MITF - A controls branching morphogenesis and nephron endowment.
ABSTRACT: Congenital nephron number varies widely in the human population and individuals with low nephron number are at risk of developing hypertension and chronic kidney disease. The development of the kidney occurs via an orchestrated morphogenetic process where metanephric mesenchyme and ureteric bud reciprocally interact to induce nephron formation. The genetic networks that modulate the extent of this process and set the final nephron number are mostly unknown. Here, we identified a specific isoform of MITF (MITF-A), a bHLH-Zip transcription factor, as a novel regulator of the final nephron number. We showed that overexpression of MITF-A leads to a substantial increase of nephron number and bigger kidneys, whereas Mitfa deficiency results in reduced nephron number. Furthermore, we demonstrated that MITF-A triggers ureteric bud branching, a phenotype that is associated with increased ureteric bud cell proliferation. Molecular studies associated with an in silico analyses revealed that amongst the putative MITF-A targets, Ret was significantly modulated by MITF-A. Consistent with the key role of this network in kidney morphogenesis, Ret heterozygosis prevented the increase of nephron number in mice overexpressing MITF-A. Collectively, these results uncover a novel transcriptional network that controls branching morphogenesis during kidney development and identifies one of the first modifier genes of nephron endowment.
Project description:Branching morphogenesis of the epithelial ureteric bud forms the renal collecting duct system and is critical for normal nephron number, while low nephron number is implicated in hypertension and renal disease. Ureteric bud growth and branching requires GDNF signaling from the surrounding mesenchyme to cells at the ureteric bud tips, via the Ret receptor tyrosine kinase and coreceptor Gfr?1; Ret signaling up-regulates transcription factors Etv4 and Etv5, which are also critical for branching. Despite extensive knowledge of the genetic control of these events, it is not understood, at the cellular level, how renal branching morphogenesis is achieved or how Ret signaling influences epithelial cell behaviors to promote this process. Analysis of chimeric embryos previously suggested a role for Ret signaling in promoting cell rearrangements in the nephric duct, but this method was unsuited to study individual cell behaviors during ureteric bud branching. Here, we use Mosaic Analysis with Double Markers (MADM), combined with organ culture and time-lapse imaging, to trace the movements and divisions of individual ureteric bud tip cells. We first examine wild-type clones and then Ret or Etv4 mutant/wild-type clones in which the mutant and wild-type sister cells are differentially and heritably marked by green and red fluorescent proteins. We find that, in normal kidneys, most individual tip cells behave as self-renewing progenitors, some of whose progeny remain at the tips while others populate the growing UB trunks. In Ret or Etv4 MADM clones, the wild-type cells generated at a UB tip are much more likely to remain at, or move to, the new tips during branching and elongation, while their Ret-/- or Etv4-/- sister cells tend to lag behind and contribute only to the trunks. By tracking successive mitoses in a cell lineage, we find that Ret signaling has little effect on proliferation, in contrast to its effects on cell movement. Our results show that Ret/Etv4 signaling promotes directed cell movements in the ureteric bud tips, and suggest a model in which these cell movements mediate branching morphogenesis.
Project description:Nephrons, the functional units of the kidney, develop from progenitor cells (cap mesenchyme [CM]) surrounding the epithelial ureteric bud (UB) tips. Reciprocal signaling between UB and CM induces nephrogenesis and UB branching. Although low nephron number is implicated in hypertension and renal disease, the mechanisms that determine nephron number are obscure. To test the importance of nephron progenitor cell number, we genetically ablated 40% of these cells, asking whether this would limit kidney size and nephron number or whether compensatory mechanisms would allow the developing organ to recover. The reduction in CM cell number decreased the rate of branching, which in turn allowed the number of CM cells per UB tip to normalize, revealing a self-correction mechanism. However, the retarded UB branching impaired kidney growth, leaving a permanent nephron deficit. Thus, the number of fetal nephron progenitor cells is an important determinant of nephron endowment, largely via its effect on UB branching.
Project description:Glial cell line-derived neurotrophic factor signaling through the Ret receptor tyrosine kinase is crucial for ureteric bud branching morphogenesis during kidney development, yet few of the downstream genes are known. Here we show that the ETS transcription factors Etv4 and Etv5 are positively regulated by Ret signaling in the ureteric bud tips. Mice lacking both Etv4 alleles and one Etv5 allele show either renal agenesis or severe hypodysplasia, whereas kidney development fails completely in double homozygotes. We identified several genes whose expression in the ureteric bud depends on Etv4 and Etv5, including Cxcr4, Myb, Met and Mmp14. Thus, Etv4 and Etv5 are key components of a gene network downstream of Ret that promotes and controls renal branching morphogenesis.
Project description:There is strong evidence from human and animal models that exposure to maternal hyperglycemia during in utero development can detrimentally affect fetal kidney development. Notwithstanding this knowledge, the precise effects of diabetic pregnancy on the key processes of kidney development are unclear due to a paucity of studies and limitations in previously used methodologies. The purpose of the present study was to elucidate the effects of hyperglycemia on ureteric branching morphogenesis and nephrogenesis using unbiased techniques. Diabetes was induced in pregnant C57Bl/6J mice using multiple doses of streptozotocin (STZ) on embryonic days (E) 6.5-8.5. Branching morphogenesis was quantified ex vivo using Optical Projection Tomography, and nephrons were counted using unbiased stereology. Maternal hyperglycemia was recognised from E12.5. At E14.5, offspring of diabetic mice demonstrated fetal growth restriction and a marked deficit in ureteric tip number (control 283.7 ± 23.3 vs. STZ 153.2 ± 24.6, mean ± SEM, p<0.01) and ureteric tree length (control 33.1 ± 2.6 mm vs. STZ 17.6 ± 2.7 mm, p = 0.001) vs. controls. At E18.5, fetal growth restriction was still present in offspring of STZ dams and a deficit in nephron endowment was observed (control 1246.2 ± 64.9 vs. STZ 822.4 ± 74.0, p<0.001). Kidney malformations in the form of duplex ureter and hydroureter were a common observation (26%) in embryos of diabetic pregnancy compared with controls (0%). Maternal insulin treatment from E13.5 normalised maternal glycaemia but did not normalise fetal weight nor prevent the nephron deficit. The detrimental effect of hyperglycemia on ureteric branching morphogenesis and, in turn, nephron endowment in the growth-restricted fetus highlights the importance of glycemic control in early gestation and during the initial stages of renal development.
Project description:MicroRNAs (miRNAs) are a large and growing class of small, non-coding, regulatory RNAs that control gene expression predominantly at the post-transcriptional level. The production of most functional miRNAs depends on the enzymatic activity of Dicer, an RNase III class enzyme. To address the potential action of Dicer-dependent miRNAs in mammalian kidney development, we conditionally ablated Dicer function within cells of nephron lineage and the ureteric bud-derived collecting duct system. Six2Cre-mediated removal of Dicer activity from the progenitors of the nephron epithelium led to elevated apoptosis and premature termination of nephrogenesis. Thus, Dicer action is important for maintaining the viability of this critical self-renewing progenitor pool and, consequently, development of a normal nephron complement. HoxB7Cre-mediated removal of Dicer function from the ureteric bud epithelium led to the development of renal cysts. This was preceded by excessive cell proliferation and apoptosis, and accompanied by disrupted ciliogenesis within the ureteric bud epithelium. Dicer removal also disrupted branching morphogenesis with the phenotype correlating with downregulation of Wnt11 and c-Ret expression at ureteric tips. Thus Dicer, and by inference Dicer-dependent miRNA activity, have distinct regulatory roles within different components of the developing mouse kidney. Furthermore, an understanding of miRNA action may provide new insights into the etiology and pathogenesis of renal cyst-based kidney disease.
Project description:Branching morphogenesis of the ureteric bud is integral to kidney development; establishing the collecting ducts of the adult organ and driving organ expansion via peripheral interactions with nephron progenitor cells. A recent study suggested that termination of tip branching within the developing kidney involved stochastic exhaustion in response to nephron formation, with such a termination event representing a unifying developmental process evident in many organs. To examine this possibility, we have profiled the impact of nephron formation and maturation on elaboration of the ureteric bud during mouse kidney development. We find a distinct absence of random branch termination events within the kidney or evidence that nephrogenesis impacts the branching program or cell proliferation in either tip or progenitor cell niches. Instead, organogenesis proceeds in a manner indifferent to the development of these structures. Hence, stochastic cessation of branching is not a unifying developmental feature in all branching organs.
Project description:While the genetic control of renal branching morphogenesis has been extensively described, the cellular basis of this process remains obscure. GDNF/RET signaling is required for ureter and kidney development, and cells lacking Ret are excluded from the tips of the branching ureteric bud in chimeric kidneys. Here, we find that this exclusion results from earlier Ret-dependent cell rearrangements in the caudal Wolffian duct, which generate a specialized epithelial domain that later emerges as the tip of the primary ureteric bud. By juxtaposing cells with elevated or reduced RET activity, we find that Wolffian duct cells compete, based on RET signaling levels, to contribute to this domain. At the same time, the caudal Wolffian duct transiently converts from a simple to a pseudostratified epithelium, a process that does not require Ret. Thus, both Ret-dependent cell movements and Ret-independent changes in the Wolffian duct epithelium contribute to ureteric bud formation.
Project description:Fibroblast growth factor receptor 2 (Fgfr2) signaling is critical in maintaining ureteric branching architecture and mesenchymal stromal morphogenesis in the kidney. Fibroblast growth factor receptor substrate 2alpha (Frs2alpha) is a major docking protein for Fgfr2 with downstream targets including Ets variant (Etv) 4 and Etv5 in other systems. Furthermore, global deletion of Frs2alpha causes early embryonic lethality. The purpose of the study was to determine the role of Frs2alpha in mediating Fgfr2 signaling in the ureteric epithelium. To that end, we generated mice with conditional deletion of Frs2alpha in the ureteric epithelium (Frs2alpha(UB-/-)) and mice with point mutations in the Frs2alpha binding site of Fgfr2 (Fgfr2(LR/LR)). Frs2alpha(UB-/-) mice developed mild renal hypoplasia characterized by decreased ureteric branching morphogenesis but maintained normal overall branching architecture and had normal mesenchymal stromal development. Reduced nephron endowment in postnatal mutant mice was observed, corresponding with the reduction in branching morphogenesis. Furthermore, there were no apparent renal abnormalities in Fgfr2(LR/LR) mice. Interestingly, Etv4 and Etv5 expression was unaltered in Frs2alpha(UB-/-) mice, as was Sprouty1, an antagonist of Frs2alpha signaling. However, Ret and Wnt11 (molecules critical for ureteric branching morphogenesis) mRNA levels were lower in mutants vs. controls. Taken together, these findings suggest that Fgfr2 signals through adapter molecules other than Frs2alpha in the ureteric epithelium. Furthermore, Frs2alpha may transmit signals through other receptor kinases present in ureteric epithelium. Finally, the renal hypoplasia observed in Frs2alpha(UB-/-) mice is likely secondary to decreased Ret and Wnt11 expression.
Project description:Adamts18 encodes a secreted metalloprotease restricted to branch-tip progenitor pools directing the morphogenesis of multiple mammalian organs. Adamts18 was targeted to explore a potential role in branching morphogenesis. In the kidney, an arborized collecting system develops through extensive branching morphogenesis of an initial epithelial outgrowth of the mesonephric duct, the ureteric bud. Adamts18 mutants displayed a weakly penetrant phenotype: duplicated ureteric outgrowths forming enlarged, bi-lobed kidneys with an increased nephron endowment. In contrast, Adamts18 mutants showed a fully penetrant lung phenotype: epithelial growth was markedly reduced and early secondary branching scaled to the reduced length of the primary airways. Furthermore, there was a pronounced delay in the appearance of differentiated cell types in both proximal and distally positions of the developing airways. Adamts18 is closely related to Adamts16. In the kidney but not the lung, broad epithelial Adamts16 expression overlaps Adamts18 in branch tips. However, compound Adamts16/18 mutants displayed a comparable low penetrance duplicated ureteric phenotype, ruling out a possible role for Adamts16 as a functional modifier of the Adamts18 kidney phenotype. Given the predicted action of secreted Adamts18 metalloprotease, and broad expression of Adamts18 in branching organ systems, these findings suggest distinct requirements for matrix modelling in the morphogenesis of epithelial networks.