Expression of Bblhx3, a LIM-homeobox gene, in the development of amphioxus Branchiostoma belcheri tsingtauense

Amphioxus Bblhx3 was identified as a LIM-homeobox gene expressed in gastrulae. Structural analysis suggested that it is a member of lhx3 but not of lhx1 gene group. Whole mount in situ hybridization revealed, that expression of Bblhx3 was initiated at the early gastrula stage and continued at least until 10-day larvae. Expression of Bblhx3 first appeared in the vegetal and future dorsal area in initial gastrulae and became restricted to the endoderm during gastrulation. In neurulae and early larvae, Bblhx3 was expressed in the developing neural tube, the notochord and preoral pit lineage. In 10-day larvae, Bblhx3 was expressed only in the preoral pit. This expression pattern is apparently distinct from that of vertebrate lhx3 genes that are not expressed during gastrulation. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Sequence characterisation and expression of homeobox HOX A7 in the multi-potential erythroleukaemic cell line TF-1

Homeobox gene expression was examined in the erythroleukaemic cell line TF-1. Expression of a number of HOX A, B and C genes, including HOX A7 was detected. Expression of this gene has not previously been reported in erythroleukaemic cell lines. A 2.1 kb full length cDNA of the HOX A7 gene was cloned. The predicted amino acid sequence C-terminal to the homeodomain consists of an alanine-rich region and a strongly negatively charged domain consisting entirely of aspartic and glutamic acid residues.

Chromosomal mapping of ANTP class homeobox genes in amphioxus: piecing together ancestral genomes

Homeobox genes encode DNA-binding proteins, many of which are implicated in the control of embryonic development. Evolutionarily, most homeobox genes fall into two related clades: the ANTP and the PRD classes. Some genes in ANTP class, notably Hox, ParaHox, and NK genes, have an intriguing arrangement into physical clusters. To investigate the evolutionary history of these gene clusters, we examined homeobox gene chromosomal locations in the cephalochordate amphioxus, Branchiostoma floridae. We deduce that 22 amphioxus ANTP class homeobox genes localize in just three chromosomes. One contains the Hox cluster plus AmphiEn, AmphiMnx, and AmphiDll. The ParaHox cluster resides in another chromosome, whereas a third chromosome contains the NK type homeobox genes, including AmphiMsx and ArnphiTlx. By comparative analysis we infer that clustering of ANTP class homeobox genes evolved just once, during a series of extensive cis-duplication events of genes early in animal evolution. A trans-duplication event occurred later to yield the Hox and ParaHox gene clusters on different chromosomes. The results obtained have implications for understanding the origin of homeobox gene clustering, the diversification of the ANTP class of homeobox genes, and the evolution of animal genomes.

Dispersal of NK homeobox gene clusters in amphioxus and humans

The Drosophila melanogaster genome has six physically clustered NK-related homeobox genes in just 180 kb. Here we show that the NK homeobox gene cluster was an ancient feature of bilaterian animal genomes, but has been secondarily split in chordate ancestry. The NK homeobox gene clusters of amphioxus and vertebrates are each split and dispersed at two equivalent intergenic positions. From the ancestral NK gene cluster, only the Tlx-Lbx and NK3-NK4 linkages have been retained in chordates. This evolutionary pattern is in marked contrast to the Hox and ParaHox gene clusters, which are compact in amphioxus and vertebrates, but have been disrupted in Drosophila.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

The homeobox gene HLXB9 is upregulated in a morphological subset of poorly differentiated hepatocellular carcinoma

The prognostic outcome for hepatocellular carcinoma (HCC) remains poor. Disease progression is accompanied by dedifferentiation of the carcinoma, a process that is not well understood. The aim of this study was to get more insight into the molecular characteristics of dedifferentiated carcinomas using high throughput techniques. Microarray-based global gene expression analysis was performed on five poorly differentiated HCC cell lines compared with non-neoplastic hepatic controls and a set of three cholangiolar carcinoma (CC) cell lines. The gene with the highest upregulation was HLXB9. HLXB9 is a gene of the homeobox genfamily important for the development of the pancreas. RT-PCR confirmed the upregulation of HLXB9 in surgical specimens of carcinoma tissue, suggesting its biological significance. Interestingly, HLXB9 upregulation was primary observed in poorly differentiated HCC with a pseudoglandular pattern compared with a solid pattern HCC or in moderate or well-differentiated HCC. Additional the expression of translated HLXB9, the protein HB9 (NCBI: NP_001158727), was analyzed by western blotting. Expression of HB9 was only detected in the cytoplasm but not in the nuclei of the HCC cells. For validation CC were also investigated. Again, we found an upregulation of HLXB9 in CC cells accompanied by an expression of HB9 in the cytoplasms of these tumor cells, respectively. In conclusion, homeobox HLXB9 is upregulated in poorly differentiated HCC with a pseudoglandular pattern. The translated HB9 protein is found in the cytoplasm of these HCC and CC. We therefore assume HLXB9 as a possible link in the understanding of the development of HCC and CC, respectively.

The bovine aristaless-like homeobox 4 (ALX4) as a candidate gene for syndactyly

The ALX4 (aristaless-like homeobox 4) gene encodes a paired-type homeodomain transcriptional activator and plays a major role in anterior-posterior pattern formation during limb development. Here, the cloning, genomic structure and expression of the bovine ortholog of the ALX4 gene are reported. The bovine ALX4 gene consists of four exons and is located on BTA15q28-->q29 in a region syntenic to HSA11p11.2. The transcribed ALX4 mRNA encodes a 397-amino-acid protein showing a paired-type homeodomain and a C-terminal stretch of amino acids known as the OAR- or aristaless domain. The predicted protein shares 92.5% identity to human and mouse ALX4 proteins and all three species share almost complete identity in the conserved domains. ALX4 expression was detected by reverse transcriptase polymerase chain reaction in bovine fetal limb bones. The ALX4 gene was evaluated as a candidate gene for bovine syndactyly which has been mapped on the telomeric region of cattle chromosome 15. Sequencing of the four exons with flanking sequences of the bovine ALX4 gene from a panel of 14 affected animals belonging to German Holstein, German Fleckvieh and crossbreds, and 27 unaffected individuals from German Holstein revealed five silent SNPs within the coding region out of eleven SNPs in total. Four SNPs were polymorphic in the affected animals, but in comparison to the genotyped unaffected individuals the genotype distribution showed no evidence for an association to the phenotype. Therefore our data indicate that the ALX4 gene can probably be excluded as candidate gene for bovine syndactyly in the examined animals.

THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

Functional studies of homeobox gene {\it Cart1\/} during mouse development

Cart1 is a paired-class homeobox-containing gene that is expressed in head mesenchyme, branchial arches, limb buds, and various cartilages during embryogenesis. To understand the role of Cart1 during mammalian development, I generated Cart1-mutant mice by gene targeting in mouse embryonic stem cells. Cart1-homozygous mutants were born alive but all died soon after birth. Most had acrania (absence of the cranial vault) and meroanencephaly (absence of part of the brain). In situ hybridization studies showed that Cart1 is expressed specifically in forebrain mesenchyme but not in midbrain or hindbrain mesenchyme nor in the neural tube. Developmental studies revealed a transient deficiency of forebrain mesenchyme cells due to apoptosis associated with a delay in neural tube closure in that region. Subsequently, the forebrain region became filled with mesenchyme and closed, however, the midbrain neural tube region never initiated closure and remained open. These results suggest that Cart1 is required for the survival of forebrain mesenchyme and that its absence disrupts cranial neural tube morphogenesis by blocking the initiation of closure in the midbrain region, and this ultimately leads to the generation of lethal craniofacial defects. Prenatal treatment of Cart1 homozygous mutants with folic acid suppressed the development of the acrania/meroanencephaly phenotype. Thus, Cart1 mutant mice provide a novel animal model for understanding the cellular, molecular, and genetic etiology of neural tube defects and for the development of prenatal therapeutic protocols using folic acid. ^

Functional studies of the homeobox gene {\it Goosecoid\/} during mouse embryogenesis

A fundamental question in developmental biology is to understand the mechanisms that govern the development of an adult individual from a single cell. Goosecoid (Gsc) is an evolutionarily conserved homeobox gene that has been cloned in vertebrates and in Drosophila. In mice, Gsc is first expressed during gastrulation stages where it marks anterior structures of the embryo, this pattern of expression is conserved among vertebrates. Later, expression is observed during organogenesis of the head, limbs and the trunk. The conserved pattern of expression of Gsc during gastrulation and gain of function experiments in Xenopus suggested a function for Gsc in the development of anterior structures in vertebrates. Also, its expression pattern in mouse suggested a role in morphogenesis of the head, limbs and trunk. To determine the functional requirement of Gsc in mice a loss of function mutation was generated by homologous recombination in embryonic stem cells and mice mutant for Gsc were generated.^ Gsc-null mice survived to birth but died hours after delivery. Phenotypic analysis revealed craniofacial and rib cage abnormalities that correlated with the second phase of Gsc expression in the head and trunk but no anomalies were found that correlated with its pattern of expression during gastrulation or limb development.^ To determine the mode of action of Gsc during craniofacial development aggregation chimeras were generated between Gsc-null and wild-type embryos. Chimeras were generated by the aggregation of cleavage stage embryos, taking advantage of two different Gsc-null alleles generated during gene targeting. Chimeras demonstrated a cell-autonomous function for Gsc during craniofacial development and a requirement for Gsc function in cartilage and mesenchymal tissues.^ Thus, during embryogenesis in mice, Gsc is not an essential component of gastrulation as had been suggested in previous experiments. Gsc is required for craniofacial development where it acts cell autonomously in cartilage and mesenchymal tissues. Gsc is also required for proper development of the rib cage but it is dispensable for limb development in mice. ^

Function and regulation of the Pem homeobox gene in vivo

Pem, a member of the PEPP homeobox family, is expressed in somatic cells in male and female reproductive tissues. In the adult murine testis, Pem is specifically expressed in Sertoli cells, where it is restricted to stages IV–VIII of the seminiferous epithelial cycle. To identify Pem's function in Sertoli cells, transgenic mice were generated that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in an increase in double-strand DNA breaks in preleptotene spermatocytes and single-strand DNA breaks in elongating spermatids. My results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells. Three additional transgenic mouse containing varying lengths of the Pem male-specific promoter (Pp) were generated to identify the sequences responsible for regulating Pem expression in the testis and epididymis. My analysis suggests that there are at least two regulatory regions in the Pem Pp. In the testis, region II directs androgen-dependent expression specifically in Sertoli cells whereas region I fine-tunes stage-specific expression by acting as a negative regulator. In the epididymis, region II confers androgen-dependent, developmentally-regulated expression in the caput whereas region I prevents inappropriate expression in the corpus. I also report the identification and characterization of two human PEPP family members related to Pem that I have named hPEPP1 and hPEPP2. The hPEPP1 and hPEPP2 homeodomains are more closely related to PEPP subfamily homeodomains than to any other homeodomain subfamily. Both genes are localized to the specific region of the human X chromosome that shares synteny with the region on the murine X chromosome containing three PEPP homeobox genes, Pem, Psx-1, and Psx-2. hPEPP1 and hPEPP2 mRNA expression is restricted to the testis but is aberrantly expressed in tumor cells of different origins, analogous to the expression pattern of Pem but not of Psx-1 or Psx-2. Unlike all known PEPP members, neither hPEPP1 nor hPEPP2 are expressed in placenta, which suggests that the regulation of the PEPP family has undergone significant alteration since the split between hominids and rodents. ^

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^