Our goal is to elucidate the genetic and cellular mechanisms controlling the formation of the ureteric bud (UB) and its growth and branching to give rise to the renal collecting duct (CD) system. Defects in UB forma- tion, growth and branching lead to abnormalities such as renal agenesis, hypoplasia and reduced nephron number, obstruction, hydroureter/hydronephrosis and vesicoureteral reflux, conditions associated with chronic renal disease. Furthermore, the number of nephrons is extremely variable in "normal" humans, and low nephron number is thought to promote the progression of renal diseases and hypertension. One cause of low nephron number is a branching defect that reduces the number of UB tips. Signaling by GDNF through the Ret receptor tyrosine kinase is critical for UB formation and branching. To understand how GDNF and Ret regulate these processes, we identified genes that are upregulated by GDNF. Among these genes were two closely related ETS transcription factors, Etv4 and Etv5, which are themselves jointly required for kidney development. Our central hypothesis is that in response to Ret, Etv4 and Etv5 regulate a set of genes that carry out diverse cellular functions, together promoting branching morphogenesis.
Aim 1 is to investigate the specific effects of Etv4/Etv5 expression on the behaviors of UB cells during branching morphogenesis. Specifically, we test the hypothesis that cell migration underlies the patterned epithelial growth that occurs during UB branching morphogenesis, and that Etv4/5 promote this migration. We use genetic, single-cell, gain-of-function and loss-of function methods to manipulate expression of Etv4 and Etv5, coupled with fluorescent cell labeling and time lapse microscopy of renal organ cultures. Etv4 and Etv5 are transcriptional regulators, so to understand their functions in kidney development we need to identify the "target genes" that they regulate.
Aim 2 is to define the targets of Etv4 and Etv5 through microarray screens of mutant kidneys (identifying genes requiring Etv4/5 for normal expression), ChIP-Seq to define Etv4/5 binding sites in the genome, and further studies to validate downstream genes. This will elaborate the gene regulatory network that controls UB branching morphogenesis, and will advance our understanding of the causes of urological/renal birth defects in mouse models and potentially humans.
Aim 3 examines the potential role in renal development and hypertension of two secreted proteases, Adamts16 and Adamts18, which are likely targets of Etv4/5. While the substrates and in vivo functions of Adamts16 and 18 are unknown, their similar sequences and renal expression suggest they may serve redundant functions in UB branching. Furthermore, Adamts16 has been linked to hypertension in rodents and humans. We hypothesize that the absence of Adamts16 and Adamts18 contributes to the UB branching defects in Etv4/5 mutants. Lack of Adamts16 and/or 18 may cause reduced nephron number and hyper- tension. We test this by examining kidney development, nephron number and blood pressure in mutant mice.
Normal development of the kidney during fetal life is important for the organ to achieve its normal size, number of nephrons (blood-filtering units) and connections to the bladder. Defects in fetal organ development may promote the progression of renal and urological diseases and hypertension. This proposal investigates the genetic mechanisms that control how the kidney grows to the correct size and shape, and how the proper number of nephrons and urine-collecting ducts is formed.
|Costantini, Frank; Watanabe, Tomoko; Lu, Benson et al. (2011) Dissection of embryonic mouse kidney, culture in vitro, and imaging of the developing organ. Cold Spring Harb Protoc 2011:pdb.prot5613|
|Michos, Odysse; Cebrian, Cristina; Hyink, Deborah et al. (2010) Kidney development in the absence of Gdnf and Spry1 requires Fgf10. PLoS Genet 6:e1000809|
|Krawchuk, Dayana; Weiner, Shoshana J; Chen, You-Tzung et al. (2010) Twist1 activity thresholds define multiple functions in limb development. Dev Biol 347:133-46|
|Costantini, Frank; Kopan, Raphael (2010) Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development. Dev Cell 18:698-712|
|Costantini, Frank (2010) GDNF/Ret signaling and renal branching morphogenesis: From mesenchymal signals to epithelial cell behaviors. Organogenesis 6:252-62|
|Kuure, Satu; Cebrian, Cristina; Machingo, Quentin et al. (2010) Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis. PLoS Genet 6:e1001176|
|Kuure, Satu; Chi, Xuan; Lu, Benson et al. (2010) The transcription factors Etv4 and Etv5 mediate formation of the ureteric bud tip domain during kidney development. Development 137:1975-9|
|Lu, Benson C; Cebrian, Cristina; Chi, Xuan et al. (2009) Etv4 and Etv5 are required downstream of GDNF and Ret for kidney branching morphogenesis. Nat Genet 41:1295-302|
|Chi, Xuan; Michos, Odysse; Shakya, Reena et al. (2009) Ret-dependent cell rearrangements in the Wolffian duct epithelium initiate ureteric bud morphogenesis. Dev Cell 17:199-209|
|Chi, Xuan; Hadjantonakis, Anna-Katerina; Wu, Zaiqi et al. (2009) A transgenic mouse that reveals cell shape and arrangement during ureteric bud branching. Genesis 47:61-6|