA interference (RNAi) is a genetic tool that disrupts the expression of selected genes by delivering dsRNA into a specific tissue of an organism, resulting in a gene knockdown. We apply RNAi methods to the marine polychaete Streblospio benedicti, which is a model system for studying evolutionary and developmental biology that currently has no established methods for gene expression manipulation. Here, we describe a RNAi gene knockdown methodology using two different approaches depending on developmental stage. We fed bacteria expressing dsRNA to early swimming larvae and microinjected in vitro transcribed dsRNA in juveniles. We used two genes for testing: R-opsin and F-actin. For both developmental stages, gene knockdown was assessed using quantitative real-time PCR. We also visualized RNA expression reduction in larvae using hybridization chain reaction in situ hybridization. We show that both delivery methods of larval feeding and juvenile microinjection are sufficient to achieve RNAi gene knockdown, although the specific effect can vary by gene and timing.
Fibroblasts mediate tissue repair after damage, but aberrant fibroblast behavior in response to injury can result in impaired wound healing. Severe burn injury often results in tissue scarring, but the underlying mechanisms by which fibroblasts respond to burn injury and the role of inflammation in fibrosis are not well understood. Here, we have developed fluorescent reporters of collagen-expressing mesenchymal cells, enabling real-time imaging of fibroblasts during homeostatic development and in response to burn injury using larval zebrafish. We find that fibroblasts derived from the mesenchyme respond to burn injury by engaging in a maturation process, characterized by the expression of vimentin, which is reminiscent of larval development. In burned tissue, fibroblast maturation is perturbed by prolonged neutrophil infiltration, resulting in disorganized extracellular matrix (ECM) and delayed ECM remodeling, which can be rescued by neutrophil depletion. This work adds to our understanding of fibroblast development in zebrafish and shows that collagen-expressing mesenchymal cells regulate ECM remodeling in coordination with immune cells during burn wound healing.
