Development

Muscle loading is required for embryonic tendon growth; however, the underlying mechanisms that regulate tendon development downstream of mechanical cues remain unidentified. Although tendons in muscle paralysis models are structurally and functionally inferior, whether these differences arise from cell or matrix deficits remains unclear. Analysis of muscular dysgenesis embryos by atomic force microscopy showed that structural and functional deficits in paralyzed tendon arise in part from reduced proliferation and collagen fibril disorganization. Bulk and single cell transcriptional analyses reveal that both collagenous and non-collagenous extracellular matrix components, as well as cytoskeletal and actomyosin-associated proteins, are dysregulated in mdg tendons, whereas tendon markers remain unchanged. Surprisingly, we find that an arrest of TGF{beta} signaling occurs during normal embryonic tendon growth and that TGF{beta} signaling is abnormally prolonged in paralyzed embryos. We also show for the first time, that specification of the epitenon depends on muscle contraction. Together, these findings establish cell and molecular requirements for muscle contraction in embryonic tendon development. TeaserMuscle contraction is required for embryonic tendon development through regulation of TGF{beta} signaling, epitenon formation, and matrix organization.

A central unresolved question in development biology is how systems of overwhelming complexity arise from relatively few families of growth factors. Compounding this issue, signaling pathways often show signal convergence, where many ligands interact with fewer receptors, which then signal through a single second messenger complex. Here we investigate this question in the context of bone morphogenetic protein (BMP) signaling and its role directing dorsal spinal cord development, focusing on two receptor-regulated (R) Smads, Smad1 and Smad5. Multiple models have been proposed for their mode of action from acting redundantly through combined signal strength, to having distinct activities that drive different fate outcomes. We sought to distinguish between these models by generating CRISPR-edited Smad1 and Smad5 null mouse embryonic stem cell (ESC) lines to dissect the cell fate of activities of individual R-Smads, with a resolution not possible in vivo. Using a directed differentiation protocol for dorsal interneurons (dI), together with bioinformatic analyses, we have defined the roles of the R-Smads at key decision points along the dI specification timeline. Together, these findings support a model in which Smad1 and Smad5 play largely distinct roles in dorsal spinal cord development. While both R-Smads can activate canonical BMP-responsive transcriptional targets, they asymmetrically contribute to cell fate specification. Smad1 plays a restricted role, while Smad5 has a dominant role, regulating dorsal progenitor transcriptional dynamics and reiteratively directing the dorsal-most dI fates.

The discovery of neuromesodermal progenitors (NMPs) -- a bipotent progenitor population in the tailbud that gives rise to traditionally ectodermal and mesodermal tissues -- has disrupted the classical view that progenitors of the three distinct germ layers are exclusively segregated during gastrulation. However, until now the notion of lineage restriction of the endoderm to traditional gastrointestinal and respiratory tissues has largely remained intact. Here, we describe our discovery of a unique subpopulation in the chick endoderm that initially lines the ventral surface of the posterior organizer (Hensens node), but at the trunk-to-tail developmental switch, undergoes an FGF-dependent epithelial-to-mesenchymal transition, invading the tailbud and subsequently differentiating into a remarkably broad range of cell types including somites, notochord, and neural tube. Strikingly, ablation of this endodermal cell population results in a severe ([~]50%) reduction in axis elongation rate. Through single cell RNA sequencing and in situ hybridization chain reaction, we conclude that these cells lose their endodermal identity upon ingression, giving rise to NMPs that are biased toward mesodermal fates. Lineage tracing reveals that the node endoderm harbors a mixed multipotent population of progenitor cells capable of generating progeny that span endoderm and mesoderm or endoderm and ectoderm. These findings illustrate a previously unappreciated endodermal source of NMPs, and further demonstrates the breakdown of traditional lineage restriction of germ layers in the posterior embryo.

Interoceptive paraneurons are neuron-like cells located within internal epithelial cell surfaces that sense internal stimuli to evoke specific behavioral or physiological responses. The elucidation of terminal differentiation programs of paraneurons is expected to provide insights into how epithelial cells acquire neuron-like feature during development and possibly also over evolutionary time. We define here transcriptional programs that control the terminal differentiation of an interoceptive paraneuron class in the nematode C. elegans, called uv1. The uv1 cells sense mechanosensory inputs in the uterus and signal via the HSN neurons to modulate egg-laying behavior. We show that like in canonical neurons, the neuron-like secretory features of uv1 are controlled by a combination of CUT homeobox genes, while the combinatorial terminal gene battery that defines the unique functional features of uv1 is jointly controlled by a combination of at least three transcription factors, a LIM homeodomain (LIN-11), a SoxD (EGL-13) and a Pax family (EGL-38) protein. These factors act in a terminal selector-type manner to jointly co-regulate the many distinct uv1-paraneuron specific molecular features, such as sensory receptors, neuromodulatory receptors and neuropeptides, as well as uv1s tyraminergic identity. Our findings demonstrate notable similarities in the dichotomous architecture of gene regulatory programs of neurons and paraneurons.

In humans, retinal-neuron death, optic-nerve injuries, and associated neurodegenerative diseases, such as glaucoma and age-related macular degeneration, often lead to permanent vision loss. While the capacity for regeneration is low in the human nervous system, including the retina, some non-mammalian vertebrate species, including zebrafish, are capable of endogenous neuronal regeneration after injury. Unlike mammals, zebrafish do not form a scar that inhibits axonal and neuronal regeneration after injury. Rather, they harbor neural progenitor and stem-cell populations allowing regeneration of entire parts of the nervous system and restoration of tissue integrity and function. In the zebrafish retina, cycling neural progenitor cells of the ciliary marginal zone and quiescent resident neural stem cells (the latter of which are also called Muller glial cells) participate in neuronal regeneration following different types of injury. In this study, we report the identification of a novel, additional cellular source participating in neuronal regeneration of neurons in the zebrafish retina after genetic ablation of retinal ganglion cells. Before injury, these progenitor cells express molecular markers of neural-crest-cell and/or fibroblast identity, such as sox10, pdgfrb, and eya2, while after neuronal ablation they also express proneural factors including the ascl1a and olig2 genes. Combining genetic ablation of neurons with photoconversion or Cre/Lox-dependent genetic lineage tracing of sox10-expressing cells, we demonstrated that these cells can differentiate into post-mitotic retinal neurons in the ganglion cell layer (GCL) in the absence of cell proliferation. We also showed, surprisingly, that this progenitor population locally produces insulin mRNA, and that insulin signaling is involved in the accumulation of mesenchymal-derived neural progenitors in the GCL and in their subsequent transdifferentiation into RGCs. This work reveals an unexpected and novel cellular mechanism of transdifferentiation, dependent on a neural-crest-derived mesenchymal cell population, participating in neuronal regeneration in the zebrafish retina. The discovery of this plastic cell population could potentially lead to new strategies to promote the formation of new neurons in the mammalian retina.

Zebrafish possess three distinct sources of retinal progenitors that produce identical photoreceptor subtypes throughout life. All photoreceptor progenitors simultaneously express multiple transcription factors specifying different identities, requiring mechanisms to repress alternative fates. Disruption of the tbx2 paralogues, tbx2a or tbx2b, resulted in a cell-fate switch of sws1 cones into rods. Here, we demonstrate that tbx2b was necessary for sws1 cone differentiation during embryogenesis and outgrowth at the retinal margin, but tbx2a was necessary during photoreceptor regeneration. Transgenic overexpression of Tbx2b was not sufficient to drive the sws1 cone fate or sws1 opsin expression. Rather, Tbx2b repressed the synergistic activity of Nrl and Crx at the rhodopsin promoter. Targeting the transcription factor thr{beta}2 on wildtype and tbx2 mutant backgrounds revealed a hierarchy wherein early progenitors have the potential to be respecified from lws cones into sws1 cones or rods. But late progenitors are limited to either the sws1 cone or rod fate. These data support a model in which transcriptional repressors, like tbx2a and tbx2b, orchestrate progression through competency states.

Embryo polarization is critical for the first cell fate segregation. While mechanisms underlying its initiation have been described, the intrinsic signaling pathways that regulate this process remain poorly understood. Here, we show that mouse embryonic stem cells, when aggregated under defined conditions, recapitulate the first lineage segregation to generate trophectoderm (TE)-like cell populations and undergo self-organized morphogenesis into blastocyst-like structures. In the blastoid-forming medium, we identify CHIR99021 is essential for the generation of blastoids from both ESCs and totipotent-like cells. CHIR99021 promotes cell polarization and TE differentiation by activating the WNT/{beta}-catenin pathway and upregulating associated genes. Consistent with this, genetic ablation of {beta}-catenin abolished the cell polarization and disrupted blastoid formation from ESCs, a defect that was restored by {beta}-catenin overexpression. Moreover, {beta}-catenin depletion compromised cell polarization in natural embryos. Collectively, this study establishes the Wnt/{beta}-catenin as a critical regulator initiating polarization in vitro and in mouse early embryo development.

Robust execution of developmental cell fates requires precise spatiotemporal control of the fate-defining regulators. In Caenorhabditis elegans, temporal patterning of larval hypodermal fates is governed by the heterochronic gene regulatory network, in which microRNAs act as major post-transcriptional regulators by silencing temporal transcripts through 3'UTR-dependent repression. Here, we investigate lin-66, which encodes a nematode-specific cold shock domain protein previously implicated in heterochronic regulation and reported to associate with the miRISC effector protein AIN-1. Using targeted domain mutations and genetic analysis, we show that LIN-66 activity in the hypodermal cell-fate patterning requires its cold shock domain. Loss of lin-66 causes persistent expression of LIN-14 and LIN-28, two early temporal regulators in the hypodermal seam cells that are canonical microRNA targets. Analysis indicates that lin-66 function in seam-cell fate patterning does not depend on the native 3'UTR sequences of lin-14 or lin-28, distinguishing its activity from canonical microRNA repression.. Consistent However, consistent with the a broad functional overlap between LIN-66 function and microRNA-mediated regulation, hypodermal lin-66 loss-of-function phenotypes are strongly enhanced by mutations in alg-1 and ain-1/2, which encode components of the microRNA-induced silencing complex. Moreover, loss of lin-66 enhances phenotypes in mutants sensitized for microRNA activity outside the hypodermis. Together, these findings identify LIN-66 as a cold shock domain-dependent post-transcriptional regulator that safeguards developmental timing by limiting persistence of early fate regulators through mechanisms that intersect with, but are partly separable from, canonical 3'UTR-mediated microRNA repression.

Competence is the capacity of a cell or tissue to respond to a specific inducing signal from a neighbouring tissue, by changing its fate in a specific direction. Neural induction is the process by which the epiblast of the early embryo responds to signals from the organizer (the tip of the primitive streak in amniotes) by forming a neural plate. Here we study why three regions of the early chick embryo lack competence to respond to neural induction by a grafted organizer: the outer anterior area opaca and the posterior area opaca at primitive streak stages (HH3+-4-), and the inner anterior area opaca at head process stage (HH5), in comparison with the competent anterior inner area opaca at HH3+-4-.Molecular analysis of these tissues, and their temporal dynamics following exposure to the organizer, reveals several differences. Among them, increased BMP and decreased ERK signalling characterise the non-competent regions. Inhibition of BMP can restore competence to HH5 epiblast; a combination of BMP-inhibition with ERK-stimulation by FGF8 can confer competence to the outer area opaca, whereas none of these can endow posterior epiblast with competence for neural induction. We conclude that spatiotemporal competence of epiblast for neural induction is regulated by several mechanisms, including extracellular signals.

In canonical developmental patterning, the embryo is exposed to gradients of signaling activators that elicit different cellular responses depending on the activators concentration. Recent optogenetic studies of terminal ERK signaling downstream of Torso receptor tyrosine kinase in the early Drosophila embryo reveal that even a brief, 5-minute ERK stimulus is sufficient to rescue the development of larval "tail" structures. Here, we reveal components of the molecular network that defines this sensitive developmental fate response. We find that low ERK doses produce sustained Abdominal-B (Abd-B) expression comparable to that of wild-type embryos. Abd-B expression is adjacent to, but non-overlapping with, two other transcriptional repressors: the ERK effector Tailless (Tll) and the gap gene Giant (Gt). Analysis of gene expression patterns in response to optogenetic perturbations suggests that the Tll-dependent repression of gt constitutes the sensitive ERK-responsive step: even low tll expression leads to potent repression of gt in nearby regions, with Abd-B expression arising in a stripe between the tll and gt domains. Our work suggests that the spectrum of phenotypes produced through optogenetic manipulation can be used to define how robust patterning can arise from low doses of inductive signals. HighlightsO_LIA very low dose of receptor tyrosine kinase signaling in the early embryo induces tail formation many hours later. C_LIO_LITransient ERK activity results in stable Abd-B expression, beginning as a stripe in nuclear cycle 14. C_LIO_LIOptogenetic ERK inputs induce a spectrum of ectopic phenotypes that reveal mutually exclusive expression of Tailless, Giant, and Abd-B in the posterior. C_LIO_LITailless-dependent repression of giant is the sensitive ERK-responsive step. C_LI

The mature kidney contains approximately one million nephrons, and defects arising during nephron development can result in lifelong renal impairment, often culminating in kidney failure and transplantation. Nephric tubule formation requires coordinated epithelial processes, including polarity, adhesion, signaling, and vesicle transport; however, how these processes are integrated during kidney morphogenesis remains unclear. Dynamin binding protein (Dnmbp) is a multi-domain scaffolding protein expressed in human kidneys that is involved in several cellular processes. Using the Xenopus embryonic kidney, we previously demonstrated that Dnmbp is essential for nephrogenesis, yet the mechanisms by which it influences nephron development remain undefined. Here, we identify Dnmbp as a novel interacting partner of the Wnt/planar cell polarity effector Daam1. The interaction between Daam1 and Dnmbp was independently identified in two yeast two-hybrid screens, biochemically verified, and supported by structural modeling predictions of a Daam1-Dnmbp complex. In developing Xenopus laevis kidneys, Dnmbp localized to punctate structures associated with E-cadherin-rich cell-cell contacts. Dnmbp depletion significantly reduced junctional E-cadherin localization in both epithelializing and mature nephric tubules without affecting total E-cadherin levels, indicating a role in E-cadherin recruitment or stabilization at adherens junctions. Furthermore, expression of human DNMBP rescued the junctional defects, confirming the specificity of the loss-of-function phenotype. Together, these findings identify Dnmbp as an essential regulator of kidney development and support a model in which Dnmbp provides a mechanistic link between Wnt/PCP signaling, Cdc42 activation, and adherens junction formation during nephrogenesis.

HighlightsO_LILamin-A and lamin-B1 are essential for midgestational embryogenesis. C_LIO_LILamin-A/B1 are required for proper yolk sac endoderm (YSE) gene regulation. C_LIO_LILamin-A/B1 maintain LADs organization and chromatin interactions in YSE. C_LIO_LILamin-A/B1 and YSE transcription factors support proper YSE gene expression. C_LI Lamins are intermediate filament proteins functioning as ubiquitous structural components of the nuclear lamina that interact with and organize the Lamina-Associated chromatin Domains (LADs). LADs remodel during development and lamins maintain LADs and gene expression profile specific to a given cell type. How ubiquitous lamins achieve cell-type-specific functions during development remains unknown. We show lamin-A and -B1 are required for mouse midgestational embryogenesis and maintain LADs, 3D chromatin interactions, and gene expression in the yolk sac endoderm (YSE). Both lamin-regulated genes and remodeled LADs in YSE cells contain binding motifs of YSE-relevant transcription factors. By analyzing changes in chromatin interactions upon lamin-A and -B1 knockout, we reveal that chromatin neighborhoods maintained by these lamins can influence gene expression orchestrated by YSE-relevant transcription factors. Our findings explain how the ubiquitously expressed lamins can collaborate with lineage-relevant transcription factors to maintain LADs and gene expression programs in specific cell types.

How regenerative capacity originates during development remains poorly understood, even in vertebrates with exceptional adult regenerative ability. Using the axolotl, we identify a defined embryonic window between stages 30 and 34 during which the tail region transitions from a regeneration-incompetent to a regeneration-competent state. Amputations across staged embryos reveal that earlier embryos entirely fail to regenerate, whereas later embryos regenerate functional tails. Notably, tail stumps from nonregenerating embryos can recover the ability to regenerate when reamputated at later stages, demonstrating that early regenerative failure does not permanently impair regenerative capacity. This differs from the transient refractory period described in Xenopus, where regenerative competence is lost and reacquired around the end of tail outgrowth, and indicates that staged acquisition of regenerative competence is a broadly shared but mechanistically distinct feature of amphibian development. To determine whether this transition reflects changes in progenitor composition, we analysed the single-cell transcriptional landscapes of axolotl tail buds across this window. Tail bud progenitors, including neuromesodermal progenitors, persist through the transition, indicating that the onset of regenerative competence is unlikely to be explained by the loss of embryonic progenitors. Finally, using Tbxt (Brachyury) crispant axolotls with severe axial defects, we show that tail regeneration occurs effectively despite earlier abnormal embryonic tail development, with functional uncoupling of the mechanisms of tail development and regeneration. This framework provides new opportunities for identifying the drivers of regenerative competence and understand why this capacity is lost in other vertebrate species.

Directed cell migration is a vital process that depends on the combined activities of attractive and repulsive cues. As it is essential for normal development, the precise identity of guidance signals and the underlying molecular and cellular mechanisms is being rigorously investigated. In a Drosophila embryo, PGC migration is orchestrated by non-cell autonomous repulsive and attractive cues, controlled by Wunen(s) - Wunen and Wunen2 and, HMGCoA-reductase (Hmgcr), respectively. Hedgehog (Hh), a PGC attractant, is potentiated by Hmgcr. We demonstrate that Wunen(s) employ both nonautonomous and autonomous modes to inhibit Hh signaling. Consistently, in embryos maternally compromised for wunen, mesodermal cells and PGCs accumulate excess Hh, leading to precocious clumping of the PGCs. This behaviour is reminiscent of PGC-specific loss of patched (ptc) - the Hh receptor and an antagonist of Smoothened (Smo), a G protein-coupled receptor (GPCR), involved in Hh signal transduction. Consistently, Wunen(s) inhibit membrane localization of Smo. Conversely, simultaneous overexpression of wunen mitigates PGC scattering induced by ectopic hmgcr expression. Finally, unbiased lipidomics of embryonic extracts after maternal knockdown of wunen confirms disruptions in lipid metabolism. We discuss the mechanistic underpinnings of Wunen(s) involvement in repressing Hh signalling to engineer PGC migration.

Epithelial tubes are an essential component of many organ systems. The formation of their lumens depends on the close coordination of epithelial polarity, the apical actin cytoskeleton, and apical junctions, yet the mechanisms that organize the apical cytoskeleton and junctions downstream of polarity remain poorly understood. Here, we identify the Ste20 family kinase GCK-4, the single C. elegans ortholog of the mammalian kinases LOK and SLK, as a critical regulator of intestinal lumen formation. GCK-4 localizes to the apical membrane during early lumen formation and to microvillar tips as these structures develop. Its loss results in a lethal cystic lumen phenotype, without disrupting the establishment of epithelial polarity, and in occasional failure to maintain attachment between the pharynx and intestine. Lumens in gck-4 mutant animals show irregular junction patterning, severely impaired apical accumulation of both actin and the membrane-actin linker Ezrin/Radixin/Moesin protein ERM-1, and microvilli atrophy. In Drosophila and mammalian cells, the GCK-4 orthologs are thought to organize the apical actin network largely through phosphorylation of ERM proteins. In contrast, ERM-1 phosphorylation is only partially reduced in gck-4 mutants and is not abolished, indicating that GCK-4 acts through additional targets and is not the principal ERM-1 kinase in the intestine. Together, these findings identify GCK-4 as a key regulator of apical actin and junction organization during lumen formation, and demonstrate that Ste20 kinases can control apical cytoskeletal architecture at least partially independently of ERM phosphorylation.

The development of C. elegans gonad involves the coordinated action of diverse biochemical factors and physical forces. The precise roles and interconnections of these diverse components remain poorly understood. TOCA-2, the C. elegans ortholog of mammalian TOCA-1 (Transducer of CDC42 Dependent Actin Assembly) is an F-BAR domain protein known to play important roles in oocyte maturation and embryogenesis through membrane remodelling and regulation of actin cytoskeleton. Here we demonstrate that TOCA-2 is actively involved in maintaining the structural integrity and function of the C. elegans gonad. toca-2(null) animals exhibit pronounced architectural defects with particularly strong perturbations on the dorsal side of the gonad. Phalloidin staining and cytoplasmic particle velocity analysis revealed that the actomyosin corset surrounding the common rachis in the syncytial germline is severely disorganized in the mutants. This disorganization leads to significant disruptions in cytoplasmic flow across different regions of the syncytium. Together, our findings quantitatively highlight mechanisms underlying gonad morphogenesis and maintenance, establishing TOCA-2 as a key regulator of these processes. This work also provides a framework for positioning TOCA-2 within broader biochemical pathways governing organogenesis and other developmental processes dependent on actomyosin dynamics in C. elegans.

The enteric nervous system (ENS) is the main branch of the peripheral nervous system that innervates the gastrointestinal tract controlling vital functions. It arises during embryogenesis via migration and differentiation of neural crest-derived ENS progenitors. Perturbation of these processes, caused by mutations in key signalling pathway components and transcription factors, prevents progenitor colonisation of the distal gut causing aganglionic phenotypes and enteric neuropathies such as Hirschsprung (HSCR) disease. While animal models implicate Notch signalling in ENS specification, its role in human ENS progenitor cell fate decisions remains unclear. Here, we employ a human pluripotent stem cell-based model to show that Notch signalling regulates the tempo of ENS progenitor differentiation. Quantitative modelling of our in vitro data supports a branching lineage model marked by an early pro-neurogenic bias; Notch signalling attenuation accelerates differentiation coincident with a shift toward increased gliogenesis. Furthermore, we establish that Notch signalling influences human ENS progenitor migration. Together, these findings provide mechanistic insights into how Notch signalling disruption may contribute to the pathogenesis of human intestinal aganglionosis. SUMMARY STATEMENTIn vitro generation of human enteric nervous system (ENS) cells and quantitative modelling reveal that Notch signalling regulates ENS progenitor differentiation rates and migration.

Uterine gland development and function is essential for reproduction and womens health, yet the epithelial cell states and signaling interactions that govern gland fate specification are not well understood. Here, integration of single cell and spatial transcriptomics with organoid culture, lineage tracing, and genetic and hormonal perturbation models were used to define mechanisms regulating postnatal uterine epithelial differentiation. A developmentally restricted epithelial plasticity state was identified that precedes luminal and glandular cell lineage segregation and is accompanied by dynamic reorganization of stromal-epithelial communication during uterine differentiation. Pseudotime analysis revealed progressive acquisition of gland-associated programs, including forkhead box A2 (Foxa2), retinoic acid metabolic genes, and epithelial estrogen receptor alpha (Esr1) expression. Functional studies revealed that ESR1 acquisition and retinoic acid signaling suppress the multilayered organoid phenotype associated with epithelial plasticity, thereby promoting epithelial specification and lineage commitment. Moreover, neonatal hormonal perturbation of adenogenesis and conditional deletion of Foxa2 abolished this organoid phenotype. Together, these findings demonstrate that ESR1 acquisition, retinoic acid signaling and FOXA2-dependent glandular differentiation each restrict a transient epithelial plasticity state, coupling the loss of developmental plasticity to the emergence of the glandular lineage.

Germ granules are condensates in germ plasm, a specialized cytoplasm that segregates to the embryonic germline. In Drosophila, translation of nanos mRNA occurs at the surface of germ granules, suggesting that the granules promote translation. In C. elegans, however, germ (P) granules are not essential for Nanos expression. Using single-molecule imaging in C. elegans embryos, we map the distribution of translating and non-translating molecules of the Nanos homolog nos-2 and two other maternal mRNAs enriched in P granules. In early germline blastomeres, these mRNAs are not translated and distribute between the cytoplasm and P granules. At translation onset, mRNA molecules in the cytoplasm are translated, while most mRNA molecules in the P granules remain non-translating. nos-2 translation requires a rise in the concentration of the RNA-binding protein POS-1, which occurs independently of P granules. Consistent with low translation inside the granules, P granules are depleted of ribosomes and 43S pre-initiation complexes. Our observations suggest that germ granules promote Nanos protein expression by concentrating Nanos mRNA in germline precursors, but do not directly promote translation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/735846v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1875b06org.highwire.dtl.DTLVardef@16919f6org.highwire.dtl.DTLVardef@1278c27org.highwire.dtl.DTLVardef@1628645_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisGerm granules are condensates proposed to regulate the translation of mRNAs like Nanos that code for germ cell fate determinants. Using single-molecule imaging in C. elegans embryos, this study shows that P granule scaffolds concentrate mRNAs in germline precursors, but do not control the activity of translational regulators. - P granules concentrate mRNAs but are depleted of ribosomes and 43S pre-initiation complexes - Translation occurs mainly in the cytoplasm where ribosomes are most abundant - nanos translation onset is timed by a rise in POS-1, which counteracts the repressor SPN-4; both enrich in P granules but act independently.

Human pluripotent stem cells (hPSCs) provide a powerful platform for modeling early human embryonic development. Here, we investigate the mechanisms underlying mesodermal heterogeneity using a minimal directed differentiation system that simultaneously generates paraxial (PXM), intermediate (IM) and lateral plate mesoderm (LPM) populations. Single-cell RNA sequencing across defined time points during hPSC differentiation revealed a temporal sequence of lineage specification with LPM emerging first, followed by PXM and IM differentiation. Ligand-receptor and differential gene expression analyses identified BMP4 as a key regulator enriched in LPM-associated clusters versus mesoderm progenitors (MPs) that hold PXM and IM precursors. Whereas LPM cells cluster with an early BMP4 signal, IM clusters are associated with later BMP4. Moreover, these early and late BMP4 signals regulate this lineage specification potentially through distinct downstream pathways. Leveraging this insight, we established a stepwise protocol combining early BMP inhibition with subsequent BMP4 supplementation, suppressing initial LPM fate to efficiently induce IM from a mixed MP population. Longer culture of these selective IM progenitors promotes more mature nephrogenesis. Moreover, we demonstrate that during early differentiation high levels of BMP4 can still redirect MPs to more lateroventral fates, illustrating a degree of plasticity within the mesoderm lineage. Together, our results define a temporal framework for BMP4 signaling in mesoderm fate determination and provide a strategy for selective mesoderm differentiation from hPSCs. HIGHLIGHTSO_LIDevelopment of a minimal 2D differentiation platform allows for heterogenous mesoderm formation. C_LIO_LITemporal BMP4 signaling differentially directs mesoderm fates, with early exposure favoring LPM and late exposure promoting IM identity. C_LIO_LILPM cells arise first while later mesoderm progenitors hold both IM and PXM-fated cells. C_LIO_LISequential BMP modulation promotes IM and enhances nephrogenesis. C_LI