Conservation of Sox4 gene structure and expression during chicken embryogenesis

Abstract

While mutations in Sox4, a member of the SRY-like HMG box gene family, have been associated with a variety of human disorders and embryonic defects in the mouse, the structure and developmental expression of Sox4 in the avian embryo has not been described. We have isolated and characterized the chicken Sox4 gene. The chicken Sox4 gene shows a high degree of sequence homology with the mouse and human Sox4 genes, particularly in the HMG-like DNA binding domain and at the carboxy terminus. Furthermore, our in situ hybridization studies document an expression pattern during embryonic development that is very similar to that described for the mouse, particularly with regards to expression in the developing heart. However, abundant expression was also detected in tissues of neural crest origin including pharyngeal arch and craniofacial mesoderm, supporting a potential primary role in neural crest cardiac pathology previously detected in Sox4 mutant mice. Furthermore, a reciprocal pattern of Sox4 and Sox11 expression in the developing neural tube was detected in the chicken compared to that seen in the mouse. These studies suggest that Sox4 plays an important and conserved role in the embryonic development of these structures in the chicken as well as in the mouse and lay the foundation for future studies of the role of Sox4 during critical events of organogenesis.

Introduction

Sox (SRY-like HMG box) genes encode a family of transcription factors which are crucial for embryonic development. Sox proteins are required for a variety of developmental processes, including sex determination, neural crest cell development, development of the central nervous system, chondrogenesis, lens development, cardiac development, and hematopoiesis (reviewed in Wegner, 1999). Given this diversity of developmental roles, it is not surprising that mutations of Sox genes have been associated with a variety of human disorders. For example, mutations in SRY and SOX9 lead to sex reversal and gonadal dysgenesis Berta et al., 1990, Jager et al., 1990, Wegner, 1999; mutations in SOX9 are associated with campomelic dysplasia (Wagner et al., 1994), while mutations in SOX10 have been associated with Waardenburg-Hirschsprung syndrome (Pingault et al., 1998). SOX3 is a candidate gene for several human X-linked mental retardation syndromes (Laumonnier et al., 2002).

All Sox proteins share a conserved HMG-like DNA binding domain, and are thus members of the HMG domain superfamily. The Sox family can be further subdivided into groups A-H based on sequence similarity of the HMG domains. Although the sequences are more divergent outside the HMG domain, members of the groups often share other structural and functional motifs (Bowles et al., 2000). Additionally, there is evidence that Sox proteins belonging to the same group may have overlapping functions (Downes and Koopman, 2001). Indeed, redundancy appears to be a common feature of processes controlled by Sox gene expression.

Sox4, a member of the C subgroup of Sox proteins, was first identified as a transcription factor required for B- and T-lymphocyte differentiation (Schilham and Clevers, 1998 and references therein). The expression pattern of Sox4 during mouse embryogenesis has been analyzed by both whole mount and section in situ hybridization studies which show that Sox4 is expressed in the mesenchyme of the branchial arches, the trachea and esophagus Schilham et al., 1996, Ya et al., 1998, and the nervous system (Cheung et al., 2000). Later in embryonic development, Sox4 is expressed in the embryonic growth plate where its expression is regulated via the parathyroid hormone and parathyroid hormone-related protein receptor (Reppe et al., 2000). Like Sox5, Sox6, and Sox9, Sox4 may have a role in chondrogenesis as well (Sekiya et al., 2002).

Sox4 is also expressed in the developing endocardial cushions. When the Sox4 gene was disrupted in mice, a role in cardiac development was revealed. Sox4 deficient embryos die at ED14 of valvular insufficiency. Histological analysis of mutant embryos revealed dysplasia of the semilunar valves along with a large outlet ventricular septation defect Schilham et al., 1996, Ya et al., 1998. Additionally, Sox4 deficient mice exhibit arrest of B-cells development at the pro-B-cell stage (Schilham et al., 1996). Thus, Sox4 appears to be critical for normal development and maturation of the endocardial cushions and for normal B-cell maturation, however, its function(s) in these processes is not yet clear.

In addition to its crucial role in embryonic development, Sox4 likely has functions outside the embryonic period. It's expression is induced by estrogens and progestins in the female reproductive tract as well as in normal breast tissue and breast cancer cells Graham et al., 1999, Hunt and Clarke, 1999. It is expressed in salivary adenoid cystic carcinoma cells (Frierson et al., 2002) and medulloblastomas (Lee et al., 2002) and upregulated during apoptosis induced by protaglandins in hepatocellular carcinoma cells (Ahn et al., 1999). It is upregulated when human marrow stromal cells are to induced differentiate into osteoblasts by BMP-2 (Locklin et al., 2001), and is induced by perinatal asphyxia in rats (Lubec et al., 2002). Thus, Sox4 appears to have functions in a wide variety of developmental, physiological, and pathological processes.

Because of its accessibility to surgical manipulation and treatment with exogenous modifiers of developmental function (viral vectors, function blocking antibodies, antisense oligonucleotides, etc.) the chick embryo has historically been a useful model for studying various aspects of development. In particular, the avian system has been useful in the analysis of later stages of cardiac development, such as semilunar valve development, that are not easily accessible in murine models. However, the characterization of the avian Sox4 gene has not been previously described. In order to facilitate to use of the avian model to study the role of Sox4 in cardiac development, we have cloned the chick Sox4 gene and have used in situ hybridization to compare its expression patterns with those described in the mouse.