HOXD10 Expression Research Articles

HOXA13 and HOXD13 expression during development of the syndactylous digits in the marsupial Macropus eugenii

BackgroundKangaroos and wallabies have specialised limbs that allow for their hopping mode of locomotion. The hindlimbs differentiate much later in development but become much larger than the forelimbs. The hindlimb autopod has only four digits, the fourth of which is greatly elongated, while digits two and three are syndactylous. We investigated the expression of two genes, HOXA13 and HOXD13, that are crucial for digit patterning in mice during formation of the limbs of the tammar wallaby.ResultsWe describe the development of the tammar limbs at key stages before birth. There was marked heterochrony and the hindlimb developed more slowly than the forelimb. Both tammar HOXA13 and HOXD13 have two exons as in humans, mice and chickens. HOXA13 had an early and distal mRNA distribution in the tammar limb bud as in the mouse, but forelimb expression preceded that in the hindlimb. HOXD13 mRNA was expressed earlier in the forelimb than the hindlimb and was predominantly detected in the interdigital tissues of the forelimb. In contrast, the hindlimb had a more restricted expression pattern that appeared to be expressed at discrete points at both posterior and anterior margins of the limb bud, and was unlike expression seen in the mouse and the chicken.ConclusionsThis is the first examination of HOXA and HOXD gene expression in a marsupial. The gene structure and predicted proteins were highly conserved with their eutherian orthologues. Interestingly, despite the morphological differences in hindlimb patterning, there were no modifications to the polyalanine tract of either HOXA13 or HOXD13 when compared to those of the mouse and bat but there was a marked difference between the tammar and the other mammals in the region of the first polyserine tract of HOXD13. There were also altered expression domains for both genes in the developing tammar limbs compared to the chicken and mouse. Together these findings suggest that the timing of HOX gene expression may contribute to the heterochrony of the forelimb and hindlimb and that alteration to HOX domains may influence phenotypic differences that lead to the development of marsupial syndactylous digits.

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

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 D10 Gene, a Candidate Tumor Suppressor, Is Downregulated through Promoter Hypermethylation and Associated with Gastric Carcinogenesis

Homeobox D10 (HoxD10 ) gene plays a critical role in cell differentiation and morphogenesis during development. However, the function of HoxD10 in tumor progression remains largely unknown. We demonstrate that the expression of HoxD10 is commonly downregulated in gastric cancer tissues (n = 33) and cell lines (n = 8) relative to normal stomach tissues. Functionally, reexpression of HoxD10 results in significant inhibition of cell survival, induction of cell apoptosis, and impairment of cell migration and invasion. Moreover, ectopic expression of HoxD10 suppresses gastric tumor growth in a mouse xenograft model. To identify target candidates of HoxD10, we performed cDNA microarray and showed that HoxD10 regulates multiple downstream genes including IGFBP3. Reintroduction of HoxD10 transcriptionally upregulates IGFBP3, activates caspase 3 and caspase 8, and subsequently induces cell apoptosis. Methylation specific PCR revealed that HoxD10 promoter DNA was hypermethylated in gastric cancer cell lines. Additionally, 5-aza demethylation treatment could transiently reactivate the expression of HoxD10 in gastric cancer cells. HoxD10 promoter methylation frequently was detected in gastric cancer tissues obtained from endoscopic biopsies (85.7%, 24/28) and surgically resected samples (82.6%, 57/69). Intestinal metaplasia tissues showed a 60% methylation rate (18/30), but no detectable methylation in normal stomach tissues (0%, 0/10). Taken together, our results suggest that HoxD10 functions as a candidate tumor suppressor in gastric cancer, which is inactivated through promoter hypermethylation.

Enforced expression of the transcription factor HOXD3 under the control of the Wnt1 regulatory element modulates cell adhesion properties in the developing mouse neural tube

HOX genes expressed in a specific spatial and temporal manner play a crucial role in determining the body plan during the early development of vertebrates. In adult tissues, many HOX genes participate in normal hematopoiesis and carcinogenesis. We previously found that overexpression of the homeobox gene HOXD3 alters expression levels of cell adhesion molecules in human cancer cell lines. Here, we have investigated whether HOXD3 expression is related to the cell adhesion processes during mouse development focusing on dorsal midline cells or roof-plate cells of the neural tube and neural crest cells. We created transgenic mouse embryos, in which HOXD3 is expressed in the dorsal midline under the control of the Wnt1 regulatory element, and analyzed these embryos at embryonic day 10.5-13.5. In HOXD3-expressing transgenic embryos, although neural crest-derived structures in the trunk region appeared to be normal, striking abnormalities were found in the neural tube. In transgenic embryos expressing the lacZ gene under the control of the Wnt1 regulatory element, expression of lacZ was restricted to roof-plate cells within the neural tube. By contrast, in HOXD3-expressing transgenic embryos, expression of HOXD3 was not only located in the dorsal neural tube, but also had spread inside the ventricular zone in more ventral regions of the neural tube. These findings show that the HOXD3 transgene is expressed more broadly than the Wnt1 gene is normally expressed. Expression of both Wnt1 and Msx1, marker genes in the roof plate, was further extended ventrally in HOXD3-expressing embryos than in normal embryos, suggesting that expression of the HOXD3 transgene expands the roof plate ventrally within the neural tube. In the ventricular zone of HOXD3-expressing embryos at embryonic day 10.5, we observed an increase in the number of mitotic cells and failure of interkinetic nuclear migration of progenitor cells. Furthermore, in HOXD3-expressing embryos at embryonic day 12.5, the ventricular zone, in which progenitor cells became more loosely connected to each other, was composed of a large number of cells that did not express N-cadherin. Our results indicate that expression of HOXD3 is closely associated with modulation of cell-adhesive properties during embryonic development.

Heparin Impairs Angiogenesis through Inhibition of MicroRNA-10b

Heparin, which has been used as an anticoagulant drug for decades, inhibits angiogenesis, whereas thrombin promotes tumor-associated angiogenesis. However, the mechanisms underlying the regulation of angiogenesis by heparin and thrombin are not well understood. Here, we show that microRNA-10b (miR-10b) is down-regulated by heparin and up-regulated by thrombin in human microvascular endothelial cells (HMEC-1). Overexpression of miR-10b induces HMEC-1 cell migration, tube formation, and angiogenesis, and down-regulates homeobox D10 (HoxD10) expression via direct binding of miR-10b to the putative 3' UTR of HoxD10. In addition, HMEC-1 cell migration and tube formation are induced by HoxD10 knockdown, whereas angiogenesis is arrested when HoxD10 expression is increased after anti-miR-10b or heparin treatments. Furthermore, expression of miR-10b and its transcription factor Twist are up-regulated by thrombin, whereas HoxD10 expression is impaired by thrombin. Using quartz crystal microbalance analysis, we show that heparin binds to thrombin, thereby inhibiting thrombin-induced expression of Twist and miR-10b. However, the expression of miR-10b is not attenuated by heparin any more after thrombin expression is silenced by its siRNA. Interestingly, we find that heparin attenuates miR-10b expression and induces HoxD10 expression in vivo to inhibit angiogenesis and impair the growth of MDA-MB-231 tumor xenografts. These results provide insight into the molecular mechanism by which heparin and thrombin regulate angiogenesis.

Comparative Analysis of 3D Expression Patterns of Transcription Factor Genes and Digit Fate Maps in the Developing Chick Wing

Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.

The sequence, structure and evolutionary features of HOTAIR in mammals

BackgroundAn increasing number of long noncoding RNAs (lncRNAs) have been identified recently. Different from all the others that function in cis to regulate local gene expression, the newly identified HOTAIR is located between HoxC11 and HoxC12 in the human genome and regulates HoxD expression in multiple tissues. Like the well-characterised lncRNA Xist, HOTAIR binds to polycomb proteins to methylate histones at multiple HoxD loci, but unlike Xist, many details of its structure and function, as well as the trans regulation, remain unclear. Moreover, HOTAIR is involved in the aberrant regulation of gene expression in cancer.ResultsTo identify conserved domains in HOTAIR and study the phylogenetic distribution of this lncRNA, we searched the genomes of 10 mammalian and 3 non-mammalian vertebrates for matches to its 6 exons and the two conserved domains within the 1800 bp exon6 using Infernal. There was just one high-scoring hit for each mammal, but many low-scoring hits were found in both mammals and non-mammalian vertebrates. These hits and their flanking genes in four placental mammals and platypus were examined to determine whether HOTAIR contained elements shared by other lncRNAs. Several of the hits were within unknown transcripts or ncRNAs, many were within introns of, or antisense to, protein-coding genes, and conservation of the flanking genes was observed only between human and chimpanzee. Phylogenetic analysis revealed discrete evolutionary dynamics for orthologous sequences of HOTAIR exons. Exon1 at the 5' end and a domain in exon6 near the 3' end, which contain domains that bind to multiple proteins, have evolved faster in primates than in other mammals. Structures were predicted for exon1, two domains of exon6 and the full HOTAIR sequence. The sequence and structure of two fragments, in exon1 and the domain B of exon6 respectively, were identified to robustly occur in predicted structures of exon1, domain B of exon6 and the full HOTAIR in mammals.ConclusionsHOTAIR exists in mammals, has poorly conserved sequences and considerably conserved structures, and has evolved faster than nearby HoxC genes. Exons of HOTAIR show distinct evolutionary features, and a 239 bp domain in the 1804 bp exon6 is especially conserved. These features, together with the absence of some exons and sequences in mouse, rat and kangaroo, suggest ab initio generation of HOTAIR in marsupials. Structure prediction identifies two fragments in the 5' end exon1 and the 3' end domain B of exon6, with sequence and structure invariably occurring in various predicted structures of exon1, the domain B of exon6 and the full HOTAIR.

Abstract 243: Functional dissection of hoxD genes in regulation of neuroblastoma cell proliferation and differentiation

Abstract Neuroblastoma is a common malignant tumor in children, arising in the sympathetic nervous system. The tumor cell differentiation state influences clinical outcome, and induction of differentiation has been considered as a promising therapeutic strategy for neuroblastoma. Retinoid acid (RA) is an effective inducer of neuroblastoma cell differentiation and has been used in clinic for treatment of high-risk neuroblastomas. However, resistance to RA presents a major obstacle to successful RA-based therapy. Therefore, identification of target genes that mediate the anti-neoplastic activity of RA is of paramout importance, as targeting these genes offers opportunities to bypass resistance to RA. A variety of experimental evidence has linked RA action to the activation of HOX genes, which function as master regulators of development and cell fate specification. Particularly, it has been shown that RA induces several HOXD genes in neuroblastoma cells including HOXD1, D8 and D9. Our microarray analysis confirmed the predominant upregulation of HOXD genes in the human neuroblastoma cell line BE(2)-C cells following RA treatment. To investigate the functional significance of the RA-induced upregulation of HOXD genes, we conducted a comprehensive functional analysis of HOXD genes in regulation of neuroblastoma cell proliferation and differentiation. The HOXD cluster contains 9 genes (HOXD1, D3, D4, and D9-13) that are positioned from 3’ to 5’ within the cluster, with HOXD1 at the 3’ end and HOXD13 the 5’ end. We established BE(2)-C-derived cell lines with inducible expression of individual HOXD genes. Induction of HOXD1, D12 or D13 had no significant effect on BE(2)-C cell growth and differentiation. By contrast, individual induction of other HOXD genes caused G1 arrest, which was associated with downregulation of cyclins and CDKs required for cell cycle progression through the S, G2 and M phases. Ectopic expression of HOXD3, D4, D8, D9, D10 or D11 also induced morphologic changes characteristic of neuronal differentiation. Interestingly, HOXD8 and D9 appeared to be most potent in the induction of G1 arrest and differentiation. These findings suggest that HOXD genes positioned in the middle of the cluster have higher differentiation-inducing activity compared to those positioned more 5’ and 3’ in the cluster. Thus, pharmacological activation of HOXD8 and HOXD9 may represent a therapeutic strategy for RA-resistant neuroblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 243. doi:10.1158/1538-7445.AM2011-243

Temporal changes in Hox gene expression accompany endothelial cell differentiation of embryonic stem cells

In pluripotent embryonic stem cells (ESCs), expression of the Hox master regulatory transcription factors that play essential roles in organogenesis, angiogenesis, and maintenance of differentiated tissues, is globally suppressed. We investigated whether differentiation of endothelial cells (ECs) from mouse ESCs was accompanied by activation of distinct Hox gene expression profiles. Differentiation was observed within 3 days, as indicated by the appearance of cells expressing specific endothelial marker genes (Flk-1+/VE-Cadherin+). Expression of HoxA3 and HoxD3, which drive adult endothelial cell invasion and angiogenesis, peaked at day 3 and declined thereafter, whereas expression of HoxA5 and HoxD10, which maintain a mature quiescent EC phenotype, was low at day 3, but increased over time. The temporal and reciprocal changes in HoxD3 and HoxA5 expression were accompanied by corresponding changes in expression of established downstream target genes including integrin β3 and Thrombospondin-2. Our results indicate that differentiation and maturation of ECs derived from cultured ESCs mimic changes in Hox gene expression that accompany maturation of immature angiogenic endothelium into differentiated quiescent endothelium in vivo.

Hyaluronan-CD44 Interaction Promotes c-Src-mediated Twist Signaling, MicroRNA-10b Expression, and RhoA/RhoC Up-regulation, Leading to Rho-kinase-associated Cytoskeleton Activation and Breast Tumor Cell Invasion

Dysregulation of microRNAs is observed in many cancers, including breast cancer. In particular, miR-10b appears to play an important role in tumor cell invasion and breast cancer progression. In this study, we investigated hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with c-Src kinase and the transcriptional factor, Twist, in breast tumor cells (MDA-MB-231 cells). Our results indicate that HA binding to CD44 promotes c-Src kinase activation, which, in turn, increases Twist phosphorylation, leading to the nuclear translocation of Twist and transcriptional activation. Further analyses reveal that miR-10b is controlled by an upstream promoter containing the Twist binding site(s), whereas ChIP assays demonstrate that stimulation of miR-10b expression by HA/CD44-activated c-Src is Twist-dependent in breast tumor cells. This process results in the reduction of a tumor suppressor protein (HOXD10), RhoA/RhoC up-regulation, Rho-kinase (ROK) activation, and breast tumor cell invasion. Treatment of MDA-MB-231 cells with PP2 (a c-Src inhibitor) or Twist-specific siRNAs effectively blocks HA-mediated Twist signaling events, abrogates miR-10b production, and increases HOXD10 expression. Subsequently, this c-Src/Twist signaling inhibition causes down-regulation of RhoA/RhoC expression and impairment of ROK-regulated cytoskeleton function (e.g. tumor cell invasion). To further evaluate the role of miR-10b in RhoGTPase signaling, MDA-MB-231 cells were also transfected with a specific anti-miR-10b inhibitor in order to silence miR-10b expression and block its target functions. Our results demonstrate that anti-miR-10b inhibitor not only enhances HOXD10 expression but also abrogates HA/CD44-mediated tumor cell behaviors in breast tumor cells. Taken together, these findings indicate that the HA-induced CD44 interaction with c-Src-activated Twist plays a pivotal role in miR-10b production, leading to the down-regulation of tumor suppressor protein (HOXD10), RhoGTPase-ROK activation, and tumor cell invasion. All of these events are critical prerequisite steps for the acquisition of metastatic properties by human breast cancer cells.

Abstract LB-263: Study of Hox gene expression in normal and malignant colonic tissues implicates Hox A4 and Hox D10 in the stem cell-origin of colon cancer

Abstract Our goal was to identify transcriptional regulatory mechanisms that maintain stem cell (SC) population size in normal colonic epithelium because dysregulation, following acquisition of mutations, leads to tumor SC overpopulation, tumor initiation, and colon cancer. We used microarray analysis to identify a unique gene-expression pattern for a SC-enriched population (the bottom third of the normal human crypt, where colonic SC reside). Two-color microarray analysis (18,500 genes) was used to analyze gene expression in isolated crypt subsections (top [T], middle [M], and bottom [B] thirds). About 25% of known genes were expressed (showed hybridization to RNA from colonic epithelium). Several RNA species (n=287) were preferentially expressed in one of the three crypt subsections (T, M, or B). Six of the known genes up regulated in the bottom third of the crypt mapped to the P13 kinase pathway and two were Hox genes (Hox A4 and D10). Immunohistochemistry (IHC) was then used to analyze expression of Hox A4 and Hox D10 in human colonic tissues. IHC showed that both Hox A4 and D10 are expressed in cells residing at the bottom of the normal crypt and are co-expressed with aldehyde dehydrogenase 1 (ALDH1), a SC marker in these cells. Moreover, IHC showed that Hox A4 and D10 are overexpressed in tumors compared with normal colonic epithelium. These data represent the first gene-expression profiles for sub-regions of the human colonic crypt, especially the SC-rich crypt base, which we now know has a unique gene-expression signature. Co-expression of Hox A4 and D10 with ALDH1 in normal colonocytes suggests that these Hox genes have a role in regulation of the crypt SC population size. Overexpression of Hox A4 and D10 in colon cancer suggests that these Hox genes, when dysregulated, contribute to SC overpopulation and colon tumorigenesis. Identification of the Hox gene-based mechanisms that are involved in the regulation of normal crypt SC population size and in dysregulation of neoplastic SC populations could lead to identification of SC-based targets and ways to develop new, more effective treatments for advanced colon cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-263.

Hoxd-13 expression in the development of hindgut in ethylenethiourea-exposed fetal rats

Purpose Hoxd-13, as one of the most posterior genes among Hox genes, was reported to play a critical role in the development of the most posterior alimentary canal in vertebrates. This study investigated the expression pattern of Hoxd-13 in the hindgut development of the normal and ethylenethiourea (ETU)-exposed rat embryos with anorectal malformations (ARMs) to find out the possible role of Hoxd-13 in the hindgut development and anorectal morphogenesis. Material and Method The ETU murine model of ARMs was used via ETU 1% (125 mg/kg) on gestational day (gD) 10. Embryos were harvested via cesarean delivery on gD13 to gD21. Temporal and spatial expression of Hoxd-13 was evaluated in the normal fetal rats (n = 215) and ARMs rats (n = 218) using immunohistochemistry staining, reverse transcriptase polymerase chain reaction, and Western blot analysis. Results Immunohistochemistry staining revealed that Hoxd-13 expression was confined to the epithelium of the hindgut, cloacal membrane, and urogenital sinus as well as the mesenchyme of the urorectal septum at all gestations in the normal group; however, in the ARMs group, the signal specific for Hoxd-13 was weak in the epithelium of the hindgut and cloacal membrane as well as the mesenchyme of the urorectal septum. Western blot analysis and reverse transcriptase polymerase chain reaction revealed that the level of Hoxd-13 expression was significantly decreased in the ARMs embryos compared with that in the normal embryos on gD13 to gD16 ( P < .05) rather than on gD18 to gD21. Conclusions The aberrations in spatiotemporal expression pattern of Hoxd-13 on gD13 to gD16 suggested that Hoxd-13 may be an essential inductive signal for normal development of the hindgut, and altered expression may contribute to the abnormal development of the hindgut and accordingly lead to ARMs.

Regulation of H3K27me3 and H3K4me3 during early porcine embryonic development

The epigenetic marks H3K27me3 and H3K4me3 are important repressive and permissive histone modifications, respectively, which are involved in gene regulation such as Hox gene expression during embryonic development. In this study, we investigated the global levels of these two histone modifications. We also investigated the expression of H3K27me3's methyltransferase (EZH2), EZH2 co-factors (EED and SUZ12) and demethylases (JMJD3 and UTX), as well as H3K4me3's methylases (ASH1L and MLL1) and demethylase (RBP2) in porcine pre-implantation embryos. In addition, the expression of Hox genes, HOXA2, HOXA3, HOXA7, HOXA10, HOXB4, HOXB7, HOXC8, HOXD8, and HOXD10 was investigated. We found that global levels of H3K27me3 decreased from the 1- to the 4-cell stage, corresponding to the time of major embryonic genome activation. Subsequently, the levels increased in hatched blastocysts, particularly in the trophectoderm. The expression levels of EZH2, EED, SUZ12, JMJD3, and UTX correlated well with these findings. The global levels of H3K4me3 decreased from the 1-cell to the morula stage and increased in hatched blastocysts, especially in trophectoderm. A peak in expression of ASH1L was seen at the 4-cell stage, but overall, expression of ASH1L, MLL1, and RBP2 correlated poorly with H3K4me3. HOXA3, A7, and B4 were expressed in 4-cell embryos, and HOXA7, A10, B4, and D8 were expressed in hatched blastocysts, and did not correlate well to global methylation of H3K27me3 or H3K4me3. Thus, H3K4me3 may play a role in early porcine embryonic genome activation, whereas, H3K27me3 may be involved in initial cell lineage segregation in the blastocyst.

The expression of Gli3, regulated by HOXD13, may play a role in idiopathic congenital talipes equinovarus

BackgroundIdiopathic congenital talipes equinovarus (ICTEV) is a congenital limb deformity. Based on extended transmission disequilibrium testing, Gli-Kruppel family member 3 (Gli3) has been identified as a candidate gene for ICTEV. Here, we verify the role of Gli3 in ICTEV development.MethodsUsing the rat ICTEV model, we analyzed the differences in Gli3 expression levels between model rats and normal control rats. We used luciferase reporter gene assays and ChIP/EMSA assays to analyze the regulatory elements of Gli3.ResultsGli3 showed higher expression levels in ICTEV model rats compared to controls (P < 0.05). We identified repressor and activator regions in the rat Gli3 promoter. The Gli3 promoter also contains two putative Hoxd13 binding sites. Using EMSA, the Hoxd13 binding site 2 was found to directly interact with Hoxd13 in vitro. ChIP assays of the Hoxd13-Gli3 promoter complex from a developing limb confirmed that endogenous Hoxd13 interacts with this region in vivo.ConclusionOur findings suggest that HoxD13 directly interacts with the promoter of Gli3. The increase of Gli3 expression in ICTEV model animal might result from the low expression of HoxD13.

Temporal and spatial dissection of Shh signaling in genital tubercle development

Genital tubercle (GT) initiation and outgrowth involve coordinated morphogenesis of surface ectoderm, cloacal mesoderm and hindgut endoderm. GT development appears to mirror that of the limb. Although Shh is essential for the development of both appendages, its role in GT development is much less clear than in the limb. Here, by removing Shh at different stages during GT development in mice, we demonstrate a continuous requirement for Shh in GT initiation and subsequent androgen-independent GT growth. Moreover, we investigated the Hh responsiveness of different tissue layers by removing or activating its signal transducer Smo with tissue-specific Cre lines, and established GT mesenchyme as the primary target tissue of Shh signaling. Lastly, we showed that Shh is required for the maintenance of the GT signaling center distal urethral epithelium (dUE). By restoring Wnt-Fgf8 signaling in Shh(-/-) cloacal endoderm genetically, we revealed that Shh relays its signal partly through the dUE, but regulates Hoxa13 and Hoxd13 expression independently of dUE signaling. Altogether, we propose that Shh plays a central role in GT development by simultaneously regulating patterning of the cloacal field and supporting an outgrowth signal.

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

HOX DNA-binding proteins control patterning during development by regulating processes such as cell aggregation and proliferation. Recently, a possible involvement of HOX proteins in replication origin activity was suggested by results showing that a number of HOX proteins interact with the DNA replication licensing regulator geminin and bind a characterized human origin of replication. The functional significance of these observations, however, remained unclear. We show that HOXD13, HOXD11, and HOXA13 bind in vivo all characterized human replication origins tested. We furthermore show that HOXD13 interacts with the CDC6 loading factor, promotes pre-replication complex (pre-RC) proteins assembly at origins, and stimulates DNA synthesis in an in vivo replication assay. HOXD13 expression in cultured cells accelerates DNA synthesis initiation in correlation with the earlier pre-RC recruitment onto origins during G(1) phase. Geminin, which interacts with HOXD13 as well, blocks HOXD13-mediated assembly of pre-RC proteins and inhibits HOXD13-induced DNA replication. Our results uncover a function for Hox proteins in the regulation of replication origin activity and reveal an unforeseen role for the inhibition of HOX protein activity by geminin in the context of replication origin licensing.

Evolution of digit identity in the three‐toed Italian skink Chalcides chalcides: a new case of digit identity frame shift

Digit identity in the avian wing is a classical example of conflicting anatomical and embryological evidence regarding digit homology. Anatomical in conjunction with phylogenetic evidence supports the hypothesis that the three remaining digits in the bird wing are digits 1, 2, and 3. At the same time, various lines of embryological evidence support the notion that these digits develop in positions that normally produce digits 2, 3, and 4. In recent years, gene expression as well as experimental evidence was published that supports the hypothesis that this discrepancy arose from a digit identity shift in the evolution of the bird wing. A similar but less well-known controversy has been ongoing since the late 19th century regarding the identity of the digits of the three-toed Italian skink, Chalcides chalcides. Comparative anatomy identifies these digits as 1, 2, and 3, while embryological evidence suggests their derivation from embryological positions 2, 3, and 4. Here we re-examine this evidence and add gene expression data to determine the identity of the three digits of C. chalcides. The data confirm that the adult and the embryological evidence for digit identity are in conflict, and the expression of Hoxd11 suggests that digits 1, 2, and 3 develop in positions 2, 3, and 4. We conclude that in C. chalcides, and likely in its close relatives, a digit identity frame shift has occurred, similar to the one in avian evolution. This result suggests that changes in of digit identity might be a more frequent consequence of digit reduction than previously assumed.

Establishment of canine hemangiosarcoma xenograft models expressing endothelial growth factors, their receptors, and angiogenesis-associated homeobox genes

BackgroundHuman hemangiosarcoma (HSA) tends to have a poor prognosis; its tumorigenesis has not been elucidated, as there is a dearth of HSA clinical specimens and no experimental model for HSA. However, the incidence of spontaneous HSA is relatively high in canines; therefore, canine HSA has been useful in the study of human HSA. Recently, the production of angiogenic growth factors and their receptors in human and canine HSA has been reported. Moreover, the growth-factor environment of HSA is very similar to that of pathophysiological angiogenesis, which some homeobox genes regulate in the transcription of angiogenic molecules. In the present study, we established 6 xenograft canine HSA tumors and detected the expression of growth factors, their receptors, and angiogenic homeobox genes.MethodsSix primary canine HSAs were xenografted to nude mice subcutaneously and serially transplanted. Subsequently, the expressions of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factors (bFGF), flt-1 and flk-1 (receptors of VEGF-A), FGFR-1, and angiogenic homeobox genes HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 were investigated in original and xenograft tumors by histopathology, immunostaining, and reverse transcription polymerase chain reaction (RT-PCR), using canine-specific primer sets.ResultsHistopathologically, xenograft tumors comprised a proliferation of neoplastic cells that were varied in shape, from spindle-shaped and polygonal to ovoid; some vascular-like structures and vascular clefts of channels were observed, similar to those in the original tumors. The expression of endothelial markers (CD31 and vWF) was detected in xenograft tumors by immunohistochemistry and RT-PCR. Moreover, the expression of VEGF-A, bFGF, flt-1, flk-1, FGFR-1, HoxA9, HoxB3, HoxB7, HoxD3, Pbx1, and Meis1 was detected in xenograft tumors. Interestingly, expressions of bFGF tended to be higher in 3 of the xenograft HSA tumors than in the other tumors.ConclusionWe established 6 xenograft canine HSA tumors in nude mice and found that the expressions of angiogenic growth factors and their receptors in xenograft HSAs were similar to those in spontaneous HSA. Furthermore, we detected the expression of angiogenic homeobox genes; therefore, xenograft models may be useful in analyzing malignant growth in HSA.

MicroRNA expression profiling of male breast cancer

IntroductionMicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. To test the hypothesis that there is a specific miRNA expression signature which characterizes male breast cancers, we performed miRNA microarray analysis in a series of male breast cancers and compared them with cases of male gynecomastia and female breast cancers.MethodsParaffin blocks were obtained at the Department of Pathology of Thomas Jefferson University from 28 male patients including 23 breast cancers and five cases of male gynecomastia, and from 10 female ductal breast carcinomas. The RNA harvested was hybridized to miRNA microarrays (~1,100 miRNA probes, including 326 human and 249 mouse miRNA genes, spotted in duplicate). To further support the microarray data, an immunohistochemical analysis for two specific miRNA gene targets (HOXD10 and VEGF) was performed in a small series of male breast carcinoma and gynecomastia samples.ResultsWe identified a male breast cancer miRNA signature composed of a large portion of underexpressed miRNAs. In particular, 17 miRNAs with increased expression and 26 miRNAs with decreased expression were identified in male breast cancer compared with gynecomastia. Among these miRNAs, some had well-characterized cancer development association and some showed a deregulation in cancer specimens similar to the one previously observed in the published signatures of female breast cancer. Comparing male with female breast cancer miRNA expression signatures, 17 significantly deregulated miRNAs were observed (four overexpressed and 13 underexpressed in male breast cancers). The HOXD10 and VEGF gene immunohistochemical expression significantly follows the corresponding miRNA deregulation.ConclusionsOur results suggest that specific miRNAs may be directly involved in male breast cancer development and that they may represent a novel diagnostic tool in the characterization of specific cancer gene targets.