25Retinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin 26 remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in 27 zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human 28 embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish 29 neurogenesis and has distinct roles in neural stem and progenitor cells. rbbp4 mutant neural 30 stem cells show delayed cell cycle progression and become hypertrophic. In contrast, rbbp4 31 mutant neural precursors accumulate extensive DNA damage and undergo programmed cell 32 death that is dependent on Tp53 signaling. Loss of Rbbp4 and disruption of genome integrity 33 correlates with failure of neural precursors to initiate quiescence and transition to differentiation. 34 rbbp4; rb1 double mutants show that survival of neural precursors after disruption of Rb1 is 35 dependent on Rbbp4. Elevated Rbbp4 in Rb1-deficient brain tumors might drive proliferation 36 and circumvent DNA damage and Tp53-dependent apoptosis, lending support to current 37 interest in Rbbp4 as a potential druggable target. 38 39 40 41 42 43 3 Author Summary 44 45Examining the developmental mechanisms controlling neural stem and progenitor cell behavior 46 is critical to our understanding of the processes driving brain tumor oncogenesis. Chromatin 47 remodelers and their associated adaptor proteins are thought to be key drivers of brain 48 development and disease through epigenetic regulation of gene expression and maintenance of 49 genome integrity, but knowledge of their in vivo roles in vertebrate neurogenesis is limited. The 50 chromatin remodeler adaptor protein Rbbp4 has recently been shown to function in a mouse 51 model of neuroblastoma and in glioblastoma multiforme cell resistance to the chemotherapeutic 52 temozolomide. However, an in vivo requirement for Rbbp4 in neurogenesis has only just been 53shown by isolation of a recessive lethal mutation in zebrafish rbbp4. Here we provide conclusive 54 genetic evidence that zebrafish rbbp4 is essential in neural stem and progenitor cell function 55 during development. Our data reveal for the first time in vivo that Rbbp4 prevents DNA damage 56 and activation of Tp53 signaling pathway that leads to programmed cell death. Importantly, 57 neural progenitors that are mutant for the tumor suppressor Rb1 also depend on Rbbp4 for 58 survival. Finally, we show that neural stem cells that have lost Rbbp4 cease dividing, and may 59 enter a senescent like state. Together, these observations provide novel evidence that elevated 60 expression of Rbbp4 in rb1-mutant tumors may contribute to cancer cell survival by blocking 61 senescence and/or DNA damage-induced cell death. 62 63 64 65 66 6 the lesion ( Fig 1G) that frequently co-labeled with PCNA ( Fig 1H, 1I). These results confirm 132 overexpression of Rbbp4 protein in zebrafish rb1-brain tumors, as predicted by our previous 133 RNA-Seq analysis (11). 134 135 136

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/lox system 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/lox regulated 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 in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, 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 demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-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/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.