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).
Figure 1. Evolutionary classification of mouse Mohawk (Mkx) as a new three–amino-acid loop extension (TALE) atypical homeobox gene. A: Comparison of the 63 amino acids of the homeodomains of mouse Mkx and other mouse TALE genes: Irx1, 2, 4, Pbx1, Meis1, and Tgif. Residues that are conserved among all homeodomains are highlighted in blue. The predicted organization of the homeodomain into three alpha-helices is displayed above the sequence. A “.” marks identical residues compared with Mkx. B: A neighbor-joining tree of homeodomains of Mkx and representative genes of the animal TALE classes (Irx1–6, Pbx1, Meis1, and Tgif) constructed using the evolutionary distances estimated under the Jones-Taylor-Thornton's (JTT) model in MEGA3 (Kumar et al.,2003). C: Schematic of the genomic organization of Mkx on mouse chromosome 18. Exons and introns are represented by boxes and lines, respectively. The coding regions are denoted by dark green shading and the 5′ and 3′ noncoding regions are denoted by light green shading. D: Schematic of the predicted 353 amino acid Mkx protein with the positions of the N terminal homeodomain (HD) and a putative nuclear localization signal displayed above (NLS). Roman numerals mark the predicted contribution of individual exons to the protein.
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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
|Species||Protein Accession No.||Homeodomaina||Whole Proteinb|
|Vertebrates|| || || |
|Rattus norvegicus||ENSRNOP00000025623||63/63 (100%)||314/357 (87%)|
|Pan troglodytes||ENSPTRP00000004041||63/63 (100%)||290/353 (82%)|
|Homo sapiens||AAH36207||63/63 (100%)||288/353 (81%)|
|Xenopus tropicalis||ENSXETP00000009890||63/63 (100%)||247/354 (69%)|
|Fugu rubripes||SINFRUP00000151281||63/63 (100%)||216/411 (52%)|
|Danio rerio||XP_683366||63/63 (100%)||172/287 (59%)|
|Invertebrates|| || || |
|Anopheles gambiae||ENSANGP00000012403||29/63 (46%)||38/84 (45%)|
|Drosophila melanogaster||CG11617-PA||29/63 (46%)||52/171 (30%)|
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).
Figure 2. Examination of Mkx transcript expression during mouse development by whole-mount in situ hybridization. A: In embryonic day (E) 9.0 embryos, Mkx mRNA was detected in the dorsomedial lip (DML) of the dermomyotome in the anterior somites. B: In E10.5 embryos, Mkx mRNA was present in the DML of hindlimb-level somites as well as the ventrolateral lip (VLL) of the interlimb somites. Mkx mRNA was also detected in the frontonasal mass (fnm), forelimb (fl) and hindlimb (hl). C,D: At E11.5, Mkx expression is maintained in the dorsal and ventral most aspects of the body somites and in the DML and VLL of tail somites. Mkx transcription was expressed throughout the dermomyotome of somites 0, +1, and +2 at E11.5. E: A transverse cryosection through the interlimb somites of an E10.5 embryo showed restriction to the epithelial DML and VLL of the dermomyotome. lpm, lateral plate mesoderm; mg, midgut; hl, hindlimb; nt, neural tube. Scale bars = 0.5 mm.
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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.
Figure 3. Mkx is expressed in the premyogenic cells of the somite and is downstream of paraxis. A: Embryonic day (E) 10.5 embryos were doubly stained for the expression of the +1565Myogenin/lacZ transgene and Mkx transcription. B: A transverse cryosection through the interlimb region of the embryo shows the position of Mkx in the lip region of the dermomyotome adjacent to the developing myotome. C–F: Whole-mount in situ hybridization (WISH) of E10.5 embryos with probes specific for Pax3 (C) and paraxis (D). Mkx was transcribed normally in E10.5 paraxis+/− embryos (E) but absent in the dml of paraxis−/− embryos (F). Scale bars = 0.5 mm.
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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).
Figure 4. A–F: Transcription shifts to the syndetome in the mature tail somites. Whole-mount in situ hybridization (WISH) was performed on the tail region of embryonic day (E) 12.5 and E13.5 embryos using probes specific for (A,D) Mkx, or (B,E) Scx, or (C,F) Pax3. A: At E12.5, the domain of Mkx transcription in the caudal somites shifted from the entire dml (yellow arrowhead) to the syndetome (blue arrowhead). In the intervening somites, Mkx transcription was present in both the dml and the syndetome (green arrowhead). B:Scx was transcribed in the syndetome, characterized as dorsal and ventral domains along the posterior aspect of each somite in the tail. C:Pax3 transcription was maintained throughout the dermomyotome at this stage. D,E: At E13.5, the dorsal and ventral (D) Mkx and (E) Scx transcription domains elongated and moved anteriorly in a similar manner. F: The tail of a E13.5 embryo was co-stained for expression of the +1565Myogenin/lacZ transgene and Mkx transcripts. The Mkx transcription domain remained closely associated with myotomal cells as they remodeled into the bicipital tail muscles. nt, neural tube; dmm, dorsal muscle mass; vmm, ventral muscle mass. Scale bars = 0.3 mm.
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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.
Figure 5. Mkx was transcribed in the condensing mesenchyme that prefigures the axial skeleton. A,B: Whole-mount in situ hybridization (WISH) was performed on embryonic day (E) 11.5 mouse embryos after a midsagittal cut, using antisense RNA probes specific to either Mkx (A) or Sox9 (B). Both were expressed in the developing proximal ribs (pr), the vertebral body (vb) and the notochord (n). C: A frontal section through an embryo co-stained for Mkx and the +1565Myogenin/lacZ transgene showed that Mkx is expressed throughout the condensed mesenchyme positioned between the hypaxial myotome. nt, neural tube. Scale bars = 0.5 mm.
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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.
Figure 6. Mkx transcription in the limbs. A–F: Whole-mount in situ hybridization (WISH) was performed on embryonic day (E) 12.5 limbs with probes specific for Mkx (A,D), Scx (B,E), and Sox9 (C,F). Mkx was transcribed in the region of the phalanges, similar to Scx and Sox9. Transverse sections through the limbs showed that Mkx (D) was excluded from the cartilaginous digits that expressed Sox9 (E). F: Mkx overlapped with Scx, which marks cells dorsal and ventral to the phalanges that are committed to the tendon lineage. Scale bars = 0.5 mm.
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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.
Figure 7. Transcription of Mkx in the urogenital ridge. A–F: Whole-mount in situ hybridization (WISH) was performed on embryonic day (E) 11.5 indifferent gonads (A–D) and E13.5 gonads (E,F), using antisense RNA probes specific to either Mkx (A,C,E) or Sox9 (B,D,F). Mkx was selectively transcribed in the male gonadal ridge (gnr) of E11.5 embryos and the testis cords (tc) of E13.5 embryos. Mkx was transcribed in the metanephric kidney (K) of both sexes. G: Mkx was expressed in discrete regions of the metanephric kidney at E13.5. This pattern was similar to (H) Sox9, which has been reported to be expressed in the epithelial cells at the tip of the ureteric buds (ub tips) in the kidney. m, mesonephros. Scale bars = 0.1 mm
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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.