Six4, a putative myogenin gene regulator, is not essential for mouse embryonal development.
Journal: 2001/June - Molecular and Cellular Biology
ISSN: 0270-7306
Abstract:
Six4 is a member of the Six family genes, homologues of Drosophila melanogaster sine oculis. The gene is thought to be involved in neurogenesis, myogenesis, and development of other organs, based on its specific expression in certain neuronal cells of the developing embryo and in adult skeletal muscles. To elucidate the biological roles of Six4, we generated Six4-deficient mice by replacing the Six homologous region and homeobox by the beta-galactosidase gene. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of the heterozygous mutant embryos revealed expression of Six4 in cranial and dorsal root ganglia, somites, otic and nasal placodes, branchial arches, Rathke's pouch, apical ectodermal ridges of limb buds, and mesonephros. The expression pattern was similar to that of Six1 except at the early stage of embryonic day 8.5. Six4-deficient mice were born according to the Mendelian rule with normal gross appearance and were fertile. No hearing defects were detected. Six4-deficient embryos showed no morphological abnormalities, and the expression patterns of several molecular markers, e.g., myogenin and NeuroD3 (neurogenin1), were normal. Our results indicate that Six4 is not essential for mouse embryogenesis and suggest that other members of the Six family seem to compensate for the loss of Six4.
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Mol Cell Biol 21(10): 3343-3350

<em>Six4</em>, a Putative <em>myogenin</em> Gene Regulator, Is Not Essential for Mouse Embryonal Development

+3 authors
Departments of Biology, Otolaryngology, and Pathology, Jichi Medical School, Tochigi 329-0498, Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, and Division of Cell Biology, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
Corresponding author. Mailing address: Department of Biology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. Phone: 81 (285) 58-7311. Fax: 81 (285) 44-5476. E-mail: pj.ca.ihcij@makawakk.
Present address: Department of Otolaryngology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan.
Present address: Institute for Experimental Animals, Faculty of Medicine, Kanazawa University, Kanazawa 920-8640, Japan.
Received 2000 Nov 27; Revisions requested 2001 Jan 9; Accepted 2001 Feb 21.

Abstract

Six4 is a member of the Six family genes, homologues of Drosophila melanogaster sine oculis. The gene is thought to be involved in neurogenesis, myogenesis, and development of other organs, based on its specific expression in certain neuronal cells of the developing embryo and in adult skeletal muscles. To elucidate the biological roles of Six4, we generated Six4-deficient mice by replacing the Six homologous region and homeobox by the β-galactosidase gene. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining of the heterozygous mutant embryos revealed expression of Six4 in cranial and dorsal root ganglia, somites, otic and nasal placodes, branchial arches, Rathke's pouch, apical ectodermal ridges of limb buds, and mesonephros. The expression pattern was similar to that of Six1 except at the early stage of embryonic day 8.5. Six4-deficient mice were born according to the Mendelian rule with normal gross appearance and were fertile. No hearing defects were detected. Six4-deficient embryos showed no morphological abnormalities, and the expression patterns of several molecular markers, e.g., myogenin and NeuroD3 (neurogenin1), were normal. Our results indicate that Six4 is not essential for mouse embryogenesis and suggest that other members of the Six family seem to compensate for the loss of Six4.

Abstract

Six family genes are homologues of Drosophila melanogaster sine oculis, one of the homeobox genes essential for compound eye formation (5). They are characterized by the presence of the Six domain and Six-type homeodomain in the encoded proteins, which confer specific DNA binding activity and function as transcription factors (12, 28). In mammals, six members of the family have so far been identified (24, 7, 8, 10, 12, 13, 31, 32, 34, 41; for a review, see reference 14). Each member of the family is expressed in a spatiotemporally regulated manner during embryogenesis. In mice, Six3 and Six6 are exclusively expressed in the developing forebrain and eyes (10, 22, 31, 40). In contrast, Six1, Six2, and Six5 show relatively broader expression patterns (15, 32). Six1 is expressed in the cranial and dorsal root ganglia, somites, otic and nasal placodes, branchial arches, limbs, Rathke's pouch, and nephrogenic cords (32). Six2 is expressed in head mesenchyme, branchial arches, limbs, and some mesenchymal regions surrounding the gastrointestinal tract (32). Six5 shows a broad expression in branchial arches, limb buds, telencephalon, eye, sclerotomes, and cartilages (15). Such distribution suggests that these genes play specific roles in embryogenesis.

Overexpression and misexpression experiments showed that Six3 and Six6 genes are involved in forebrain and eye organogenesis (18, 21, 30, 48). Consistently, SIX3 mutations cause holoprosencephaly, a severe malformation of the brain in humans (42). SIX5 is located immediately downstream of the CTG trinucleotide repeats whose expansion causes myotonic dystrophy (3). Downregulation of the gene was observed in myotonic dystrophy patients and is thought to be responsible for some of the symptoms of the disease such as cataracts (16, 44). In agreement with this, Six5-heterozygous and -homozygous mutant mice develop cataracts (15, 35).

Six4 was originally identified as a binding factor to the positive regulatory element of the Na,K-ATPase α1 subunit gene (Atp1a1) (12, 38). Immunostaining showed the presence of Six4 protein in some populations of neuronal cells in developing mouse embryos and developing retina (27, 29). In adult mice, Six4 protein is localized in skeletal muscles, as demonstrated by gel retardation assay (37). These observations suggest that this gene is involved in neurogenesis, myogenesis, and probably the development of other organs. In myogenesis, myogenin plays an important role along with other myogenic genes such as MyoD, Myf5, and MRF4 (33). Promoter analysis with transgenic mice demonstrated that the MEF3 site in the promoter region is essential for myogenin expression (37). Mouse Six1 and Six4 proteins are present in the developing somites in which myogenin expression is activated and bind to the MEF3 site in the myogenin promoter, as shown by gel retardation assay (37). Furthermore, Six4 can activate the myogenin gene promoter alone or in synergy with the specific cofactor, Eya, through direct binding to the MEF3 site in cultured cells (28). These results support the notion that Six4 is one of the genes that control myogenesis through activation of myogenin. In addition, Eya1, one of the specific cofactors of Six4, is implicated in branchio-oto-renal syndrome, a dominantly inherited disorder characterized by hearing loss and branchial arch and renal anomalies in humans (1). Eya1-deficient mice lack ears and kidneys, and heterozygous mutant mice show hearing loss and renal anomalies, as seen in human branchio-oto-renal syndrome (46). Because immunostaining showed the presence of Six4 protein in acoustic ganglia and otic vesicles (29), it is possible that Six4 is involved in the development of the ear in association with Eya1. Nevertheless, the biological function of Six4 in development is not clear, due to the lack of natural mutants, knockout models, and overexpression experiments. To access the biological function of Six4, we generated Six4-deficient mice and analyzed phenotypic changes both in adults and in embryos. We found no apparent changes in morphology and expression patterns of some marker genes in Six4-deficient mice. Functionally, hearing ability was normal. The reason for the lack of phenotype in Six4-deficient mice and the possible compensation among Six family genes is discussed.

ACKNOWLEDGMENTS

We thank F. Relaix for discussion and critical reading of the manuscript, S. J. Tapscott for providing mouse NeuroD cDNA, Q. Ma for providing neurogenin1 cDNA, and T. Yagi for providing pMC1DTpA. We also thank M. Yamakado, K. Ikeda, and S. Sato for discussion and H. Ohto and M. Kikuchi for technical assistance.

This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan and from the Ministry of Health and Welfare of Japan and by the Jichi Medical School Young Investigator Award.

ACKNOWLEDGMENTS

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