Dataset Information

Xenopus laevis kidney-induced animal caps nanoLC-MS/MS - Xenopus Ssbp2 is required for embryonic pronephros morphogenesis and terminal differentiation.

ABSTRACT: The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays critical roles in kidney development, regulating target gene expression by forming multiprotein complexes with LIM binding protein 1 (Ldb1). However, few Lhx1-Ldb1 cofactors have been identified for this organ formation. By tandem- affinity purification from kidney-induced Xenopus animal caps, we identified single-stranded DNA binding protein 2 (Ssbp2) to interact with the Ldb1-Lhx1 complex. Ssbp2 is expressed in the Xenopus pronephros, and knockdown prevents normal morphogenesis and differentiation of the glomus and the convoluted renal tubules. We demonstrate a role for a member of the Ssbp family in kidney organogenesis and provide evidence of a fundamental function for the Ldb1-Lhx1-Ssbp transcriptional complexes in embryonic development.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

TISSUE(S): Embryo, Early Embryonic Cell

SUBMITTER: Maria Cirio

LAB HEAD: Mairia Cecilia Cirio

PROVIDER: PXD041761 | Pride | 2023-10-24

REPOSITORIES: Pride

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			Action	DRS
	OTHER_FILE_Subset_Database.fasta	Fasta
	RAW_FILE_ProtXML	Other
	SEARCH_FILE_Animal_Caps_Peptide_and_Protein_Prophet.xml	Xml
	checksum.txt	Txt

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Xenopus Ssbp2 is required for embryonic pronephros morphogenesis and terminal differentiation.

Cervino Ailen S AS Collodel Mariano G MG Lopez Ivan A IA Roa Carolina C Hochbaum Daniel D Hukriede Neil A NA Cirio M Cecilia MC

Scientific reports 20231004 1

The nephron, functional unit of the vertebrate kidney, is specialized in metabolic wastes excretion and body fluids osmoregulation. Given the high evolutionary conservation of gene expression and segmentation patterning between mammalian and amphibian nephrons, the Xenopus laevis pronephric kidney offers a simplified model for studying nephrogenesis. The Lhx1 transcription factor plays several roles during embryogenesis, regulating target genes expression by forming multiprotein complexes with L ...[more]

PMID: 37794075

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Project description:The goal of this study is to investigate the molecular mechanism of lhx1 on regulation of pronephros formation during the early embryonic development. In the vertebrate embryo the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. The animal cap cells can be induced by treatment of activin and retinoic acid to differentiate into pronephros tissue. In this study we investigated the role of Lhx1 in differentiation of pronephros by depleting lhx1 in the organ culture system. We generated the gene expression profile of early pronephros tissue, and demonstrated that expression of genes from all the kidney domains is affected by the absence of lhx1. Taken together our results highlight an essential role for Lhx1 in pronephros formation. lhx1 is involved in driving specification of intermediate mesoderm into nephrogenic mesenchyme. Lhx1 is initially expressed throughout the entire intermediate mesoderm. To determine the role of lhx1 pronephros formation, we performed a microarray analysis using an explant culture system. Xenopus tissue explants can be surgically isolated and cultured under specific conditions to be driven towards many distinct tissue types. Formation of pronephric cell fates is induced by culturing isolated explants in the presence of Activin and RA (AcRA). Treatment of dissected explants of stage 9 blastulae embryos with 10ng/ml Activin and 1x10-4 M retinoic acid can induce differentiation of the pluripotent ectoderm into pan-kidney tissue. For this experiment, both blastomeres of 2-cell embryos were injected with a total of 800pg lhx1 DEED-AS. Explants were dissected and treated with AcRA and expression of pax8 at stage 15 (based on timing of paired control whole embryos) was analyzed. We observed a lack of induction of pax8 expression in lhx1-depleted explants under AcRA treatment conditions in which expression of this gene is normally induced. Based on this observation, microarray analysis was carried out to identify genes whose expression is affected by the absence of lhx1. Explants of injected embryos with 800pg of lhx1 DEED-AS were dissected, treated with pronephric tissue inductive conditions (AcRA) and harvested after 24 hours incubation at 14C (Fig. S5B). The sibling control embryos reached stage 12.5. Explants from uninjected embryos +AcRA and -AcRA as well as explants from DEED injected embryos -AcRA were also harvested. Approximately 12 caps were pooled for each RNA preparation and the analysis was performed using triplicates.

Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate and proton secreting cells or with dominant negative human Mastermind (HMM) to induce the formation of ectopic multi-ciliate and proton secreting cells. Results show which genes are up or down-regulated when HMM is compared to ICD. Specialized epithelial cells in the amphibian skin play important roles in ion transport, but how they arise developmentally is largely unknown. Here we show that proton-secreting cells (PSCs) differentiate in the X. laevis larval skin soon after gastrulation, based on the expression of “kidney” specific form of the H+v-ATPase that localizes to the plasma membrane, orthologs of the Cl-/HCO3- antiporters ae1 and pendrin, and two isoforms of carbonic anhydrase. Like PSCs in other species, we show that the expression of these genes is likely to be driven by an ortholog of foxi1, which is also sufficient to promote the formation of PSC precursors. Strikingly, the PSCs form in the skin as two distinct subtypes that resemble the alpha- and beta-intercalated cells of the kidney. The alpha-subtype expresses ae1 and localizes H+v-ATPases to the apical plasma membrane, whereas the beta-subtype expresses pendrin and localizes the H+v-ATPase cytosolically or basolaterally. These two subtypes are specified during early differentiation, and can be distinguished based on the expression of ubp1, a transcription factor in the grainyhead family. Finally, we show that ectopic expression ubp1 is sufficient to promote the differentiation of beta-subtypes while repressing alpha-subtypes. These results have implications for how PSCs are specified in vertebrates and become functionally heterogeneous. 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD or HMM. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 22 RNA was harvested from explants and used as the starting material for arrays.