Physical interactions between Gsx2 and Ascl1 balance progenitor expansion versus neurogenesis in the mouse lateral ganglionic eminence

Abstract

The Gsx2 homeodomain transcription factor promotes neural progenitor identity in the lateral ganglionic eminence (LGE), despite upregulating the neurogenic factor Ascl1. How this balance in maturation is maintained is unclear. Here, we show that Gsx2 and Ascl1 are co-expressed in subapical progenitors that have unique transcriptional signatures in LGE ventricular zone (VZ) cells. Moreover, whereas Ascl1 misexpression promotes neurogenesis in dorsal telencephalic progenitors, the co-expression of Gsx2 with Ascl1 inhibits neurogenesis. Using luciferase assays, we found that Gsx2 reduces the ability of Ascl1 to activate gene expression in a dose-dependent and DNA bindingindependent manner. Furthermore, Gsx2 physically interacts with the basic helix-loop-helix (bHLH) domain of Ascl1, and DNA-binding assays demonstrated that this interaction interferes with the ability of Ascl1 to bind DNA. Finally, we modified a proximity ligation assay for tissue sections and found that Ascl1-Gsx2 interactions are enriched within LGE VZ progenitors, whereas Ascl1-Tcf3 (E-protein) interactions predominate in the subventricular zone. Thus, Gsx2 contributes to the balance between progenitor maintenance and neurogenesis by physically interacting with Ascl1, interfering with its DNA binding and limiting neurogenesis within LGE progenitors.

Conserved Gsx2/Ind homeodomain monomer versus homodimer DNA binding defines regulatory outcomes in flies and mice

“…Binding reactions containing the indicated DNA probes and protein concentrations listed in each Figure legend were mixed and incubated in the dark at room temperature for 10 min prior to gel electrophoresis. EMSAs were imaged via a Li-Cor Odyssey CLx scanner, and quantified using the Li-Cor image studio software as described previously (Roychoudhury et al 2020).…”

“…5xUAS sites (highlighted in green), Gsx2-binding sites (highlighted red for M sites and blue for D sites), and a minimal promoter (gray) were synthesized by GenScript and subcloned into pGL3-basic luciferase (Promega) between KpnI and NcoI sites. The Gsx2 pCDNA6 construct has been previously described (Roychoudhury et al 2020). The Gal4-VP16 coding DNA was cloned into pCDNA6 between KpnI and XbaI sites.…”

“…For example, fly Ind is necessary for the production of intermediate column neuroblasts and the specification of their neuronal progeny (Weiss et al 1998). In the mouse LGE, Gsx2 is necessary for the generation of secondary proliferative (i.e., subventricular zone) progenitors that are restricted to the neuronal lineage and specified to generate a variety of neuronal subtypes, including striatal projection neurons and olfactory bulb interneurons (Toresson and Campbell 2001;Waclaw et al 2009;Pei et al 2011;Roychoudhury et al 2020). Accordingly, Gsx2-null mouse mutants have significantly diminished basal ganglia (i.e., striatum) and olfactory bulb structures (Corbin et al 2000;Toresson et al 2000;Toresson and Campbell 2001;Yun et al 2001Yun et al , 2003.…”

Single Cell Enhancer Activity Maps Neuronal Lineages in Embryonic Mouse Basal Ganglia

“…This zone also had high expression of Hes5 and Fabp7 , which mark radial glia (Feng et al, 1994; Kageyama et al, 2008) (Figure S7E,H). Other genes strongly marked both VZ zones (Eisenstat et al, 1999; Petryniak et al, 2007; Roychoudhury et al, 2020), including Lhx2, Rest , and Rgcc (Figure S7B-D). Genes like Ascl1 , Gsx2 and Dlx2 began expression in the VZ as scattered cells both in the UMAP plot and by ISH (Figures 5I; S7J,K).…”

“…Gsx1 showed perhaps the most specific SVZ1 periventricular expression by ISH and was largely confined to cl-9 (Figure 5F). Cells in this ‘isthmus’ cluster also expressed high levels of known SVZ genes ( Ascl1 , Gsx2 and Hes6 ) (Long et al, 2009; Porteus et al, 1994; Roychoudhury et al, 2020) and neurogenic transition markers ( Btg2 ) (Haubensak et al, 2004) (Figure S7J-L). Gadd45g , a marker of intermediate progenitors in the cortex (Yuzwa et al, 2017), was also highest in cl-9.…”