Developmental Cell

Placenta progenitor cells, also known as trophoblasts, initially specify at very low oxygen (O2) concentrations. Across their differentiation path in the uterine microenvironment, they encounter a wide range of O2 levels. Despite previous efforts, dissecting how these rapid and dynamic O2 levels are sensed by trophoblast stem cells and transduced via hypoxia inducible factors (HIFs) has been challenging and has led to conflicting conclusions. This is at least in part due to the lack of tractable and reliable methods to model human placental development. Here, by recapitulating the dynamic O2 levels of the uterine microenvironment, and by genetically ablating HIF1 in human trophoblast organoids, we found that O2 availability and HIF pathway independently control trophoblast lineage specification, maturation and function. Specifically, low O2 levels promote expansion of extravillous trophoblast (EVT) progenitors independently of HIF1, while HIF1 is necessary for EVT invasion regardless of O2 availability. Altogether, our results reveal a dual regulatory framework that disentangles the role of O2 from that of HIF1, offering a revised view of how O2 availability regulates early human placental development.

The regulation and coordination of developmental processes involves the secretion of morphogens and membrane carriers, including extracellular vesicles, which facilitate their transport over long distance. The long-range activity of the Hedgehog morphogen is conveyed by extracellular vesicles. However, the site and the molecular basis of their biogenesis remains unknown. By combining fluorescence and electron microscopy combined with genetics and cell biology approaches, we investigated the origin and the cellular mechanisms underlying extracellular vesicle biogenesis, and their contribution to Drosophila wing disc development, exploiting Hedgehog as a long-range morphogen. We show that microvilli of Drosophila wing disc epithelium are the site of generation of small extracellular vesicles that transport Hedgehog across the tissue. This process requires the Prominin-like protein, whose activity, together with interacting cytoskeleton components and lipids, is critical for maintaining microvilli integrity and function in secretion. Our results provide the first evidence that microvilli-derived extracellular vesicles contribute to Hedgehog long-range signaling activity highlighting their physiological significance in tissue development in vivo.

Nascent vascular networks adapt to the increasing metabolic demands of growing tissues by expanding via angiogenesis. As vascular networks expand, blood vessels remodel, progressively refining vascular connectivity to generate a more haemodynamically efficient network. This process is driven by interplay between endothelial cell (EC) signalling and blood flow. While much is known about angiogenesis, considerably less is understood of the mechanisms underlying vessel remodelling by blood flow. Here we employ the zebrafish sub-intestinal venous plexus (SIVP) to characterise the mechanisms underlying blood flow-dependent remodelling. Using live imaging to track ECs we show that blood flow controls SIVP remodelling by coordinating collective migration of ECs within the developing plexus. Blood flow opposes continuous ventral EC migration within the SIVP and is required for regression of angiogenic sprouts to support plexus growth. Sprout regression occurs by coordinated polarisation and migration of ECs from non-perfused leading sprouts, which migrate in opposition to blood flow and incorporate into the SIV. Sprout regression is compatible with low blood flow and is dependent upon vegfr3/flt4 function under these conditions. Collectively, these studies reveal how blood flow sculpts a developing vascular plexus by coordinating EC migration and balancing vascular remodelling via vegfr3/flt4.

During tissue regeneration, proliferation, dedifferentiation, and reprogramming are necessary to restore lost structures. However, it is not fully understood how metabolism intersects with these processes. Chicken embryos can regenerate their retina through retinal pigment epithelium (RPE) reprogramming when treated with fibroblast factor 2 (FGF2). Using transcriptome profiling, we uncovered extensive regulation of gene sets pertaining to proliferation, neurogenesis, and glycolysis throughout RPE-to-neural retina reprogramming. By manipulating cell media composition, we determined that glucose, glutamine, or pyruvate are sufficient to support RPE reprogramming identifying glycolysis as a requisite. Conversely, the induction of oxidative metabolism by activation of pyruvate dehydrogenase induces Epithelial-to-mesenchymal transition (EMT), while simultaneously blocking the activation of neural retina fate. We also identify that EMT is partially driven by an oxidative environment. Our findings provide evidence that metabolism controls RPE cell fate decisions and provide insights into the metabolic state of RPE cells, which are prone to fate changes in regeneration and pathologies, such as proliferative vitreoretinopathy.

The endoderm is the cell lineage which gives rise in the embryo to the organs of the respiratory and gastrointestinal system. Uniquely, endodermal tissue does not just derive from descendants of the embryo proper (the epiblast) but instead arises from their gradual incorporation into an extraembryonic substrate (the visceral endoderm). Given the configuration of the early embryo, such a paradigm requires epiblast endodermal progenitors to negotiate embryonic compartments with very diverse epithelial character, a developmental contingency reflected by the fact that key early endodermal markers such as Foxa2 and Sox17 have been consistently found to be embedded within gene programmes involved in epithelialisation. To explore the underlying cell biology of embryonic endoderm precursors, and to explore the relationship between endoderm development, epithelial identity, and extraembryonic mixing, we leveraged Gastruloids, in vitro models of early development. These self-organising three-dimensional aggregates of mouse embryonic stem cells do not possess an extraembryonic component, nor do they appear to display typical tissue architecture. Yet, they generate cells expressing endodermal markers. By tracking these cells throughout in vitro development, we highlight a persistent and uninterrupted pairing between epithelial and endodermal identity, with FoxA2+/Sox17+ endoderm progenitors never transitioning through mesenchymal intermediates and never leaving the epithelial compartment in which they arise. We also document the dramatic morphogenesis of these progenitors into a macroscopic epithelial primordium extending along the entire anterior-posterior axis of the Gastruloid. Finally, we find that this primordium correctly patterns into broad domains of gene expression, and matures cells with anterior foregut, midgut, and hindgut identities within 7 days of culture. We thus postulate that Gastruloids may serve as a potential source of endodermal types difficult to obtain through classical 2D differentiation protocols.

The lineage and developmental trajectory of a cell are key determinants of cellular identity. Yet, the functional relevance of deriving a specific cell type from ontologically distinct progenitors, remains an open question. In the case of the vascular system, blood and lymphatic vessels are composed of endothelial cells (ECs) that differentiate and diversify to cater the different physiological demands of each organ. While lymphatic vessels have been shown to originate from multiple cell sources, lymphatic ECs (LECs) themselves seem to have a unipotent cell fate. In this work we uncover a novel mechanism of blood vessel formation through transdifferentiation of LECs. Using advanced long-term reiterative imaging and lineage-tracing of ECs in zebrafish, from embryonic development through adulthood, we reveal a hitherto unknown process of LEC-to-BEC transdifferentiation, underlying vascularization of the anal fin (AF). Moreover, we demonstrate distinct functional implications for deriving AF vessels from either LECs or BECs, uncovering for the first time a clear link between cell ontogeny and functionality. Molecularly, we identify Sox17 as a negative regulator of lymphatic fate specification, whose specific expression in AF LECs suppresses its lymphatic cell fate. Finally, we show that akin to the developmental process, during adult AF regeneration the vasculature is re-derived from lymphatics, demonstrating that LECs in the mature fish retain both potency and plasticity for generating specialized blood vessels. Overall, our work highlights a novel mechanism of blood vessel formation through LEC trans-differentiation, and provides the first in vivo evidence for a link between cell ontogeny and functionality in ECs.

Hepatocytes have a unique multiaxial polarity with several apical and basal surfaces. The prevailing model for the emergence of this multipolarity and the coordination of lumen formation between adjacent hepatocytes is based on asymmetric cell division. Here, investigating polarity generation in liver cell progenitors, the hepatoblasts, during liver development in vivo and in vitro, we found that this model cannot explain the observed dynamics of apical lumen formation in the embryonic liver. Instead, we identified a new mechanism of multi-axial polarization: We found that polarization can be initiated in a cell-autonomous manner by re-positioning apical recycling endosomes (AREs) to the cell cortex via fibronectin sensing through Integrin V. Using live cell imaging we showed that this process repeats, leading to multiaxial polarity independently of cell division. We found that establishment of oriented trafficking leads to secretion of the metalloprotease MMP13, allowing neighboring hepatoblasts to synchronize their polarization by sensing extracellular matrix (ECM) distribution and enabling lumen opening. Finally, active remodeling of ECM in proximity of nascent apical surfaces closes a positive feedback loop of polarization, whereas disruption of this loop by either blocking MMP13 or downregulating Integrin V prevents formation of the bile canaliculi network. Integration of this feedback loop into a simple mathematical model reproduces the observed dynamics of bile canaliculi network formation during liver development quantitatively. Our combined findings thus suggest a new mechanism of polarization coupling to self-organization at the tissue scale.

Embryogenesis is commonly viewed through a tree model of cell differentiation, which fails to capture the spatiotemporal modulation of cell multipotency underlying morphogenesis. In this study we profile the multipotency landscape of the embryo, using in vivo single-cell clonal lineage tracing of mouse embryos traced from neurulation until mid-gestation, combined with a machine learning tool that categorizes individual clones into lineages based on shared transcriptional context. This revealed a previously unrecognized continuous, embryo-wide gradient of clonal fate biases, in which anatomical position and clonal composition are mutually predictive. Comparing clonal lineages revealed gene regulatory networks underlying the dynamic biasing of cells towards specific fates by spatial transcription factor programs. However, mosaic combinatorial perturbations targeting the Hedgehog pathway generated clones in which positional identity was mismatched with clonal composition, demonstrating that extrinsic signals can override the axial patterning system underlying clonal fate biases. Altogether, our work demonstrates an effective practical approach for dissecting mechanisms of lineage specification and has implications for stem cell engineering.

The creation of a microvilli-rich apical luminal domain is a key event in the development of epithelial tissues. De novo lumenogenesis, in which epithelial cells establish apical identity by directing apical cargo to an apical membrane initiation site (AMIS), has been studied extensively. However, the mechanisms that govern the formation of the AMIS and its progression into a luminal precursor remain poorly understood. In this study, we employed super-resolution imaging, light and electron microscopy, and time-resolved proximity proteomics to explore the spatial, temporal, and molecular processes involved in apical lumen initiation in MDCK-II cells. Interestingly, we discovered that, in both 2D and 3D cultures, the formation of the apical cell cortex begins with the fusion of large intracellular apical precursor organelles called vacuolar apical compartments (VACs) at developing cell junctions. Time-resolved proteomics and high-resolution imaging indicate that the exocytosis of VACs and lumen initiation is temporally coordinated with the formation and maturation of the apical junctional complex. Furthermore, we report that loss of the Crumbs complex proteins Pals1 or PatJ perturbs the organisation of the apical cell cortex and impedes VAC exocytosis at the AMIS, resulting in severe defects in lumen morphogenesis. Overall, our findings define a temporally resolved protein network for de novo cell polarization and highlight Pals1/PatJ as key regulators of apical domain initiation. We further propose that VACs represent a previously unrecognized apical transport carrier that plays a crucial role in the rapid establishment of apical domain identity.

Organ formation and homeostasis require the coordination of cell-cell adhesion, epithelial cell polarity and orientation of cell division to organize epithelial tissue architecture. We have previously identified proximity protein networks acting downstream of members of the EPH family of tyrosine kinase receptors and found within these networks an enrichment of components associated with cell morphogenesis and cell-cell junctions. Here, we show that two EPH receptors, EPHA1 and EPHB4, are localized to the basolateral domain of Caco-2 cells in spheroidal cultures. Depletion of either EPHA1 or EPHB4 disrupts spheroid morphogenesis, without affecting cell polarity, but via randomizing mitotic spindle orientation during cell division. Strikingly, EPHA1 and EPHB4 exert this function independently of their catalytic activity but still requiring EFN ligand binding. Consistent with this, the most abundantly expressed EPHB4 ligand in Caco-2 cells, EFNB2, is also compartmentalized at the basolateral domain in spheroids, and is required for epithelial morphogenesis. Taken together, our data reveal a new role for EPHRs in epithelial morphogenesis.

Robust tissue growth is orchestrated by the precise coordination of cell death and cell proliferation. Our previous study found that in the developing wing pouches of Drosophila Minute/+ animals, both cell death and compensatory cell proliferation are dramatically increased, which contributes to robust growth of mutant tissue. The induction of this cell-turnover depends on activation of JNK signaling, although the mechanism by which JNK activation induces cell-turnover remained unclear. Here, we show that JNK-mediated elevation of exocytosis in dying cells is crucial for triggering cell-turnover in M/+ wing morphogenesis. Mechanistically, elevated JNK signaling in dying cells upregulates exocytosis-related genes and Wingless (Wg), leading to enhanced Wg secretion. Furthermore, this exocytosis-mediated Wg secretion generally occurs downstream of JNK signaling, regardless of the genetic background. Overall, our findings provide mechanistic insights into robust tissue growth through the orchestration of cell-turnover, which is primarily governed by JNK-mediated exocytosis during Drosophila Minute/+ wing morphogenesis.

How complex three-dimensional (3D) organs coordinate cellular morphogenetic events to achieve the correct final form is a central question in development. The question is uniquely tractable in the late Drosophila pupal retina where cells maintain stereotyped contacts as they elaborate the specialized cytoskeletal structures that pattern the apical, basal and longitudinal planes of the epithelium. In this study, we combined cell type-specific genetic manipulation of the cytoskeletal regulator Abelson (Abl) with 3D imaging to explore how the distinct cellular morphogenetic programs of photoreceptors and interommatidial pigment cells coordinately organize tissue pattern to support retinal integrity. Our experiments revealed an unanticipated intercellular feedback mechanism whereby correct cellular differentiation of either cell type can non-autonomously induce cytoskeletal remodeling in the other Abl mutant cell type, restoring retinal pattern and integrity. We propose that genetic regulation of specialized cellular differentiation programs combined with inter-plane mechanical feedback confers spatial coordination to achieve robust 3D tissue morphogenesis.

Trophoblasts within the fetal membrane primarily serve as protective barriers, shielding the developing fetus from the external environment, while those in chorionic villi facilitate interactions between the maternal and fetal compartments. Although these distinct trophoblast populations perform specialized roles based on their placental location, the mechanisms governing their development and differentiation remain largely unknown. In this study, we derived trophoblast organoids from both the smooth chorion of fetal membranes and chorionic villi from matched human placentas to create parallel in vitro models of these distinct trophoblast populations. Using comparative transcriptional profiling with both bulk and single-cell RNA sequencing, we identified subtle transcriptional variations, while overall gene expression patterns and cellular composition remained highly conserved. Comparative single-cell RNA sequencing and differentiation trajectory analysis of organoids derived from chorion and villous tissues, along with their respective tissue counterparts, revealed region-specific gene expression patterns that were only partially conserved in organoids. The similarity between these in vitro models suggests that regional differences in trophoblast expression observed in vivo are driven by environmental and regional cues. These findings highlight the critical role of local environmental factors in shaping trophoblast function and offer insights into the conserved mechanisms that support placental integrity and fetal development, while emphasizing the influence of region-specific cues in vivo.

Cell differentiation and proliferation are fundamental to the development of multicellular organ-isms. While studies in various non-mammalian species show that development can proceed despite disrupted cell cycle progression, the extent to which normal cell cycle dynamics are re-quired in mammals remains unclear. Using mouse gastruloids, we examined the effects of cell cycle inhibition on development. Despite near-complete growth arrest, gastruloids still underwent symmetry breaking, elongation, and germ layer specification, indicating that core differentiation programs are robust to growth inhibition. However, microscopy and single-cell transcriptomics revealed consistent alterations in cell type proportions, including delayed differentiation and re-duced mesodermal populations. To investigate the origin of these changes, we used the differ-ential kinetics of unspliced and spliced cycling transcripts to compare proliferation rates between cell types. While differences in proliferation partly explained the imbalance, our analysis showed that cell cycle perturbations also modulate lineage-specific differentiation timing and efficiency, highlighting a regulatory role of cell cycle dynamics in mammalian development.

Blood vessels form elaborate networks depending on tissue-specific signalling pathways and anatomical structures to guide their growth. However, it is not clear which morphogenetic principles organize the stepwise assembly of the vasculature. We thus performed a longitudinal analysis of zebrafish tail fin vascular assembly, revealing the existence of temporally and spatially distinct morphogenetic processes. Initially, vein-derived endothelial cells (ECs) generated arteries in a reiterative process requiring Vascular Endothelial Growth Factor (VEGF), Notch and cxcr4a signalling. Subsequently, veins produced veins in more proximal fin regions, transforming pre-existing artery-vein loops into a three-vessel pattern consisting of an artery and two veins. A distinct set of vascular plexuses formed at the base of the fin. They differed by virtue of diameter, flow magnitude and marker gene expression. At later stages, intussusceptive angiogenesis occurred from veins in distal fin regions. In proximal fin regions, we observed new vein sprouts crossing the inter-ray tissue through sprouting angiogenesis. Together, our results reveal a surprising diversity among the mechanisms generating the mature fin vasculature and suggest that these might be driven by separate local cues.

Dissociation and reaggregation experiments in several animal systems have revealed the stunning capacity of self-organization. Reaggregated early gastrula cells (here called gastruloids) of the sea anemone Nematostella vectensis are able to regenerate with only little delay whole organisms that are virtually indistinguishable from normal developing polyps. However, the molecular control underlying the restoration of body axis and germ layers remains largely unknown. To address this, we established a standardized protocol, which reproducibly generates gastruloids developing into polys with a single body axis. Here, we show that committed mesodermal and endodermal cells are sorted out to the surface of the aggregate, where mesodermal cells form clusters of about 30 cells. At a critical time point, one mesodermal cluster immigrates, along with peripherally attached endodermal cells. Thereby, the inner germ layer and the oral-aboral axis is established in one and the same process. Functional studies demonstrated that this polarization of the endodermal cells requires a feedback loop of Notch and Wnt signaling. The dissociation of the early embryo disrupts Notch signaling in the endodermal cells, which leads to transient adoption of an endomesodermal profile, marked by the expression of the mesodermal cadherin1 until the boundaries between the germ layer identities are re-established. Our results highlight distinct morphogenetic behaviors of mesodermal and endodermal cells and the hitherto unknown role of Notch signaling in germ layer boundary formation in self-organizing gastruloids. Conservation of Notch-mediated boundary formation between endoderm and mesoderm mirrors bilaterian mechanisms, demonstrating how adoption of ancestral regulatory networks governing morphogenesis likely enabled the diversification of metazoan body plans.

The organization and maintenance of complex tissues requires emergent properties driven by self-organizing and self-limiting cell-cell interactions. We examined these interactions in the murine mammary gland. Luminal and myoepithelial subpopulations of the postnatal mammary gland arise from unipotent progenitors, but the destiny of cap cells, which enclose terminal end buds (TEB) in pubertal mice, remains controversial. Using a transgenic strain (Tg11.5kb-GFP) that specifically marks cap cells, we found ~50% of these cells undergo divisions perpendicular to the TEB surface, suggesting they might contribute to the underlying luminal cell population. To address their stemness potential we developed a lineage tracing mouse driven from the s-SHIP (11.5 kb) promoter. Induction of tdTomato (tdTom) from this promoter in vivo demonstrated that all cap cell progeny are myoepithelial, with no conversion to luminal lineage. Organoid cultures also exhibited unipotency. However, isolated cap cells cultured as mammospheres generated mixed luminal/myoepithelial spheres. Moreover, ablation of luminal cells in vivo using diphtheria toxin triggered repopulation by progeny of tdTom+ cap cells. A signaling inhibitor screen identified the TGF{beta} pathway as a potential regulator of multipotency. TGF{beta}R inhibitors or gene deletion blocked conversion to the luminal lineage, consistent with an autocrine loop in which cap cells secrete TGF{beta} to activate the receptor and promote luminal transdifferentiation. Ductal tree regeneration in vivo from isolated cap cells was much more efficient when they were pre-treated with inhibitor, consistent with more cells retaining cap cell potential prior to transplantation. Notably, in vitro transdifferentiation of cap cells was blocked by co-culture with luminal cells. Overall, these data reveal a self-limiting cell circuit through which mammary luminal cells suppress cap cell conversion to the luminal lineage.

Positional information driving limb muscle patterning is contained in lateral plate mesoderm-derived tissues, such as tendon or muscle connective tissue but not in myogenic cells themselves. The long-standing consensus is that myogenic cells originate from the somitic mesoderm, while connective tissue fibroblasts originate from the lateral plate mesoderm. We challenged this model using cell and genetic lineage tracing experiments in birds and mice, respectively, and identified a subpopulation of myogenic cells at the muscle tips close to tendons originating from the lateral plate mesoderm and derived from connective tissue gene lineages. Analysis of single-cell RNA-sequencing data obtained from limb cells at successive developmental stages revealed a subpopulation of cells displaying a dual muscle and connective tissue signature, in addition to independent muscle and connective tissue populations. Active BMP signalling was detected in this junctional cell sub-population and at the tendon/muscle interface in developing limbs. BMP gain- and loss-of-function experiments performed in vivo and in vitro showed that this signalling pathway regulated a fibroblast-to-myoblast conversion. We propose that localised BMP signalling converts a subset of lateral plate mesoderm-derived fibroblasts to a myogenic fate and establishes a boundary of fibroblast-derived myonuclei at the muscle/tendon interface to control the muscle pattern during limb development.

O_LIMouse and human pluripotent cells with multipurpose degron alleles establish versatile platforms to dissect cell type-specific functions of the pleiotropic transcription factor OTX2 C_LIO_LIOTX2 controls molecular programs required for anterior-posterior patterning of the developing gut C_LIO_LIOTX2 establishes and maintains chromatin accessibility at distinct distal gene regulatory elements in definitive endoderm C_LIO_LIOTX2 functions as a patterning factor across different germ layers and species C_LI The molecular mechanisms that drive essential developmental patterning events in the mammalian embryo remain poorly understood. To generate a conceptual framework for gene regulatory processes during germ layer specification, we analyzed transcription factor (TF) expression kinetics around gastrulation and during in vitro differentiation. This approach identified Otx2 as a candidate regulator of definitive endoderm (DE), the precursor of all gut-derived tissues. Analysis of multipurpose degron alleles in gastruloid and directed differentiation models revealed that loss of OTX2 before or after DE specification alters the expression of core components and targets of specific cellular signaling pathways, perturbs adhesion and migration programs as well as de-represses regulators of other lineages, resulting in impaired foregut specification. Key targets of OTX2 are conserved in human DE. Mechanistically, OTX2 is required to establish chromatin accessibility at candidate enhancers, which regulate genes critical to establishing an anterior cell identity in the developing gut. Our results provide a working model for the progressive establishment of spatiotemporal cell identity by developmental TFs across germ layers and species, which may facilitate the generation of gut cell types for regenerative medicine applications.

During differentiation, cells become structurally and functionally specialized, but comprehensive views of the underlying remodeling processes are elusive. Here, we leverage scRNA-seq developmental trajectories to reconstruct differentiation using two secretory tissues as a model system - the zebrafish notochord and hatching gland. First, we present an approach to integrate expression and functional similarities for gene module identification, revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second, we focused on the unfolded protein response (UPR) transducer module to study how general versus cell-type specific secretory functions are regulated. By profiling loss- and gain-of-function embryos, we found that the UPR transcription factors creb3l1, creb3l2, and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program, while xbp1 partners with bhlha15 to activate a gland-specific secretion program. Our study offers a multi-source integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions.