Antennapedia: The complexity of a master developmental transcription factor.

Hox genes encode transcription factors that play an important role in establishing the basic body plan of animals. In Drosophila, Antennapedia is one of the five genes that make up the Antennapedia complex (ANT-C). Antennapedia determines the identity of the second thoracic segment, known as the mesothorax. Misexpression of Antennapedia at different developmental stages changes the identity of the mesothorax, including the muscles, nervous system, and cuticle. In Drosophila, Antennapedia has two distinct promoters highly regulated throughout development by several transcription factors. Antennapedia proteins are found with other transcription factors in different ANTENNAPEDIA transcriptional complexes to regulate multiple subsets of target genes. In this review, we describe the different mechanisms that regulate the expression and function of Antennapedia and the role of this Hox gene in the development of Drosophila.

Potential role of exitron-containing homeobox genes in cancer.

Homeobox genes are protagonists in developmental and cancer biology, making comprehending their regulation pivotal in multiple molecular pathways. Exitrons, also known as intronic exons, are new players in the transcriptional organization, providing additional splicing variants whose functions are still vastly unknown. Exitron splicing sites were identified in eight homeobox genes, which has not been yet debated in the scientific literature. Due to the intimate connection between homeobox genes and tumorigenesis, it is worth investing more time in understanding how these less explored exitron-containing transcriptional isoforms could play a role in modulating the homeobox gene's biological functions. The perspectives devised in this article are meant to instigate fresh debates on how the transcriptional variants retaining exitrons identified in the human homeobox genes HOXA1, HOXA9, HOXD8, NKX3.1, and DLX6 can be examined in the context of tumorigenesis. This article is categorized under: Cancer > Genetics/Genomics/Epigenetics.

What snakes and caecilians have in common? Molecular interaction units and the independent origins of similar morphotypes in Tetrapoda.

Developmental pathways encompass transcription factors and cis-regulatory elements that interact as transcription factor-regulatory element (TF-RE) units. Independent origins of similar phenotypes likely involve changes in different parts of these units, a hypothesis promisingly tested addressing the evolution of the rib-associated lumbar (RAL) morphotype that characterizes emblematic animals such as snakes and elephants. Previous investigation in these lineages identified a polymorphism in the Homology region 1 [H1] enhancer of the Myogenic factor-5 [Myf5], which interacts with HOX10 proteins to modulate rib development. Here we address the evolution of TF-RE units focusing on independent origins of RAL morphotypes. We compiled an extensive database for H1-Myf5 and HOX10 sequences with two goals: (i) evaluate if the enhancer polymorphism is present in amphibians exhibiting the RAL morphotype and (ii) test a hypothesis of enhanced evolutionary flexibility mediated by TF-RE units, according to which independent origins of the RAL morphotype might involve changes in either component of the interaction unit. We identified the H1-Myf5 polymorphism in lineages that diverged around 340 Ma, including Lissamphibia. Independent origins of the RAL morphotype in Tetrapoda involved sequence variation in either component of the TF-RE unit, confirming that different changes may similarly affect the phenotypic outcome of a given developmental pathway.

Identification of Hox genes and their expression profiles during embryonic development of the emerging model organism, Macrobrachium olfersii.

Hox genes encode transcription factors that specify the body segment identity during development, including crustaceans, such as amphipods and decapods, that possess a remarkable diversity of segments and specialized appendages. In amphipods, alterations of specialized appendages have been obtained using knockout experiment of Hox genes, which suggests that these genes are involved in the evolution of morphology within crustaceans. However, studies of Hox genes in crustaceans have been limited to a few species. Here, we identified the homeodomain of nine Hox genes: labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abdA), and Abdominal-B (AbdB), and evaluated their expression by RT-qPCR and RT-PCR in the ovary, during embryonic development, and at the first larval stage (Zoea I) of the decapod Macrobrachium olfersii. The transcript levels of lab, Dfd, and ftz decreased and transcripts of pb, Scr, Antp, Ubx, abdA, and AbdB increased during embryonic development. Hox genes were expressed in mature ovaries and Zoea I larval stages, except Scr and ftz, respectively. In addition, isoforms of Dfd, Scr, Ubx, and abdA, which have been scarcely reported in crustaceans, were described. New partial sequences of 87 Hox genes from other crustaceans were identified from the GenBank database. Our results are interesting for future studies to determine the specific function of Hox genes and their isoforms in the freshwater prawn M. olfersii and to contribute to the understanding of the diversity and evolution of body plans and appendages in Crustaceans.

Upregulation of HOX genes promotes cell migration and proliferation in head and neck squamous cell carcinoma.

BACKGROUND: Expression dysregulation of HOX homeobox genes has been observed in several cancers, including head and neck squamous cell carcinoma (HNSC). Although characterization of HOX gene roles in HNSC development has been reported, there is still a need to better understand their real contribution to tumorigenesis. OBJECTIVE: The present study aimed to evaluate the contribution of the protein-coding HOX genes (HOXA10, HOXC9, HOXC10, and HOXC13) in cellular processes related to carcinogenesis and progression of the HNSC. METHODS: Expression of HOX genes was analyzed in HNSC RNA-Seq data from The Cancer Genome Atlas (TCGA) and by RT-qPCR in different tumor cell lines. siRNA-mediated knockdown of HOXA10, HOXC9, HOXC10 or HOXC13 was performed in HNSC cell lines, and predicted transcriptional targets HOX genes was analyzed by bioinformatic. RESULTS: Thirty-one out of the 39 mammalian HOX genes were found upregulated in HNSC tissues and cell lines. The HOXC9, HOXC10 or HOXC13 knockdown attenuated cell migration, and lead to downregulation of epithelial-mesenchymal transition (EMT) markers, which were predicted as transcriptional targets of these three HOX genes. Diminished colony formation and cell cycle arrest after HOXC10 or HOXC13 knockdown were also observed, corroborating the fact that there was an enrichment for genes in proliferation/cell cycle pathways. CONCLUSIONS: In summary, we revealed roles for HOXC9, HOXC10, and HOXC13 in cell migration and proliferation/cell cycle progression in HNSC cells and suggested that those HOX members contribute to HNSC development possibly by regulating tumor growth and metastasis.

Contributions of HOX genes to cancer hallmarks: Enrichment pathway analysis and review.

Homeobox genes function as master regulatory transcription factors during development, and their expression is often altered in cancer. The HOX gene family was initially studied intensively to understand how the expression of each gene was involved in forming axial patterns and shaping the body plan during embryogenesis. More recent investigations have discovered that HOX genes can also play an important role in cancer. The literature has shown that the expression of HOX genes may be increased or decreased in different tumors and that these alterations may differ depending on the specific HOX gene involved and the type of cancer being investigated. New studies are also emerging, showing the critical role of some members of the HOX gene family in tumor progression and variation in clinical response. However, there has been limited systematic evaluation of the various contributions of each member of the HOX gene family in the pathways that drive the common phenotypic changes (or "hallmarks") and that underlie the transformation of normal cells to cancer cells. In this review, we investigate the context of the engagement of HOX gene targets and their downstream pathways in the acquisition of competence of tumor cells to undergo malignant transformation and tumor progression. We also summarize published findings on the involvement of HOX genes in carcinogenesis and use bioinformatics methods to examine how their downstream targets and pathways are involved in each hallmark of the cancer phenotype.

HOX genes: potential candidates for the progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is a very aggressive cancer, considered to be a subtype of the head and neck squamous cell carcinoma (HNSCC). Despite significant advances in the understanding and treatment of cancer, prognosis of patients with LSCC has not improved recently. In the present study, we sought to understand better the genetic mechanisms underlying LSCC development. Thirty-two tumor samples were collected from patients undergoing surgical resection of LSCC. The samples were submitted to whole-genome cDNA microarray analysis aiming to identify genetic targets in LSCC. We also employed bioinformatic approaches to expand our findings using the TCGA database and further performed functional assays, using human HNSCC cell lines, to evaluate viability, cell proliferation, and cell migration after silencing of selected genes. Eight members of the homeobox gene family (HOX) were identified to be overexpressed in LSCC samples when compared to normal larynx tissue. Quantitative RT-PCR analysis validated the overexpression of HOX gene family members in LSCC. Receiver operating characteristic (ROC) statistical method curve showed that the expression level of seven members of HOX gene family can distinguish tumor from nontumor tissue. Correlation analysis of clinical and gene expression data revealed that HOXC8 and HOXD11 genes were associated with the differentiation degree of tumors and regional lymph node metastases, respectively. Additionally, siRNA assays confirmed that HOXC8, HOXD10, and HOXD11 genes might be critical for cell colony proliferation and cell migration. According to our findings, several members of the HOX genes were overexpressed in LSCC samples and seem to be required in biological processes involved in tumor development. This suggests that HOX genes might play a critical role in the physiopathology of LSCC tumors.

Selection on different genes with equivalent functions: the convergence story told by Hox genes along the evolution of aquatic mammalian lineages.

BACKGROUND: Convergent evolution has been a challenging topic for decades, being cetaceans, pinnipeds and sirenians textbook examples of three independent origins of equivalent phenotypes. These mammalian lineages acquired similar anatomical features correlated to an aquatic life, and remarkably differ from their terrestrial counterparts. Whether their molecular evolutionary history also involved similar genetic mechanisms underlying such morphological convergence nevertheless remained unknown. To test for the existence of convergent molecular signatures, we studied the molecular evolution of Hox genes in these three aquatic mammalian lineages, comparing their patterns to terrestrial mammals. Hox genes are transcription factors that play a pivotal role in specifying embryonic regional identity of nearly any bilateral animal, and are recognized major agents for diversification of body plans. RESULTS: We detected few signatures of positive selection on Hox genes across the three aquatic mammalian lineages and verified that purifying selection prevails in these sequences, as expected for pleiotropic genes. Genes found as being positively selected differ across the aquatic mammalian lineages, but we identified a substantial overlap of their developmental functions. Such pattern likely resides on the duplication history of Hox genes, which probably provided different possible evolutionary routes for achieving the same phenotypic solution. CONCLUSIONS: Our results indicate that convergence occurred at a functional level of Hox genes along three independent origins of aquatic mammals. This conclusion reinforces the idea that different changes in developmental genes may lead to similar phenotypes, probably due to the redundancy provided by the participation of Hox paralogous genes in several developmental functions.

Developmental identity versus typology: Lucy has only four sacral segments.

OBJECTIVES: Both interspecific and intraspecific variation in vertebral counts reflect the action of patterning control mechanisms such as Hox. The preserved A.L. 288-1 ("Lucy") sacrum contains five fused elements. However, the transverse processes of the most caudal element do not contact those of the segment immediately craniad to it, leaving incomplete sacral foramina on both sides. This conforms to the traditional definition of four-segmented sacra, which are very rare in humans and African apes. It was recently suggested that fossilization damage precludes interpretation of this specimen and that additional sacral-like features of its last segment (e.g., the extent of the sacral hiatus) suggest a general Australopithecus pattern of five sacral vertebrae. METHODS: We provide updated descriptions of the original Lucy sacrum. We evaluate sacral/coccygeal variation in a large sample of extant hominoids and place it within the context of developmental variation in the mammalian vertebral column. RESULTS: We report that fossilization damage did not shorten the transverse processes of the fifth segment of Lucy's sacrum. In addition, we find that the extent of the sacral hiatus is too variable in apes and hominids to provide meaningful information on segment identity. Most importantly, a combination of sacral and coccygeal features is to be expected in vertebrae at regional boundaries. DISCUSSION: The sacral/caudal boundary appears to be displaced cranially in early hominids relative to extant African apes and humans, a condition consistent with the likely ancestral condition for Miocene hominoids. While not definitive in itself, a four-segmented sacrum accords well with the "long-back" model for the Pan/Homo last common ancestor. Am J Phys Anthropol 160:729-739, 2016. © 2016 Wiley Periodicals, Inc.

Clinicopathological and immunohistochemical evaluation of oral and oropharyngeal squamous cell carcinoma in Chilean population.

In oral and oropharyngeal squamous cell carcinoma (OCSCC and OPSCC) exist an association between clinical and histopathological parameters with cell proliferation, basal lamina, connective tissue degradation and surrounding stroma markers. We evaluated these associations in Chilean patients. A convenience sample of 37 cases of OCSCC (n=16) and OPSCC (n=21) was analyzed clinically (TNM, clinical stage) and histologically (WHO grade of differentiation, pattern of tumor invasion). We assessed the expression of p53, Ki67, HOXA1, HOXB7, type IV collagen (ColIV) and carcinoma-associated fibroblast (α-SMA-positive cells). Additionally we conducted a univariate/bivariate analysis to assess the relationship of these variables with survival rates. Males were mostly affected (56.2% OCSCC, 76.2% OPSCC). Patients were mainly diagnosed at III/IV clinical stages (68.8% OCSCC, 90.5% OPSCC) with a predominantly infiltrative pattern invasion (62.9% OCSCC, 57.1% OPSCC). Significant association between regional lymph nodes (N) and clinical stage with OCSCC-HOXB7 expression (Chi-Square test P < 0.05) was observed. In OPSCC a statistically significant association exists between p53, Ki67 with gender (Chi-Square test P < 0.05). In OCSCC and OPSCC was statistically significant association between ki67 with HOXA1, HOXB7, and between these last two antigens (Pearson's Correlation test P < 0.05). Furthermore OPSCC-p53 showed significant correlation when it was compared with α-SMA (Kendall's Tau-c test P < 0.05). Only OCSCC-pattern invasion and OPSCC-primary tumor (T) pattern resulted associated with survival at the end of the follow up period (Chi-Square Likelihood Ratio, P < 0.05). Clinical, histological and immunohistochemical features are similar to seen in other countries. Cancer proliferation markers were associated strongly from each other. Our sample highlights prognostic value of T and pattern of invasion, but the conclusions may be limited and should be considered with caution (small sample). Many cases were diagnosed in the advanced stages of the disease, which suggests that the diagnosis of OCSCC and OPSCC is made late.

Genomic atolls of differentiation in coral reef fishes (Hypoplectrus spp., Serranidae).

Because the vast majority of species are well diverged, relatively little is known about the genomic architecture of speciation during the early stages of divergence. Species within recent evolutionary radiations are often minimally diverged from a genomic perspective, and therefore provide rare opportunities to address this question. Here, we leverage the hamlet radiation (Hypoplectrus spp., brightly coloured reef fishes from the tropical western Atlantic) to characterize genomic divergence during the early stages of speciation. Transect surveys and spawning observations in Belize, Honduras and Panama confirm that sympatric barred (H. puella), black (H. nigricans) and butter (H. unicolor) hamlets are phenotypically distinct and reproductively isolated, although hybrid spawnings and individuals with intermediate phenotypes are seen on rare occasions. A survey of approximately 100 000 restriction site-associated SNPs in 126 samples from the three species across the three replicate populations reveals extremely slight genomewide divergence among species (FST  = 0.0038), indicating that ecomorphological differences and functional reproductive isolation are maintained in sympatry in a backdrop of extraordinary genomic similarity. Nonetheless, a very small proportion of SNPs (0.05% on average) are identified as FST outliers among sympatric species. Remarkably, a single SNP is identified as an outlier in repeated populations for the same species pair. A minicontig assembled de novo around this SNP falls into the genomic region containing the HoxCa10 and HoxCa11 genes in 10 teleost species, suggesting an important role for Hox gene evolution in this radiation. This finding, if confirmed, would provide a better understanding of the links between micro- and macroevolutionary processes.

Epigenetic repression of HOXB cluster in oral cancer cell lines.

OBJECTIVE: Aberrant DNA methylation is a fundamental transcriptional control mechanism in carcinogenesis. The expression of homeobox genes is usually controlled by an epigenetic mechanism, such as the methylation of CpG islands in the promoter region. The aim of this study was to describe the differential methylation pattern of HOX genes in oral squamous cell carcinoma (OSCC) cell lines and transcript status in a group of hypermethylated and hypomethylated genes. DESIGN: Quantitative analysis of DNA methylation was performed on two OSCC cell lines (SCC4 and SCC9) using a method denominated Human Homeobox Genes EpiTect Methyl qPCR Arrays, which allowed fast, precise methylation detection of 24 HOX specific genes without bisulfite conversion. RESULTS: Methylation greater than 50% was detected in HOXA11, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXC8 and HOXD10. Both cell lines demonstrated similar hypermethylation status for eight HOX genes. A similar pattern of promoter hypermethylation and hypomethylation was demonstrated for the HOXB cluster and HOXA cluster, respectively. Moreover, the hypermethylation profile of the HOXB cluster, especially HOXB4, was correlated with decreased transcript expression, which was restored following treatment with 5-aza-2'-deoxycytidine. CONCLUSIONS: The homeobox methylation profile in OSCC cell lines is consistent with an epigenetic biomarker.

The gap gene Krüppel of Rhodnius prolixus is required for segmentation and for repression of the homeotic gene sex comb-reduced.

The establishment of the anterior-posterior segmentation in insects requires the concerted action of a hierarchical gene network. Here, we study the orthologue of Krüppel gap gene in the hemipteran Rhodnius prolixus (Rp-Kr). We characterized its structure, expression pattern and function. The genomic sequence upstream of the Rp-Kr transcriptional unit shows a putative regulatory region conserved in the orthologue genes from Drosophila melanogaster and Tribolium castaneum. Rp-Kr expression is zygotic and it is expressed in the anterior half of the embryo (the posterior half of the egg) during the blastoderm stage and germ band formation; later, during germ band extension, it is expressed in a central domain, from T2 to A3. The Rp-Kr loss of function phenotypes shows disrupted thoracic and abdominal segmentation. Embryos with weak segmentation phenotypes show homeotic transformations, in which an ectopic tibial comb, typical of T1 leg, appears in T2, which correlates with the ectopic expression of Rp-sex-comb reduced in this leg.

A study of the distribution of phylogenetically conserved blocks within clusters of mammalian homeobox genes

Genome sequencing efforts of the last decade have produced a large amount of data, which has enabled whole-genome comparative analyses in order to locate potentially functional elements and study the overall patterns of phylogenetic conservation. In this paper we present a statistically based method for the characterization of these patterns in mammalian DNA sequences. We have applied this approach to the study of exceptionally well conserved homeobox gene clusters (Hox), based on an alignment of six species, and we have constructed a map of Hox cataloguing the conserved fragments, along with their locations in relation to the genes and other landmarks, sometimes showing unexpected layouts.

HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the capacity to differentiate into osteoblasts during osteogenesis. Several studies attempted to identify osteogenesis-related genes in hMSCs. Although HOX genes are known to play a pivotal role in skeletogenesis, their function in the osteogenesis of hMSCs has not yet been investigated in detail. Our aim was to characterize the expression of 37 HOX genes by multiplex RT-PCR to identify the ones most probably involved in osteogenic differentiation. The results showed that the expression patterns of four HOX genes were altered during this process. In particular, the expression levels of HOXC13 and HOXD13 were dramatically changed. Real-time PCR and Western blot analysis were performed in order to further analyze the expression of HOXC13 and HOXD13. The qRT-PCR results showed that transcription of HOXC13 was up-regulated by up to forty times, whereas that of HOXD13 was down-regulated by approximately five times after osteogenic differentiation. The Western blot results for the HOXC13 and HOXD13 proteins also corresponded well with the real-time PCR result. These findings suggest that HOXC13 and HOXD13 might be involved in the osteogenic differentiation of hMSCs.

Mutações do gene notch1 e outras alterações moleculares relacionadas à etiopatogênese das leucemias linfoblásticas agudas de linhagem t (lla-t)

As leucemias linfoblásticas agudas de célula T (LLAs-T) apresentam alta prevalência em países em desenvolvimento, justificando a realização de estudos exploratórios na população brasileira. Análises das alterações genético-moleculares têm permitido a identificação de elementos envolvidos na leucemogênese. A fusão SIL-TAL1, a expressão do gene HOX11L2, e as mutações do NOTCH1 são alterações genéticas comuns nas LLAs-T. Com o objetivo de testar a influência destas anormalidades no prognóstico das LLAs-T de pacientes brasileiros, nós analisamos uma série de 170 crianças e adultos jovens com LLA-T, com idades entre 1-21 anos, sendo 39 meninas e 131 meninos, diagnosticados entre 2001-2007. A metodologia incluiu imunofenotipagem para a definição do subtipo celular e para análise do status do CD10 e CD1a; RT-PCR para detecção do SIL-TAL1e do HOX11L2, PCR, DHPLC e seqüenciamento para a identificação de mutações no NOTCH1. O método de Kaplan-Meyer foi usado para estimar a sobrevida dos casos em 36 meses de acordo com o perfil dos marcadores analisados. Nossos resultados mostraram 36,4 % das amostras com CD10+ e 28,8 % com CD1a+. As freqüências do SIL-TAL1 e do HOX11L2 foram 24,4 % e 11,5 % respectivamente. Sobre as mutações do NOTCH1, o domínio PEST estava mutado em 8,8 % dos casos; o HD representou 16,2 % das mutações, enquanto a análise de ambos os domínios juntos demonstrou 57,4 % das mutações. Interessantemente, duas irmãs com LLA-T (não-gemelares) foram analisadas para o NOTCH1, vimos que ambas apresentaram mutações nos domínios HD e PEST nas mesmas regiões. A análise das células do SP dos pais destas irmãs não demonstrou mutações no NOTCH1. Com estes resultados, nós podemos afirmar que estas mutações são somáticas. O impacto de cada marcador na sobrevida não foi estatisticamente significante. Através destes resultados vê-se que é necessário continuar este estudo com um número maior de casos analisados para o NOTCH1, para que, assim, possamos avaliar melhor o impacto destes marcadores na patogênese das LLAs-T.

T acute lymphoblastic leukemias (T-ALL) subtype presents higher prevalence in developing countries, justifying exploring studies in a Brazilian population. The analyses of genetic-molecular alterations have allowed the identification of elements involved in leukemogenesis. The SIL-TAL1 fusion gene, the expression of HOX11L2 gene, and NOTCH1 mutations are the common genetic alterations found in T-ALL. In order to test the influence of such abnormalities on outcome of T-ALL in Brazilian patients, we carried out on analysis of a series of 170 children and young adults with T-ALL, age range 1-21 years old, being 39 girls and 131 boys consecutively diagnosed between 2001-2007. Methodology included immunophenotyping for subtype definition and analyses of CD10 and CD1a status; RT-PCR for molecular analyses of SIL-TAL1and HOX11L2, PCR, DHPLC and sequencing for NOTCH1 mutations detections. Kaplan-Meyer survival methodology was used to estimate the 36-months survival rate of T-ALL cases according to the markers analyzed. Our results show that CD10 was positive in 36.4 % of the samples and CD1a in 28.8 %. The frequency of SILTAL1 fusion gene and HOX11L2 were 24.4 % and 11.5 % respectively. Regarding NOTCH1mutations, the domain PEST was mutated in 8.8 % of the cases; the HD domain demonstrated 16.2 % of mutations, whereas the analysis of both domains together presented 57.4 % of mutations. Interesting, T-ALL in two sisters (non-twins) included in these analyses, demonstrated that they both had mutation in HD and PEST domains in the same region. The analysis of PB cells of both parents demonstrated any mutation in NOTCH1. With these results, we can confirm that NOTCH1 mutations are somatic. The impact of all markers in survival was not significant statistically. From these results it is necessary to continue the study with a larger number of cases analyzed to NOTCH1, so we can better evaluate the impact of each marker with the pathogenesis of T-ALLs.