Probes for INS

In mammalian ovaries, immature oocytes are reserved in primordial follicles until their activation for potential ovulation. Precise control of primordial follicle activation (PFA) is essential for reproduction, but how this is achieved is unclear. Here, we show that canonical wingless-type MMTV integration site family (WNT) signaling is pivotal for pre-granulosa cell (pre-GC) activation during PFA. We identified several WNT ligands expressed in pre-GCs that act in an autocrine manner. Inhibition of WNT secretion from pre-GCs/GCs by conditional knockout (cKO) of the wntless (Wls) gene led to female infertility. In Wls cKO mice, GC layer thickness was greatly reduced in growing follicles, which resulted in impaired oocyte growth with both an abnormal, sustained nuclear localization of forkhead box O3 (FOXO3) and reduced phosphorylation of ribosomal protein S6 (RPS6). Constitutive stabilization of β-catenin (CTNNB1) in pre-GCs/GCs induced morphological changes of pre-GCs from a squamous into a cuboidal form, though it did not influence oocyte activation. Our results reveal that canonical WNT signaling plays a permissive role in the transition of pre-GCs to GCs, which is an essential step to support oocyte growth.

Neural tissue maturation is a coordinated process under tight transcriptional control. We previously analyzed the kinetics of gene expression in the medial nucleus of the trapezoid body (MNTB) in the brainstem during the critical postnatal phase of its development. While this work revealed timed execution of transcriptional programs, it was blind to the specific cells where gene expression changes occurred. Here, we utilized single-cell RNA-sequencing (scRNA-Seq) to determine transcriptional profiles of each major MNTB cell type. We discerned directional signaling patterns between neuronal, glial, and vascular-associated cells (VACs) for VEGF, TGFβ, and Delta-Notch pathways during a robust period of vascular remodeling in the MNTB. Furthermore, we describe functional outcomes of the disruption of neuron-astrocyte fibroblast growth factor 9 (Fgf9) signaling. We used a conditional knockout (cKO) approach to genetically delete Fgf9 from principal neurons in the MNTB, which led to an early onset of glial fibrillary acidic protein (Gfap) expression in astrocytes. In turn, Fgf9 cKO mice show increased levels of astrocyte-enriched brevican (Bcan), a component of the perineuronal net matrix (PNN) that ensheaths principal neurons in the MNTB and the large calyx of Held (CH) terminal, while levels of the neuron-enriched hyaluronan and proteoglycan link protein 1 (Hapln1) were unchanged. Finally, volumetric analysis of vesicular glutamate transporters 1 and 2 (Vglut1/2), which serves as a proxy for terminal size, revealed an increase in CH volume in the Fgf9 cKO. Overall, we demonstrate a coordinated neuron-astrocyte Fgf9 signaling network that functions to regulate astrocyte maturation, PNN structure, and synaptic refinement.

The liver has complex interconnecting blood vessel and biliary networks, however how the vascular and biliary network form and regulate each other and liver function are not well understood. We aimed to examine the role of Heg in mammalian liver development and functional maintenance.Global (Heg-/-) or liver endothelial cell-specific deletion of Heg (Lyve1-Cre;Hegfl/fl ) mice were used to study the in vivo function of Heg in the liver. Carbon-ink anterograde and retrograde injection were used to visualize the 3-D patterning of liver portal and biliary networks, respectively. RNA sequencing, histology, molecular and biochemical assays were used to assess liver gene expression, protein distribution, liver injury response and function.Heg deficiency in liver endothelial cells led to a sparse liver vascular and biliary network. This network paucity does not compromise liver function under baseline conditions but did alter liver zonation. Molecular analysis revealed that endothelial Heg deficiency decreased expression of Wnt ligands/agonists including Wnt2, Wnt9b and Rspo3 in endothelial cells, which limits Axin2 mediated canonical Wnt signaling and the expression of cytochrome P450 enzymes in hepatocytes. Under chemical-induced stressed conditions, Heg-deficiency in liver endothelial cells protected mice from drug-induced liver injuries.Our study found that endothelial Heg is essential for the 3-D patterning of the liver vascular and indirectly regulates biliary networks and proper liver zonation via its regulation of Wnt ligand production in liver endothelial cells. The endothelial Heg-initiated changes of the liver metabolic zonation and metabolic enzyme expression in hepatocytes was functionally relevant to xenobiotic metabolism and drug induced liver toxicity.

The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression, but a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the ganglionic eminences (GEs), the source of all forebrain GABAergic neurons. By using a transgenic mouse line to enrich for VZ cells, we characterize significant transcriptional heterogeneity, both between GEs and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of VZ cells in the ventral forebrain that will aid our understanding of initial fate decisions in the forebrain.

Pancreatic development requires spatially and temporally controlled expression of growth factors derived from mesenchyme. Here, we report that in mice the secreted factor Fgf9 is expressed principally by mesenchyme and then mesothelium during early development, then subsequently by both mesothelium and rare epithelial cells by E12.5 and onwards. Global knockout of the Fgf9 gene resulted in the reduction of pancreas and stomach size, as well as complete asplenia. The number of early Pdx1+ pancreatic progenitors was reduced at E10.5, as was proliferation of mesenchyme at E11.5. Although loss of Fgf9 did not interfere with differentiation of later epithelial lineages, single-cell RNA-Sequencing identified transcriptional programs perturbed upon loss of Fgf9 during pancreatic development, including loss of the transcription factor Barx1. Lastly, we identified conserved expression patterns of FGF9 and receptors in human fetal pancreas, suggesting that FGF9 expressed by pancreatic mesenchyme may similarly affect the development of the human pancreas.

The lung alveolus is lined with alveolar type 1 (AT1) and type 2 (AT2) epithelial cells. During alveologenesis, increasing demand associated with expanding alveolar numbers is met by proliferating progenitor AT2s (pAT2). Little information exists regarding the identity of this population and their niche microenvironment. We show that during alveologenesis, Hedgehog-responsive PDGFRa(+) progenitors (also known as SCMFs) are a source of secreted trophic molecules that maintain a unique pAT2 population. SCMFs are in turn maintained by TGFβ signaling. Compound inactivation of Alk5 TβR2 in SCMFs reduced their numbers and depleted the pAT2 pool without impacting differentiation of daughter cells. In lungs of preterm infants who died with bronchopulmonary dysplasia, PDGFRa is reduced and the number of proliferative AT2s is diminished, indicating that an evolutionarily conserved mechanism governs pAT2 behavior during alveologenesis. SCMFs are a transient cell population, active only during alveologenesis, making them a unique stage-specific niche mesodermal cell type in mammalian organs.

Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent mesoderm cells in the fetal foregut. Although the recent successes in recapitulating developmental signaling in vitro has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue. Here, we describe a protocol to differentiate hPSCs into different types of organ-specific mesoderm, leveraging signaling networks and molecular markers elucidated from single-cell transcriptomics of mouse foregut organogenesis. Building on established methods, hPSC-derived lateral plate mesoderm treated with either retinoic acid (RA) or RA together with a Hedgehog (HH) agonist generates posterior or anterior foregut splanchnic mesoderm, respectively, after 4-d cultures. These are directed into organ-specific mesenchyme lineages by the combinatorial activation or inhibition of WNT, BMP, RA or HH pathways from days 4 to 7 in cultures. By day 7, the cultures are enriched for different types of mesoderm with distinct molecular signatures: 60-90% pure liver septum transversum/mesothelium-like, 70-80% pure liver-like fibroblasts and populations of ~35% respiratory-like mesoderm, gastric-like mesoderm or esophageal-like mesoderm. This protocol can be performed by anyone with moderate experience differentiating hPSCs, provides a novel platform to study human mesoderm development and can be used to engineer more complex foregut tissue for disease modeling and regenerative medicine.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.