Greb1l is essential for pronephric tubule morphogenesis in zebrafish by maintaining cell homeostasis via MAPK signaling

Abstract Mutations in the human GREB1L (growth regulation by estrogen in breast cancer 1-like) gene are associated with congenital anomalies of the kidneys and urinary tracts (CAKUT). However, its precise function and molecular mechanisms remain poorly understood. This study aimed to explore the role of Greb1l deficiency on pronephros development in zebrafish ( Danio rerio ) and to elucidate its potential molecular mechanisms. We created Greb1l-deficient zebrafish models using CRISPR/Cas9 and morpholino oligonucleotide (MO)-based methods. Maternal-zygotic greb1l homozygous mutants (MZ greb1l −/− ) exhibited human-like phenotypes, including a cystic pronephros, abnormal reproduction, pericardial edema, and skeletal deformities. Moreover, there was a significant increase in both cell proliferation and apoptosis in the cystic proximal tubules of the pronephros, indicating a disruption of cellular homeostasis. Transcriptomic sequencing of 5 dpf MZ greb1l −/− mutants revealed that the differentially expressed genes (DEGs) were significantly enriched in the cell cycle, apoptosis, and MAPK signaling pathway. Further functional validation confirmed a significant downregulation in the phosphorylation levels of key MAPK pathway proteins ERK, JNK, and p38 in the Greb1l-deficient model. By treating with the MAPK activator lipopolysaccharide (LPS), we restored the pathway’s activity, significantly rescuing the pronephric cyst phenotype and alleviating the cell proliferation/apoptosis imbalance caused by Greb1l deficiency. Our findings demonstrate that Greb1l plays a vital role in the morphogenesis of pronephric tubules, contributing to cellular equilibrium through modulation of the MAPK signaling pathway. This study sheds light on the underlying molecular mechanisms of GREB1L-associated congenital kidney diseases.