The development of the bladder trigone, the center of the anti-reflux mechanism

The urinary tract is an outflow system that conducts urine from the kidneys to the bladder via the ureters that propel urine to the bladder via peristalsis. Once in the bladder, the ureteral valve, a mechanism that is not well understood, prevents backflow of urine to the kidney that can cause severe damage and induce end-stage renal disease. The upper and lower urinary tract compartments form independently, connecting at mid-gestation when the ureters move from their primary insertion site in the Wolffian ducts to the trigone, a muscular structure comprising the bladder floor just above the urethra. Precise connections between the ureters and the trigone are crucial for proper function of the ureteral valve mechanism; however, the developmental events underlying these connections and trigone formation are not well understood. According to established models, the trigone develops independently of the bladder, from the ureters, Wolffian ducts or a combination of both; however, these models have not been tested experimentally. Using the Cre-lox recombination system in lineage studies in mice, we find, unexpectedly, that the trigone is formed mostly from bladder smooth muscle with a more minor contribution from the ureter, and that trigone formation depends at least in part on intercalation of ureteral and bladder muscle. These studies suggest that urinary tract development occurs differently than previously thought, providing new insights into the mechanisms underlying normal and abnormal development.     

A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and apoptosis

Protein phosphatase 1 (PP1) catalytic subunits typically combine with other proteins that modulate their activity, direct them to distinct substrates, or serve as substrates for PP1. More than 50 PP1-interacting proteins (PIPs) have been identified so far. Given there are approximately 10 000 phosphoproteins in mammals, many PIPs remain to be discovered. We have used arrays containing 100 carefully selected antibodies to identify novel PIPs that are important in cell proliferation and cell survival in murine fetal lung epithelial cells and human A549 lung cancer cells. The antibody arrays identified 31 potential novel PIPs and 11 of 17 well-known PIPs included as controls, suggesting a sensitivity of at least 65%. A majority of the interactions between PP1 and putative PIPs were isoform- or cell type-specific. We confirmed by co-immunoprecipitation that 9 of these proteins associate with PP1: APAF-1, Bax, E-cadherin, HSP-70, Id2, p19Skp1, p53, PCNA, and PTEN. We examined two of these interactions in greater detail in A549 cells. Exposure to nicotine enhanced association of PP1 with Bax (and Bad), but also induced inhibitory phosphorylation of PP1. In addition to p19Skp1, PP1alpha antibodies also coprecipitated cullin 1, suggesting that PP1alpha is associated with the SCF1 complex. This interaction was only detectable during the G1/S transition and S phase. Forced loss of PP1 function decreased the levels of p27Kip1, a well-known SCF1 substrate, suggesting that PP1 may rescue proteins from ubiquitin/proteasome-mediated destruction. Both of these novel interactions are consistent with PP1 facilitating cell cycle arrest and/or apoptosis.     

Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites.     

Nephric duct insertion is a crucial step in urinary tract maturation that is regulated by a Gata3-Raldh2-Ret molecular network in mice

Urinary tract development depends on a complex series of events in which the ureter moves from its initial branch point on the nephric duct (ND) to its final insertion site in the cloaca (the primitive bladder and urethra). Defects in this maturation process can result in malpositioned ureters and hydronephrosis, a common cause of renal disease in children. Here, we report that insertion of the ND into the cloaca is an unrecognized but crucial step that is required for proper positioning of the ureter and that depends on Ret signaling. Analysis of Ret mutant mice at birth reveals hydronephrosis and defective ureter maturation, abnormalities that our results suggest are caused, at least in part, by delayed insertion of the ND. We find a similar set of malformations in mutants lacking either Gata3 or Raldh2. We show that these factors act in parallel to regulate ND insertion via Ret. Morphological analysis of ND extension in wild-type embryos reveals elaborate cellular protrusions at ND tips that are not detected in Ret, Gata3 or Raldh2 mutant embryos, suggesting that these protrusions may normally be important for fusion with the cloaca. Together, our studies reveal a novel Ret-dependent event, ND insertion, that, when abnormal, can cause obstruction and hydronephrosis at birth; whether ND defects underlie similar types of urinary tract abnormalities in humans is an interesting possibility.     

Modulation of cell-cycle dynamics is required to regulate the number of cerebellar GABAergic interneurons and their rhythm of maturation

The progenitors of cerebellar GABAergic interneurons proliferate up to postnatal development in the prospective white matter, where they give rise to different neuronal subtypes, in defined quantities and according to precise spatiotemporal sequences. To investigate the mechanisms that regulate the specification of distinct interneuron phenotypes, we examined mice lacking the G1 phase-active cyclin D2. It has been reported that these mice show severe reduction of stellate cells, the last generated interneuron subtype. We found that loss of cyclin D2 actually impairs the whole process of interneuron genesis. In the mutant cerebella, progenitors of the prospective white matter show reduced proliferation rates and enhanced tendency to leave the cycle, whereas young postmitotic interneurons undergo severe delay of their maturation and migration. As a consequence, the progenitor pool is precociously exhausted and the number of interneurons is significantly reduced, although molecular layer interneurons are more affected than those of granular layer or deep nuclei. The characteristic inside-out sequence of interneuron placement in the cortical layers is also reversed, so that later born cells occupy deeper positions than earlier generated ones. Transplantation experiments show that the abnormalities of cyclin D2(-/-) interneurons are largely caused by cell-autonomous mechanisms. Therefore, cyclin D2 is not required for the specification of particular interneuron subtypes. Loss of this protein, however, disrupts regulatory mechanisms of cell cycle dynamics that are required to determine the numbers of interneurons of different types and impairs their rhythm of maturation and integration in the cerebellar circuitry.