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, 103 (46), 17284-9

A Subunit of the Mediator Complex Regulates Vertebrate Neuronal Development

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A Subunit of the Mediator Complex Regulates Vertebrate Neuronal Development

Xiaoqun Wang et al. Proc Natl Acad Sci U S A.

Abstract

The unique profiles of gene expression dictate distinct cellular identity. How these profiles are established during development is not clear. Here we report that the mutant motionless (mot), identified in a genetic screen for mutations that affect neuronal development in zebrafish, displays deficits of monoaminergic neurons and cranial sensory ganglia, whereas expression of the pan-neuronal marker Hu is largely unperturbed; GABAergic and subsets of cranial motor neurons do not appear to be deficient. Positional cloning reveals that mot encodes Med12, a component of the evolutionarily conserved Mediator complex, whose in vivo function is not well understood in vertebrates. mot/med12 transcripts are enriched in the embryonic brain and appear distinct from two other Mediator components Med17 and Med21. Delivery of human med12 RNA into zebrafish restores normality to the mot mutant and, strikingly, leads to premature neuronal differentiation and an increased production of monoaminergic neuronal subtypes in WT. Further investigation reveals that mot/med12 is necessary to regulate, and when overexpressed is capable of increasing, the expression of distinct neuronal determination genes, including zash1a and lim1, and serves as an in vivo cofactor for Sox9 in this process. Together, our analyses reveal a regulatory role of Mot/Med12 in vertebrate neuronal development.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The mot mutant displays overall normal brain patterning and expression of the pan-neuronal marker Hu. Anterior is to the left, and dorsal is up. (A, C, E, G, and I) WT. (B, D, F, H, and J) mot. (AF) In situ hybridization shows the expression of shh (A and B), pax2 (C and D), and pax6 (E and F). (GJ) Immunostaining shows the pattern of the pan-neuronal marker Hu. fb, forebrain; hb, hindbrain; mb, midbrain; MHB, midbrain hindbrain boundary. (Magnifications: A, B, and GJ, ×200; CF, ×100.)
Fig. 2.
Fig. 2.
The mot mutation disrupts the development of distinct neuronal classes in the brain. All are lateral views except G, H, and MP, which are dorsal views. (A, C, E, G, I, K, M, and O) WT. (B, D, F, H, J, L, N, and P) mot. (A and B) Whole-mount in situ hybridization shows TH+ forebrain dopaminergic neurons, hindbrain LC NA neurons, and NA arch-associated CA cells. (C and D) 5HT immunostaining shows forebrain and hindbrain 5HT neurons. (EH) Immunostaining shows Hu+ cranial sensory ganglia (E and F) and TH+ sympathetic ganglia (G and H). (IL) In situ hybridization shows gad67+ GABAergic neurons. (M and N) In situ hybridization shows phox2a+ ocular and trochlear and hindbrain motor nuclei. (O and P) In situ hybridization with islet-1 shows hindbrain cranial motor neurons. AAC, arch-associated CA cells; C, cranial sensory neurons; OT, ocular and trochlear motor neurons; Sym, sympathetic ganglia. (Magnifications: ×400.)
Fig. 3.
Fig. 3.
Positional cloning identifies the mot gene as zebrafish med12. (A) The genetic and physical map of the mot locus on zebrafish LG 14 is shown. (B) Schematic drawing shows the structure of the predicted WT mot/med12 gene product and the truncated gene product in the motm807 mutant. L, leucine-rich domain; LS, leucine- and serine-rich domain; PQL, proline-, glutamine-, and lucine-rich domain; OPA, glutamine-rich domain. (C) DNA sequence chromatograms show the molecular nature of the motm807 mutation.
Fig. 4.
Fig. 4.
Expression of mot/med12, med17, and med21 is shown. (A and B) Maternal mot/med12 expression. (C and D) mot/med12 expression in 24-hpf WT and the mot mutant embryo. (E and F) Higher magnification view of the ≈24-hpf embryonic brain showing mot/med12 expression (Inset is sectioned image). (G and H) mot/med12 expression in 48 hpf embryos shows enrichment in clusters of cells dorsal and posterior to the eyes (black arrows), and such staining is significantly reduced in the mot mutant. White arrows indicate background staining. (IN) Expression of med17 (I, K, and M) and med21 (J, L, and N) in 24- and 48-hpf embryos. (Magnifications: ×200.)
Fig. 5.
Fig. 5.
Overexpression of mot/med12 in WT embryos is shown. All are lateral views. (A, C, E, G, I, and K) WT injected with β-gal RNA. (B, D, F, H, J, and L) WT injected with med12 RNA. (AF) TH immunostaining shows that TH+ dopaminergic neurons (B) and LC NA neurons (D) are detected precociously in med12 RNA-injected embryos; dopaminergic (F) and LC NA (D) are also increased in med12 RNA-injected embryos. (G and H) 5HT neuron increase in med12 RNA-injected embryos. (IL) Hu labeling (I and J) and GAD67 in situ hybridization (K and L) show comparable patterns between control and the med12 RNA-injected embryos. (Magnifications: AH, ×200; IL, ×100.)
Fig. 6.
Fig. 6.
mot/med12 regulates the expression of distinct neuronal determination genes. (AF) In situ hybridization shows the expression of zash1a, lim1, and dlx2 in the brain of WT (A, C, and E) and the mot mutant (B, D, and F) embryos. (GL) In situ hybridization shows the expression of zash1a, lim1, and dlx2 in WT embryos injected with β-gal RNA (G, I, and K) and human med12 mRNA (H, J, and L). (Magnifications: ×100.)
Fig. 7.
Fig. 7.
Sox9 regulates lim1 and zash1a expression in a mot/med12-dependent manner. All are lateral views of the embryonic brain. (AD) lim1 and zash1a expression in WT (A and C) and sox9asox9b double mutant (B and D) embryos. (EH) lim1 and zash1a expression in WT embryos injected with β-gal RNA (E and G) and sox9a mRNA (F and H). (IL) lim1 and zash1a expression in WT (I and K) and mot mutant (J and L) embryos injected with sox9a mRNA. (Magnifications: AH, 200×; IL, ×100.)

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