The diversity of vertebrate body plans is dizzying, yet stunning for the many things they have in common. Vertebrates have inhabited virtually every part of the earth from its coldest to warmest climates. They locomote by swimming, flying, walking, slithering, or climbing, or combinations of these behaviors. And they exist in many different sizes, from the smallest of frogs, fish and lizards to giraffes, elephants, and blue whales. Despite these differences, vertebrates follow a remarkably similar blueprint for the establishment of their body plan. Within the relatively small amount of time required to complete gastrulation, the process through which the three germ layers, ectoderm, mesoderm, and endoderm are created, the embryo also generates its body axis and is simultaneously patterned. For the length of this axis, the genes that distinguish the neck from the rib cage or the trunk from the sacrum are the Hox genes. In vertebrates, there was evolutionary pressure to maintain this set of genes in the organism. Over the past decades, much has been learned regarding the regulatory mechanisms that ensure the appropriate expression of these genes along the main body axes. Genetic functions continue to be explored though much has been learned. Much less has been discerned on the identity of co-factors used by Hox proteins for the specificity of transcriptional regulation or what downstream targets and pathways are critical for patterning events, though there are notable exceptions. Current work in the field is demonstrating that Hox genes continue to function in many organs long after directing early patterning events. It is hopeful continued research will shed light on remaining questions regarding mechanisms used by this important and conserved set of transcriptional regulators.

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy.

Filamin B (FLNB) plays an important role in skeletal development. Mutations in FLNB can lead to skeletal malformation such as an abnormal number of ossification centers, indicating that the skeletal segmentation in the embryonic period may be interfered with. We established a mouse model with the pathogenic point mutation FLNB NM_001081427.1: c.4756G > A (p.Gly1586Arg) using CRISPR-Cas9 technology. Micro-CT, HE staining and whole skeletal preparation were performed to examine the skeletal malformation. In situ hybridization of embryos was performed to examine the transcription of HOX genes during embryonic development. The expression of FLNB was downregulated in FLNBG1586R/G1586R and FLNBWT/G1586R mice, compared to FLNBWT/WT mice. Fusions in tarsal bones were found in FLNBG1586R/G1586R and FLNBWT/G1586R mice, indicating that the skeletal segmentation was interfered with. In the embryo of FLNBG1586R/G1586R mice (E12.5), the transcription levels of HOXD10 and HOXB2 were downregulated in the carpal region and cervical spine region, respectively. This study indicated that the loss-of-function mutation G1586R in FLNB may lead to abnormal skeletal segmentation, and the mechanism was possibly associated with the downregulation of HOX gene transcription during the embryonic period.

Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila, which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia (Antp) and Ultrabithorax (Ubx). Here, we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis-regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non-cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling.

During embryonic development, the timing of events at the cellular level must be coordinated across multiple length scales to ensure the formation of a well-proportioned body plan. This is clear during somitogenesis, where progenitors must be allocated to the axis over time whilst maintaining a progenitor population for continued elaboration of the body plan. However, the relative importance of intrinsic and extrinsic signals in timing progenitor addition at the single-cell level is not yet understood. Heterochronic grafts from older to younger embryos have suggested a level of intrinsic timing whereby later staged cells contribute to more posterior portions of the axis. To determine the precise step at which cells are delayed, we performed single-cell transcriptomic analysis on heterochronic grafts of somite progenitors in the chicken embryo. This revealed a previously undescribed cell state within which heterochronic grafted cells are stalled. The delayed exit of older cells from this state correlates with expression of posterior Hox genes. Using grafting and explant culture, we find that both Hox gene expression and the migratory capabilities of progenitor populations are intrinsically regulated at the population level. However, by grafting varied sizes of tissue, we find that small heterochronic grafts disperse more readily and contribute to more anterior portions of the body axis while still maintaining Hox gene expression. This enhanced dispersion is not replicated in explant culture, suggesting that it is a consequence of interaction between host and donor tissue and thus extrinsic to the donor tissue. Therefore, we demonstrate that the timing of cell dispersion and resulting axis contribution is impacted by a combination of both intrinsic and extrinsic cues.

OBJECTIVE: Understanding the regulatory role of homeobox (HOX) and mutated genes in the progression of head and neck cancers is essential, although their interaction remains elusive. This study aims to decipher the critical regulation of mutation driven effects on homeobox genes to enhance our understanding of head and neck cancer progression.
METHODS: Genomic mutation data from The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma were analyzed using VarScan2 for somatic variant detection. Mutational clustering, driver mutation identification, and cancer signaling pathway analysis were performed using the OncodriveCLUST method. Harmonizome datasets were retrieved to identify critical cancer driver genes affecting HOX genes. The effects of HPV infection on HOX and mutated genes were assessed using the oncoviral database. Altered pathway activity due to the effects of cancer drivers on HOX genes was analyzed with Gene Set Cancer Analysis. Functional enrichment analysis of gene ontology biological processes and molecular functions was conducted using the ClusterProfiler R package.
RESULTS: Significant alterations in HOX genes were observed in head and neck cancer cohorts with mutated TP53, FAT1, and CDKN2A. HOX genes were identified as functionally downstream targets of TP53, signifying transcriptionally mediated regulation. The interaction between HOX genes and mutated TP53, FAT1, and CDKN2A dysregulated the epithelial-to-mesenchymal transition, cell cycle, and apoptosis pathways in head and neck cancer progression.
CONCLUSIONS: The interplay between cancer driver genes and HOX genes is pivotal in regulating the oncogenic processes underlying the pathogenesis of head and neck squamous cell carcinoma.

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.

The homeobox A10 (HOXA10) gene is known to be related to endometriosis; however, due to a lack of knowledge/evidence in the pathogenesis of endometriosis, the mechanisms that link HOXA10 to endometriosis still need to be clarified. This review addresses the difference in the expression of the HOXA10 gene in endometriotic women versus non-endometriotic women across populations by country and discusses its influences on women\'s fertility. An organized search of electronic databases was conducted in Scopus, ScienceDirect, PubMed, and Web of Science. The keywords used were (HOXA10 OR \"homeobox A10\" OR PL OR HOX1 OR HOX1H OR HOX1.8) AND (\"gene expression\") AND (endometriosis). The initial search resulted in 623 articles, 10 of which were included in this review. All ten papers included in this study were rated fair in terms of the quality of the studies conducted. The expression of the HOXA10 gene was found to be downregulated in most studies. However, one study provided evidence of the downregulation and upregulation of HOXA10 gene expression due to the localization of endometriotic lesions. Measuring the expression of the HOXA10 gene in women is clinically essential to predicting endometriosis, endometrial receptivity, and the development of pinopodes in the endometrium during the luteal phase.

This study examined the anomalous variations of the ventral process of C6 in modern E. ferus caballus. The aim was to provide an incremental grading protocol measuring the absence of the caudal ventral tubercle (CVT) in this ventral process. The findings revealed the most prevalent absent CVT (aCVT) was left unilateral (n = 35), with bilateral (n = 29) and right unilateral (n = 12). Grading was determined in equal increments of absence 1/4, 2/4, 3/4, with 4/4 representing a complete aCVT in 56/76, with a significance of p = 0.0013. This also applied to bilateral specimens. In those C6 osseous specimens displaying a 4/4 grade aCVT, 41/56 had a partial absence of the caudal aspect of the cranial ventral tubercle (CrVT). Here, grading absent CrVTs (aCrVT) followed similarly to aCVTs, though 4/4 was not observed. The significance between 4/4 grade aCVTs and the presentation of an aCrVT was left p = 0.00001 and right p = 0.00018. In bilateral specimens, C6 morphologically resembled C5, implying a homeotic transformation that limited the attachment sites for the cranial and thoracal longus colli muscle. This potentially diminishes function and caudal cervical stability. Therefore, it is recommended that further studies examine the morphological extent of this equine complex vertebral malformation (ECVM) as well as its interrelationships and genetic code/blueprint.