Abstract
Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.
Highlights
The hypothalamic-pituitary axis regulates stress responses, growth, reproduction and energy homeostasis
Our forward genetic study in zebrafish suggests that loss of usp39 results in aberrant rb1 mRNA splicing, which likely causes elevated expression of its target e2f4, a key regulator known to have oncogenic activity when overexpressed
We established that e2f4 upregulation is a main factor responsible for the adenohypophyseal cell lineage hyperplasia observed in the zebrafish usp39 mutant
Summary
The hypothalamic-pituitary axis regulates stress responses, growth, reproduction and energy homeostasis. Neuropeptides released from the hypothalamus via the hypophyseal portal plexus control synthesis and secretion of anterior pituitary hormones [1]. Different pituitary cell types secrete hormones that regulate postnatal growth (growth hormone, GH), lactation (prolactin, PRL), metabolism (thyroid stimulating hormone, TSH), stress (adrenocorticotrophic hormone, ACTH), pigmentation (melanocytestimulating hormone, aMSH), sexual development and reproduction (luteinizing hormone, LHb, and follicle stimulating hormone, FSHb) [2]. Central and peripheral signals including hypothalamic stimulatory hormones, growth factors and estrogen cause pituitary hyperplasia, genetic instability, subsequent monoclonal growth expansion and tumor formation [3]. How developmental or acquired signals elicit plastic change in pituitary cell growth resulting in hyperplasia or benign adenomas is not fully understood [6]
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