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Keywords:

  • double homeobox gene;
  • Duxbl;
  • myogenesis;
  • reproductive development;
  • mouse embryo

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We identified and cloned a mouse double homeobox gene (Duxbl), which encodes two homeodomains. Duxbl gene, a tandem triplicate produces two major transcripts, Duxbl and Duxbl-s. The amino acid sequences of Duxbl homeodomains are most similar to those of human DUX4 protein, associated with facioscapulohumeral muscular dystrophy. In adult tissues, Duxbl is predominantly expressed in female reproductive organs and eyes, and slightly expressed in brain and testes. During gonad development, Duxbl is expressed from embryonic to adult stages and specifically expressed in oocytes and spermatogonia. During embryonic development, Duxbl is transcribed in limbs and tail. However, Duxbl proteins were only detected in trunk and limb muscles and in elongated myocytes and myotubes. In C2C12 muscle cell line, Duxbl expression pattern is similar to differentiated marker gene, Myogenin, increased in expression from 2 days onward in differentiating medium. We suggest that Duxbl proteins play regulatory roles during myogenesis and reproductive developments. Developmental Dynamics 239:927–940, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Homeobox genes encode transcription factors that regulate embryonic development programs including organogenesis, axis formation, and limb development (McGinnis and Krumlauf, 1992; Boncinelli, 1997). Their products regulate the expressions of target genes in tissue- and spatiotemporal-specific manners through conserved DNA-binding motifs called homeodomains. Homeodomains have three α-helical segments of which the third constitutes the main DNA recognition site and binds to the major groove of DNA (Gehring et al., 1994). This sequence-specific binding allows homeodomain proteins to activate or repress the expression of a battery of downstream target genes. The correct expressions of homeodomain proteins in adult tissues including liver, kidney, and intestine are important for the regulations of cellular morphogenesis, growth, and differentiation (Cillo et al., 2001).

Different homeobox genes are classified through similarities in amino acid sequences within their homeodomains and the other coding regions of their gene products (Galliot et al., 1999; Holland and Takahashi, 2005). The paired (PRD) class is divided into two subclasses: the PAX subclass and the PAXL subclass (Holland et al., 2007). The PAX subclass homeodomain proteins have a conserved 130-amino-acid DNA-binding domain, the paired domain, upstream of their homeodomains (Bopp et al., 1986). The PAX gene family is an ancient and remarkably conserved gene family, which plays key roles in the formations of tissues and organs during embryogenesis. PAX3 and PAX7 mark myogenic progenitor cells and regulate their behaviors and entries into the program of skeletal muscle differentiation (Buckingham and Relaix, 2007). PAX6 is required for eye formation in vertebrates and its homologues in invertebrates, such as eyeless in Drosophila also play a crucial role in eye formation (Kozmik, 2005). The PAXL-subclass homeodomain proteins show significant sequence similarities (55–75%) to PRD-class homeodomain proteins but lack the paired domain, and contain a glutamine residue at position 9 of the third helix in their homeodomains (Burglin, 1994). Another common feature of PAXL homeobox genes is the presence of an additional intron within their homeoboxes, between the region that encodes position 46 and 47 homeodomain amino acid residues. Many PAXL homeobox genes, such as Rax (retinal homeobox), Arx (aristaless-related homeobox), and Vsx (visual system homeobox), are expressed in the nervous system and during brain or eye morphogenesis (Mathers et al., 1997; Miura et al., 1997; Ohtoshi et al., 2001). They play critical roles during embryonic developments.

The human double homeobox (DUX) genes encode two PAXL-subclass homeodomains. The DUX genes are present in multiple polymorphic copies with a 3.3-kilobase (kb) tandem repeat scattered in human heterochromatins (Ding et al., 1998; Gabriels et al., 1999; Beckers et al., 2001). The 3.3-kb dispersed DUX repeats in the D4Z4 locus of chromosome 4 (DUX4) have been found to be associated with the facioscapulohumeral muscular dystrophy (FSHD), the third most common form of inherited muscular dystrophy (Wijmenga et al., 1992; van Deutekom et al., 1993; Hewitt et al., 1994). It has been hypothesized that the larger DUX4 copy numbers in nonaffected individuals are associated with an inhibitory chromatin structure preventing gene expressions, and the inhibition is relieved by the shorter DUX4 arrays found in FSHD patients (Winokur et al., 1994; Tupler and Gabellini, 2004). The coding region of human DUX4 gene shows evolutionary conservation (Clapp et al., 2007) and DUX4 protein can be detected in primary myoblasts extracted from FSHD patients (Belayew, 2004; Kowaljow et al., 2007). Previously, DUX4 protein is identified to have pro-apoptotic activity (Kowaljow et al., 2007) and is found to be a transcriptional activator of PITX1 gene (Dixit et al., 2007). DUX4 expression recapitulates key features of FSHD molecular phenotype, including repression of MyoD and its target genes, and then diminished myogenic differentiation (Bosnakovski et al., 2008). However, the mechanism(s) that causes FSHD phenotype remain unclear. Furthermore, the in vivo functions of DUX4 protein, especially in normal tissues, remain unknown. Other human double homeobox genes are previously identified using PRD-class homeoboxes as query sequences to search human genome sequences, and they have been assigned into four paralogous groups including DUXA, DUXB, DUXC, and DUXB-like (Duxbl; Booth and Holland, 2007; Clapp et al., 2007). The molecular structures of their transcripts and the expression patterns and functions of their protein products have not been provided.

Recently, a novel mouse double homeobox gene has been reported as Duxl (Kawazu et al., 2007) and Duxbl (Clapp et al., 2007), respectively. This gene has been shown to play a critical role in CD4/CD8 double negative thymocyte development (Kawazu et al., 2007), but its detailed genomic structure, major transcript(s), expression patterns, and protein product(s) are not available. Here, we characterize the genomic structure of this mouse double homeobox gene, Duxbl, and suggest that the Duxbl gene is the mouse ortholog of human DUX4 gene. The Duxbl protein is found expressed in adult tissues, including reproductive tissues, eyes, brain, but not in muscle. However, during embryo development, Duxbl is seen expressed in differentiated myocytes. The spatiotemporal expression patterns of Duxbl are also analyzed during gonad developments, and Duxbl is specifically expressed in germ cells, including oocytes and spermatogonia. Duxbl is predicted to play important regulatory roles during myogenesis and reproductive developments.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Genomic Structure and RNA Transcripts of Duxbl Genes

To identify novel homeobox genes involved in early embryonic development, we screened a murine embryonic stem cell cDNA library by degenerate RT-PCR (Wang et al., 2003; Li et al., 2006). One of the genes identified contained a sequence similar to a Riken full-length cDNA clone (1110051B16 Rik) previously reported as Duxl (Kawazu et al., 2007) and Duxbl (Clapp et al., 2007), respectively. However, the genomic structure, expression profiles, protein product(s) of this gene, and in vivo function(s) of this gene product(s) are all unknown. Based on the sequence in the database (1110051B16Rik), the putative translated protein of this gene, Duxbl, contains two helix-turn-helix domains weakly similar to the known homeodomains. Analysis of the genomic structure of Duxbl gene based on information in Ensembl and NCBI databases localized it to the mouse chromosome 14A3, downstream of the Cphx gene (previously named as Eso-1, Li et al., 2006) and mapped to 24.82m (Gene ID: 48502; Fig. 1A). However, we discovered two other downstream Duxbl genes mapped to 24.96m (Gene ID: 72675) and 25.1m (Gene ID: 72672; Fig. 1A). These two downstream Duxbl genes are identical to each other, and share approximately 95% sequence identity with the upstream one (Gene ID: 48502). During analysis of the genomic structure of Duxbl, we observed that in addition to Duxbl, Plac9, and Cphx genes in mouse chromosome 14A3 are all duplicated. Previous reports suggested that Plac9 and Cphx genes play important roles in mouse reproduction (Galaviz-Hernandez et al., 2003; Li et al., 2006). This near-telomeric cluster of reproductive genes in rodents may constitute an evolutionary advantage (Paillisson et al., 2005) and may come from a mechanism for protecting genes against mutation (Clapp et al., 2007). The location of these Duxbl genes in a near-telomeric cluster neighboring two reproductive genes (Plac9 and Cphx) suggests that Duxbl genes also play a role in mouse reproduction.

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Figure 1. Genomic structure and RNA transcripts of Duxbl genes. A: The tandem triplicate of mouse Duxbl genes and their surrounding genes (Plac9 and Cphx) are present on mouse chromosome 14A3. The lengths of spaces between each Duxbl gene and RNA transcripts are indicated. The ORFs of Duxbl and Duxbl-s transcripts are shown in gray and the regions encoding homeodomains are in black. B: Comparison of the predicted amino acid sequences of Duxbl and Duxbl-s proteins. The identical amino acid residues are in bold and the homeodomains are boxed. The different amino acid residues in helix 3 of homeodomain II (H2) are in italics. C: Alignment of amino acid sequences of two Duxbl homeodomains (in bold) with those of other predicted homeodomains of double and paired-like homeobox genes. The names of gene products and their homeodomains are listed in the left. Dashes represent sequences identical to those of Duxbl homeodomains. Identity indicates the percentage of sequence identity to Duxbl homeodomain. The asterisks (*) indicate amino acid residues predicted to be involved in sequence-specific DNA binding. D: Amino acid sequences of homeodomains of human DUX4 and mouse Duxbl proteins. The basic amino acid residues in two terminal regions of homeodomains are shown in bold. The total numbers of lysine and arginine residues in two ends are shown on the right.

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According to our data of rapid amplification of cDNA ends (RACE) and the sequences of expressed sequence tag (EST) clones in databases, the upstream Duxbl gene (Gene ID: 48502) is composed of five exons spanning 6,390 bp. More than one transcriptional start sites for this Duxbl were identified by 5′ RACE analysis. The major transcript is 2,055 bp in length (Fig. 1A) and was deposited in the NCBI GeneBank, accession number EF472598. Although there are different transcriptional initiation sites found in these Duxbl transcripts, they contain the same open reading frame (ORF) of 1,053 bp and a putative polyadenylation signal, AATAAA, at position 2,037 to 2,042. For the two downstream Duxbl genes (Gene ID: 72675 and 72672), minor differences in the intron 4 sequences between them and the upstream Duxbl gene (Gene ID: 48502) result in producing an additional minor transcript, Duxbl-s, composed of four exons (Fig. 1A). More than one transcriptional start sites for Duxbl-s transcripts were also identified by 5′ RACE, and they also encode one identical protein. The most abundant Duxbl-s transcript is 1,653 bp in length with an ORF of 606 bp, and a putative polyadenylation signal, AATGAA, at position 1,566 to 1,571 (accession number EU257807). The proposed translational start sites for Duxbl and Duxbl-s transcripts, both correspond to the Kozak consensus site surrounding their start codons (Kozak, 1996). Generations of Duxbl and Duxbl-s transcripts may result from different promoter usage and/or alternative splicing during development.

Analyzing ORFs of the major Duxbl and minor Duxbl-s transcripts indicates that the putative Duxbl and Duxbl-s proteins contain 350 and 201 amino acids, respectively (Fig. 1B). The N-terminal 139 amino acid residues of Duxbl and Duxbl-s proteins are identical and contain one domain similar to known homeodomains (homeodomain I, H1). The C terminus of Duxbl protein contains the other homeodomain (homeodomain II, H2); however, Duxbl-s protein does not because it lacks helix 3, the sequence-specific recognition helix (Fig. 1B). Thus, the major product (Duxbl protein) of these three Duxbl genes contains two homeodomains, so they are double homeobox genes. Furthermore, the coding regions of all homeodomains for Duxbl and Duxbl-s proteins contain an interrupting intron between homeodomain codon 46 and 47. This is a common feature of PAXL-subclass homeodomain proteins (Burglin, 1994). In addition, the amino acid sequences of Duxbl homeodomains share the highest similarities with known PAXL homeodomains (Fig. 1C). Therefore, these three Duxbl genes belong to PAXL-subclass homeobox gene family and they are also mouse double homeobox genes.

Searching for Putative Duxbl Ortholog

Although the genomic structure of Duxbl is more similar to human DUXA gene than human DUX4 gene, the predicted amino acid sequences of homeodomains for Duxbl and DUX4 proteins are more similar (H1: 42% identity; H2: 67% identity) than those of Duxbl and DUXA proteins (H1: 35% identity; H2: 53% identity; Fig. 1C). Recently, a mouse representative of D4Z4 on chromosome 10 was identified and named as Dux (Clapp et al., 2007) and mDUX (Bosnakovski et al., 2009), respectively. However, sequence identities of the predicted protein and DUX4 homeodomains (H1: 36% identity; H2: 56% identity) are also lower than those of Duxbl and DUX4. Based on these sequence identities, we suggest that the human ortholog of Duxbl gene is DUX4 gene but not DUXA gene as previously reported (Kawazu et al., 2007). Furthermore, comparison of total amino acid sequences of Duxbl protein with those of its predicted ortholog shows that Duxbl protein shares the highest similarity with its rat ortholog (RGD1311053; 80% identity; data not shown). The homeodomain sequences of Duxbl H1 and H2 also exhibit the highest similarity with those of its rat ortholog (93% and 91% identities) followed by its human ortholog (42% and 67% identities; Fig. 1C). Comparisons of homeodomain sequences of Duxbl and other PAXL-subclass homeodomain proteins show identities of only 30 to 45% (Fig. 1C). This result indicates that Duxbl is a special member of the PAXL-subclass homeobox gene family.

Otherwise, the sequence identity between Duxbl H1 and H2 is only 43% (Fig. 1C). We found that Duxbl H1 binds DNA in a nonspecific manner but Duxbl H2 specifically binds a palindromic sequence (Tsai et al., unpublished data). The large differences in amino acid residues and DNA binding properties between Duxbl H1 and H2 suggest that two Duxbl homeodomains may bind and regulate different downstream genes through different mechanisms.

Duxbl Expression Pattern in Adult Tissues

Although many double homeobox genes are found in humans, only DUX4 proteins have been detected in vivo in myoblasts from FSHD patients (Belayew, 2004; Kowaljow et al., 2007). However, the in vivo expression pattern of DUX4 protein is still unknown. Accordingly, we first detected their major and minor transcripts, Duxbl and Duxbl-s, of these three Duxbl genes in various adult tissues by reverse transcriptase-polymerase chain reactions (RT-PCRs). Both Duxbl and Duxbl-s transcripts are predominantly expressed in adult eye, brain, and reproductive organs including ovary, uterus, placenta and testis (Fig. 2A). We next performed Western blotting to decipher in vivo expressions of Duxbl proteins in adult mouse tissues using affinity-purified homemade Duxbl polyclonal antibodies. A 38-kDa protein band indicating Duxbl protein is predominantly detected in adult eye, brain and ovary (Fig. 2B). The results of Duxbl expression patterns in adult tissues by RT-PCRs (Fig. 2A) and Western blotting (Fig. 2B) are complementary, because we only detected Duxbl proteins in adult tissues with strong Duxbl transcript signals. However, Duxbl-s protein is not detected in these adult tissues by our polyclonal antibodies. This may result from the much lower expression levels of Duxbl-s transcripts than Duxbl transcripts in these tissues (Fig. 2A).

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Figure 2. Expression profiles of Duxbl and Duxbl-s transcripts and Duxbl protein. A: The expression levels of Duxbl and Duxbl-s transcripts in adult mouse tissues were analyzed by reverse transcriptase-polymerase chain reactions (RT-PCRs). Expression level of actin was used as an internal control. B, brain; H, heart; Lu, lung; Li, liver; Sp, spleen; Ki, kidney; Mu, skeletal muscles; Ut, uterus; Eye, eye; Pl, placenta; Te, testis; Ov, ovary. B: Purified Duxbl fusion protein containing N-terminal 6 histidine residues (6His-Duxbl) and equal-amount of total proteins extracted from indicated adult tissues were analyzed by Western blotting using affinity-purified Duxbl polyclonal antibodies. C: Analyzing the specificity of purified Duxbl polyclonal antibodies. The purified Duxbl polyclonal antibodies were preincubated with purified GST-H1 and GST-H2 fusion proteins (Ab +H1+H2) or with GST proteins only (Ab +GST). Total proteins extracted from adult ovaries and HeLa cells transfected with Duxbl-V5 fusion protein expression vectors (Duxbl-V5), or purified 6His-Duxbl fusion proteins were analyzed by Western blotting using two different pretreated antibodies. Position of 38-kDa protein bands representing Duxbl proteins was indicated by arrow.

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Blocking assays for preincubations of polyclonal antibodies with purified glutathione S-transferase (GST) proteins or GST-H1 and GST-H2 fusion proteins were used to verify the specificity and activity of our Duxbl polyclonal antibodies. After preincubation with GST-H1 and GST-H2 fusion proteins, the purified Duxbl polyclonal antibodies did not recognize the previously identified Duxbl proteins in adult ovary and overexpressed Duxbl-V5 fusion proteins (Fig. 2C). However, antibodies preincubated with GST proteins could recognize Duxbl proteins in adult ovary, overexpressed Duxbl-V5 fusion proteins, and purified 6His-Duxbl fusion proteins. This result verifies the activity and specificity of our homemade affinity-purified Duxbl polyclonal antibodies.

Duxbl Expression Patterns in Gonads

Because Duxbl is predominantly expressed in adult ovary and slightly expressed in adult testis (Fig. 2A), we examined Duxbl expression levels in embryonic and postnatal gonads by RT-PCRs. In female gonads, Duxbl transcripts are observed from embryonic day 12.5 (E12.5) until birth (Fig. 3A). After birth, Duxbl expressions in the ovaries are found to be high until adulthood. In male gonads, Duxbl transcripts are also detected from E12.5 until birth (Fig. 3B). After birth, Duxbl expressions peaked at day 7 and returned to low levels from day 21 until adulthood. Furthermore, the expression patterns of Duxbl-s transcripts are similar to but lower than those of Duxbl transcripts (Fig. 3A,B). In addition, low levels of Duxbl transcripts are also detected in the mesonephros (data not shown). Because no Duxbl expression is detected in mouse Leydig (TM3), Sertoli (TM4), and spermatocyte (GC-2) cell lines (data not shown), we suggest that Duxbl expresses in germ cells. To verify this suggestion, we performed quantitative RT-PCRs in male gonads. Results of real-time RT-PCRs confirm the increases of Duxbl expressions after birth. Duxbl expressions in male testes peak at day 7 (Fig. 3C), while male gonocytes exit cell cycle arrest and enter a wave of spermatogonia proliferation. Duxbl expressions were seen decreasing from day 15 to adulthood. Before birth, there is a Duxbl expression peak with a moderate expression level observed around E13.5, a time for rapid proliferations of male germ cells. After this time, the proliferations of male germ cells slow down and enter mitotic arrest at around E16.5 (Olaso and Habert, 2000). Results of quantitative RT-PCRs suggest high Duxbl expressions in proliferating male germ cells including gonocytes and spermatogonia.

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Figure 3. Analyses of expression levels and profiles of Duxbl and Duxbl-s transcripts during gonad developments. A,B: The expression levels of Duxbl and Duxbl-s transcripts in female (A) and male (B) gonad developments were analyzed by reverse transcriptase-polymerase chain reactions (RT-PCRs). Total RNAs were extracted from ovaries (A) or testes (B) from different embryonic days (E12.5 to E19.5), newborn mice (NB), and mice at different postnatal days (7d to 56d). Expression levels of actin were used as internal controls. C: Analysis of Duxbl expression levels in testes by real-time RT-PCRs. The relative expression levels of Duxbl to GADPH transcripts in testes from different embryonic and postnatal days were determined. The relative expression levels were shown after comparison with the expression level of 7-day-old testes.

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We further characterized Duxbl expressions in adult testis by in situ hybridizations to decipher the cellular localizations of Duxbl transcripts. In adult testis sections, strong Duxbl transcript signals are only detected in spermatogonia from stage X to XII seminiferous tubules (Fig. 4A). However, we did not detect any Duxbl signal in postmeiotic cells of testis including spermatocytes and spermatids. The Duxbl expressions in spermatogonia and the absence in other postmeiotic cells are also confirmed by RT-PCRs of germ cells isolated from distinctive stages (data not shown). Results of in situ hybridizations in adult testis (Fig. 4A) are identical to results of RT-PCRs from testis cell lines, because we could not detect Duxbl expression in Leydig and Sertoli cells, and in round spermatids (data not shown). Pang and his coworkers also identified differential expressions of 1110051B16Rik gene in spermatogonia by microarray and quantitative RT-PCR at different stages of spermatogenesis (Pang et al., 2003). Furthermore, we performed in situ hybridizations to find cells that specifically express Duxbl in adult ovary. In adult ovary sections, weak expressions of Duxbl transcripts are found in the oocytes of primordial follicles, but strong expressions are seen in the oocytes of primary and secondary follicles (Fig. 4B). These results indicate that Duxbl are specifically expressed in oocytes during oogenesis and in spermatogonia during spermatogenesis.

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Figure 4. Identifying Duxbl transcripts in gonads by in situ hybridizations. A,B: The localizations of Duxbl transcripts were analyzed in adult testis (A) and 10-day-old ovary (B). Adjacent sections were labeled with digoxigenin-labeled anti-sense (left panel) and sense probes (middle panel), or stained with hematoxylin and eosin (H&E, right panel). Duxbl transcripts were only detected in spermatogonia (indicated by arrows) of testis (A) and in oocyte of primordial (Pr), primary (P), and secondary (S) follicles in ovary (B). Scale bars = 50 μm.

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We next examined the in vivo localizations of Duxbl proteins in 2-week-old and adult ovaries by immunohistochemistry using homemade affinity-purified Duxbl polyclonal antibodies. Results of immunostained ovary sections show that Duxbl proteins are specifically present in oocytes of primordial, primary, secondary, and antral follicles (Fig. 5). These results of in vivo Duxbl protein localizations are consistent with results of in situ hybridizations (Fig. 4B), because we obtain oocyte-specific signals of Duxbl transcripts and Duxbl proteins in the same types of follicles in both cases.

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Figure 5. Identifying Duxbl proteins in 2-week-old and adult ovaries by immunohistochemical analysis. A–D: Duxbl proteins in 2-week-old (A,B) and adult (C,D) mouse ovary sections were probed using affinity-purified Duxbl polyclonal antibodies (A–C) and normal rabbit immunoglobulin G (D), respectively. Duxbl proteins are specifically present in oocytes of primordial (Pr), primary (P), secondary (S), and antral follicles (AnF) as indicated. Scale bars = 50 μm.

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Subcellular Localizations of Duxbl and Duxbl-s Proteins

We used the same homemade Duxbl polyclonal antibodies to examine the subcellular localizations of overexpressed epitope-tagged Duxbl and Duxbl-s proteins, respectively. Results of immunofluorescence reveal that the overexpressed Duxbl-V5 and FLAG-Duxbl-s fusion proteins are both restricted to the nuclei of transfected cells (Fig. 6). We observed the same subcellular localizations of Duxbl and Duxbl-s fusion proteins using commercial anti-V5 and anti-FLAG monoclonal antibodies, respectively (data not shown). These results indicate that both overexpressed Duxbl and Duxbl-s proteins contain the nuclear localization signal (NLS). Although the amino acid residues of homeodomains from various homeodomain proteins show a high degree of conservation, NLSs of homeodomain proteins are not identical. A common theme of their NLSs is the basic amino acid residues at two ends of homeodomains (Ploski et al., 2004; Ostlund et al., 2005). The first (H1) and the second (H2) homeodomains of Duxbl protein contain 8 and 6 lysine/arginine residues at their two ends, respectively (Fig. 1D). In addition, the human DUX4 protein also contains 6 and 7 lysine/arginine residues at two ends of its H1 and H2, respectively (Fig. 1D). Previously, DUX4 protein has been shown to transactivate PITX1 expression (Dixit et al., 2007). Because Duxbl and Duxbl-s proteins contain similar lengths of basic residues as DUX4 protein at two ends of their homeodomains (Fig. 1D) and both of them localize in the nuclei (Fig. 6), they might transactivate downstream gene(s) as DUX4 protein. However, overexpressing Duxbl proteins in C2C12 cells could not transactivate Pitx1 expression (data not shown).

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Figure 6. A,B: Subcellular localizations of overexpressed Duxbl and Duxbl-s proteins. Duxbl-V5 (A) and FLAG-Duxbl-s (B) fusion proteins were overexpressed in HeLa cells by transfection of Duxbl-V5 and FLAG-Duxbl-s expression vectors, respectively. The overexpressed Duxbl-V5 or FLAG-Duxbl-s proteins were detected by affinity-purified anti-Duxbl polyclonal antibodies (left panel). Cell nuclei were stained with DAPI (middle panel). Images of left and middle panels were merged (right panel).

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Duxbl Expression Patterns in Embryonic Development

Because Duxbl transcripts are predominantly restricted in oocytes of ovary (Fig. 4B), Duxbl expressions during embryonic development were further determined. Duxbl transcripts are first detected in unfertilized eggs then continued to blastocysts but not in cumulus cells (Fig. 7A). After implantation, Duxbl expressions in embryos are seen decreased from embryonic day 11.5 (E11.5) to 17.5 (E17.5), and Duxbl-s shows similar expression pattern (Fig. 7B). Furthermore, whole-mount in situ hybridizations were used to identify Duxbl transcripts in developing mouse embryos from E9.5 to E12.5. The Duxbl transcripts are detected in forelimb, hindlimb, and tail beginning from E9.5 and maintained to E12.5 (Fig. 7C–E). Duxbl expressions in limbs were further detected by section in situ hybridization and Alcian blue staining. Duxbl signals are localized in muscle cells (data not shown). Furthermore, in vivo Duxbl protein expressions were analyzed by immunohistochemistry. Strong Duxbl protein signals are detected in muscle cells of limbs at E13.5 embryo (Fig. 7F), while myotubes begin to form. Especially, Duxbl protein signals are detected in the fiber-like muscle cells but not in the mononuclear cells expressing MyoD proteins (Fig. 7G). These results indicate that Duxbl proteins are expressed in limbs and tail during embryo development and in differentiated myocytes but not in myoblasts.

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Figure 7. Identifying Duxbl expressions during mouse development by reverse transcriptase-polymerase chain reaction (RT-PCR), whole mount in situ hybridization, and immunohistochemical analysis. A,B: Expressions of Duxbl and Duxbl-s transcripts in preimplantation (A) and postimplantation (B) embryos were analyzed by RT-PCRs. A: Cu, cumulus cells; UF, unfertilized eggs; F, fertilized eggs; 2C, two-cell embryos; MO, morula; BL, blastocysts. B: Total RNAs were extracted from postimplantation embryos at embryonic day 11.5 (E11.5), 13.5 (E13.5), and 17.5 (E17.5), respectively. Amplifications without RNA served as negative controls (−). C–E: Identifying Duxbl expressions in different embryos at embryonic day 9.5 (E9.5), 11.5 (E11.5), and 12.5 (E12.5) by whole-mount in situ hybridizations, respectively. t: tail; fl: forelimb; hl: hindlimb. F,G: Identifying Duxbl and MyoD protein expressions in dorsal sections through forelimb of 13.5-day embryo by immunohistochemical analysis using anti-Duxbl (F) and anti-MyoD (G) antibodies, respectively. The boxed regions in small figures were amplified. H: Analyzing Duxbl and MyoD expression levels by RT-PCRs during embryonic and postnatal limb developments. Total RNAs were extracted from limbs at embryonic day 12.5 (E12.5), 15.5 (E15.5), and 17.5 (E17.5), and at postnatal day 3 (3d) and adult, respectively. Expression levels of actin were used as internal controls. Scale bars = 50 μm.

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Duxbl Expressions During Limb Development

During muscle development in limbs, muscle progenitor cells from somite migrate into limb bud, where they proliferate, express myogenic determination factors and subsequently differentiate into skeletal muscles. MyoD and myogenin (MyoG) are first detected in the proximal regions of both hindlimb and forelimb at embryonic day (E) 11.5 embryo and later accumulated in the differentiated muscle masses, continuously expressed in fetal skeletal muscles (Sassoon et al., 1989). Although expressions of Duxbl and MyoD are both continuous from E12.5 to postnatal stage (3d) in limbs by RT-PCR analyses (Fig. 7H), Duxbl expressions in limbs are largely increased from E12.5 to E15.5 and maintained in high level to postnatal (3d) stage, but almost undetectable in adult muscles by RT-PCR analyses (Fig. 7H). These results suggest that Duxbl expressions are low in myoblast proliferation stage but increase largely following myotube formation, so Duxbl may play a role in myogenesis during embryonic limb development.

Duxbl Protein Expressions in Trunk Myogenesis

During embryogenesis, skeletal muscles in the trunk and limbs are both derived from somites (Tajbakhsh and Buckingham, 2000). During myotome development, MyoG and MyoD are detectable at E10.5 (Cusella-De Angelis et al., 1992), while terminally differentiated muscle cells could already be identified in the myotome. To decipher the Duxbl protein expressions during myotome development in advance, we used immunohistochemistry on parasagittal sections of E11.5 embryos. Duxbl protein is not detected in somite (Fig. 8A). However, the myoblast marker gene products, MyoD proteins, are expressed in the myotomes. To further confirm the expressions of Duxbl proteins during skeletal myogenesis in the trunk and limb, we used immunohistochemistry on transverse sections of embryo at E11.5 with antibodies react with MyoD and Duxbl proteins, respectively. In transverse sections of the same embryo, MyoD-positive cells are also observed in myoblasts of developing limbs and somite. However, no Duxbl-expressing cells are present in transverse sections of the same E11.5 embryos (data not shown). In further embryonic development, Duxbl is seen expressed in the epaxial, hypaxial, and forelimb muscles in E12.5 embryo (Fig. 8B), similar to MyoD (Fig. 8C). However, the intensities of Duxbl proteins are less than MyoD proteins in all muscle-forming regions, especially in limb muscles. After comparing stained cell morphology, Duxbl proteins are obviously stained in fewer elongated myocytes and immature myofibrils (Fig. 8B). However, MyoD proteins are detected in mononuclear myocytes (Fig. 8C). These results suggest that Duxbl expressions are low in limb myoblast proliferation stage, embryonic day 12.5. After myoblast proliferation stage, at E13.5 embryo, myoblasts have clearly acquired a spindle-shaped morphology, and considerably more and longer myofibrils have now been formed. In E13.5 embryonic transverse sections, Duxbl proteins are expressed in increased number of cells, virtually including all of the newly formed primary fibers, present in both body wall and limbs (Fig. 9A). Strong Duxbl signals are detected in elongated myocytes and multinuclear myotubes (Fig. 9A). The distribution and intensity of Duxbl expressions are both similar to those of myosin heavy chain (MHC; Kablar et al., 1997). MHC is expressed exclusively in terminally differentiated myotubes and is detectable in the intercostal muscles of the trunk and the muscle anlagen of forelimb. The expressions of both Duxbl and MHC are increased in E13.5 embryo, in which stage the myotube formations are observed.

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Figure 8. Identifying Duxbl and MyoD protein expressions in embryonic muscles. A–C: Duxbl and MyoD proteins were detected on parasagittal sections of 11.5-day embryo (A) and transverse sections of 12.5-day embryo (B,C) by immunohistochemistry. A: High magnifications of the boxed regions in the left panels were shown in the right. B,C: High magnification of the boxed regions in the left panels of epaxial (E), hypaxial (H), and limb (L) muscles were shown in the middle and right panels as indicated. FL, forelimb; NT, neural tube. Scale bars = 50 μm.

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Figure 9. Identifying Duxbl and MyoD protein expressions in developing muscles. Duxbl (A) and MyoD (B) proteins were detected on transverse sections of 13.5-day embryo by immunohistochemistry. A,B: High magnification of the boxed regions in the left panels of epaxial (E), hypaxial (H), and limb (L) muscles were shown in the middle and right panels as indicated. FL, forelimb. Scale bars = 50 μm.

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Duxbl Expressions During C2C12 Cell Differentiation

To verify that Duxbl is expressed in differentiated myocytes but not in myoblasts during skeletal muscle formation, we examined Duxbl expressions during in vitro differentiations of C2C12 cells. Duxbl transcripts are slightly detected in confluent myocytes, and seen significantly increased in differentiated cells following 2 days in differentiation medium as MyoG (Fig. 10A). Because Duxbl exhibits similar expression patterns as MyoG during C2C12 cell differentiation, we suggest that Duxbl also play a role in myoblast differentiation and/or fusion. Our double immunofluorescence data also show the presence of Duxbl protein signals in differentiating cells and clearly located in the nuclei of multinuclear myotubes (Fig. 10B), similar to results of in vivo immunohistochemistry analyses (Figs. 7F, 8B, 9A). However, MyoD proteins are stained strongly in small nuclear myoblasts and myocytes, while only slightly in myotubes (Fig. 10B). From the above results, we conclude that Duxbl is expressed in myotubes but not in proliferating myoblasts during in vitro differentiation of myoblasts and in vivo skeletal muscle formation. Because the expression of Duxbl is downstream of MyoD and seems parallel to MyoG, whether expression of Duxbl influences MyoD or MyoG or other muscle specific genes to affect myogenesis need further characterizations. C2C12 can be used as a model cell line to identify the molecular mechanism of Duxbl influencing myoblast differentiation or how Duxbl interact with the myogenic regulatory factors to regulate myogenesis.

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Figure 10. Identifying Duxbl, MyoD, and MyoG expressions in differentiated and undifferentiated C2C12 cells. A: Expression levels of Duxbl, MyoD, and MyoG transcripts were determined by reverse transcriptase-polymerase chain reactions (RT-PCRs) during C2C12 differentiation. Total RNAs were extracted from proliferating myoblasts (PMB), confluence myoblasts (CMB), and differentiating myocytes cultured in differentiation medium for 2, 3, and 5 days (D2, D3, D5), respectively. Expression levels of actin were used as internal controls. B: Double immunofluorescence analysis of Duxbl and MyoD expressions in C2C12 cells. C2C12 cells were incubated in growth medium as proliferating myoblasts (PMB) and in differentiation medium for 6 days (D6) as differentiating myocytes, respectively. They were analyzed by double immunofluorescence with anti-Duxbl and anti-MyoD antibodies. Signals of Duxbl proteins were detected with Texas red-conjugated secondary antibodies (Duxbl) and signals of MyoD were detected with fluorescein isothiocyanate (FITC) -conjugated secondary antibodies (MyoD). Cell nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI). Scale bars = 50 μm.

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In brief, Duxbl is a mouse double homeobox gene that contains introns. The Duxbl homeodomain exhibits the maximum identities to those of human DUX4 gene. From the homeodomain similarity, we suggest that Duxbl is the ortholog of human DUX4, which is the candidate gene to cause FSHD. However, the exact expression pattern of DUX4 in human development or during myogenesis is not detected and might be undetectable, because DUX4 protein cannot be obtained from normal adult tissues. Studying the expression profile of a mouse double homeobox gene, Duxbl, can facilitate the understanding of the functions of double homeobox genes including DUX4 during development. In addition, the characterization of Duxbl expression during myogenesis might help in understanding the molecular mechanism of FSHD. During embryo development, Duxbl is predominantly expressed in differentiated myocytes during embryo skeletal myogenesis. The myofibers of epaxial, hypaxial, and limb express Duxbl proteins, first, from E12 embryo. However, Duxbl is barely detectable in the adult limb muscle. Because DUX4 protein can be detected in primary myoblasts extracted from FSHD patients but not from normal people, we suggest that an increase in the expression of embryo protein, mDux in adult tissue may develop a muscular dystrophy with features characteristic of the human disease. In addition to skeletal myogenesis, Duxbl is also expressed in germ cells, especially in oocytes and spermatogonia, during gonad development. Our results suggest that Duxbl is important in germ cell development. Our future experiments would be directed toward characterizing the exact function of this gene using transgenic or conditional knockout analysis.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Animals

Mice were maintained in a specific pathogen-free environment at the animal housing in Chung Shan Medical University. All experimental procedures were conducted in accordance with the guidelines of the institutional animal committee and received protocol number 401.

RNA Isolations and RT-PCRs

Total RNAs were extracted using Trizol reagent (Life Technologies) from various mouse tissues, embryos at different stages, and various mouse cell lines. Quantities and purities of RNAs were determined by ultraviolet absorbance (DU 800; Beckman Coulter) and by gel electrophoresis. Five or two micrograms each of total RNAs were reverse transcribed using Superscript II system (Life Technologies) and oligo-dT primer. P1/GSP1 (P1, 5′-GAGCTGCAGTACTGGCCTACTG-3′; GSP1, 5′-CTGGGAGGACTGAAGTAGTGTGGT-3′) and P1/GSP2 (GSP2, 5′-ATGATTATGCAGGTCTGATGTG-3′) primer pairs were used to determine the expressions of Duxbl and Duxbl-s transcripts (Fig. 1A), respectively. As positive controls, the expression of β-actin gene was detected using the following primers: Actin F: 5′-GAGACCTTCAACACCCCAGC-3′ and Actin R: 5′-AGGAAGGCTGGAAAAGAGCC-3′.

Cloning the Full-Length cDNAs

Because Duxbl and Duxbl-s transcripts are predominantly expressed in adult ovary, total ovary RNAs were used to characterize the full-length Duxbl and Duxbl-s transcripts. The 5′ and 3′ end sequences of Duxbl and Duxbl-s transcripts were obtained by 5′ and 3′ RACE-PCRs using the SMART RACE cDNA amplification kit (Clontech), respectively. Total RNAs were reversely transcribed using 5′ or 3′ CDS primers. The resultant cDNA products were subjected to PCR for generating the 5′ and 3′ Duxbl and Duxbl-s cDNA fragments, respectively, using transcript-specific primers (GSP1, GSP2, or GSP3: 5′-AGCAGGAGCAGGATAAACCTAGAGTTAAAGA-3′) and UPM primer. The PCR products were then subjected to nested PCR using transcript-specific primers (GSP1 or GSP2) and nested universal primer A. Finally, the PCR products were analyzed on 1.2% agarose gels, subcloned into pGEM-T Easy vectors (Promega) and then sequenced.

Real-Time PCRs

Total RNAs were extracted from testes at different stages using TRI Reagent (Sigma-Aldrich). Primers for real-time PCRs were designed for mouse Duxbl and GAPDH transcripts. The forward primer for Duxbl was 5′-GCATCTCTGAGTCTCAAATTATGACTTG-3′, and the reverse primer was 5′-GCGTTCTGCTCCTTCTAGCTTCT-3′. The forward primer for GAPDH was 5′-TGTGTCCGTCGTGGATCTGA-3′, and the reverse primer was 5′-CCTGCTTCACCACCTTCTTG-3′. Complementary DNAs were synthesized from RNA samples using Superscript II RNase H-minus reverse transcriptase (Invitrogen) according to the manufacturer's protocol, and then cDNAs were used as templates for real-time PCR assays using the ABI Prism 7000 Sequence Detection System (Applied Biosystems). Threshold (Ct) values for Duxbl and GADPH transcripts were determined using Prism SDS software version 1.0 (Applied Biosystems).

Expressions and Purifications of Duxbl Homeodomain Fusion Proteins

The coding regions of Duxbl N-terminal homeodomain (H1) and C-terminal homeodomain (H2) were PCR amplified, gel-purified, and then subcloned into pGEX-6P-2 expression vectors (Amersham). After induction, the expressed glutathione-S-transferase (GST) fusion proteins, GST-H1 and GST-H2, were purified using Glutathione Sepharose beads (Amersham). The forward primer for H1 homeodomain was 5′-CGGAATTCTTGAGCTGAGCTGCAGT-3′, and the reverse primer was 5′-CCCTCGAGCTATGCAAACTCTGCT TG-3′. The forward primer for H2 homeodomain was 5′-CGGAATTCTTAGAGTTAAAGAAGCTAGAAG-3′, and the reverse primer was 5′-GCGGCCGCTTA AGTTTTCTGAGTGTTCTGTCC-3′.

Production of Anti-Duxbl Polyclonal Antibodies and Western blotting

The Duxbl coding region was PCR amplified, gel-purified, and then inserted into the pAE expression vector (Ramos et al., 2004). The Duxbl fusion proteins containing 6 histidine residues at the N-terminus (6His-Duxbl) were overexpressed and then purified using His-bind resin (Novagen), before being used to immunize rabbits for generation of anti-serum against Duxbl proteins. These anti-Duxbl polyclonal antibodies were purified by affinity chromatography using 6His-Duxbl fusion proteins. To test the immunospecificity of purified anti-Duxbl antibodies, we performed a blocking assay using antibodies pretreated with purified GST-H1 and GST-H2 proteins or GST protein only. To detect Duxbl protein in various mouse tissues, equal amount of total proteins extracted from various adult mouse tissues were separated in 12% sodium dodecyl sulfate-polyacrylamide gels and electro-transferred onto polyvinylidene fluoride membranes (Millipore). Expression levels of Duxbl proteins were then determined by Western blotting using purified anti-Duxbl polyclonal antibodies.

Whole-Mount In Situ and Section In Situ Hybridizations

Expressions of the Duxbl gene were analyzed by in situ hybridizations using a 660-bp Duxbl cDNA fragment (265-925), which encodes the coding region of Duxbl protein. The 660-bp Duxbl cDNA fragments (position 265-925) were subcloned into pGEM-T Easy vectors. The sense and anti-sense riboprobes were prepared by in vitro transcriptions using SP6 and T7 RNA polymerases with digoxigenin (Dig)-UTP (Boehringer Mannheim), respectively. Some serial sections were stained with hematoxylin and eosin (Sigma-Aldrich).

Testes from adult mice and ovaries from 10-day-old mice were collected and fixed in 4% paraformaldehyde for in situ hybridizations. They were dehydrated, embedded in paraffin, and then serially sectioned. Five- or 7-μm sections were cut and counterstained with methyl green. These sections were then mounted before observation. For whole-mount in situ hybridizations, embryos were rehydrated and bleached in PBT containing 6% hydrogen peroxide. Whole-mount in situ hybridizations of embryos was performed as previously described (Correia and Conlon, 2001).

Immunohistochemistry

Embryos of FVB mice and adult ovaries were fixed in 4% paraformaldehyde and embedded in paraffin according to standard protocols. Adjacent 6-μm sections were used for comparative analysis. Sections were deparaffinized and rehydrated, and some of them were stained with hematoxylin and eosin. Sections were incubated with a 1:300 dilution of purified anti-Duxbl polyclonal antibodies or with 1:500 dilution of mouse monoclonal anti-MyoD antibody clone 5.8A (IMGENEX). After washing, sections were incubated with a 1:200 dilution of biotinylated goat anti-rabbit IgG or biotinylated goat anti-mouse IgG followed by incubation with horse radish peroxidase-streptavidin complexes. Positive signals were visualized by incubation with diaminobenzidine (DAB), a kit from Molecular Probes, and sections were then lightly counter-stained with methyl green (Sigma-Aldrich). Negative controls consisted of identical reactions with normal rabbit immunoglobulin G as the primary antibodies.

Cell Cultures

C2C12 cells were kept in DMEM supplemented with 10% fetal bovine serum. Differentiation in C2C12 cells was induced by replacing the medium with differentiation medium (2% horse serum in DMEM). HeLa cells were kept in alpha-MEM supplemented with 10% fetal calf serum, 1% nonessential amino acids, and 1% sodium pyruvate.

Subcellular Localizations of Duxbl and Duxbl-s Proteins

The coding regions of Duxbl and Duxbl-s were inserted into pcDNA3.1/V5-His (Life technologies) and pFLAG-CMV2 (Sigma-Aldrich) to produce the Duxbl-V5 and FLAG-Duxbl-s expression vectors, respectively. The expression vectors were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After transfection, cells were immunostained with a 1:500 dilution of purified anti-Duxbl polyclonal antibodies and then a 1:200 dilution of fluorescein isothiocyanate (FITC) -conjugated goat anti-rabbit immunoglobulin. After immunostaining, cell nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The fluorescent signals were obtained by fluorescence microscopy (Axioplan, Zeiss).

Immunocytochemistry

Expressions of Duxbl and MyoD proteins in C2C12 cells were examined by immunocytochemistry. C2C12 myoblasts were grown in 4-well chamber slides and were allowed to differentiate into myotubes. The cells were then fixed in 4% paraformaldehyde followed by permeable with 0.1% Triton. Cells were blocked with 1% bovine serum albumin in phosphate buffered saline (PBS). C2C12 cells were immunostained with rabbit anti-Duxbl (1:500) polyclonal antibodies and mouse monoclonal anti-MyoD (1:500) antibody. Cells were washed with PBS, incubated with FITC-conjugated chicken anti-mouse IgG antibodies and Texas red-conjugated goat anti-rabbit IgG antibodies at a 1:300 dilution, and then counterstained for nuclei with DAPI. The fluorescent signals were obtained by fluorescence microscopy.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
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