Development

During mouse embryogenesis, three sequential waves of cell polarization occur in the 8-cell-morula trophectoderm (TE), E3.5-E4.5 primitive endoderm (PrE), and E4.5-E5.5 epiblast (Epi), driving cell fate determination and morphogenic events that lay the foundation for embryonic development. However, it is not clear whether a general regulator that functions in all three waves of polarization exist. Here, we demonstrate that transcriptional factor THAP11 is such a general regulator. Using a novel mouse model combining genetic knockout and an inducible degron system, we demonstrate that stage-specific acute THAP11 depletion causes the failure of the three waves cell polarization, resulting in defective blastocyst formation, PrE maturation, and Epi lumenogenesis, respectively. Mechanistically, THAP11 does not directly regulate pluripotency genes but drive polarization through both stage-specific and conserved chromatin-bindings that commonly activate polarity programs, including Golgi vesicle transport. THAP11 activates polarity genes by promoting chromatin accessibility at their promoters. THAP11, together with stage-specific regulators, sequentially paces the timing of cell polarization. Collectively, our study establishes THAP11 as a general regulator of embryonic morphogenesis.

How progenitors can faithfully self-assemble to build new organs in adulthood is a central unsolved problem in regeneration biology. The planarian eye is a model for uncovering principles of de novo organogenesis during regeneration. In this process, pluripotent adult stem cells specify into ovo+ migratory progenitors which target to the eye field due to Wnt and FGFRL signals produced from bodywall muscle, then differentiate into photoreceptor neurons (PRNs) and pigmented optic cup (OC) cells, followed by organization of these two cell types in an opposing pattern that completes eye organ maturation. We used scRNA-seq to characterize eye progenitor transcriptomes during regeneration, followed by FISH validation, and RNAi screening to identify regulators of eye regeneration. This uncovered a function for the discoidin domain receptor ddr-1 in preventing the premature terminal differentiation of migratory eye progenitors. DDRs encode collagen receptors, and bodywall muscle is the major source of collagen in planarians, suggesting eye progenitors likely interface with muscle ECM for their proper deployment in regeneration. We further identified a novel eye morphogenesis phenotype from knockdown of tmem132-1, causing failed heterotypic adhesion between eye cell types. TMEM132 factors are conserved cell-surface receptors that mediate adhesion and regulate migration, and human TMEM132 mutations are associated with several neurobehavioral diseases. Therefore, planarian tmem132-1 likely directly regulates the capture and assembly of migratory progenitors into mature eyes during regeneration. Together, these results define new steps in the eye regeneration pathway and suggest ancient roles for DDR and TMEM132 in progenitor migration and coordinated organ assembly.

Regenerating tissues like the axolotl (Ambystoma mexicanum) limb draw from many cell types, but isolating specific cell populations remains a bottleneck. Standard approaches rely on enzymatic dissociation and fluorescence-activated cell sorting (FACS), which are slow, costly, and require specialized instrumentation. We adapted INTACT (Isolation of Nuclei Tagged in specific Cell Types) to the axolotl, generating a transgenic line (INTACT-olotl) expressing Sun1-sfGFP in limb connective tissue, the population that carries limb positional information, dedifferentiates following injury, and forms the majority of the regenerative blastema. Combining an optimized nuclei isolation protocol with anti-GFP nanobody capture enriched labeled nuclei from ~34.6% of input to 80-89% purity. Relative to dissociation and FACS, this recovers roughly 30-fold more target nuclei in less than half the time and without a cytometer. Bead-bound nuclei are directly applied to combinatorial-barcoding snRNA-seq, eliminating fluorescence-activated nuclei sorting (FANS). Profiling INTACT+ connective tissue during TGF{beta} inhibition revealed a loss of extracellular matrix remodeling with upregulated pro-fibrotic and inflammatory gene programs, resembling an unresolved wound environment. These changes reverse and regeneration resumes upon drug washout, indicating that functional TGF{beta} signaling is required for the transition from wound resolution to blastema formation.

Pericyte specification and expansion must be coordinated with vascular growth to establish a functional cerebrovascular network, yet how endothelial signals control these distinct developmental transitions remains incompletely understood. Here, using genetic manipulation, single-cell transcriptomic analysis, and longitudinal in vivo imaging in zebrafish, we identify ligand-specific endothelial Notch signaling as a mechanism coordinating brain pericyte development. Endothelial Jag2b is required for efficient pericyte specification from pericerebral progenitor pools and subsequently promotes Notch-dependent pericyte proliferation and population expansion. By contrast, endothelial Dll4 restrains pericyte proliferation and functionally opposes Jag2b-dependent expansion, demonstrating that distinct endothelial Notch ligands generate different developmental outputs. Endothelial Notch activation increases jag2b expression, and genetic interaction analyses establish Jag2b as an important mediator of Notch-dependent pericyte expansion. Moreover, genetic disruption of piezo1 reduces brain pericyte abundance, whereas endothelial jag2b expression rescues this phenotype, placing Jag2b downstream of Piezo1-dependent hemodynamic signaling. Together, these findings reveal a ligand-specific endothelial Notch program that integrates hemodynamic cues with endothelial-pericyte communication to coordinate the specification and expansion of brain pericytes during cerebrovascular development.

Hematopoietic stem cells (HSCs) arise from the hemogenic endothelium in the dorsal aorta through the endothelial-to-hematopoietic transition, a tightly regulated developmental process requiring coordinated signaling from the developing hematopoietic niche. Wnt and Notch signaling are essential for HSC specification, with Wnt functioning upstream of Notch; however, the molecular mechanisms by which these pathways are coordinated during HSC development remain poorly understood. Here, we identify Disheveled 2 (Dvl2), a key intracellular Wnt scaffold protein, as an essential regulator of HSC identity specification in zebrafish. Loss of dvl2 impaired HSC specification and reduced Notch signaling in the dorsal aorta, despite normal canonical Wnt activity in the developing endothelium, suggesting that Dvl2 regulates HSC specification independently of endothelial beta-catenin signaling. Hemogenic endothelial-specific expression of the Notch intracellular domain rescued HSC defects in dvl2 morphants, demonstrating that Dvl2 functions upstream of Notch activation in the hemogenic endothelium during HSC specification. In addition, loss of dvl2 reduced primitive macrophages and neutrophil populations. Restoration of primitive myeloid cells through gata1a knockdown rescued both endothelial Notch signaling and HSC defects, demonstrating that Dvl2 is also required for the supportive immune cells that induce HSC identity. Together, these findings reveal that Dvl2 coordinates hemogenic endothelial and immune niche cues to control HSC identity specification.

The adult hematopoietic system is established during embryogenesis through the generation of hematopoietic stem and progenitor cells (HSPCs) within specialized transient niches, yet the signaling mechanisms that coordinate this process remain poorly understood. Here, we identify a developmental role for kainate receptors (KARs), glutamate-gated ion channels classically associated with excitatory neurotransmission, in coordinating primitive hematopoiesis and hemogenic niche formation in zebrafish. Loss of the KAR subunits encoded by grik1b or grik5 depletes primitive blood cells and disrupts hemogenic niche organization. These defects are accompanied by endothelial oxidative stress and impaired Notch signaling, which together reduce HSPC emergence and limit the clonal diversity of the adult hematopoietic stem cell pool. Shifting primitive hematopoietic output away from erythropoiesis and toward myelopoiesis restored macrophage niche colonization, endothelial Notch activity, and HSPC formation in KAR-deficient embryos. Moreover, hemogenic cell-specific Notch reactivation rescued HSPC production, demonstrating that KAR-dependent primitive hematopoiesis regulates this process through endothelial Notch signaling. Lastly, conserved KAR expression in endothelial and hematopoietic populations across zebrafish, mouse, and human suggests that these receptors may regulate blood development across multiple cellular components. Together, these findings reveal that glutamate receptor signaling extends beyond its well-established functions in neuronal transmission to coordinate the developmental processes that establish the blood system.

During mammalian gastrulation, progenitors of craniofacial and cardiopharyngeal muscles arise from anterior mesoderm adjacent to foregut endoderm, and they are controlled by gene regulatory networks and signalling pathways distinct from those of trunk mesoderm. While in vitro models capture aspects of posterior and heart development, a spatially organised interface between foregut-like endoderm and cranial-mesoderm-like tissue has not been described. Here we introduce a dual-aggregate gastruloid model, formed by fusing two mouse embryonic stem cell aggregates patterned under distinct Nodal, FGF, and Wnt signalling. These structures reproducibly establish an anterior-posterior axis and position a Foxa2/Sox17/Cdh1 foregut-like epithelium proximal to Pax1/Pax9 pharyngeal-like endoderm and Tbx1 cranial-mesoderm-like cells, resembling the E8.5 pharyngeal region. Reciprocal chimeras and Foxa2 loss-of-function indicate that Foxa2 endoderm is required for Tbx1 expression in adjacent anterior mesoderm. Pharmacological perturbation of Hedgehog disrupts Foxa2 and Tbx1 expression. The model provides a tractable platform for dissecting endoderm-mesoderm signalling and congenital craniofacial disease mechanisms.

Body axis specification is essential for morphogenesis, yet how axial patterning is dynamically coordinated remains unclear. In Hydra, Wnt/{beta}-catenin signaling is central to axial patterning, but its spatiotemporal dynamics are not well understood. Since nuclear {beta}-catenin mediates Wnt-dependent transcription, its localization provides a relevant readout for pathway dynamics. Here, we track {beta}-catenin abundance and nuclear localization in vivo during regeneration from bisected Hydra, tissue rings, and small tissue fragments. Following bisection, nuclear {beta}-catenin is detected exclusively at the regenerating oral end, where a broad signal appears and then declines before focusing at the future head. Tissue rings, lacking both head and foot, exhibit similar oral-aboral asymmetry. In regenerating fragments, the initial response often spans nearly the entire tissue, including the future foot region, followed by an additional peak before stabilizing at the future head. Overall, our results show that the spatial restriction of nuclear {beta}-catenin correlates with the divergence of regenerative dynamics toward head or foot formation, while the trajectories leading to these outcomes vary across different initial tissue configurations.

Mechanosensory hair cells in the cochlea of the inner ear are essential for sound detection. Previous studies have shown that loss of the Notch ligand Jagged1 (JAG1) disrupts maintenance of prosensory progenitors, from which hair cells and their surrounding supporting cells derive. However, the molecular and cellular mechanisms by which JAG1 regulates prosensory progenitor maintenance are poorly understood, and the Notch receptors that mediate this function in the developing cochlea remain unknown. To address this, we generated Jag1 conditional knockout mice and characterized their prosensory deficits using transcriptomic and phenotypic analyses. We show that JAG1 loss abolishes prosensory expression of CDKN1B (p27Kip1) and reduces expression of essential neurotrophic factors, thereby disrupting the stereotyped pattern of cell-cycle exit and innervation within the prosensory domain. Furthermore, conditional knockout of Notch1 and Notch2 (Notch1/2) receptors in supporting cells phenocopied the Jag1 deletion phenotype, showing both a severe reduction in outer hair cell formation and a disrupted pattern of cell-cycle exit and innervation, thereby confirming their essential roles in mediating the prosensory functions of JAG1.

During oocyte growth, the marked increase in cell size is accompanied by robust RNA polymerase II (Pol II) transcription, generating transcripts that are either translated into proteins or stored as part of the maternal transcriptome. While the RNA decay machinery is known to critically regulate the maternal transcriptome during oocyte maturation, its contribution during the growth phase remains poorly understood. We previously identified an oocyte-specific transcription machinery dependent on the TATA-binding protein paralog TBPL2, which is required for oocyte growth. To investigate how the absence of TBPL2-mediated transcription affects the cellular state of the oocyte, we characterized the phenotypic consequences of the Tbpl2-/- mutation in growing oocytes. Our data reveal that both nuclear and cytoplasmic organizations are profoundly disrupted in the absence of TBPL2-mediated transcription. Given that cytoplasmic organization is closely linked to RNA storage capacity, we examined the expression and localization of proteins involved in mRNA regulation, stability and storage. We observed a marked impairment in the redistribution of these key factors in Tbpl2-/- oocytes. To assess the impact of TBPL2 on RNA storage, we analyzed poly(A) tail length and found a global increase in polyadenylation in mutant oocytes. Gene-specific poly(A) tail analyses revealed differential mRNA changes, with shortened tails for a downregulated transcript and elongated tails for an upregulated one, suggesting stabilization of the latter. Collectively, our findings indicate that TBPL2-mediated transcription is essential for maintaining proper cellular organization in growing oocytes, at least in part by ensuring the functional integrity of the RNA decay machinery.

How progenitor mitotic timing is coupled to the number of neurons in a nervous system remains poorly understood, in part because most model systems have large, variable progenitor pools that obscure this relationship. The non-vertebrate chordate Ciona robusta offers a tractable alternative: its larval nervous system arises from an invariant lineage, including four Bipolar Tail Neurons (BTNs) that are reproducibly generated from the caudal neural plate border. Here we investigate the developmental function of the single Ciona ortholog belonging to the leucine zipper tumor suppressor (Lzts) gene family, whose vertebrate members are implicated in cell-cycle regulation and tumor suppression. In Ciona, Lzts is expressed in several embryonic tissues, including differentiating BTNs. Tissue-specific CRISPR/Cas9 knockout of Lzts in the BTN lineage produced supernumerary BTNs, without expanding the embryonic Neurog+ domain, indicating that Lzts acts downstream of BTN specification to limit neuron number. Lzts1 promotes Cdk1 activity via stabilization of Cdc25C, co-expression of Ciona Cdc25C or a non-phosphorylatable Cdk1/2/3 mutant rescued normal BTN number in Lzts crispants, proving the existence of an ancestral Lzts-Cdc25-Cdk1 circuit controlling neural cell cycle. These findings establish Ciona Lzts as a regulator of mitotic tempo determining the BTN number and suggest that this cell-cycle regulatory axis links proliferation to neurogenesis across chordates in vivo.

Many proteins required for embryogenesis are expressed in multiple tissues and across developmental time, often with tissue- and stage-specific roles. To resolve where and when proteins function, we developed a tissue-targeted auxin-inducible degradation toolkit for C. elegans embryogenesis. We generated a plasmid set compatible with single-copy insertion or high-copy extrachromosomal array generation that employs tissue-specific promoters (active in the germline, intestine, muscle, pharynx, neuroblasts, neurons, sensory neurons, epidermis, and pan soma) to drive an operon expressing an optimized auxin-dependent plant F-box factor TIR1 and, in most cases, a fluorescent histone. Using a ubiquitous nuclear envelope marker as a test substrate, we identified single-copy insertions and arrays that exhibit efficient auxin-dependent, tissue-specific target degradation. We also establish a workflow that ensures all functionally important isoforms of a target are tagged and that degradation reproduces the loss-of-function phenotype prior to tissue-specific analysis. Applying the toolkit to the broadly expressed Rho kinase LET-502, we find that degradation in the epidermis, but not in other embryonic tissues, disrupts elongation. Unexpectedly, temporal analysis revealed that LET-502 is required for late as well as early elongation.

Butterfly wing scales are intricately structured, cuticle-derived, extensions of single scale cells. Their highly ordered structures confer a range of functions, including producing structural color, which depends upon unknown molecular mechanisms controlling the secretion and ordering of an extracellular matrix (ECM). To find the genes required for patterning the ECM we compare scale nanostructure development between structurally colored blue scales and non-iridescent black scales on the forewings of the butterfly Heliconius sara using transmission electron microscopy, RNA-seq and proteomics. We first determined that structural differences between the two scale types are established from about half-way through pupal development and then investigated differences in relevant genes and proteins between scale types from this stage onwards. We show that knockouts of the genes coding for zona pellucida (ZP) domain proteins, miniature and trinity, and the cuticle protein gene, Cpr128 produce distinct effects on scale morphology and structure formation. In particular, some highly unusual scale morphologies are observed in miniature knockout individuals, suggesting a previously undescribed role of ZP domain proteins in structuring the ECM of scale cells and in mediating the mechanochemical interactions needed to produce the complex extracellular structures important for biological function in this system.

Human hair follicle engineering is hindered by the rapid loss of inductive competence in culture-expanded dermal papilla (DP) fibroblasts. Here we show that bone morphogenetic protein 6 (BMP6) maintains and restores DP-associated characteristics while promoting follicular epithelial differentiation by restricting keratinocyte (KC) proliferation and inducing hair follicle-associated marker expression. Human follicle transcriptomics further linked BMP activity to DP identity and follicular epithelial differentiation. Genetic mouse studies demonstrate that BMP6 in Sox2-positive DP cells contributes to normal anagen onset, progression, and maintenance. Gain- and loss-of-function studies in KC-DP organoids show that BMP6 promotes epithelial-mesenchymal organization and spatially patterned follicular structures. In vivo, topical BMP6 promotes anagen onset, whereas BMP6 overexpression restores the hair follicle-generating capacity of late-passage human DP fibroblasts. Together, these findings identify BMP6 as a compartment-dependent regulator that enhances human DP competence and promotes epithelial differentiation, supporting regenerative hair follicle formation and engineering with culture-expanded DP cells.

Gastrulation begins when the epiblast epithelium breaks symmetry to form the primitive streak, but current in vitro models neither fully capture this process nor separate its mechanical and chemical inputs. Here, we introduce LightBMP, an optogenetic platform that enables spatiotemporally programmable BMP4 expression in human pluripotent stem cells. Dose and duration control reveal a sustained low-BMP regime, inaccessible to recombinant ligand, that specifies mesoderm. Spatially programmed BMP4 sources in uniform cultures mimic the radial organization of 2D gastruloids, showing that mechan-ical context and edge sensing are not required for patterning. In micropatterned colo-nies, light-defined sources break symmetry to generate a stripe-shaped, primitive-streak-like domain where cells acquire multiple mesodermal fates and undergo directed migration. Together, these results show that spatial control of a single morphogen source is sufficient to drive symmetry breaking and primitive-streak-like patterning, independent of edge constraints.

During mammalian nervous system development, multipotent progenitor cells differentiate into hundreds of distinct neuronal subtypes. Neuronal subtype terminal differentiation is regulated by transcription factors that directly control the acquisition and maintenance of terminal identity. Using the mouse retina as a model system, we show that the transcription factor ISL1 is required for the terminal differentiation and maintenance of starburst amacrine cells (SACs), the sole cholinergic neuron subtype in the retina. We conditionally deleted Isl1 selectively from amacrine and horizontal cell precursors, allowing us to assess its cell-autonomous role in SAC development without disrupting its critical function in bipolar cell and retinal ganglion cell development. We find that ISL1 is not required for initial SAC specification but is required for their terminal differentiation. Isl1-deficient SACs retain a GABAergic amacrine cell identity but fail to adopt many SAC-specific features including expression of genes required for cholinergic transmission. In the absence of ISL1, SACs fail to migrate properly, with both OFF- and ON- SACs localizing to the inner nuclear layer. Deletion of Isl1 from post-migratory SACs at later stages of development revealed that it is also required for maintaining SAC identity and proper dendrite stratification. Loss of Isl1 from SACs ultimately caused abnormal development of the direction selective circuit. Together, these findings establish that ISL1 is required for the induction and maintenance of SAC-specific identity from a generic amacrine state.

Sexually dimorphic behaviors arise from underlying differences in neural circuit architecture between the sexes, yet the molecular mechanisms that establish these structural differences remain poorly understood. In Caenorhabditis elegans, the sex-shared interneuron AVG exhibits sexually dimorphic synaptic connectivity, offering a model for investigating the development of sexually dimorphic neuronal morphology. Here, we identified that AVG displays male-specific neurite branching and demonstrated that the conserved axon guidance cue UNC-6/Netrin and its receptor UNC-40/DCC are essential for this structural remodeling. Cell-specific manipulation of sexual identity revealed that male identity confers branching competence to AVG. Loss of UNC-6 or UNC-40 abolished branch formation, whereas loss of the alternate Netrin receptor UNC-5/Unc5 had no detectable effect. Cell-specific rescue experiments showed that UNC-40 functions in AVG through its UNC-6 binding domains. Furthermore, genetic ablation of the male-specific presynaptic neuron PCA, which expresses unc-6, diminished AVG branch length, establishing PCA as a candidate local source of UNC-6. Together, our findings support a model in which presynaptic UNC-6/Netrin cues from male-specific neurons promote dimorphic neurite branching in sex-shared neurons, illustrating how evolutionarily conserved signaling molecules are redeployed during sexual maturation to sculpt sex-specific neural circuits.

NANOG is a conserved regulator of epiblast identity and is essential for hypoblast development and embryo survival after the blastocyst stage in mice. However, its role during post-blastocyst development remains poorly understood in non-rodent mammals. Here, we investigated the function of NANOG during ovine embryogenesis using CRISPR/Cas9-mediated gene ablation. NANOG ablation did not affect blastocyst formation or the initial development of the epiblast. However, NANOG-deficient blastocysts contained significantly fewer SOX17+ hypoblast cells by day (D) 8, followed by reduced hypoblast migration during early post-hatching stages in D12 in vitro embryos. These defects were transient, as hypoblast development was comparable between NANOG-deficient and wild-type conceptuses during subsequent in vivo conceptus elongation from embryonic day (E) 11 onwards. In contrast, although extraembryonic membranes development and conceptus elongation proceeded normally, NANOG-deficient conceptuses lacked SOX2+ epiblast cells and failed to form an embryonic disc (ED) from E11 onwards. These findings demonstrate that NANOG is dispensable for hypoblast development but is required for epiblast maintenance and ED formation during conceptus elongation in sheep.

Hair cell (HC) loss in the auditory organ (cochlea) is irreversible and represents the leading cause of deafness in humans. The absence of HC regeneration is primarily due to the limited plasticity of cochlear supporting cells (SCs). Using a novel doxycycline-inducible TRIM71 transgenic mouse model, this study demonstrates that overexpression of the RNA-binding protein TRIM71 promotes spontaneous cochlear HC regeneration in neonatal mice. Experiments in cochlear organoids indicate that TRIM71 reprograms SCs into progenitor-like cells and that this reprogramming is further enhanced by coactivation of follistatin (FST). FST augments TRIM71-induced increases in chromatin accessibility at progenitor and HC-specific gene loci, facilitating their transcriptional activation in SCs. Notably, FST also enhances TRIM71-induced reactivation of Sall4, which, like Trim71, is highly expressed in early otic progenitors. Using cochlear organoids and explants, we show that the early otic transcription factor SALL4 is required for TRIM71-induced HC formation. By contrast, SALL4 overexpression inhibits HC formation by maintaining reprogrammed SCs in an undifferentiated, proliferative state. These findings establish TRIM71 as an activator of HC regeneration in vivo, provide mechanistic insights into TRIM71's role in SC reprogramming, and identify SALL4 as its essential effector.

During embryogenesis, the cortical and medullary compartments of the adrenal gland emerge from two distinct sources: intermediate mesoderm-derived steroidogenic progenitors that comprise the cortex, and migratory neural crest-derived catecholaminergic progenitors that give rise to the medulla. The distinct cellular origins and paracrine signaling environments that foster induction, differentiation, and/or migration of cortical and medullary layers present challenges to recapitulating their co-emergence and incorporation in a human pluripotent stem cell (hPSC) based system. To address this, we developed an approach for modeling the adreno-gonadal primordium (AGP) in compound organoids that are organized along a gradient of NR5A1 expression and which unexpectedly incorporate SOX10-expressing Schwann cell precursors that ultimately generate chromaffin cells. Deconstruction of paracrine signaling within AGP-like organoids (AGPLOs) identified a fibroblast growth factor 9 (FGF9)-driven mechanism that expands bridge cell-like chromaffin progenitors and correlates to paracrine signaling cues encountered by physiological correlates along their migration corridor to the cortex in vivo. Combination of NR5A1-expressing cells with chromaffin cell clusters generated corticomedullar-like assembloids (CMLAs) that generated steroids and catecholamines and were responsive to adrenocorticotropic hormone. This work establishes a platform for modeling the reciprocal signaling relationships that drive adrenal gland development and function in health and disease and provides an approach for generating hPSC-derived adrenal cells/tissues that can be applied therapeutically.