The anterior pituitary controls key biological processes, including growth, metabolism, reproduction and stress responses through distinct cell types that each secrete specific hormones. The anterior pituitary cells show a remarkable level of cell type plasticity that mediates the shifts in hormone producing cell populations that are required to meet organismal needs. The molecular mechanisms underlying pituitary cell plasticity are not well understood. Recent work has implicated the pituitary stem cell populations and specifically, the mRNA binding proteins of the Musashi family in control of pituitary cell type identity. In this study we have identified the target mRNAs that mediate Musashi function in the adult mouse pituitary and demonstrate the requirement for Musashi function in vivo. Using Musashi RNA immunoprecipitation, we identify a cohort of 1184 mRNAs that show specific Musashi binding. Identified Musashi targets include the Gnrhr mRNA, which encodes the gonadotropin releasing hormone receptor (GnRHR), and the Fshb mRNA, encoding follicle-stimulating hormone (FSH). Reporter assays reveal that Musashi functions to exert repression of translation of the Fshb mRNA, in addition to the previously observed repression of the Gnrhr mRNA. Importantly, mice engineered to lack Musashi in gonadotropes demonstrate a failure to repress translation of the endogenous Gnrhr and Fshb mRNAs during the estrous cycle and display abberant ovarian follicle developement. The range of identified target mRNAs suggests that, in addition to these key gonadotrope proteins, Musashi may exert broad regulatory control over the pituitary proteome in a cell-type specific manner.

Description of the data and file structure

This supplemental data set includes supporting figures and Tables as cited in the primary manuscript. These include Author generated RNA sequencing data (see Gene Expression Omnibus (GEO) under accession number GSE199098) with differential mRNA enrichment in Musashi vs control IgG immunoprecipitation performed as described in primary manuscript.

Supp. Figure S1- additional bioinformatic analyses performed as described in accompanying supplementary figure legends.

Supp. Figure S2 - validation of mouse genotypes

Supp. Figure S3 - additional charaacterization of mouse gonadotropin levels, mRNA and protein

Supplemental Tables 1-7 - include all recovered mRNAs (Supp. Table 1); mRNAs enriched in Musashi1/2 over IgG control immunoprecipitations log2FC>1 and adj P <0.1 (Supp. Table 2); mRNA enriched in Musashi immunoprecipitations apparently lacking MBEs (Supp. Table 3); pituitary lineage-restricted Musashi target mRNAs (Supp. Table 4); ; Musashi target mRNA comaprison to other published studies (Supp. Table 5); and GO analysis of mRNAs common to Msi1 or MSi2 across species and tissue types (Supp. Table 6).

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