Expression Patterns Of Hox Genes Research Articles

The expression pattern of Wnt6, Wnt10A, and HOXA13 during regenerating tails of Gekko Japonicus

Wnt signal is crucial to correctly regenerate tissues along the original axis in many animals. Lizards are able to regenerate their tails spontaneously, while the anterior-posterior axis information required for the successful regeneration is still elusive. In this study, we investigated the expression pattern of Wnt ligands and HOX genes during regeneration. The results of in situ hybridization revealed that Wnt6 and Wnt10A mRNA levels are higher in wound epithelium (WE) than that in blastema during regeneration. In addition, we showed that Wnt agonist positively regulated the expression of HOXA13 in cultured blastema cells, while did not show similar effect on that of HOXB13, HOXC13 and HOXD13. Finally, we found that HOXA13 showed a gradient level along the anterior-posterior axis of regenerated blastema, with higher level at the caudal end. These data proposed that Wnt6, Wnt10A and HOXA13 might play an important role in establishing distal position for regeneration.

Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells

Abstract HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 & HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 & SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5599.

Upregulation of Hox genes leading to caste-specific morphogenesis in a termite

BackgroundIn social insects, interactions among colony members trigger caste differentiation with morphological modifications. In termite caste differentiation, caste-specific morphologies (such as mandibles in soldiers, genital organs in reproductives or wings in alates) are well developed during post-embryonic development under endocrine controls (e.g., juvenile hormone and ecdysone). Since body part-specific morphogenesis in caste differentiation is hormonally regulated by global factors circulated throughout the body, positional information should be required for the caste-specific and also body part-specific morphogenesis. To identify factors providing the positional information, expression and functional analyses of eight Hox genes were carried out during the three types of caste differentiation (i.e., soldier, neotenic and alate differentiation) in a termite, Hodotermopsis sjostedti.ResultsSpatio-temporal patterns of Hox gene expression during caste differentiation were elucidated by real-time qPCR, showing the caste-specific upregulations of Hox genes during the differentiation processes. Among eight Hox genes, Deformed (Dfd) was upregulated specifically in mandibles in soldier differentiation, abdominal-A (abd-A) and Abdominal-B (Abd-B) were upregulated in the abdomen in neotenic differentiation, while Sex-comb reduced (Scr) and Antennapedia (Antp) were upregulated during alate differentiation. Furthermore, RNAi knockdown of Dfd in soldier differentiation and of abd-A and Abd-B in neotenic differentiation distorted the modifications of caste-specific morphologies.ConclusionsGene expression and functional analyses in this study revealed that, in the caste differentiation in termites, upregulation of Hox genes provide positional identities of body segments, resulting in the caste-specific morphogenesis. The acquisition of such developmental modifications would have enabled the evolution of sophisticated caste systems in termites.

Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube.

Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in the specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here, we optimized a single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild-type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate that each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies that rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the retinoic acid response.

Sf3b4 regulates chromatin remodeler splicing and Hox expression

SF3B proteins form a heptameric complex in the U2 small nuclear ribonucleoprotein, essential for pre-mRNA splicing. Heterozygous pathogenic variants in human SF3B4 are associated with head, face, limb, and vertebrae defects. Using the CRISPR/Cas9 system, we generated mice with constitutive heterozygous deletion of Sf3b4 and showed that mutant embryos have abnormal vertebral development. Vertebrae abnormalities were accompanied by changes in levels and expression pattern of Hox genes in the somites. RNA sequencing analysis of whole embryos and somites of Sf3b4 mutant and control litter mates revealed increased expression of other Sf3b4 genes. However, the mutants exhibited few differentially expressed genes and a large number of transcripts with differential splicing events (DSE), predominantly increased exon skipping and intron retention. Transcripts with increased DSE included several genes involved in chromatin remodeling that are known to regulate Hox expression. Our study confirms that Sf3b4 is required for normal vertebrae development and shows, for the first time, that like Sf3b1, Sf3b4 also regulates Hox expression. We propose that abnormal splicing of chromatin remodelers is primarily responsible for vertebral defects found in Sf3b4 heterozygous mutant embryos.

Transcriptional Analysis of the Endostyle Reveals Pharyngeal Organ Functions in Ascidian.

The endostyle is a pharyngeal organ with an opening groove and cilia in invertebrate chordates (amphioxus and ascidian) and cyclostomate (lamprey), serving as a filter-feeding tract and thyroid-secreting location. Emerging evidence implies its complex cellular composition and potentially versatile functions. Multiple cell types in the endostyle have been thought to be progenitors of complex organs in advanced vertebrates. To describe the expression profile and the potential functions, bulk RNA sequencing on the endostyle in ascidian Styela clava was conducted and distinct markers were selected by multileveled comparative analysis. Transcriptional data assay and qRT-PCR-verified results showed the regional expression patterns of Hox genes in the longitudinal axis. Organ-specific markers of the endostyle was proposed by comparing expression with the main organs of the ascidian. A cross-species transcriptional profile projection between the endostyle and organs from Danio rerio and Homo sapiens indicates a robust homogenous relationship to the thyroid and digestive system of the endostyle. The high similarity between the endostyle and the head kidney in zebrafish/the bone marrow in human implies uniquely profound functions of the pharyngeal organ in proto-vertebrates. Our result revealed that the transcriptional profile of the human parathyroid gland was similar to the ascidian endostyle, indicating the evolutionary origin of vertebrate hormone secretion organs.

Gene Loss may have Shaped the Cnidarian and Bilaterian Hox and ParaHox Complement.

Hox and ParaHox transcription factors are important for specifying cell fates along the primary body axes during the development of most animals. Within Cnidaria, much of the research on Hox/ParaHox genes has focused on Anthozoa (anemones and corals) and Hydrozoa (hydroids) and has concentrated on the evolution and function of cnidarian Hox genes in relation to their bilaterian counterparts. Here we analyze together the full complement of Hox and ParaHox genes from species representing all four medusozoan classes (Staurozoa, Cubozoa, Hydrozoa, and Scyphozoa) and both anthozoan classes (Octocorallia and Hexacorallia). Our results show that Hox genes involved in patterning the directive axes of anthozoan polyps are absent in the stem leading to Medusozoa. For the first time, we show spatial and temporal expression patterns of Hox and ParaHox genes in the upside-down jellyfish Cassiopea xamachana (Scyphozoa), which are consistent with diversification of medusozoan Hox genes both from anthozoans and within medusozoa. Despite unprecedented taxon sampling, our phylogenetic analyses, like previous studies, are characterized by a lack of clear homology between most cnidarian and bilaterian Hox and Hox-related genes. Unlike previous studies, we propose the hypothesis that the cnidarian-bilaterian ancestor possessed a remarkably large Hox complement and that extensive loss of Hox genes was experienced by both cnidarian and bilaterian lineages.

Echiuran Hox genes provide new insights into the correspondence between Hox subcluster organization and collinearity pattern.

In many bilaterians, Hox genes are generally clustered along the chromosomes and expressed in spatial and temporal order. In vertebrates, the expression of Hox genes follows a whole-cluster spatio-temporal collinearity (WSTC) pattern, whereas in some invertebrates the expression of Hox genes exhibits a subcluster-level spatio-temporal collinearity pattern. In bilaterians, the diversity of collinearity patterns and the cause of collinearity differences in Hox gene expression remain poorly understood. Here, we investigate genomic organization and expression pattern of Hox genes in the echiuran worm Urechis unicinctus (Annelida, Echiura). Urechis unicinctus has a split cluster with four subclusters divided by non-Hox genes: first subcluster (Hox1 and Hox2), second subcluster (Hox3), third subcluster (Hox4, Hox5, Lox5, Antp and Lox4), fourth subcluster (Lox2 and Post2). The expression of U. unicinctus Hox genes shows a subcluster-based whole-cluster spatio-temporal collinearity (S-WSTC) pattern: the anterior-most genes in each subcluster are activated in a spatially and temporally colinear manner (reminiscent of WSTC), with the subsequent genes in each subcluster then being very similar to their respective anterior-most subcluster gene. Combining genomic organization and expression profiles of Hox genes in different invertebrate lineages, we propose that the spatio-temporal collinearity of invertebrate Hox is subcluster-based.

Disruption of CTCF Boundary Remodels Chromatin Domain and Reprograms HOX Gene Transcription in Acute Myeloid Leukemia

The organization of the mammalian genome into separate domains either facilitates or restricts interactions between regulatory elements, e.g. enhancers and promoters, for normal gene regulation. CTCF insulator constrains the temporal HOX gene expression patterns within the confined chromatin domain for normal development. However, dysregulation of HOX genes is also a common feature of acute myeloid leukemia (AML). The molecular mechanisms of aberrant HOX expression and associated AML pathogenesis remain unclear. Here, we found that CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) acts as a chromatin boundary to establish and maintain AML specific aberrant HOX gene expression patterns and is essential for pathogenesis of myeloid malignancies.To unbiasedly and systematically investigate the mechanism underlying HOX gene regulation in AML, we employed a pooled CRISPR-Cas9 genetic knockout library screening to interrogate intergenic regulatory and chromatin boundary elements in all 4 HOX gene loci. Through this screening, we identified the CBS7/9 chromatin boundary located between HOXA7 and HOXA9 genes that is critical for maintaining aberrant expression of posterior HOXA genes including oncogene HOXA9 and HOXA10 in primary AML patients and cells. To further confirm the role of CBS7/9 in HOX gene regulation, two single-guide RNAs (sgRNAs) flanking 47 base pairs (bps) of the core CTCF motif located between HOXA7 and HOXA9 geneswere employed to guild Cas9 for deletion of the CBS7/9 boundary in 3 different subtypes of AML cells containing MLL rearrangements or NPM1C+ mutation. The defected CBS7/9 resulted in a decrease in CTCF binding to the caudal HOXA domain and an inhibition of posterior HOXA gene expression.To further delineate the role of CBS7/9 chromatin boundary in HOX gene organization and regulation, we then carried out genome wide ChIP-seq, ATAC-seq, 4C, and RNA-seq analysis to examine the changes in chromatin domain organization and corresponding gene expression patterns compared wild-type control and the CBS7/9 KO AML cells. Disruption of CBS7/9 boundary resulted in a spreading of repressive H3K27me3 domain into the caudal HOXA domain that subsequently impaired AML associated active TAD domain by altering enhancer/promoter chromatin accessibility and reducing ectopic interactions among the posterior HOXA genes. Consistent with the role of CBS7/9 boundary in HOX locus chromatin structure and organization, impairment of CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid specific transcriptome profiles important for pathogenesis of myeloid malignancies suggesting that the CBS7/9 boundary mediated ectopic HOX expression patterns might be essential for pathogenesis of myeloid malignancies. To further test this possibility, the CBS 7/9 boundary was deleted in both MLL rearranged AML cells and primary patient samples by the CRISPR-Cas9 mediated genome editing and the edited cells were transplanted into NSG mice for the development of human AML in mice. Disruption of CBS chromatin boundary in the HOXA locus reduced human leukemic blast development and invasion as well as enhanced survival of both transplanted AML cell xenograft and patient derived xenograft (PDX) mouse models. Thus, our finding demonstrated that CTCF boundary not only constrains normal gene expression program for memory of cell identity, but also plays a role in maintaining oncogenic transcription program for leukemic transformation. Alteration of CTCF mediated topological boundaries in the oncogene loci provides therapeutic approaches for targeting oncogenic expression program and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Disregulation of CTCF Boundary at the HOX Locus Enhances Sensitity to Multiple Drug Treatment in Myeloid Malignancies

Dysregulation of HOX genes is a dominant mechanism of leukemic transformation by altering self-renewal and differentiation properties of HS/PCs leading to leukemic transformation. The overexpression of posterior HOXA9 gene is also a poor prognostic marker in leukemia patients. Recent studies revealed that CTCF insulator constrains temporal HOX gene expression patterns within confined chromatin domains suggesting that CTCF mediated chromatin boundary activity may involve in regulating HOX gene patterns during AML prognosis.To investigate the role of CTCF in aberrant HOX gene expression and AML prognosis, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the core CTCF motif located between HOXA7 and HOXA9 genes (CBS7/9) in different subtype of AML cells with either MLL-rearrangement or NPM1 mutation. RNA-seq analysis revealed that transcription factors, HOXA9 , RUNX1 , and ZEB1 , as well as MAPK signaling pathway, all of them are involved in AML survival, prognosis and chemo-resistance, are significantly dowregulated in response to the CBS7/9 boundary deletion. Given that these genes and pathways are involved in AML prognosis and chemo-resistance, we then examined whether perturbation of CBS7/9 chromatin boundary resulted in alteration of drug resposes of AML cells. Both WT AML cells and the CBS7/9 defected AML cells derived from MLL-AF9 rearranged MOLM-13 and NPM mutated OCI-AML3 cells were treated with 1 mM of Ara-C or 100 nM of bromodomain (BET) inhibitors. Compared to the WT controls, the defected CBS7/9 boundary resulted in a significantly increase in AML sensitivity to both Ara-C and BET inhibitor treatment in both MOLM-13 and OCI-AML3 cells as evidence by significantly reducing percentage of proliferating cells.To further examine if treatment related drug sensitivity in AML cells is depended on the levels of HOXA9 expression, we overexpressed HOXA9 cDNA in the CBS7/9 deleted MOLM-13 and OCI-AML3 cells. Gain-of-function (GOF) expression of HOXA9 in the CBS7/9 defected AML cells rescued and restored the resistance to the Ara-C treatment of the CBS7/9 KO cells to the levels compatible to that of the WT control MOLM-13 and OCI-AML3 cells, but HOXA9 overexpression did not rescued the resistance of the CBS7/9 KO AML cells to the BET inhibitor treatment. The data suggest that HOXA9 is directly associated with cytotoxic drug Ara-C resistance, but not BET inhibitor mediated drug responses. KO of CBS7/9 specifically decreased enrichment of H3K9ac and H4K5ac, as well as recruitment of BRD4 to the posterior HOXA domain. Furthermore, overexpression of HOXA9 gene in the CBS7/9 KO AML cells indeed rescued the mRNA transcripts affected by CBS7/9 deletion to the levels compatible to that of WT control MOLM-13 or OCI-AML3 cells. Among rescued genes, ZEB1, STAT5A, and RUNX1 play essential roles in pathogenesis of myeloid malignancies and chemo-drug sensitivity.Finally, in order to further explore biological role of CTCF boundaries in AML pathogenesis, we analyze the whole genome sequencing data of 183 primary AML patients obtained from public available Cosmic Database for somatic mutations occurred in the CTCF binding sites. Several core CTCF motifs in the HOXA locus, including CBS5/6 and CBS7/9 boundaries, are highly mutated in AML patients. Thus, disruption of CTCF boundary located between HOXA7 and HOXA9 genes perturbs HOXA9 oncogene transcription and regulates chemo- or epigenetic drug sensitivity of acute myeloid leukemia. Alteration of CTCF boundaries in the oncogene loci may provide a novel therapeutic target for treatment of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Evolutionary Conservation of Hox Genes in Vertebrate Brain Development

Hox genes, their conserved derivatives, and the pathways responsible for their expression have been extensively studied in the fruit fly, Drosophila melanogaster;the experimentation done in the Drosophila model system has given developmental biologists tools to better understand the role and significance of Hox genes and their derivatives in anterior-posterior axis determination in the Drosophila embryo. Along with this, Drosophila research opened up the door to investigation on the conservation of Hox genes between vertebrates and invertebrates. Comparative embryology in mice, chickens, pufferfish, and zebrafish have shown conserved Hox gene expression patterns specifically along the anterior-posterior axis. Recently, comparative analysis performed on dorsal-ventral axis formation showed that patterning and segmentation of the spinal cord is influenced by the action of Hox genes as well. This review will briefly consider the evolution of the vertebrate brain and the evolution and conservation of Hox genes in regulating hindbrain patterning and spinal cord development.

Recapitulating Developmental Condensation and Constructing Self-organised Cartilaginous Tissue for Cartilage Regeneration.

To develop a novel chondrocyte condensation culture strategy recapitulating developmental condensation and construct self-organised cartilaginous tissue for cartilage regeneration. Cell-condensation aggregate (CCA) was generated using the condensation culture method by sequential cell seeding. The chondrification capacities and biocompatibilities of CCA were assessed by comparison with the cell-scaffold complex (CSC), which was constructed by cell-scaffold coculture. Preclinical studies including implantation into nude mice subcutaneously and cartilage defect repair in rabbits were performed. CCA constructed by condensation culture exhibited a morphology of self-organised cartilaginous tissue. Meanwhile, the condensation culture inhibited or abolished expression of HOX genes including HOXC4 and HOXD8, which was partially consistent with developmental HOX gene expression patterns and associated with enhanced regeneration capacities. Compared with CSC, CCA showed a higher capacity for chondrification and regeneration of rabbit cartilage defects. The therapeutic assessments indicate that CCA is an efficient therapeutic tool for cartilage regeneration, providing a new strategy for tissue engineering by mimicking developmental events.

Exploring the biological domain of the neural embryonic stem cell test (ESTn): Morphogenetic regulators, Hox genes and cell types, and their usefulness as biomarkers for embryotoxicity screening

Animal-free assessment of compound-induced developmental neurotoxicity will most likely be based on batteries of multiple in vitro tests. The optimal battery is built by combining tests with complementary biological domains that together ideally cover all relevant toxicity pathways. Thus, biological domain definition, i.e. which biological processes and cell types are represented, is an important assay characteristic for determining the place of assays in testing strategies. The murine neural embryonic stem cell test (ESTn) is employed to predict the developmental neurotoxicity of compounds. The aim of this study was to explore the biological domain of ESTn according to three groups of biomarker genes of early (neuro)development: morphogenetic regulators, Hox genes and cell type markers for the ectodermal and neural lineages. These biomarker groups were selected based on their crucial regulatory role in (neuro)development. Analysis of these genes in a series of previously generated whole transcriptome datasets of ESTn showed that at day 7 in culture cell differentiation resembled hindbrain/branchial/thoracic development between E6.5-E12.5 in vivo, with subsequent development into a mixed cell culture containing different neural subtypes, astrocytes and oligodendrocytes by day 13. In addition, the selected biomarkers showed common and distinct responses to compound exposure. Monitoring the biological domain of ESTn through gene expression patterns of morphogenetic regulators, Hox genes and cell type markers proved instrumental in providing mechanistic understanding of compound effects on neural differentiation in ESTn, and can aid in positioning of the test in a battery of complementary in vitro tests in integrated approaches to testing and assessment.

Neuroendocrine prostate cancer has distinctive, non-prostatic HOX code that is represented by the loss of HOXB13 expression

HOX gene-encoded homeobox proteins control body patterning during embryonic development; the specific expression pattern of HOX genes may correspond to tissue identity. In this study, using RNAseq data of 1019 human cancer cell lines that originated from 24 different anatomic sites, we established HOX codes for various types of tissues. We applied these HOX codes to the transcriptomic profiles of prostate cancer (PCa) samples and found that the majority of prostate adenocarcinoma (AdPCa) samples sustained a prostate-specific HOX code whereas the majority of neuroendocrine prostate cancer (NEPCa) samples did not, which reflects the anaplastic nature of NEPCa. Also, our analysis showed that the NEPCa samples did not correlate well with the HOX codes of any other tissue types, indicating that NEPCa tumors lose their prostate identities but do not gain new tissue identities. Additionally, using immunohistochemical staining, we evaluated the prostatic expression of HOXB13, the most prominently changed HOX gene in NEPCa. We found that HOXB13 was expressed in both benign prostatic tissues and AdPCa but its expression was reduced or lost in NEPCa. Furthermore, we treated PCa cells with all trans retinoic acid (ATRA) and found that the reduced HOXB13 expression can be reverted. This suggests that ATRA is a potential therapeutic agent for the treatment of NEPCa tumors by reversing them to a more treatable AdPCa.

Hox-Positive Adult Mesenchymal Stromal Cells: Beyond Positional Identity.

Homeotic genes (Hox) are universal regulators of the body patterning process in embryogenesis of metazoans. The Hox gene expression pattern (Hox code) retains in adult tissues and serves as a cellular positional identity marker. Despite previously existing notions that the Hox code is inherent in all stroma mesenchymal cells as a whole, recent studies have shown that the Hox code may be an attribute of a distinct subpopulation of adult resident mesenchymal stromal cells (MSC). Recent evidence allows suggesting a “non-canonical” role for Hox gene expression which is associated with renewal and regeneration in postnatal organs after damage. In tissues with high regenerative capacity, it has been shown that a special cell population is critical for these processes, a distinctive feature of which is the persistent expression of tissue-specific Hox genes. We believe that in the postnatal period Hox-positive subpopulation of resident MSC may serve as a unique regenerative reserve. These cells coordinate creation and maintenance of the correct structure of the stroma through a tissue-specific combination of mechanisms. In this article, we summarize data on the role of resident MSC with a tissue-specific pattern of Hox gene expression as regulators of correct tissue reconstruction after injury.

How do signaling and transcription factors regulate both axis elongation and Hox gene expression along the anteroposterior axis?

In vertebrates, vertebral primordia, called somites, are formed from the head to the tail along the anteroposterior axis of the body during development. The presomitic mesoderm (PSM), which differentiates into somites, is formed by continuous supply of new cells derived from the caudal lateral epiblast (CLE), resulting in body axis posterior elongation. Previous studies of mutants identified genes for posterior extension and vertebral patterning along the anteroposterior axis. Hox gene has been extensively investigated for its expression pattern and transcriptional regulation. In recent years, to elucidate the mechanism that controls the expression patterns of Hox genes, researchers have not only searched for enhancer regions and the transcription factors that bind to them but have also investigated chromatin structure, epigenetics and non-coding RNA associated with Hox gene expression. These new findings reveal that the previously identified genes essential for posterior body axis elongation of the embryo determine positional information along the anteroposterior axis by induction of Hox genes via enhancer regions. In this review, we focus on genes that control posterior elongation and vertebral patterning along the anteroposterior axis in the PSM and CLE. We first describe the mechanism of maintenance of the stem cell-like cell populations at the CLE, which is essential for the posterior elongation of the embryo. Next, the factors involved in posterior region formation and patterning of the vertebra are described. Finally, we discuss the regulatory mechanism of Hox gene expression and the mechanism that is responsible for the differences in skeletal pattern between species.

Hox gene expression during development of the phoronid Phoronopsis harmeri

BackgroundPhoronida is a small group of marine worm-like suspension feeders, which together with brachiopods and bryozoans form the clade Lophophorata. Although their development is well studied on the morphological level, data regarding gene expression during this process are scarce and restricted to the analysis of relatively few transcription factors. Here, we present a description of the expression patterns of Hox genes during the embryonic and larval development of the phoronid Phoronopsis harmeri.ResultsWe identified sequences of eight Hox genes in the transcriptome of Ph. harmeri and determined their expression pattern during embryonic and larval development using whole mount in situ hybridization. We found that none of the Hox genes is expressed during embryonic development. Instead their expression is initiated in the later developmental stages, when the larval body is already formed. In the investigated initial larval stages the Hox genes are expressed in the non-collinear manner in the posterior body of the larvae: in the telotroch and the structures that represent rudiments of the adult worm. Additionally, we found that certain head-specific transcription factors are expressed in the oral hood, apical organ, preoral coelom, digestive system and developing larval tentacles, anterior to the Hox-expressing territories.ConclusionsThe lack of Hox gene expression during early development of Ph. harmeri indicates that the larval body develops without positional information from the Hox patterning system. Such phenomenon might be a consequence of the evolutionary intercalation of the larval form into an ancestral life cycle of phoronids. The observed Hox gene expression can also be a consequence of the actinotrocha representing a “head larva”, which is composed of the most anterior body region that is devoid of Hox gene expression. Such interpretation is further supported by the expression of head-specific transcription factors. This implies that the Hox patterning system is used for the positional information of the trunk rudiments and is, therefore, delayed to the later larval stages. We propose that a new body form was intercalated to the phoronid life cycle by precocious development of the anterior structures or by delayed development of the trunk rudiment in the ancestral phoronid larva.

Evolutionary lability in Hox cluster structure and gene expression in Anolis lizards.

Hox genes orchestrate development by patterning the embryonic axis. Vertebrate Hox genes are arranged in four compact clusters, and the spacing between genes is assumed to be crucial for their function. The genomes of squamate reptiles are unusually rich and variable in transposable elements (TEs), and it has been suggested that TE invasion is responsible for the Hox cluster expansion seen in snakes and lizards. Using de novo TE prediction on 17 genomes of lizards and snakes, I show that TE content of Hox clusters are generally 50% lower than genome‐wide TE levels. However, two distantly related lizards of the species‐rich genus Anolis have Hox clusters with a TE content that approaches genomic levels. The age distribution of TEs in Anolis lizards revealed that peaks of TE activity broadly coincide with speciation events. In accordance with theoretical models of Hox cluster regulation, I find that Anolis species with many TEs in their Hox clusters show aberrant Hox gene expression patterns, suggesting a functional link between TE accumulation and embryonic development. These results are consistent with the hypothesis that TEs play a role in developmental processes as well as in evolutionary diversifications.

365 Multivalent chromatin looping orchestrates cellular reprogramming for advanced gene therapy

The discovery of cellular plasticity, an epigenetic phenomenon by which differentiated cells alter their identity through reprogramming, dedifferentiation or transdifferentiation has led to a paradigm shift in regenerative medicine for treatment of genetic diseases and injuries. Current surgical methods pose high mortality rates making stem cell-based therapy an attractive option. HOX genes, a set of transcription factor genes, play key roles during stem cell renewal, multilineage differentiation and reprogramming of differentiated cells. Delineating the molecular mechanisms of HOX genes regulation in diverse cellular identities will provide clues to modulate cell fate for development of novel, more effective stem cell-based treatments. Recent studies highlight that a cell identity is determined not only by specific gene expression profile and posttranslational modifications but largely by its characteristic three-dimensional (3D) genomic organization. Chromatin maintains a high-order topology organizing genes into structural domains through long-range chromatin looping that is essential for controlled gene regulation. Existing techniques to study the link between chromatin looping and gene regulation require either large alterations of the linear DNA sequence or prior knowledge of loop-mediating factors. We have pioneered a technology to form multivalent chromosomal contacts and loops in 3D space at will. Previous studies revealed that primary adult fibroblasts retain several HOX gene expression patterns similar to that in embryonic cells. We will affect chromosomal remodeling using our new multiplexed chromatin looping system to examine how 3D architectural reorganization of chromatin at the Hox loci in human dermal fibroblasts can alter cellular identities (via reprogramming and transdifferentiation) with the potential for reproducible and efficient clinical-grade cell production. This study will provide insights for linking chromatin architecture to cutaneous gene expression that affects cellular plasticity in both adult and embryonic cells that may have transformative outcomes for regenerative medicine.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.