Total RNA (1-1.5?g) was retrotranscribed using first-strand cDNA MMLV-retrotranscriptase (Invitrogen) and random primers. markers are upregulated in cerebellar ventricular zone progenitors. Our evidence sheds light on the domain-specific roles played by ZFP423 in different aspects of PC progenitor development, and at the same time strengthens the emerging notion that an impaired DDR may be a key factor in the pathogenesis of JS and other ciliopathies. gene mutations/deletions have been diagnosed with JS, CVH, nephronophthisis (NPHP) and other signs of ciliopathy (Chaki et al., 2012). Although ZFP423 has been convincingly implicated in the cilium-mediated Fgfr2 response to sonic hedgehog (SHH) during cerebellar granule cell (GC) proliferation (Hong and Hamilton, 2016), our observations clearly point to an additional key role for this protein in PC development long before the onset of GC clonal expansion. Incidentally, GC clonal expansion relies on SHH released by PCs starting before birth (Dahmane and Ruiz-i-Altaba, 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999), so that the final number of GCs is heavily influenced by the total number of postmitotic PCs. encodes a 30 zinc-finger nuclear protein that works both as a scaffold and as a transcription factor, cooperating with multiple regulatory molecules. Through a domain spanning zinc fingers 9-20, ZFP423 acts a co-activator in BMP (Hata et al., 2000) and Notch (Masserdotti et al., 2010) signaling pathways. Although the role of BMP signaling in granule cell development has been clearly established (reviewed by Roussel and Hatten, 2011), its involvement in PC development can only be partially inferred from the analysis of conditional SMAD4-null mice, although SMAD4 is not Madecassoside exclusively a BMP signaling transducer (Massagu, 2000). These mice display a marked decrease in the number of PCs and parvalbumin-positive interneurons (Zhou et al., 2003). As regards Notch, its importance in the genesis of PCs has been characterized through both constitutive (Ltolf et al., 2002) and conditional mutants (Machold et al., 2007) that exhibit a massive decrease in PC number. Moreover, through a C-terminal Madecassoside domain spanning zinc fingers 28-30, ZFP423 interacts with EBF transcription factors (Tsai and Reed, 1997, 1998), which are involved in cerebellar development (Croci et al., 2006, 2011) and molecular patterning of the cerebellar cortex (Chung et al., 2008, 2009). To date, the null mutation is the only genetic manipulation thus far shown to subvert PC subtype specification (Croci et al., 2006). acts to repress the zebrin II+ phenotype in late-born PCs (Chung et al., 2008). Thus, the possible interaction of ZFP423 with these regulatory signals in the context of PC development remains a relevant unanswered question. Importantly, ZFP423/ZNF423 also interacts with Poly ADP-ribose polymerase 1 (PARP1) through zinc fingers 9-20 (Ku et al., 2006) and with centrosomal protein 290 (CEP290) through an N-terminal domain (Chaki et al., 2012). PARP1 is a double-stranded (ds) DNA-damage sensor that recruits MRE11 and ataxia-telangectasia mutated (ATM) to sites of DNA damage. CEP290 is a centrosomal protein mutated in JS and NPHP, the loss of which causes enhanced DNA-damage signaling, DNA breaks, replication stress and supernumerary centrioles (Slaats et al., 2015). Because a successful DNA-damage response (DDR) requires a tight control over cell cycle checkpoints, we postulated Madecassoside that defective DNA-damage signaling might delay cell cycle progression, contributing to the hypoplastic cerebellum seen in mutant mice and patients alike. Interestingly, recent evidence supports the notion of a broad role for ciliopathy genes in the DDR: in fact, both CEP290 and NEK8 mutations lead to an accumulation of DNA damage due to disturbed replication forks (Choi et al., 2013; Slaats et al., 2015). Furthermore, increased DNA-damage signaling has been detected in CEP164-, ZNF423- and SDCCAG8-associated renal cells (Airik et al., 2014; Chaki et al., 2012). In the present paper, we describe the results of a detailed analysis of two allelic in-frame deletion mouse lines, each characterized by nullisomy for a distinct functionally characterized protein-protein.