The Rhox5 homeobox gene regulates the region-specific expression of its paralogs in the rodent epididymis

The mechanisms by which the region-specific expression patterns of clustered genes evolve are poorly understood. The epididymis is an ideal organ to examine this, as it is a highly segmented tissue that differs significantly in structure between closely related species. Here we examined this issue through analysis of the rapidly evolving X-linked reproductive homeobox (Rhox) gene cluster, the largest known homeobox gene cluster in metazoans. In the mouse, we found that most Rhox genes are expressed primarily in the caput region of the epididymis, a site where sperm mature and begin acquiring forward motility. This region-specific expression pattern depends, in part, on the founding member of the Rhox cluster--Rhox5--as targeted mutation of Rhox5 greatly diminishes the expression of several other family members in the caput region. In the rat, Rhox5 expression switches from the caput to the site of sperm storage: the cauda. All Rhox genes under the control of Rhox5 in the mouse epididymis display a concomitant change in their regional expression in the rat epididymis. Our results lead us to propose that widespread changes in the region-specific expression pattern of genes over evolutionary time can be the result of alterations of one or only a few master regulatory genes.     

Remarkable expansions of an X-linked reproductive homeobox gene cluster in rodent evolution

Rhox is a recently identified cluster of 12 X-linked homeobox genes in mice. The expression pattern of Rhox genes during postnatal testis development corresponds to their chromosomal position, much like the colinear gene regulation of the Hox gene clusters during animal embryonic development. We here report the identification of 18 additional Rhox genes and 3 pseudogenes in mice. Comparative analyses of the mouse, rat, human, dog, cow, opossum, and chicken genomes suggest that the Rhox cluster originated in the common ancestor of primates and rodents. It subsequently underwent two remarkable expansions, first in the common ancestor of mice and rats and then in mice. Positive selection promoting amino acid substitutions was detected in some young Rhox genes, suggesting adaptive functional diversification. The recent expansions of the Rhox cluster provide an opportunity to study the mechanism and origin of colinear gene regulation, but they may also undermine the utility of mouse models for understanding the development and physiology of the human reproductive system.     

The rhox homeobox gene family shows sexually dimorphic and dynamic expression during mouse embryonic gonad development

Reproductive capacity is fundamental to the survival of all species. Consequently, much research has been undertaken to better understand gametogenesis and the interplay between germ cells and the somatic cell lineages of the gonads. In this study, we have analyzed the embryonic expression pattern of the X-linked gene family Reproductive homeobox genes on the X chromosome (Rhox) in mice. Our data show that eight members of the Rhox gene family are developmentally regulated in sexually dimorphic and temporally dynamic patterns in the developing germ cells during early gonadogenesis. These changes coincide with critical stages of differentiation where the germ cells enter either mitotic arrest in the testis or meiotic arrest in the ovary. Finally, we show that Rhox8 (Tox) is the only member of the Rhox gene family that is expressed in the somatic compartment of the embryonic gonads. Our results indicate that the regulation of Rhox gene expression and its potential function during embryogenesis are quite distinct from those previously reported for Rhox gene regulation in postnatal gonads.     

High-level expression, polyclonal antibody preparation and sub-cellular localization analysis of mouse Rhox5 protein

Mouse reproductive homeobox on the X chromosome (Rhox) is a novel homeobox gene cluster. Rhox5, also called Pem, belongs to the beta subcluster of Rhox. Codon analysis indicated that the cDNA contains 16% of codons rarely used in Escherichia coli. To achieve high-level expression of Rhox5, the coding sequence of Rhox5 was amplified and subcloned into the prokaryotic expression vector pET22b (+) in order to produce 6His-tagged fusion protein in the modified BL21 (DE3) cells, namely Rosetta2 (DE3) cells. The 6His-tagged Rhox5 was expressed efficiently in Rosetta2 (DE3), compared with marginal expression in BL21 (DE3). The fusion protein amounted to 16% of the total bacterial proteins after induction with 0.4mM IPTG for 1.5h at 37 degrees C. After purification, Rhox5-6His was used to immunize New Zealand white rabbits following standard protocol. The homemade antiserum could detect both endogenous Rhox5 protein expressed in eukaryotic cells (Cos-7) and exogenous GFP-Rhox5 protein. Furthermore, the antiserum was used to determine the localization of Rhox5 in NIH3T3 cells using an immunofluorescence technique. The results demonstrated that Rhox5 was localized predominantly in the nucleus. Preparation of the anti-Rhox5 polyclonal antibody will facilitate further functional study of Rhox5.     

shRNA Off-Target Effects In Vivo: Impaired Endogenous siRNA Expression and Spermatogenic Defects

RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA—Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.     

Homeobrain, a novel paired-like homeobox gene is expressed in the Drosophila brain

The homeobrain (hbn) gene is a new paired-like homeobox gene which is expressed in the embryonic brain and the ventral nerve cord. Expression of homeobrain initiates during the blastoderm stage in the anterior dorsal head primordia and the gene is persistently expressed in these cells which form parts of the brain during later embryonic stages. An additional weaker expression pattern is detected in cells of the ventral nerve cord from stage 11 on. The homeodomain in the Homeobrain protein is most similar to the Drosophila proteins DRx, Aristaless and Munster. In addition, the localized brain expression patterns of homeobrain and DRx resemble each other. Two other homeobox genes, orthopedia and DRx are clustered in the 57B region along with homeobrain. The current evidence indicates that homeobrain, DRx and orthopedia form a homeobox gene cluster in which all the members are expressed in specific embryonic brain subregions.     

Mdfic与Rhox5蛋白相互作用的关键结构域鉴定

Mdfic（MyoD family inhibitor domain containing）是一个新发现的含有MyoD抑制素结构域（I-mfa domain）的转录调控因子,可能在肌细胞的分化过程中发挥重要作用. 小鼠Rhox5为同源异型框基因,隶属于Rhox基因簇（reproductive homeobox on the X chromosome genes cluster）β亚簇.在前期证实Mdifc能结合Rhox5蛋白的基础上,进一步鉴定两者相互作用的关键结构域.生物信息学分析Mdfic 的氨基酸序列,PCR方法扩增Mdfic A截短型片段（第72～247位氨基酸残基）,含保守的I-mfa结构域; 双向酵母双杂交和体外GST-Pull down结果表明,该截短型片段可以与Rhox5蛋白结合,且结合力度较完整的Mdfic蛋白强; 将Mdfic A片段划分为两段: Mdfic B（72～191 aa, 不含I-mfa结构域）和Mdfic C（191～247 aa, 含I-mfa结构域）.结果表明,含保守I-mfa结构域的Mdfic C截短型片段丧失了与Rhox5蛋白结合的能力,而不含I-mfa结构域的Mdfic B截短型片段可以结合Rhox5蛋白. 鉴于Mdfic蛋白的非I-mfa结构域在Rhox5/Mdfic结合中发挥关键作用, Rhox5与Mdfic的结合可能进一步调控由Mdfic的I-mfa结构域参与的其他转录因子（如MyoD）的调控,三者形成一个复杂的调控网络,共同参与肌细胞发生及分化的调控.     

Expression of a homologue of the fushi tarazu (ftz) gene in a cirripede crustacean

In Metazoa, Hox genes control the identity of the body parts along the anteroposterior axis. In addition to this homeotic function, these genes are characterized by two conserved features: They are clustered in the genome, and they contain a particular sequence, the homeobox, encoding a DNA-binding domain. Analysis of Hox homeobox sequences suggests that the Hox cluster emerged early in Metazoa and then underwent gene duplication events. In arthropods, the Hox cluster contains eight genes with a homeotic function and two other Hox-like genes, zerknullt (zen)/Hox3 and fushi tarazu (ftz). In insects, these two genes have lost their homeotic function but have acquired new functions in embryogenesis. In contrast, in chelicerates, these genes are expressed in a Hox-like pattern, which suggests that they have conserved their ancestral homeotic function. We describe here the characterization of Diva, the homologue of ftz in the cirripede crustacean Sacculina carcini. Diva is located in the Hox cluster, in the same position as the ftz genes of insects, and is not expressed in a Hox-like pattern. Instead, it is expressed exclusively in the central nervous system. Such a neurogenic expression of ftz has been also described in insects. This study, which provides the first information about the Hoxcluster in Crustacea, reveals that it may not be much smaller than the insect cluster. Study of the Diva expression pattern suggests that the arthropod ftz gene has lost its ancestral homeotic function after the divergence of the Crustacea/Hexapoda clade from other arthropod clades. In contrast, the function of ftz during neurogenesis is well conserved in insects and crustaceans.     

LIM同源框基因家族与神经系统

同源框基因是指一类含有同源序列的基因,它编码的蛋白质作为转录调节因子调节细胞的发育和分化,控制基因的表达形式。ＬＩＭ同源框基因不仅含有同源框基因也含有编码ＬＩＭ结构域的保守序列。