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Development
2004 Apr 01;1318:1679-89. doi: 10.1242/dev.01055.
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Evolution of neural precursor selection: functional divergence of proneural proteins.
Quan XJ
,
Denayer T
,
Yan J
,
Jafar-Nejad H
,
Philippi A
,
Lichtarge O
,
Vleminckx K
,
Hassan BA
.
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How conserved pathways are differentially regulated to produce diverse outcomes is a fundamental question of developmental and evolutionary biology. The conserved process of neural precursor cell (NPC) selection by basic helix-loop-helix (bHLH) proneural transcription factors in the peripheral nervous system (PNS) by atonal related proteins (ARPs) presents an excellent model in which to address this issue. Proneural ARPs belong to two highly related groups: the ATONAL (ATO) group and the NEUROGENIN (NGN) group. We used a cross-species approach to demonstrate that the genetic and molecular mechanisms by which ATO proteins and NGN proteins select NPCs are different. Specifically, ATO group genes efficiently induce neurogenesis in Drosophila but very weakly in Xenopus, while the reverse is true for NGN group proteins. This divergence in proneural activity is encoded by three residues in the basic domain of ATO proteins. In NGN proteins, proneural capacity is encoded by the equivalent three residues in the basic domain and a novel motif in the second Helix (H2) domain. Differential interactions with different types of zinc (Zn)-finger proteins mediate the divergence of ATO and NGN activities: Senseless is required for ATO group activity, whereas MyT1 is required for NGN group function. These data suggest an evolutionary divergence in the mechanisms of NPC selection between protostomes and deuterostomes.
Fig. 1. The proneural activities of Atonal-related proteins. (A-D) Whole-mount in situ hybridisation with an N-tubulin probe to visualise neurogenesis in Xenopus embryos at stage 15. (A) Uninjected embryos. (B) 500 pg Ngn1 mRNA. (C) 500 pg Ato mRNA. (D) 500 pg Math1 mRNA. (E) Part of a wild-type fly wing showing no sensory bristles along the AP axis. (F) A uasMath1/+; dppGal4/+ wing. (G) A uasngn1/+; dppGal4/+ wing. (H) Quantitative analysis of the number of ectopic bristles per fly induced by expression of MATH1 or NGN1 using dppGal4 driver, `n' is the number of flies counted. (I-K) Third instar larval (L3) wing discs stained with anti-β-GAL (green) and proneural antibodies (red). (I) An A101/TM6 wing disc. (J) A uasato,dppGal4/A101 wing disc, anti-ATO (red) and anti-β-GAL (green). (K) A uasngn1, dppGal4/A101 wing disc, anti-NGN1 (red), anti-β-GAL (green). (L-N) L3 wing discs stained with anti-ASE. (L) A wild-type fly (CS) wing disc. (M) A uasato,dppGal4/TM6 wing disc. (N) A uasngn1, dppGal4/TM6 wing disc. (O,P) Late stage embryos stained with 22C-10 (green) and anti-NGN1 (red). (O) A CS embryo. (P) A daGal4/uasngn1 embryo. (Q) A uasngn1;Gal4-7,ato1/ato1 L3 eye disc stained with anti-SENS. (Inset) A uasngn1;Gal4-7,ato1/TM6 L3 eye disc stained with anti-SENS revealing the R8 cells.
Fig. 4. NGN proteins and ATO proteins interact with different Znfinger proteins. (A) A scutellum of a uassens/+; C5Gal4/+ fly. Some ectopic microchaetes are indicated by arrows. (B) Ectopic microchaete on a uasMath1/+; C5Gal4/+ fly scutellum. (C) Ectopic microchaete on a uassens/+; C5Gal4/uasngn1 fly scutellum. (D) A scutellum of uassens/+; uasMath1/+; C5Gal4/+ fly. (E) Quantitative analysis of the effect of SENS on NGN1 and MATH1. (F-K) Detection of N-tubulin expression via whole-mount in situ hybridization in stage 19 Xenopus embryos, injected or co-injected with different mRNAs into a single blastomere at two-cell stage. (F) 250 pg X-MyT1 mRNA. (G) 250 pg Ngn1 mRNA. (H) 1000 pg Math1 mRNA. (I) 250 pg X-MyT1 and 250 pg Ngn1 mRNAs. (J) 250 pg X-MyT1 and 1000 pg Ato mRNAs. (K) 250 pg X-MyT1 and 1000 pg Math1 mRNAs.
Fig. 2. Differential encoding of proneural activity in the bHLH domains of NGN proteins and ATO proteins. (A) The basic domains of ATO (red) and NGN1 (purple). Group-specific amino acids are in green. (B) Schematic representation of NGNbATO with exchanged amino acids in red. (C) Quantitative analysis of proneural activity of misexpressed ATO, NGN1, NGNbATO in a wild-type background. (Inset) A wing from a uasngnbato/+; dppGal4/+ fly. (D) Schematic representation of ATObNGN with the exchanged amino acids in purple. (E,F) N-tubulin stained Xenopus embryo at stage 19, injected with different mRNAs into one cell (right side) of two cell-stage embryos. (E) 1000 pg of Ato mRNA. (F) 1000 pg of AtobNGN mRNA. (G) Schematic representation of NGNH2ATO and ATOH2NGN. (H) Quantitative analysis of proneural activity of misexpressed ATO (blue), NGN1 (dark pink), NGNH2ATO (light pink). (I) N-tubulin stained Xenopus embryo at stage 19, injected with 1000 pg of AtoH2NGN mRNA into one cell of two-cell stage embryos.
Fig. 5. NGNbATO and ATObNGN have reversed interactions with Zn-finger proteins. (A-C) uasngnbato/+; dppGal4/+ L3 wing disc stained with (A) anti-NGN1 (red) and (B) anti-SENS (green). (C) A merged image of A and B shows that misexpression of NGNbATO induces SENS. (D) Quantitative analysis of the SENS effect on NGNbATO. (E-H) Injected Xenopus embryos at stage 19, stained with N-tubulin. (E) 100 pg AtobNGN. (F) 100 pg AtobNGN and 250 pg X-MyT1. (G) 100 pg AtoH2-NGN. (H) 100 pg of AtoH2-NGN and 250 pg X-MyT1 mRNA.
