Search

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/loxsystem has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/loxregulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron inhdac1, rbbp4, andrb1. Active, gene off orientationhdac1-UFlip-Offandrb1-UFlip-Offintegration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientationrbbp4-UFlip-Onandrb1-UFlip-Onintegration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs ofloxPandlox2272sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstraterbbp4-UFlip-Onandrb1-UFlip-Ongene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP inrbbp4-UFlip-Off, or 2A-KalTA4 inrb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/loxresponsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

Retinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish neurogenesis and has distinct roles in neural stem and progenitor cells. rbbp4 mutant neural stem cells show delayed cell cycle progression and become hypertrophic. In contrast, rbbp4 mutant neural precursors accumulate extensive DNA damage and undergo programmed cell death that is dependent on Tp53 signaling. Loss of Rbbp4 and disruption of genome integrity correlates with failure of neural precursors to initiate quiescence and transition to differentiation. rbbp4; rb1 double mutants show that survival of neural precursors after disruption of Rb1 is dependent on Rbbp4. Elevated Rbbp4 in Rb1-deficient brain tumors might drive proliferation and circumvent DNA damage and Tp53-dependent apoptosis, lending support to current interest in Rbbp4 as a potential druggable target.Author SummaryExamining the developmental mechanisms controlling neural stem and progenitor cell behavior is critical to our understanding of the processes driving brain tumor oncogenesis. Chromatin remodelers and their associated adaptor proteins are thought to be key drivers of brain development and disease through epigenetic regulation of gene expression and maintenance of genome integrity, but knowledge of their in vivo roles in vertebrate neurogenesis is limited. The chromatin remodeler adaptor protein Rbbp4 has recently been shown to function in a mouse model of neuroblastoma and in glioblastoma multiforme cell resistance to the chemotherapeutic temozolomide. However, an in vivo requirement for Rbbp4 in neurogenesis has only just been shown by isolation of a recessive lethal mutation in zebrafish rbbp4. Here we provide conclusive genetic evidence that zebrafish rbbp4 is essential in neural stem and progenitor cell function during development. Our data reveal for the first time in vivo that Rbbp4 prevents DNA damage and activation of Tp53 signaling pathway that leads to programmed cell death. Importantly, neural progenitors that are mutant for the tumor suppressor Rb1 also depend on Rbbp4 for survival. Finally, we show that neural stem cells that have lost Rbbp4 cease dividing, and may enter a senescent like state. Together, these observations provide novel evidence that elevated expression of Rbbp4 in rb1-mutant tumors may contribute to cancer cell survival by blocking senescence and/or DNA damage-induced cell death.