The proneural bHLH genes Mash1, Math3 and NeuroD are required for pituitary development

in Journal of Molecular Endocrinology
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Multiple signaling molecules and transcription factors are required for pituitary development. Activator-type bHLH genes Mash1, Math, NeuroD (Neurod) and Neurogenin (Neurog) are well known as key molecules in neural development. Although analyses of targeted mouse mutants have demonstrated involvement of these bHLH genes in pituitary development, studies with single-mutant mice could not elucidate their exact functions, because they cooperatively function and compensate each other. The aim of this study was to elucidate the roles of Mash1, Math3 and NeuroD in pituitary development. Mash1;Math3;NeuroD triple-mutant mice were analyzed by immunohistochemistry and quantitative real-time RT-PCR. Misexpression studies with retroviruses in pituisphere cultures were also performed. The triple-mutant adenohypophysis was morphologically normal, though the lumen of the neurohypophysis remained unclosed. However, in triple-mutant pituitaries, somatotropes, gonadotropes and corticotropes were severely decreased, whereas lactotropes were increased. Misexpression of Mash1 alone with retrovirus could not induce generation of hormonal cells, though Mash1 was involved in differentiation of pituitary progenitor cells. These data suggest that Mash1, Math3 and NeuroD cooperatively control the timing of pituitary progenitor cell differentiation and that they are also required for subtype specification of pituitary hormonal cells. Mash1 is necessary for corticotroph and gonadotroph differentiation, and compensated by Math3 and NeuroD. Math3 is necessary for somatotroph differentiation, and compensated by Mash1 and NeuroD. Neurog2 may compensate Mash1, Math3 and NeuroD during pituitary development. Furthermore, Mash1, Math3 and NeuroD are required for neurohypophysis development. Thus, Mash1, Math3 and NeuroD are required for pituitary development, and compensate each other.

 

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    Mash1 and NeuroD expression is increased in double-mutant pituitaries. Expression of Mash1 was significantly upregulated in Math3; NeuroD double-mutant pituitaries compared with the control (A). n = 6 (control) and 3 (double mutants). Expression of NeuroD was detected in the periluminal area of the intermediate lobe and upregulated in the anterior lobe of Mash1; Math3 double-mutant pituitaries (C) compared with the control (B). A, anterior lobe; I, intermediate lobe. Bar, 25 µm (B and C). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0090.

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    Delayed pituitary differentiation in Mash1; Math3; NeuroD triple-mutant embryos. The mutant adenohypophysis was morphologically normal, but the lumen of the neurohypophysis remained unclosed (A and B). TUNEL+ cells were not significantly increased (C and D). In the mutant anterior pituitary lobes, Sox2+ cells were increased compared with the control (E and F, bracket). The proportions of Sox2+ cells were significantly increased in triple-mutant pituitaries compared with the control (G). n = 3 (control), and 3 (triple mutants). A, anterior lobe; I, intermediate lobe; P, posterior lobe; TKO, triple knockout. Bar, 50 µm (A, B, C, D, E and F). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0090.

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    Math3 is required for Pit1-lineage differentiation and compensated by Mash1 and NeuroD. At E17.5, GH+ cells were decreased in Math3-null pituitaries compared with the control (A and B). GH+ cells were more prominently decreased and scarcely detected in Mash1;Math3;NeuroD triple-mutant pituitaries (C). Quantification of GH+ cells (D). GH+ cells were significantly decreased in Math3-null pituitaries compared with the control. GH+ cells were more prominently decreased in Mash1; Math3; NeuroD triple-mutant pituitaries (D). n = 8 (control), 5 (Math3 mutants), and 3 (triple mutants). GH mRNA levels were significantly reduced in Math3 single-mutant pituitaries compared with the control (E). GH mRNA levels were further reduced in Mash1; Math3; NeuroD triple-mutant pituitaries compared with Math3 single-mutant embryos (E). n = 6 (control), 5 (Math3 mutants), and 5 (triple mutants). In Mash1; Math3; NeuroD triple-mutant mice, Pit1 mRNA levels were significantly up-regulated compared with the control (F). n = 6 (control), 5 (Math3 mutants), and 5 (triple mutants). In Mash1; Math3; NeuroD triple-mutant embryos, PRL mRNA levels were significantly up-regulated (G). n = 5 (control), 3 (Math3 mutants), and 3 (triple mutants). PRL+ cells were increased in Math3-null pituitaries compared with the control (H and I, open arrowheads). PRL+ cells were more prominently increased in Mash1; Math3 double-mutant pituitaries (J, open arrowheads). In Mash1; Math3; NeuroD triple-mutant embryos, TSH mRNA levels were not changed compared with the control (K). n = 6 (control), 4 (Math3 mutants), and 4 (triple mutants). TSH+ cells were not changed in Math3 single-mutant and Mash1;Math3; NeuroD triple-mutant pituitaries compared to the control (L, M and N). A, anterior lobe; I, intermediate lobe; TKO, triple knockout. Bar, 50 µm (A, B, C, H, I, J, L, M and N). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0090.

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    Mash1 is required for POMC lineage differentiation and compensated by Math3 and NeuroD. At E17.5, POMC+ cells were decreased in Mash1-null pituitaries compared with the control (A and B). POMC+ cells were more prominently decreased in Mash1; Math3; NeuroD triple-mutant pituitaries (C). Quantification of POMC+ cells (D). POMC+ cells were significantly decreased in Mash1-null pituitaries compared with the control. POMC+ cells were more prominently decreased in Mash1; Math3; NeuroD triple-mutant pituitaries (D). n = 5 (control), 3 (Mash1 mutants), and 3 (triple mutants). In Mash1; Math3; NeuroD triple-mutant mice, POMC mRNA levels were significantly down-regulated compared with the control (E). n = 5 (control), 4 (Mash1 mutants), and 4 (triple mutants). A, anterior lobe; I, intermediate lobe; TKO, triple knockout. Bar, 50 µm (A, B and C). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0090.

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    Mash1, Math3 and NeuroD cooperatively control gonadotrope differentiation. At E17.5, FSH+ cells were significantly decreased in Mash1; Math3; NeuroD triple-mutant pituitaries compared to the control (A, B and C, open arrowheads). n = 7 (control), and 3 (triple mutants). LH+ cells were also significantly decreased in Mash1; Math3; NeuroD triple-mutant pituitaries compared to the control (D, E and F, open arrows). n = 7 (control), and 3 (triple mutants). FSH and LH mRNA levels were significantly reduced in Mash1 single-mutant pituitaries compared with the control (G and H). FSH and LH mRNA levels were further reduced in Mash1; Math3; NeuroD triple-mutant pituitaries compared with the control (G and H). n = 5 (control), 4 (Mash1 mutants), and 5 (triple mutants). SF-1 mRNA levels were not changed between control and Mash1; Math3; NeuroD triple-mutant pituitaries (I). n = 5 (control), and 4 (triple mutants). Neurog2 mRNA levels were significantly increased in Mash1; Math3; NeuroD triple-mutant pituitaries (J). n = 3 (control), and 3 (triple mutants). Bar, 50 µm (A, B, D and E). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0090.

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    Misexpression studies with retroviruses in pituisphere cultures. All pMX-GFP-Mash1-infected cells coexpressed Mash1 and GFP (A, B, C and D). When the control virus pMX-GFP was applied, virus-infected cells did not express POMC (E, F, G and H). When pMX-GFP-Mash1 was applied, virus-infected cells did not express POMC (I, J, K and L), though a few virus-infected cells expressed POMC (M, N, O and P, arrowheads). When the control virus pMX-GFP was applied, almost all virus-infected cells strongly expressed Sox2 (Q, R, S and T). However, when pMX-GFP-Mash1 was applied, expression level of Sox2 was decreased in some of virus-infected cells (U, V, W and X, arrows). Bars, 5 µm (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W and X).

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    Misexpression studies with retroviruses in pituisphere cultures. When pMX-GFP-Math3 was applied, virus-infected cells did not express GH (A, B, C and D) though a few virus-infected cells expressed GH (E, F, G and H, arrowheads). Bars, 5 µm (A, B, C, D, E, F, G and H). A full colour version of this figure is available at https://doi.org/10.1530/JME-18-0090.

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