4. DISCUSSION
4.2 Functional characterization of the Sox15 gene and its role in muscle differentiation
4.2.1 Introduction and expression of the Sox15 gene
Sox genes encode a group of proteins that carry a DNA binding HMG domain implicated in transcriptional regulation (Pevny and Lovell-Badge 1997). Sox genes are expressed in various phases of embryonic development and cell differentiation in a manner linked to cell specification. Genes which belong to Sox family show a variety of functions concerning with development, including sex determination, early embryogenesis, neural development, chondrogenesis, etc. The functions of Sox family genes are summarized in table 4.1.
Sox proteins interact with DNA through the HMG domain, which is a 79-amino acid protein motif. Because Sox HMG domains share a number of conserved amino acid residues, HMG box sequences of the Sox genes have been identified by PCR-based cloning.
Sox proteins were initially registered and classified by the deduced amino acid sequence of the HMG domain alone. To date, over 20 Sox proteins and their genes have been identified in vertebrates and they have been grouped into Groups A-I, Group G being assigned to Sox15. Our current data concurring Sox15 provide direct evidence for a specific role of Sox15 in regulation of myoblast proliferation, differentiation and regeneration.
The Sox15 gene is specifically expressed in proliferating myoblasts. Detailed expression analysis of the distribution of Sox15 mRNA was conducted using RT-PCR and Northern blot analysis. The RT-PCR analysis revealed that the Sox15 is ubiquitously expressed in adult tissues. However, Northern blot analysis demonstrated that the Sox15 was expressed exclusively in embryonic stem (ES) cells and proliferating primary myoblasts. No Sox15 transcript was detectable in 20 µg of total RNA from several adult mouse tissue samples.
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There are two explanations for the discrepancy between the results obtained by RT-PCR and Northern blot analysis. (1) The Sox15 is expressed at high level in the ES cells and myoblast and at low level in the adult tissues. (2) The expression of the Sox15 is restricted to the embryonic and adult stem cells. Based on the relatively low number of stem cells in the adult tissues, the Sox15 transcript could be only detected using the RT-PCR assay.
Immunostaining using the monospecific anti-Sox15 antibody revealed that the Sox15 was down-regulated after myogenic differentiation. Furthermore, Sox15 was detected in nucleus of proliferating C2C12 mouse myoblast, which is a continuous cell line originally derived from myogenic stem cells. In contrast, no Sox15 was detected in non-muscle cell line as primary fibroblasts and Swiss3T3 cell line. These results clearly demonstrate that Sox15 is highly expressed in proliferated myogenic stem cells and down-regulated after the fusion and differentiation of the myogenic cells. It remains to be addressed, whether the Sox15 is expressed at high level in other adult stem cells such as spermatogonia, hematopoietic and neural stem cells.
An alternative method to determine the specific expression of the Sox15 in different precursor cells is the induction of ES cells to differentiation and immunostaining the resulted precursor cells with anti-Sox15 antibody and different antibodies, which recognize specific protein in different stem cells.
(Hiraoka et al. 1998) reported that SOX20 is highly expressed in fetal testis and down-regulated in adult testis. These results led to suggest that its murine homolog Sox15 has a potential role in the sex determination or testis development. However, the genotyping PCR, combining with the Sry gene PCR, of 10 Sox15-/- mice showed that there was no sex reversal like in Sox9 knock-out mice. Indeed, immunohistological section of Sox15-/- testis revealed normal structure of testis (data not shown).
Based on the increased expression of Sox15 in proliferating primary myoblasts and down regulation of the gene after myogenic differentiation lead us to consider the hypothesis that Sox15 may have some roles in proliferation and differentiation of myogenic cells and regeneration of skeletal muscle. To confirm this hypothesis we have investigated the following points:
1. The proliferation and differentiation potential of primary Sox15-/- myogenic cells 2. The expression pattern of myogenic factors that regulate the proliferation and
differentiation of myogenic cells in the primary Sox15-/- myoblasts
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3. Role of the Sox15 in skeletal muscle regeneration and determine whether the impaired skeletal muscle regeneration shown in Sox15-/- mice is due to decreased number of the satellite cells in the skeletal muscle.
4. Effect of the dystrophin (mdx) and Sox15 deficiency in the double mutant mice on the skeletal muscle development.
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Table 4.1: Functions of Sox genes in development (adapted by Bowles et al. 2000).
Mammalian testis-determining gene.
Expressed in early developing CNS in Drosophila (also calledSoxNeuro). Neuroectoderm expression may be controlled by the zygotic dorsoventral patterning genes (dpp, sog, brk, twi).
Expressed in mouse embryonic CNS, lens; putative neural differentiation gene. Knockout mice show microphthalmia, cataracts, seizures.
Expressed in pluripotent lineages of preimplantation mouse embryo, developing CNS, lens.
Expressed in mouse embryonic CNS, lens. No direct functional data. May act Expressed in early CNS and lens in zebrafish. No direct functional data.
Also called dichaete. Involved in early embryo segmentation andbrain development and is required for the correct differentiation of the hindgut.
May specify a subset of ventral interneurons in the spinal cord and neuronal subtypes in the brain.
Transcriptional repressor.
Expressed in developing CNS. Transcriptional repressor.
Roles in cardiac outflow tract development and B-cell development revealed by phenotype of knockout mice.
Expressed in maturing neurons in CNS, also in PNS and sites of epithelial–mesenchymal interaction.
Involved in oligodendrocyte differentiation.
Not known.
Expressed in CNS and many other tissues. No direct functional data.
Expressed in oocytes. Not studied in detail. No direct functional data.
Also called COG-2 (connection of gonad); regulates late-stage uterine seam cell differentiation and fusion.
Expressed during spermatogenesis and chondrogenesis. Involvement in chondrogenesis supported by interaction with SOX9. Homo- and heterodimerization with SOX6.
Expressed during spermatogenesis and chondrogenesis, and in CNS. Involvement in chondrogenesis supported by interaction with SOX9. May act redundantly with SOX5.
Expressed in developing arteries and thymus, and widely in adult human tissues.
Expressed in embryonic ovary and brain. Homodimerization.
Also called Sox100B. Expressed in large intestinal cells, in basophilic cells in the midgut, in the Malpighian tubules, and at the posterior cap of gonadal mesoderm.
Expressed in fetal CNS, brain, branchial arches, limb, heart, dorsal root ganglia, and testes. Deleted in ATR-16 patient. No direct functional data.
Key regulator of chondrogenesis and sex determination, roles also in heart, kidney, and brain development, as revealed by phenotype of campomelic dysplasia patients. Cells lacking SOX9 cannot form chondrocytes.
Regulator of neural crest cell differentiation. Mutation leads to neurocristopathy in humans and mice.
Not known.
Regulator of endoderm development and spermatogenesis. Role in endoderm induction shown by protein function interference in Xenopus.
Blood vessel and hair follicle development, as demonstrated by mutations in ragged mice.
Expressed in fetal testes but expression and function not studied in detail.
Inhibitor of myoblast differentiation, as revealed by experiments involving ultured myoblasts.
Not known.
Expressed in germ cells of embryonic testes.
Expressed in late blastula, gastrula, and neural tissues. Dominant negative xperiments in Xenopus demonstrate role in neural induction.
Expressed in early developing CNS in Drosophila (also calledSoxNeuro). Neuroectoderm expression may be controlled by the zygotic dorsoventral patterning genes (dpp, sog, brk, twi).
Expressed in mouse embryonic CNS, lens; putative neural differentiation gene. Knockout mice show microphthalmia, cataracts, seizures.
Expressed in pluripotent lineages of preimplantation mouse embryo, developing CNS, lens.
Expressed in mouse embryonic CNS, lens. No direct functional data. May act Expressed in early CNS and lens in zebrafish. No direct functional data.
Also called dichaete. Involved in early embryo segmentation andbrain development and is required for the correct differentiation of the hindgut.
May specify a subset of ventral interneurons in the spinal cord and neuronal subtypes in the brain.
Transcriptional repressor.
Expressed in developing CNS. Transcriptional repressor.
Roles in cardiac outflow tract development and B-cell development revealed by phenotype of knockout mice.
Expressed in maturing neurons in CNS, also in PNS and sites of epithelial–mesenchymal interaction.
Involved in oligodendrocyte differentiation.
Not known.
Expressed in CNS and many other tissues. No direct functional data.
Expressed in oocytes. Not studied in detail. No direct functional data.
Also called COG-2 (connection of gonad); regulates late-stage uterine seam cell differentiation and fusion.
Expressed during spermatogenesis and chondrogenesis. Involvement in chondrogenesis supported by interaction with SOX9. Homo- and heterodimerization with SOX6.
Expressed during spermatogenesis and chondrogenesis, and in CNS. Involvement in chondrogenesis supported by interaction with SOX9. May act redundantly with SOX5.
Expressed in developing arteries and thymus, and widely in adult human tissues.
Expressed in embryonic ovary and brain. Homodimerization.
Also called Sox100B. Expressed in large intestinal cells, in basophilic cells in the midgut, in the Malpighian tubules, and at the posterior cap of gonadal mesoderm.
Expressed in fetal CNS, brain, branchial arches, limb, heart, dorsal root ganglia, and testes. Deleted in ATR-16 patient. No direct functional data.
Key regulator of chondrogenesis and sex determination, roles also in heart, kidney, and brain development, as revealed by phenotype of campomelic dysplasia patients. Cells lacking SOX9 cannot form chondrocytes.
Regulator of neural crest cell differentiation. Mutation leads to neurocristopathy in humans and mice.
Not known.
Regulator of endoderm development and spermatogenesis. Role in endoderm induction shown by protein function interference in Xenopus.
Blood vessel and hair follicle development, as demonstrated by mutations in ragged mice.
Expressed in fetal testes but expression and function not studied in detail.
Inhibitor of myoblast differentiation, as revealed by experiments involving ultured myoblasts.
Not known.
Expressed in germ cells of embryonic testes.
Expressed in late blastula, gastrula, and neural tissues. Dominant negative xperiments in Xenopus demonstrate role in neural induction.
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