Caspase activity is required for nephrogenesis in the developing mouse metanephros

Programmed cell death is a mechanism through which organisms get rid of unwanted cells and is thought to be an important process in organogenesis. Although large-scale cell death is observed in the developing kidney, the precise roles of cell death in kidney organogenesis remain to be elucidated. To address this question, we prevented cell death in metanephric explants by applying caspase inhibitors. Administration of caspase inhibitors (Z-D-CH2DCB and Ac-DEVD-CHO) effectively prevented the cell death that is normally observed in nondifferentiating mesenchymal cells. Both ureteric bud branching and nephrogenesis were prevented by caspase inhibition. Our results suggest that caspases are crucial in kidney organogenesis and cell death in the nondifferentiating mesenchyme.     

Cadherin-6 is required for zebrafish nephrogenesis during early development

We performed functional analyses of cadherin-6 (cdh6) in zebrafish nephrogenesis using antisense morpholino oligonucleotide (MO) inhibition combined with in situ hybridization. We have cloned a zebrafish homolog (accession number AB193290) of human K-cadherin (CDH6), which showed 6063% identity and 7678% similarity to the human, mouse, chicken and Xenopus homologs. Whole-mount in situ hybridization showed that cdh6 is expressed in the pronephric ducts and nephron primordia in addition to the central and peripheral nervous systems. Expression of cdh6 in the pronephric ducts was first detected at 14 hours post-fertilization (hpf) and increased to 24 hpf. Embryos injected with MOs directed against cdh6 (cdh6MOs) showed developmental defects, including a small head, body axis curvature, short yolk extension and a short bent tail by 30 hpf and edema appeared in the thorax by 42 hpf. Such defects and edema became more marked by 52 hpf and most of the affected embryos died by 5 days post-fertilization. Embryos injected with cdh6MOs were subjected to in situ hybridization with probes for the pronephric markers, wt1 and pax2.1, to examine disturbed development of the anterior region of the pronephric ducts and the nephron primordia. Histological studies showed malformation of the pronephros as abnormally fused glomerulus primordia, fused or abnormally bent pronephric tubule anlagen and coarctated pronephric ducts. These results suggest that cdh6 plays pivotal roles in the development of the pronephros in zebrafish embryos.     

BRCA2 is required for neurogenesis and suppression of medulloblastoma

Defective DNA damage responses in the nervous system can result in neurodegeneration or tumorigenesis. Despite the importance of DNA damage signalling, the neural function of many critical DNA repair factors is unclear. BRCA2 is necessary for homologous recombination repair of DNA and the prevention of diseases including Fanconi Anemia and cancer. We determined the role of BRCA2 during brain development by inactivating murine Brca2 throughout neural tissues. In striking contrast to early embryonic lethality after germ-line inactivation, Brca2(LoxP/LoxP);Nestin-cre mice were viable. However, Brca2 loss profoundly affected neurogenesis, particularly during embryonic and postnatal neural development. These neurological defects arose from DNA damage as Brca2(LoxP/LoxP);Nestin-cre mice showed extensive gammaH2AX in neural tissue and p53 deficiency restored brain histology but lead to rapid formation of medulloblastoma brain tumors. In contrast, loss of the Atm kinase did not markedly attenuate apoptosis after Brca2 loss, but did partially restore cerebellar morphology, supporting a genomic surveillance function for ATM during neurogenesis. These data illustrate the importance of Brca2 during nervous system development and underscore the tissue-specific requirements for DNA repair factors.     

Fibroblast growth factor signaling is required for the proliferation and patterning of progenitor cells in the developing anterior pituitary

The proliferation and patterning of progenitor cells in the anterior pituitary require signals derived from the neuroepithelium of the juxtaposed infundibulum. The infundibulum expresses Fibroblast growth factor (Fgf) 8 and Fgf 18, and FGFs can mimic some of the activities of the infundibulum. The requirement for FGF signaling during growth and patterning of the anterior pituitary has not, however, been established. By blocking FGF receptor signaling in explants of the anterior pituitary cultured in vitro we provide evidence that FGF signaling derived from the infundibulum is required for the proliferation and patterning of progenitor cells in the anterior pituitary.     

Mouse CD24 is required for homeostatic cell renewal

Under physiological conditions, some adult tissues retain a capacity for self-renewal. This property is attributable to the proliferation and differentiation of stem, transit-amplifying, and differentiating cells, which are regulated by cell-cell or cell-matrix interactions or by secreted factors. By gain and loss of function experiments, we demonstrate the involvement of mouse CD24 (mouse cluster of differentiation 24), which is a glycosyl phosphatidylinositol (GPI)-anchored cell-surface glycoprotein, in the regulation of homeostatic cell renewal. BrdU incorporation observations, at optical and electron-microscopic levels, have revealed increased cell proliferation in the developing brain and in the epithelia of mCD24-deleted mice. We have observed ectopic proliferative cells in the suprabasal layers of the mutant skin leading to a general disruption of basal and suprabasal layers. By contrast, ectopic mCD24 expression mediated by retroviral infection of the embryonic brain leads to a decreased number of clusters of cells generated in the progeny. Together, these results and our previous published data indicate that mCD24 contributes to the regulation of the production of differentiated cells by controlling the proliferation/differentiation balance between transit-amplifying and committed differentiated cells.     

Grhl2 is required in nonneural tissues for neural progenitor survival and forebrain development

Grainyhead‐like genes are part of a highly conserved gene family that play a number of roles in ectoderm development and maintenance in mammals. Here we identify a novel allele of Grhl2, cleft‐face 3 (clft3), in a mouse line recovered from an ENU mutagenesis screen for organogenesis defects. Homozygous clft3 mutants have a number of phenotypes in common with other alleles of Grhl2. We note a significant effect of genetic background on the clft3 phenotype. One of these is a reduction in size of the telencephalon where we find abnormal patterns of neural progenitor mitosis and apoptosis in mutant brains. Interestingly, Grhl2 is not expressed in the developing forebrain, suggesting this is a survival factor for neural progenitors exerting a paracrine effect on the neural tissue from the overlying ectoderm where Grhl2 is highly expressed. genesis 53:573–582, 2015. © 2015 Wiley Periodicals, Inc.     

Six3 is required for ependymal cell maturation

Ependymal cells are part of the neurogenic niche in the adult subventricular zone of the lateral ventricles, where they regulate neurogenesis and neuroblast migration. Ependymal cells are generated from radial glia cells during embryonic brain development and acquire their final characteristics postnatally. The homeobox gene Six3 is expressed in ependymal cells during the formation of the lateral wall of the lateral ventricles in the brain. Here, we show that Six3 is necessary for ependymal cell maturation during postnatal stages of brain development. In its absence, ependymal cells fail to suppress radial glia characteristics, resulting in a defective lateral wall, abnormal neuroblast migration and differentiation, and hydrocephaly.     

LGR4 is required for the cell survival of the peripheral mesenchyme at the embryonic stages of nephrogenesis

In mice, homozygous Lgr4 inactivation results in hypoplastic kidneys. To understand better the role of LGR4 in kidney development, we performed an analysis of kidneys in Lgr4-/- embryos. We stained Lgr4-/- kidneys with anti-WT1 and anti-Cleaved Caspase3 antibodies at E16.5, and observed that the structures of the cap mesenchyme were disrupted and that apoptosis increased. In addition, the expression of PAX2, an anti-apoptotic factor in kidney development, was also significantly decreased at E16.5. We found that the LGR4 defect caused an increase in apoptosis in the peripheral mesenchyme during kidney development.     

TGFbeta superfamily signals are required for morphogenesis of the kidney mesenchyme progenitor population

The TGFbeta superfamily plays diverse and essential roles in kidney development. Gdf11 and Bmp4 are essential for outgrowth and positioning of the ureteric bud, the inducer of metanephric mesenchyme. During nephrogenesis, Bmp7 is required for renewal of the mesenchyme progenitor population. Additionally, in vitro studies demonstrate inhibitory effects of BMPs and TGFbetas on collecting duct branching and growth. Here, we explore the predicted models of TGFbeta superfamily function by cell-specific inactivation of Smad4, a key mediator of TGFbeta signaling. Using a HoxB7cre transgene expressed in ureteric bud and collecting duct, we find that development of the collecting duct is Smad4 independent. By contrast, removal of Smad4 in nephrogenic mesenchyme using the Bmp7(cre/+) allele leads to disorganization of the nephrogenic mesenchyme and impairment of mesenchyme induction. Smad4-deficient metanephric mesenchyme does not display defects in inducibility in LiCl or spinal cord induction assays. However, in situ hybridization and lineage analysis of Smad4 null mesenchyme cells at E11.5 show that the nephrogenic mesenchyme does not aggregate tightly around the ureteric bud tips, but remains loosely associated, embedded within a population of cells expressing markers of both nephrogenic mesenchyme and peripheral stroma. We conclude that the failure of recruitment of nephrogenic mesenchyme leaves a primitive population of mesenchyme at the periphery of the kidney. This population is gradually depleted, and by E16.5 the periphery is composed of cells of stromal phenotype. This study uncovers a novel role for TGFbeta superfamily signaling in the recruitment and/or organization of the nephrogenic mesenchyme at early time-points of kidney development. Additionally, we present conclusive genetic lineage mapping of the collecting duct and nephrogenic mesenchyme.     

Presenilins are required for the formation of comma- and S-shaped bodies during nephrogenesis

Mammalian presenilins consist of two highly homologous proteins, PSEN1 and PSEN2, which share redundant activities in Notch processing and signaling. To bypass the early lethality of the Psen1- and Psen2-double (PSEN) null embryos, we used a human PSEN1 transgene to rescue the somite patterning defects in PSEN-null animals and to allow a determination of the function of presenilins in late embryogenesis. We report here that expression of the human PSEN1 transgene supported the survival of PSEN-null embryos to the perinatal stage. However, presenilin deficiency in the kidney led to severe nephrogenesis defects and virtually no comma- or S-shaped bodies, or mature glomeruli were formed. We document that the mesenchyme was induced which could further progress to renal vesicles in the PSEN-null kidney, indicating that the presenilins are not essential for the inductive interactions and mesenchyme to epithelium transition. However, renal vesicles failed to pattern to form proximal tubules and glomerular epithelium. A presenilin-dependent, signaling-competent form of Notch1 was detected in mesenchymal derivatives but not in the ureteric buds of wild-type mice. Consistent with an obligatory role of presenilins in Notch processing and activation, the active form of Notch1 and its downstream target Hesr1 were absent in the PSEN-null kidney. Importantly, sustained Notch1 signaling was required for the maintenance of Notch ligand Jag1 expression. These results identify presenilins as one determinant of renal vesicle patterning in the developing mouse kidney, and we hypothesize that they act through the Notch signaling pathway.     

Cathepsin L is required for endothelial progenitor cell-induced neovascularization

Infusion of endothelial progenitor cells (EPC), but not of mature endothelial cells, promotes neovascularization after ischemia. We performed gene expression profiling of EPC and endothelial cells to identify genes that might be important for the neovascularization capacity of EPC. Notably, the protease cathepsin L (CathL) was highly expressed in EPC as opposed to endothelial cells and was essential for matrix degradation and invasion by EPC in vitro. CathL-deficient mice showed impaired functional recovery following hind limb ischemia, supporting the concept of a crucial role for CathL in postnatal neovascularization. Infused CathL-deficient progenitor cells neither homed to sites of ischemia nor augmented neovascularization. Forced expression of CathL in mature endothelial cells considerably enhanced their invasive activity and sufficed to confer their capacity for neovascularization in vivo. We concluded that CathL has a critical role in the integration of circulating EPC into ischemic tissue and is required for EPC-mediated neovascularization.     

Gamma-secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney

Notch signaling is involved in pronephros development in Xenopus and in glomerulogenesis in mice. However, owing to early lethality in mice deficient for some Notch pathway genes and functional redundancy for others, a role for Notch signaling during early stages of metanephric development has not been defined. Using an antibody specific to the N-terminal end of gamma-secretase-cleaved Notch1, we found evidence for Notch1 activation in the comma and S-shaped bodies of the mouse metanephros. We therefore cultured mouse metanephroi in the presence of a gamma-secretase inhibitor, N-S-phenyl-glycine-t-butyl ester (DAPT), to block Notch signaling. We observed slightly reduced ureteric bud branching but normal mesenchymal condensation and expression of markers indicating that mesenchyme induction had occurred. However, fewer renal epithelial structures were observed, with a severe deficiency in proximal tubules and glomerular podocytes, which are derived from cells in which activated Notch1 is normally present. Distal tubules were present but in reduced numbers, and this was accompanied by an increase in intervening, non-epithelial cells. After a transient 3-day exposure to DAPT, proximal tubules expanded, but podocyte differentiation failed to recover after removal of DAPT. These observations suggest that gamma-secretase activity, probably through activation of Notch, is required for maintaining a competent progenitor pool as well as for determining the proximal tubule and podocyte fates.     

Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development

Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process are unclear. We demonstrate that the Six2-expressing cap mesenchyme represents a multipotent nephron progenitor population. Six2-expressing cells give rise to all cell types of the main body of the nephron during all stages of nephrogenesis. Pulse labeling of Six2-expressing nephron progenitors at the onset of kidney development suggests that the Six2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some Six2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, Six2 functions cell autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking Six2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that Six2 activity cell-autonomously regulates a multipotent nephron progenitor population.