| Experiment Id | GSE245506 | Name | Netrin-1 directs vascular patterning and maturity in the developing kidney |
| Experiment Type | RNA-Seq | Study Type | WT vs. Mutant |
| Source | GEO | Curation Date | 2024-06-20 |
| description | The kidneys intricate vascular system supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin-1 (Ntn1) is a secreted ligand critical for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature, and differentiating nephrons. Together, our findings highlight the significance of netrin-1 to proper vascularization and kidney development. Whole kidneys were collected at E15.5 from 3 separate Ntn1fl/fl x Foxd1GC/+;Ntn1fl/+ crosses. Three kidneys were collected of each genotype: Foxd1GC/+;Ntn1fl/+ (heterozygous), Foxd1GC/+;Ntn1fl/fl (homozygous), and Ntn1fl/fl (wildtype). Kidneys were placed in RNAlater (Thermo Fisher) until all samples were collected. RNA was extracted with TRIzol (Invitrogen). RNA was sent to Cancer Genetics Inc (Morrisville, NC) for sequencing and data processing. RNA samples were converted to sequencing libraries using the TruSeq Stranded mRNA kit (Illumina Cat. #20020595). Resulting libraries were assessed for quality via analysis on a 2100 Bioanalyzer (Agilent Technologies) and quantitated by qPCR (KAPA Library Quantification; Roche: KK4824, Cat# 07960140001). Normalized and pooled libraries were sequenced on the Illumina Nextseq 550 system. Alignment and analysis were performed following a modified Tuxedo workflow. Briefly, alignment was done with STAR aligner followed by cufflinks/cuffmerge for assembled transcripts. FPKM was calculated with CuffDiff and analyzed with the R package |
