Probes for INS

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions-near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3βHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

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.

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.

Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions.