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Homeobox genes are a superfamily of transcription factors that regulate the spatial organization of the embryonic body plan, cellular identity, proliferation, and differentiation during organogenesis (Carroll et al.,2001). Typical homeobox genes contain a conserved 180 base pair sequence that encodes a 60 amino acid DNA binding domain, called the homeodomain. X-ray crystallography and NMR studies have shown that the homeodomain forms three alpha-helices, with the first two helices in parallel and the third at an angle that allows it to fit into the major groove of DNA. Five amino acids, tryptophan 48 (W48), phenylalanine 49 (F49), asparagine 51 (N51), arginine 53 (R53), and lysine/arginine 55 (K/R55) found in helix III, are conserved among all known homeobox genes and are predicted to be important in DNA binding (Bürglin,1994). Greater than 200 homeodomain-containing genes have been identified in the mammalian genome and are divided into seven subgroups: Hox, extended Hox, Paired, NK, LIM, Pou, and atypical, based on sequence similarity within the homeodomain (Burglin,1994; Banerjee-Basu and Baxevanis,2001; Tupler et al.,2001). Identifying new homeobox genes and their function is essential to unraveling the regulation of basic developmental processes.
The Iroquois class (IRO) of genes belong to the three–amino-acid loop extension (TALE) superclass of atypical homeobox genes, characterized by three additional amino acids in the loop region between helix I and helix II of the homeodomain. The IRO genes were first identified in Drosophila as a complex of three clustered and highly related genes, araucan, caupolican, and mirror, that control the transcription of the proneural genes, specify wing identity, and act as dorsal selector genes in the eye (Leyns et al.,1996; Gómez-Skarmeta et al.,1996; Grillenzoni et al.,1998; Kehl et al.,1998; Cavodeassi et al.,1999; Diez del Corral et al.,1999). Murine orthologs, Iroquois 1–6 (Irx1–6), are expressed broadly during development, including the central nervous system, ventricles of the heart, somites, lungs, gonads, and cartilage (Bosse et al.,1997; Bellefroid et al.,1998; Bao et al.,1999; Cohen et al.,2000; Houweling et al.,2001). There is evidence that they promote proneural gene transcription, patterning of the neural tube and ventricular-specific transcription in the heart (Bao et al.,1999; Gómez-Skarmeta and Modolell,2002). A unique feature of the Irx homeodomain is an alanine at position 50 (Ala50) of the recognition helix (helix III), which is believed to participate in determining DNA binding specificity (Hanes and Brent,1989; Stepchenko et al.,1997; Duan and Nilsson,2002). All Irx proteins also possess a highly conserved acidic motif (IRO Box) in their C-terminal region (Bürglin,1997; McNeill et al.,1997).
In these studies, we identify Mohawk (Mkx) as novel homeobox gene that is most-closely related to the Irx genes. Divergence within the homeodomain and the absence of an IRO Box suggest that Mkx should be considered a new atypical TALE homeobox class. During embryonic mouse development, Mkx is transcribed in the discrete premyogenic cell populations of the somite, the condensing prechondrogenic mesenchymal cells of the axial skeleton, the pretendenous cells of the tail and limbs, the testis cords of the developing male gonad, and the metanephric kidney. On the basis of the specificity in the tissues in which Mkx is expressed and the high degree of overlap in expression with transcription factors paraxis, Pax3, Sox9, and scleraxis, Mkx is predicted to play an important regulatory role during development.
RESULTS AND DISCUSSION
Identification and Classification of the Mohawk Homeobox Gene
To identify related members of the Irx genes, the amino acid sequence of the Irx2 homeodomain was used to search the mouse genome in the GenBank Database using tblastn (Gish and States,1993). The outcome of this search included a previously undescribed mouse gene (GenBank mRNA accession no. NM_177595 and protein accession no. NP_808263) that we have subsequently named Mohawk (Mkx). The nucleotide sequence of Mkx was confirmed by sequencing cDNA generated from embryonic day (E) 10.5 mouse embryos. A tblastn search of the mouse genome using the full-length Mkx protein as the query showed that members of the IRO class were the most-closely related genes. Alignment of the predicted amino acid sequence of the Mkx homeodomain with the homeodomains of Irx1, 2, and 4, and representative murine members of the TALE class PBC (Pbx1; Nourse et al.,1990), MEIS (Meis1; Moskow et al.,1995), and TGIF (Tgif; Bertolino et al.,1995) revealed that the greatest sequence homology lay within helix III of the Irx genes (82% identity, 14/17 residues), including the Ala50 (Fig. 1A). Over the entire Mkx homeodomain, however, the highest sequence homology was only 56% (35 identical residues with Irx2). Furthermore, Mkx shared less sequence similarity outside the homeodomain with the Irx genes, and did not contain the IRO Box found in all members of the Irx genes (data not shown).
The PBC class of genes has been shown previously to be the next closest relatives to the IRO genes (Bürglin,1997). A phylogenetic analysis of the homeodomain amino acid sequences revealed a closer evolutionary relationship of the Mkx homeodomain with the Irx homeodomains (evolutionary distance less than one substitution per site) as compared with Pbx1, Meis1, and Tgif, all of which show higher sequence similarity with each other than with the Irx and Mkx homeodomains (Fig. 1A,B). In any case, the Mkx homeodomain is distantly related to the Irx homeodomains, because it shows an evolutionary distance >0.7 substitutions per site when compared with Irx homeodomains. Therefore, it may be considered to be in its own TALE class, which we refer to as the MKX class.
The genomic organization of Mkx in the mouse genome was examined using the University of Santa Clara Genome Browser (Karolchik et al.,2003). The gene is located at qA1 on chromosome 18, which is distinct from the two Irx gene clusters, IrxA and IrxB, found on chromosomes 13 and 8, respectively (Houweling et al.,2001). Mkx consists of seven exons that span 69,755 bases and code for a 353 amino acid protein (Fig. 1C). The homeodomain is encoded by exon III and IV (Fig. 1D). Exon II and III encode a putative bipartite nuclear localization signal.
A tblastn search of the Ensembl and GenBank databases, using the mouse Mkx homeodomain sequence, revealed predicted orthologs in both vertebrate and invertebrate species. The homeodomain was completely conserved at the amino acid level across all the vertebrate orthologs examined (Table 1; for protein sequence alignment, see Supplementary Material, which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat). Among the whole protein, the degree of sequence conservation varied from 87% identity in Rattus norvegicus to 47% identity in Danio rerio. Regression of sequence identity with time of species divergence (between mouse and other species listed in Table 1; see Hedges and Kumar,2003) indicates that sequence identity has decayed at a rate of five amino acids per 100 million years in vertebrates, when the whole protein is considered. Among invertebrates, we found proteins with weak sequence homology (46% identity) in the homeodomain in the Anopheles gambiae and Drosophila melanogaster genomes (Table 1). This finding is a lower percent similarity than is normally seen between invertebrate and vertebrate orthologs of homeobox genes (Bürglin,1994). However, when using the invertebrate homeodomain sequences as the queries in a tblastn search of the mouse genome, Mkx was found to be the most-related murine protein.
Table 1. Predicted Protein Sequence Identity Comparing Mouse Mkx to its Orthologs
A pairwise alignment between the predicted amino acid sequence of the homeodomain from the mouse Mkx and each ortholog, using ClustalW v1.82 (Chenna et al.,2003). Identity is expressed as the number of identical amino acids over the total number of amino acids in the homeodomain.
A pairwise alignment between the whole predicted amino acid sequences of mouse Mkx and each ortholog, using bl2seq using blastp (Tatusova and Madden,1999). Identity is expressed as the number of identical amino acids over the total number of amino acids in high-scoring segment pairs.
Dynamic Mkx Expression in the Somites
Whole-mount in situ hybridization (WISH) analysis with an Mkx-specific digoxigenin-labeled antisense RNA probe was used to examine Mkx transcription in developing mouse embryos between E9.0 to E11.5. A dominant feature of Mkx transcription was a dynamic pattern in the somites of embryos beginning at E9.0 (Fig. 2). Mkx was transcribed in the dorsal region of the dermomyotome of the most-anterior somites of E9.0 embryos (Fig. 2A). This transcription extended to the tail somites by E10.5 (Fig. 2B). A second ventral domain of somite expression was noted in E10.5 embryos, with the strongest staining in the interlimb region (Fig. 2B). In E11.5 embryos, Mkx transcription persisted in these discrete populations in the dorsal and ventral aspects of the dermomyotome of body and tail somites (Fig. 2C,D). Transverse sections through the interlimb region of an E10.5 embryo revealed that expression was limited to the epithelial dorsomedial lip (DML) and ventrolateral lip (VLL) of the dermomyotome (Fig. 2E).
The DML and VLL of the dermomyotome in the maturing somites have been characterized as the sites of self-renewing premyogenic cell populations that act as a source of cells for the expanding myotome (Christ and Ordahl,1995; Cossu et al.,1996; Denetclaw et al.,1997; Venters and Ordahl,2002). Cells at the VLL are also the source of a highly migratory cell population that establishes the appendicular, hypoglossal, and diaphragm muscles (Ordahl and Le Douarin,1992; Denetclaw and Ordahl,2001). To examine the association of the Mkx transcription domain to myoblasts and myocytes of the myotome, WISH was performed on mouse embryos carrying the muscle-specific +1565Myogenin/lacZ transgene (Cheng et al.,1992). In the thoracic region of E10.5 embryos, Mkx transcription in the DML was closely associated with the dorsal aspect of the myotome (Fig. 3A). Transverse sections revealed that Mkx-expressing cells in the epithelial DML are adjacent to the myotome but are not found within the myotome (Fig. 3B). It is interesting to note that the Berkeley Drosophila Genome Project (BDGP) found that the Drosophila melanogaster ortholog of Mkx (Table 1) is expressed in the embryonic/larval muscle system (Tomancak et al.,2002). This finding suggests a conserved role for Mkx in regulating myogenesis in vertebrates and invertebrates.
Little is known about the signals involved in specifying cells to the premyogenic fate or regulating the morphological events associated with the epithelial to mesenchymal transition and migration into the myotome. The transcription factors Pax3 and paraxis have been demonstrated to regulate the specification of premyogenic cells, proliferation, migration into the limbs and the regulation of their epithelial state (Franz et al.,1993; Goulding et al.,1994; Williams and Ordahl,1994; Daston et al.,1996; Burgess et al.,1996; Tajbakhsh et al.,1997; Wilson-Rawls et al.,1999; Schubert et al.,2001; Wiggan et al.,2002). A comparison of Mkx transcription with paraxis and Pax3 revealed an overlapping expression pattern in the DML of the somites of E10.5 embryos, while the VLL expressed only Mkx and Pax3 (Fig. 3C,D). In E10.5 embryos deficient for paraxis, Mkx transcription was absent in the DML but was unchanged in the VLL and nonsomitic regions such as the forelimb bud (Fig. 3E,F). We have shown previously that paraxis differentially regulates premyogenic populations in the DML and VLL of the dermomyotome (Wilson-Rawls et al.,1999). Pax3 transcription in the DML was dependent on paraxis, whereas transcription in the subpopulation of cells in the VLL that migrate to give rise to the hypaxial muscles in the limbs, tongue, diaphragm, and ventral midline, were unaffected. Because the pattern of Mkx in the paraxis mutant was similar to that of Pax3, this draws some intriguing parallels between the two genes in their regulation by paraxis.
The maturation of the tail somites in E12.5 embryos is associated with the remodeling of the myotome from a continuous sheet of myocytes into distinct dorsal and ventral muscle masses. These muscle groups become the short intrinsic and extrinsic bicipital muscles that span adjacent vertebrae of the tail (Shinohara,1999). Before the remodeling of the myotome (E11.5), Mkx and Pax3 were transcribed in the DML and VLL in the dermomyotome (Fig. 3). In E12.5 tails, Mkx expression shifted from the dorsal and ventral aspects of the somite to two distinct domains along the posterior edge of the somite (Fig. 4A). This shift occurred over six somites, with a somite in the middle expressing equally in both domains. The new domain of expression overlapped with the transcription of the tendon-specific scleraxis gene that defines the syndetome compartment of the somite (Cserjesi et al.,1995; Schweitzer et al.,2001) and not with the dermomyotome-specific gene Pax3 (Fig. 4B,C). The Mkx transcription domain became long and thin and crossed segmental boundaries in E13.5 embryos (Fig. 4D). The same morphological changes were observed in the scleraxis transcription domain, suggesting that these cells are forming the tendons that connect to the bicipital tail muscles (Fig. 4E). This suggestion was supported by the alternating pattern of +1565Myogenin/lacZ expression in the myotome and Mkx transcription (Fig. 4F).
The expression of Mkx in the syndetome during somite maturation predicts a second role for the gene in regulating the tendons that form junctions with the fetal muscles. It has been demonstrated previously that FGFs secreted from the myotome are essential to stimulate scleraxis transcription in the syndetome (Brent et al.,2003). The expression of Mkx in both the premyogenic populations of the dermomytome and syndetome raises the possibility of additional regulatory links between the myogenic and tendon lineages.
Mkx transcription was examined in the body wall of E11.5 embryos that had been bisected midsagittally and eviscerated. Under these conditions, Mkx mRNA was detected in the vertebral bodies and notochord, ventral to the neural tube (Fig. 5A). Stripes of transcripts were observed in the condensing mesenchyme of the proximal ribs that form from the posterior sclerotome (Fig. 5A). This was confirmed by examining the expression of Sox9, a prechondrogenic mesenchymal marker at the same developmental stage, which is transcribed in all of these tissues (Wright et al.,1995; Fig. 5B). Furthermore, coronal sections through an embryo doubly stained for +1565Myogenin/lacZ expression and Mkx showed that Mkx was expressed throughout the discrete condensing mesenchyme of the proximal ribs and not the muscle or tendons (Fig. 5C). Mkx transcripts were also found in the frontonasal mass beginning at E10.5 (Fig. 2B). Cells in this region give rise to skeletal elements of the face, including the forehead, nasal cartilage, and philtrum (Richman and Tickle,1989). Thus, Mkx is expressed in the condensing prechondrogenic mesenchyme of the axial skeleton, predicting a role for this gene in regulating the early events in chondrogenesis.
Expression of Mkx in the Limb Bud
Based on the expression of Mkx in the tendons and prechondrogenic cells derived from the somites, transcription was examined in the limbs. At E12.5, Mkx was strongly expressed in the autopod in a pattern that overlaps with the forming phalanges and metacarpals, in a pattern similar to scleraxis and Sox9 (Fig. 6A–C). Transverse sections revealed that Mkx transcription was superficial to the condensing mesenchyme, marked by Sox9 transcription, which was different than what was observed in the developing axial skeleton (Fig. 6D,F). The pattern of Mkx transcription was more similar to scleraxis, suggesting that Mkx-positive cells in the limbs at this stage may be in the tendon lineage (Fig. 6E). However, the broader expression pattern of Mkx predicts that the gene may be involved in other developmental processes in this region, including chondrogenesis or myogenesis.
Mkx Transcription in the Gonads and Kidney
The gonads are derived from bipotential cells that appear along the ventromedial surface of the urogenital ridge around E10.5 in the mouse. Sexual determination occurs through the male-specific transcription of Sry (sex-determining region, Y chromosome) and Sox9 in the gonadal ridge, which leads to differentiation of Sertoli cells and the production of anti-Mullerian hormone (Gubbay et al.,1990; Sinclair et al.,1990; Kent et al.,1996; Morais de Silva et al.,1996). We examined the transcription of Mkx in male and female indifferent urogenital ridges isolated from E11.5 embryos using WISH (Fig. 7A–D). Mkx transcription was present throughout the male gonadal ridge and absent in the female gonadal ridge (Fig. 7A,C), this expression pattern overlapped that of Sox9 (Fig. 7B,D). In E13.5 gonads, Mkx transcription was restricted to the testis cords of the male gonad, similar to that of Sox9 (Fig. 7E,F). The testis cords are formed from the aggregation of cells that will differentiate into Sertoli cells and are the site of the primordial germ cells. The expression of Mkx in these cells and its absence in the female gonad suggest that the gene may play a role in regulating Sertoli cell differentiation and/or sex determination.
The metanephric kidney develops from the ureteric bud of the mesonephric duct and the adjacent metanephric mesenchyme. The ureteric bud invades the mesenchyme and undergoes repeated rounds of branching in response to mesenchymal signals. Mesenchyme at the tip of the ureteric bud condenses to give rise to nephrons that will drain into the collecting ducts generated through branching. At E11.5, Mkx was expressed diffusely in the newly forming kidney of both males and females (Fig. 7A,C). Expression became restricted to the tips of the ureteric buds by E13.5 (Fig. 7G). Similar to the gonads, the expression pattern overlapped with Sox9, which has been reported to be expressed in the epithelium at the distal tip of the ureteric buds (Pepicelli et al.,1997; Fig. 7H).
In summary, Mohawk defines a new class of TALE atypical homeobox genes that is highly conserved among vertebrates. Analysis of the Mkx expression in the mouse embryo revealed transcription in developmentally important regions that give rise to skeletal muscle, tendons, cartilage, male gonads, and the ureteric buds of the kidney. In each of these cell types, the expression of Mkx preceded differentiation, suggesting that Mkx participates in the early events that lead to differentiation. Changes in cell morphology associated with an increase in cell–cell contact are common in these tissues. Cell aggregation occurs during the condensation of prechondrogenic mesenchyme, formation of the male sex cords, and the tendons (Wezeman,1998; Delise et al.,2002; Moreno-Mendoza et al.,2003). In the dermomyotome and ureteric buds, Mkx-positive cells are maintained in an epithelial state associated with growth and differentiation of the lips of the dermomyotome and branching of the ureter (Christ and Ordahl,1995; Davies et al.,1999). This finding raises the possibility that Mkx may act as a morphogenic regulator of cell adhesion. This possibility is supported by the observation that Mkx lay downstream of paraxis, a regulator of the mesenchyme-to-epithelia transition of the somite. We also note a striking spatial and temporal overlap in transcription between Mkx and Sox9 in prechondrogenic cells, the testis cords, and the ureteric buds of the kidneys. Sox9 is required for differentiation of chondrocytes and is a critical regulator of sexual differentiation of the male gonad (Sinclair et al.,1990; Kent et al.,1996; Morais de Silva et al.,1996; de Crombrugghe et al.,2000; Akiyama et al.,2002). Similarly, Mkx transcription overlaps with Scleraxis, which is expressed in the tendon precursors, and Sertoli cells (Brown et al., 1999; Muir et al.,2005). Based on these observations, Mkx is predicted to participate in these regulatory pathways. However, further mechanistic studies will be needed to understand the potential contribution of Mkx to the existing regulatory pathways in these cells.
Intercrosses and Genotyping
Mice carrying the +1565Myogenin/lacZ transgene (Cheng et al.,1992) were maintained as a homozygous colony. Paraxis null mice were described previously (Burgess et al.,1996) and were maintained as a heterozygous breeding stock for the null allele (paraxis+/−). Paraxis−/− and paraxis+/+ embryos were generated from heterozygous crosses. The genotype was determined by a polymerase chain reaction (PCR) strategy using primers specific to the 3′ recombination arm (upstream primer, 5′-ACCACCAAGCGAAAACATC-3′; downstream primer, 5′-CAAGAGGAAGGAACCAGA-3′). To determine the sex of the embryos from which E11.5 urogenital ridges were isolated, genomic DNA isolated from the embryo's head was used to screen for the presence (male) or absence (female) of the Y chromosome by PCR amplification of the Sry gene locus (Hogan et al.,1994).
WISH on tissues were performed in the automated InsituPro (Intavis, LLC, San Marcos, CA), as described previously (Belo et al.,1997). In experiments where tissues were examined for both Mkx transcription and β-galactosidase expression from the +1565Myogenin/lacZ transgene, the β-galactosidase staining was performed first. Tissues were dissected in DPBS and fixed in 0.8% paraformaldehyde, 0.2% glutaraldehyde in DPBS at 4°C. Tissues were washed in DPBS and then incubated in lacZ stain (5 mM KFerrocyanide, 5 mM KFerricyanide, 2 mM MgCl2, 1 mg/ml X-Gal, in DPBS) at 37°C. This was followed by washing in DPBS and postfixation in 4% formaldehyde in DPBS at 4°C.
Antisense digoxigenin-labeled RNA probes were generated using a PCR product specific for each gene. Gene-specific templates were amplified by RT-PCR from total RNA extracted from E9.5 mouse embryos (Rhee et al.,2003). Gene-specific primers were designed using Primer3 (Rozen and Skaletsky,2000) and then modified by adding the T7 RNA polymerase binding site sequence (5′-CTAATACGACTCACTAT AGGGAGA-3′) to the 5′ end of the downstream primer. Mouse Mkx upstream primer 5′-GAGCCGTGCTTTTTGAAGAC-3′ and downstream primer 5′-TACTTGGGCGGTGACACATA-3′; mouse paraxis upstream primer 5′-ACCTTCTGTCTCAGCAACCA-3′ and downstream primer 5′-CCCCGATTTGCTCACATACT-3′. A tenth of the 25-μl PCR reaction product was directly added to an in vitro transcription reaction with digoxigenin RNA labeling mix (Roche Applied Sciences, Indianapolis, IN) as described in Johnson et al. (2001). The Pax3 probe was generated as described previously (Wilson-Rawls et al.,2000).
Cryosections on WISH Stained Tissues
Tissues were washed in DPBS and then equilibrated in 5% sucrose in DPBS at room temperature and then 15% sucrose in DPBS at 4°C. Tissues were then equilibrated in a solution of 15% sucrose and 7.5% gelatin in DPBS at 37°C overnight. The tissues were embedded in the same solution and frozen in liquid nitrogen. The block was sectioned on a cryostat kept at −30°C at a thickness of 25 μm and collected on gelatin-subbed slides. The slides were incubated in warm DPBS to remove the gelatin and then dehydrated in an ethanol series and mounted with coverslips.
We thank the Evolutionary and Functional Genomics Center of the Arizona Biodesign Institute for the contribution of the InsituPro, Dr. Miles Orchinik for his assistance in the generation and visualization of the cryosections, and Sarah Parsons and Leann Chavez for their technical assistance. We also thank Dr. Sudhir Kumar for his valuable discussion. J.A.R. and J.W.R. are supported by grants from the National Science Foundation and the Muscular Dystrophy Association (0131726).