Figure 6

The SIP1 expression pattern, RNAi knockdown of sip1 and SIP1:: GFP fusion proteins. (a) Schematic drawing shows the gene span, transcript, protein structure and reagents related to sip1 (upper panel). TIP-N: Tuftelin-interacting protein N terminal domain; G-patch: domain enriched with highly conserved glycines; GCFC: domain containing a sequence similar to a GC-rich sequence DNA-binding factor, transcriptional repressor and histone-interacting proteins. Three SIP1:: GFP fusion proteins were used in this study, including the full-length SIP1:: GFP, SIP1ΔC:: GFP and SIP1ΔN:: GFP (bottom panel). (b–d) SIP1:: sfGFP expression (green; the transgenic fly was obtained from Vienna Drosophila Resource Center, VDRC318488) was enriched in neuroblasts. Estimation of the relative SIP1:: sfGFP expression level in neuroblast and neurons can be found in Supplemental Fig. 7. The plasma membrane (outer) and the nuclear membrane (inner) of neuroblasts are indicated by arrows and arrowheads, respectively (labeled in magenta by mCD8:: RFP driven by worniu-GAL4 (wor)). (e–g) RNAi knockdown of sip1 using a GAL4 line expressed in neuroblasts, worniu-GAL4 (wor), but not using a pan-neuronal GAL4 line, synaptobrevin-GAL4 (syb), resulted in aberrant brain morphology, including overall smaller brain size, abnormal neuropil architectures (the AL was indicated by arrows). Excitatory and inhibitory neurons were visualized by choline acetyltransferase (Chat; green) and γ-aminobutyric acid (GABA; magenta) staining. A reduction of GABA-positive neuronal number dorsolateral to the AL was observed in wor > sip1^RNAi knockdown samples (dashed circles; wild-type: 123.5 ± 13.3, n = 6; wor > sip1^RNAi: 39.3 ± 11.3, n = 6; syb > sip1^RNAi: 127.3 ± 24.3, n = 6). (h–j) The Nuwa-associated neurogenesis defect in the P¹⁰²⁵²³ mutation was rescued by the full-length sip1::GFP transgene, but not sip1ΔN::GFP or sip1ΔC::GFP transgenes. Neuropils were revealed by Brp staining (blue). Scale bar: 10 μm.