Radial glia regulate vascular patterning around the developing spinal cord.

Vascular networks surrounding individual organs are important for their development, maintenance, and function; however, how these networks are assembled remains poorly understood. Here we show that CNS progenitors, referred to as radial glia, modulate vascular patterning around the spinal cord by acting as negative regulators. We found that radial glia ablation in zebrafish embryos leads to excessive sprouting of the trunk vessels around the spinal cord, and exclusively those of venous identity. Mechanistically, we determined that radial glia control this process via the Vegf decoy receptor sFlt1: sflt1 mutants exhibit the venous over-sprouting observed in radial glia-ablated larvae, and sFlt1 overexpression rescues it. Genetic mosaic analyses show that sFlt1 function in trunk endothelial cells can limit their over-sprouting. Together, our findings identify CNS-resident progenitors as critical angiogenic regulators that determine the precise patterning of the vasculature around the spinal cord, providing novel insights into vascular network formation around developing organs.

Coordination of signaling and tissue mechanics during morphogenesis of murine intestinal villi: a role for mitotic cell rounding.

Efficient digestion and absorption of nutrients by the intestine requires a very large apical surface area, a feature that is enhanced by the presence of villi, fingerlike epithelial projections that extend into the lumen. Prior to villus formation, the epithelium is a thick pseudostratified layer. In mice, villus formation begins at embryonic day (E)14.5, when clusters of mesenchymal cells form just beneath the thick epithelium. At this time, analysis of the flat lumenal surface reveals a regular pattern of short apical membrane invaginations that form in regions of the epithelium that lie in between the mesenchymal clusters. Apical invaginations begin in the proximal intestine and spread distally, deepening with time. Interestingly, mitotically rounded cells are frequently associated with these invaginations. These mitotic cells are located at the tips of the invaginating membrane (internalized within the epithelium), rather than adjacent to the apical surface. Further investigation of epithelial changes during membrane invagination reveals that epithelial cells located between mesenchymal clusters experience a circumferential compression, as epithelial cells above each cluster shorten and widen. Using a computational model, we examined whether such forces are sufficient to cause apical invaginations. Simulations and in vivo data reveal that proper apical membrane invagination involves intraepithelial compressive forces, mitotic cell rounding in the compressed regions and apico-basal contraction of the dividing cell. Together, these data establish a new model that explains how signaling events intersect with tissue forces to pattern apical membrane invaginations that define the villus boundaries.

Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi.

In the adult intestine, an organized array of finger-like projections, called villi, provide an enormous epithelial surface area for absorptive function. Villi first emerge at embryonic day (E) 14.5 from a previously flat luminal surface. Here, we analyze the cell biology of villus formation and examine the role of paracrine epithelial Hedgehog (Hh) signals in this process. We find that, before villus emergence, tight clusters of Hh-responsive mesenchymal cells form just beneath the epithelium. Cluster formation is dynamic; clusters first form dorsally and anteriorly and spread circumferentially and posteriorly. Statistical analysis of cluster distribution reveals a patterned array; with time, new clusters form in spaces between existing clusters, promoting approximately four rounds of villus emergence by E18.5. Cells within mesenchymal clusters express Patched1 and Gli1, as well as Pdgfrα, a receptor previously shown to participate in villus development. BrdU-labeling experiments show that clusters form by migration and aggregation of Hh-responsive cells. Inhibition of Hh signaling prevents cluster formation and villus development, but does not prevent emergence of villi in areas where clusters have already formed. Conversely, increasing Hh signaling increases the size of villus clusters and results in exceptionally wide villi. We conclude that Hh signals dictate the initial aspects of the formation of each villus by controlling mesenchymal cluster aggregation and regulating cluster size.

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells.

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.

Separate and distinctive roles for Wnt5a in tongue, lingual tissue and taste papilla development.

Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor.

Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis.

The cellular mechanisms that drive growth and remodeling of the early intestinal epithelium are poorly understood. Current dogma suggests that the murine fetal intestinal epithelium is stratified, that villi are formed by an epithelial remodeling process involving the de novo formation of apical surface at secondary lumina, and that radial intercalation of the stratified cells constitutes a major intestinal lengthening mechanism. Here, we investigate cell polarity, cell cycle dynamics and cell shape in the fetal murine intestine between E12.5 and E14.5. We show that, contrary to previous assumptions, this epithelium is pseudostratified. Furthermore, epithelial nuclei exhibit interkinetic nuclear migration, a process wherein nuclei move in concert with the cell cycle, from the basal side (where DNA is synthesized) to the apical surface (where mitosis takes place); such nuclear movements were previously misinterpreted as the radial intercalation of cells. We further demonstrate that growth of epithelial girth between E12.5 and E14.5 is driven by microtubule- and actinomyosin-dependent apicobasal elongation, rather than by progressive epithelial stratification as was previously thought. Finally, we show that the actin-binding protein Shroom3 is crucial for the maintenance of the single-layered pseudostratified epithelium. In mice lacking Shroom3, the epithelium is disorganized and temporarily stratified during villus emergence. These results favor an alternative model of intestinal morphogenesis in which the epithelium remains single layered and apicobasally polarized throughout early intestinal development.

Gastric tuft cells express DCLK1 and are expanded in hyperplasia.

Epithelial tuft cells are named after their characteristic microtubule bundles located at the cell apex where these are exposed to the luminal environment. As such, tuft cells are found in multiple organs, including the gastrointestinal (GI) tract where the apical "tuft" is hypothesized to detect and transmit environmental signals. Thus, the goal of our study was to characterize gastric tuft cells during GI tract development, then subsequently in the normal and metaplastic adult stomach. GI tracts from mouse embryos, and newborn and postnatal mice were analyzed. Tuft cells were identified by immunohistochemistry using acetylated-α-tubulin (acTub) antibody to detect the microtubule bundle. Additional tuft cell markers, e.g., doublecortin-like kinase 1 (DCLK1), were used to co-localize with acTub. Tuft cells were quantified in human gastric tissue arrays and in mouse stomachs with or without inflammation. In the developing intestine, tuft cells in both the crypts and villi expressed all markers by E18.5. In the stomach, acTub co-localized with DCLK1 and other established tuft cell markers by E18.5 in the antrum, but not until postnatal day 7 in the corpus, with the highest density of tuft cells clustered at the forestomach ridge. Tuft cell numbers increased in hyperplastic human and mouse stomachs. In the adult GI tract, the tuft cell marker acTub co-expressed with DCKL1 and chemosensory markers, e.g.,TRPM5. In summary, tuft cells appear in the gastric antrum and intestine at E18.5, but their maximal numbers in the corpus are not achieved until after weaning. Tuft cell numbers increase with inflammation, hyperplasia, and metaplasia.

Paracrine Hedgehog signaling in stomach and intestine: new roles for hedgehog in gastrointestinal patterning.

BACKGROUND & AIMS: Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine, and colon. METHODS: Hedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed. RESULTS: Hedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include the following: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the 2 layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; and after birth, the muscularis mucosa and villus smooth muscle consist primarily of Hedgehog-responsive cells and Hh levels actively modulate villus core smooth muscle. CONCLUSIONS: These studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus, and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association.