Relaxed selection in evolution of genes regulating limb development gives clue to variation in forelimb morphology of cetaceans and other mammals.

Cetaceans have evolved unique limb structures, such as flippers, due to genetic changes during their transition to aquatic life. However, the full understanding of the genetic and evolutionary mechanisms behind these changes is still developing. By examining 25 limb-related protein-coding genes across various mammalian species, we compared genetic changes between aquatic mammals, like whales, and other mammals with unique limb structures such as bats, rodents and elephants. Our findings revealed significant modifications in limb-related genes, including variations in the Hox, GDF5 and Evx genes. Notably, a relaxed selection in several key genes was observed, suggesting a lifting of developmental constraints, which might have facilitated the emergence of morphological innovations in cetacean limb morphology. We also uncovered non-synonymous changes, insertions and deletions in these genes, particularly in the polyalanine tract of HOXD13, which are distinctive to cetaceans or convergent with other aquatic mammals. These genetic variations correlated with the diverse and specialized limb structures observed in cetaceans, indicating a complex interplay of relaxed selection and specific mutations in mammalian limb evolution.

The functional roles of zebrafish HoxA- and HoxD-related clusters in the pectoral fin development.

The paralogs 9-13 Hox genes in mouse HoxA and HoxD clusters are critical for limb development. When both HoxA and HoxD clusters are deleted in mice, significant limb truncation is observed compared to the phenotypes of single and compound mutants of Hox9-13 genes in these clusters. In zebrafish, mutations in hox13 genes in HoxA- and HoxD-related clusters result in abnormal morphology of pectoral fins, homologous to forelimbs. However, the effect of the simultaneous deletions of entire HoxA- and HoxD-related clusters on pectoral fin development remains unknown. Here, we generated mutants with several combinations of hoxaa, hoxab, and hoxda cluster deletions and analyzed the pectoral fin development. In hoxaa-/-;hoxab-/-;hoxda-/- larvae, the endoskeletal disc and the fin-fold are significantly shortened in developing pectoral fins. In addition, we show that this anomaly is due to defects in the pectoral fin growth after the fin bud formation. Furthermore, in the surviving adult mutants, micro-CT scanning reveals defects in the posterior portion of the pectoral fin which is thought to represent latent regions of the limb. Our results further support that the functional role of HoxA and HoxD clusters is conserved in the paired appendage formation in bony fishes.

Chamaeleo calyptratus (veiled chameleon) chromosome-scale genome assembly and annotation provides insights into the evolution of reptiles and developmental mechanisms.

The family Chamaeleonidae comprises 228 species, boasting an extensive geographic spread and an array of evolutionary novelties and adaptations, but a paucity of genetic and molecular analyses. Veiled chameleon (Chamaeleo calyptratus) has emerged as a tractable research organism for the study of squamate early development and evolution. Here we report a chromosomal-level assembly and annotation of the veiled chameleon genome. We note a remarkable chromosomal conservation across squamates, but comparisons to more distant genomes reveal GC peaks correlating with ancestral chromosome fusion events. We subsequently identified the XX/XY region on chromosome 5, confirming environmental-independent sex determination in veiled chameleons. Furthermore, our analysis of the Hox gene family indicates that veiled chameleons possess the most complete set of 41 Hox genes, retained from an amniote ancestor. Lastly, the veiled chameleon genome has retained both ancestral paralogs of the Nodal gene, but is missing Dand5 and several other genes, recently associated with the loss of motile cilia during the establishment of left-right patterning. Thus, a complete veiled chameleon genome provides opportunities for novel insights into the evolution of reptilian genomes and the molecular mechanisms driving phenotypic variation and ecological adaptation.

Comparative Hox genes expression within the dimorphic annelid Streblospio benedicti reveals patterning variation during development.

Hox genes are transcriptional regulators that elicit cell positional identity along the anterior-posterior region of the body plan across different lineages of Metazoan. Comparison of Hox gene expression across distinct species reveals their evolutionary conservation; however, their gains and losses in different lineages can correlate with body plan modifications and morphological novelty. We compare the expression of 11 Hox genes found within Streblospio benedicti, a marine annelid that produces two types of offspring with distinct developmental and morphological features. For these two distinct larval types, we compare Hox gene expression through ontogeny using hybridization chain reaction (HCR) probes for in situ hybridization and RNA-seq data. We find that Hox gene expression patterning for both types is typically similar at equivalent developmental stages. However, some Hox genes have spatial or temporal differences between the larval types that are associated with morphological and life-history differences. This is the first comparison of developmental divergence in Hox gene expression within a single species and these changes reveal how body plan differences may arise in larval evolution.

Plasticity Comparison of Two Stem Cell Sources with Different Hox Gene Expression Profiles in Response to Cobalt Chloride Treatment during Chondrogenic Differentiation.

The limited self-repair capacity of articular cartilage is a challenge for healing injuries. While mesenchymal stem/stromal cells (MSCs) are a promising approach for tissue regeneration, the criteria for selecting a suitable cell source remain undefined. To propose a molecular criterion, dental pulp stem cells (DPSCs) with a Hox-negative expression pattern and bone marrow mesenchymal stromal cells (BMSCs), which actively express Hox genes, were differentiated towards chondrocytes in 3D pellets, employing a two-step protocol. The MSCs' response to preconditioning by cobalt chloride (CoCl2), a hypoxia-mimicking agent, was explored in an assessment of the chondrogenic differentiation's efficiency using morphological, histochemical, immunohistochemical, and biochemical experiments. The preconditioned DPSC pellets exhibited significantly elevated levels of collagen II and glycosaminoglycans (GAGs) and reduced levels of the hypertrophic marker collagen X. No significant effect on GAGs production was observed in the preconditioned BMSC pellets, but collagen II and collagen X levels were elevated. While preconditioning did not modify the ALP specific activity in either cell type, it was notably lower in the DPSCs differentiated pellets compared to their BMSCs counterparts. These results could be interpreted as demonstrating the higher plasticity of DPSCs compared to BMSCs, suggesting the contribution of their unique molecular characteristics, including their negative Hox expression pattern, to promote a chondrogenic differentiation potential. Consequently, DPSCs could be considered compelling candidates for future cartilage cell therapy.

Members of an array of zinc-finger proteins specify distinct Hox chromatin boundaries.

Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.

Hox gene activity directs physical forces to differentially shape chick small and large intestinal epithelia.

Hox transcription factors play crucial roles in organizing developmental patterning across metazoa, but how these factors trigger regional morphogenesis has largely remained a mystery. In the developing gut, Hox genes help demarcate identities of intestinal subregions early in embryogenesis, which ultimately leads to their specialization in both form and function. Although the midgut forms villi, the hindgut develops sulci that resolve into heterogeneous outgrowths. Combining mechanical measurements of the embryonic chick intestine and mathematical modeling, we demonstrate that the posterior Hox gene HOXD13 regulates biophysical phenomena that shape the hindgut lumen. We further show that HOXD13 acts through the transforming growth factor ß (TGF-ß) pathway to thicken, stiffen, and promote isotropic growth of the subepithelial mesenchyme-together, these features lead to hindgut-specific surface buckling. TGF-ß, in turn, promotes collagen deposition to affect mesenchymal geometry and growth. We thus identify a cascade of events downstream of positional identity that direct posterior intestinal morphogenesis.

Irreducible Complexity of Hox Gene: Path to the Canonical Function of the Hox Cluster.

The evolution of major taxa is often associated with the emergence of new gene families. In all multicellular animals except sponges and comb jellies, the genomes contain Hox genes, which are crucial regulators of development. The canonical function of Hox genes involves colinear patterning of body parts in bilateral animals. This general function is implemented through complex, precisely coordinated mechanisms, not all of which are evolutionarily conserved and fully understood. We suggest that the emergence of this regulatory complexity was preceded by a stage of cooperation between more ancient morphogenetic programs or their individual elements. Footprints of these programs may be present in modern animals to execute non-canonical Hox functions. Non-canonical functions of Hox genes are involved in maintaining terminal nerve cell specificity, autophagy, oogenesis, pre-gastrulation embryogenesis, vertical signaling, and a number of general biological processes. These functions are realized by the basic properties of homeodomain protein and could have triggered the evolution of ParaHoxozoa and Nephrozoa subsequently. Some of these non-canonical Hox functions are discussed in our review.

Teleost Hox code defines regional identities competent for the formation of dorsal and anal fins.

The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position.

An atlas of spider development at single-cell resolution provides new insights into arthropod embryogenesis.

Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5 k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. We found differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We identified many Hox genes as markers of cell clusters, and Hox gene ohnologs were often present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in an arachnopulmonate ancestor (spiders, scorpions, and related orders). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.

Acceleration of genome rearrangement in clitellate annelids.

Comparisons of multiple metazoan genomes have revealed the existence of ancestral linkage groups (ALGs), genomic scaffolds sharing sets of orthologous genes that have been inherited from ancestral animals for hundreds of millions of years (Simakov et al. 2022; Schultz et al. 2023) These ALGs have persisted across major animal taxa including Cnidaria, Deuterostomia, Ecdysozoa and Spiralia. Notwithstanding this general trend of chromosome-scale conservation, ALGs have been obliterated by extensive genome rearrangements in certain groups, most notably including Clitellata (oligochaetes and leeches), a group of easily overlooked invertebrates that is of tremendous ecological, agricultural and economic importance (Charles 2019; Barrett 2016). To further investigate these rearrangements, we have undertaken a comparison of 12 clitellate genomes (including four newly sequenced species) and 11 outgroup representatives. We show that these rearrangements began at the base of the Clitellata (rather than progressing gradually throughout polychaete annelids), that the inter-chromosomal rearrangements continue in several clitellate lineages and that these events have substantially shaped the evolution of the otherwise highly conserved Hox cluster.

A 2024 Update on Menin Inhibitors. A New Class of Target Agents against KMT2A-Rearranged and NPM1-Mutated Acute Myeloid Leukemia.

Menin inhibitors are new and promising agents currently in clinical development that target the HOX/MEIS1 transcriptional program which is critical for leukemogenesis in histone-lysine N-methyltransferase 2A-rearranged (KMT2Ar) and in NPM1-mutated (NPM1mut) acute leukemias. The mechanism of action of this new class of agents is based on the disruption of the menin-KMT2A complex (consisting of chromatin remodeling proteins), leading to the differentiation and apoptosis of AML cells expressing KMT2A or with mutated NPM1. To date, this new class of drugs has been tested in phase I and II clinical trials, both alone and in combination with synergistic drugs showing promising results in terms of response rates and safety in heavily pre-treated acute leukemia patients. In this brief review, we summarize the key findings on menin inhibitors, focusing on the mechanism of action and preliminary clinical data on the treatment of acute myeloid leukemia with this promising new class of agents, particularly revumenib and ziftomenib.

EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE DURING THE EMERGENCE OF DIGITS AND GENITALS.

The transition from fins to limbs has been a rich source of discussion for more than a century. One open and important issue is understanding how the mechanisms that pattern digits arose during vertebrate evolution. In this context, the analysis of Hox gene expression and functions to infer evolutionary scenarios has been a productive approach to explain the changes in organ formation, particularly in limbs. In tetrapods, the transcription of Hoxd genes in developing digits depends on a well-characterized set of enhancers forming a large regulatory landscape1,2. This control system has a syntenic counterpart in zebrafish, even though they lack bona fide digits, suggestive of deep homology3 between distal fin and limb developmental mechanisms. We tested the global function of this landscape to assess ancestry and source of limb and fin variation. In contrast to results in mice, we show here that the deletion of the homologous control region in zebrafish has a limited effect on the transcription of hoxd genes during fin development. However, it fully abrogates hoxd expression within the developing cloaca, an ancestral structure related to the mammalian urogenital sinus. We show that similar to the limb, Hoxd gene function in the urogenital sinus of the mouse also depends on enhancers located in this same genomic domain. Thus, we conclude that the current regulation underlying Hoxd gene expression in distal limbs was co-opted in tetrapods from a preexisting cloacal program. The orthologous chromatin domain in fishes may illustrate a rudimentary or partial step in this evolutionary co-option.

Role of HOX genes in cancer progression and their therapeutical aspects.

HOX genes constitute a family of evolutionarily conserved transcription factors that play pivotal roles in embryonic development, tissue patterning, and cell differentiation. These genes are essential for the precise spatial and temporal control of body axis formation in vertebrates. In addition to their developmental functions, HOX genes have garnered significant attention for their involvement in various diseases, including cancer. Deregulation of HOX gene expression has been observed in numerous malignancies, where they can influence tumorigenesis, progression, and therapeutic responses. This review provides an overview of the diverse roles of HOX genes in development, disease, and potential therapeutic targets, highlighting their significance in understanding biological processes and their potential clinical implications.

The expanding roles of Nr6a1 in development and evolution.

The Nuclear Receptor (NR) family of transcriptional regulators possess the ability to sense signalling molecules and directly couple that to a transcriptional response. While this large class of proteins are united by sequence and structural homology, individual NR functional output varies greatly depending on their expression, ligand selectivity and DNA binding sequence specificity. Many NRs have remained somewhat enigmatic, with the absence of a defined ligand categorising them as orphan nuclear receptors. One example is Nuclear Receptor subfamily 6 group A member 1 (Nr6a1), an orphan nuclear receptor that has no close evolutionary homologs and thus is alone in subfamily 6. Nonetheless, Nr6a1 has emerged as an important player in the regulation of key pluripotency and developmental genes, as functionally critical for mid-gestational developmental progression and as a possible molecular target for driving evolutionary change in animal body plan. Here, we review the current knowledge on this enigmatic nuclear receptor and how it impacts development and evolution.

A Glance into the Destiny of Transcriptomic Activity, Embodied by the HOX Genes, in Neonatal and Aging Dermal Cells.

Skin is an organ having a crucial role in the protection of muscle, bone, and internal organs and undergoing continuous self-renewal and aged. The growing interest in the prevention of skin aging and rejuvenation has sparked a surge of industrial and research studies focusing on the biological and transcriptional changes that occur during skin development and aging. In this study, the aim is to identify transcriptional differences between two main types of human skin cells: the human dermal fibroblasts (HDFs) and the human epidermis keratinocytes (HEKs) isolated from 30 neonatal and 30 adults (old) skin. Through differentially expressed gene (DEG) profiling using DEseq2, 604 up-, and 769 down-regulated genes are identified in the old group. A functional analysis using Metascape Gene Ontology and Reactome pathways revealed systematic transcriptomic shifts in key skin formation and maintenance markers, alongside a distinct difference in HOX gene families crucial for embryonic development and diverse biological processes. Among the 39 human HOX gene family, ten posterior HOX genes (HOXA10, 11, 13, HOXB13, HOXC11, and HOXD9-13) are significantly downregulated, and anterior 25 genes (HOXA2-7, HOXB1-9, HOXC4-6 and 8-9, and HOXD1,3,4 and 8) are upregulated, especially in the old HDFs. The study successfully demonstrates the correlation between HOX genes and the skin aging process, providing strong evidence that HOX genes are proposed as a new marker for skin aging assessment.

SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods.

The genome editing technique CRISPR/Cas9 has led to major advancements in many research fields and this state-of-the-art tool has proven its use in genetic studies for various arthropods. However, most transformation protocols rely on microinjection of CRISPR/Cas9 components into embryos, a method which is challenging for many species. Alternatively, injections can be performed on adult females, but transformation efficiencies can be very low as was shown for the two-spotted spider mite, Tetranychus urticae, a minute but important chelicerate pest on many crops. In this study, we explored different CRISPR/Cas9 formulations to optimize a maternal injection protocol for T. urticae. We observed a strong synergy between branched amphipathic peptide capsules and saponins, resulting in a significant increase of CRISPR/Cas9 knock-out efficiency, exceeding 20%. This CRISPR/Cas9 formulation, termed SYNCAS, was used to knock-out different T. urticae genes - phytoene desaturase, CYP384A1 and Antennapedia - but also allowed to develop a co-CRISPR strategy and facilitated the generation of T. urticae knock-in mutants. In addition, SYNCAS was successfully applied to knock-out white and white-like genes in the western flower thrips, Frankliniella occidentalis. The SYNCAS method allows routine genome editing in these species and can be a game changer for genetic research in other hard to transform arthropods.

Distinct roles of the Hox genes Ultrabithorax and abdominal-A in scorpionfly embryonic proleg development.

The abdominal appendages of larval insects have a complex evolutionary history of gain and loss, but the regulatory mechanisms underlying the abdominal appendage development remain largely unclear. Here, we investigated the embryogenesis of abdominal prolegs in the scorpionfly Panorpa liui Hua (Mecoptera: Panorpidae) using in situ hybridization and parental RNA interference. The results show that RNAi-mediated knockdown of Ultrabithorax (Ubx) led to a homeotic transformation of the first abdominal segment (A1) into the third thoracic segment (T3) and changed the distributions of the downstream target Distal-less (Dll) expression but did not affect the expression levels of Dll. Knockdown of abdominal-A (abd-A) resulted in malformed segments, abnormal prolegs and disrupted Dll expression. The results demonstrate that the gene Ubx maintains an ancestral role of modulating A1 appendage fate without preventing Dll initiation, and a secondary adaptation of abd-A evolves the ability to specify abdominal segments and proleg identity. We conclude that changes in abdominal Hox gene expression and their target genes regulate abdominal appendage morphology during the evolutionary course of holometabolous larvae.

Discovery of seven hox genes in zebrafish thrombopoiesis.

Thrombopoiesis is the production of platelets from megakaryocytes in the bone marrow of mammals. In fish, thrombopoiesis involves the formation of thrombocytes without megakaryocyte-like precursors but derived from erythrocyte thrombocyte bi-functional precursor cells. One unique feature of thrombocyte differentiation involves the maturation of young thrombocytes in circulation. In this study, we investigated the role of hox genes in zebrafish thrombopoiesis to model platelet production. We selected hoxa10b, hoxb2a, hoxc5a, hoxd3a, and hoxc11b from thrombocyte RNA expression data, and checked whether they are expressed in young or mature thrombocytes. We found hoxa10b, hoxb2a, hoxc5a, and hoxd3a were expressed in both young and mature thrombocytes and hoxc11b was expressed in only young thrombocytes. We then performed knockdowns of these 5 hox genes and found hoxc11b knockdown resulted in thrombocytosis and the rest showed thrombocytopenia. To identify hox genes that could have been missed by the above datasets, we performed knockdowns 47 hox genes in the zebrafish genome and found hoxa9a, and hoxb1a knockdowns resulted in thrombocytopenia and they were expressed in both young and mature thrombocytes. In conclusion, our comprehensive knockdown study identified Hoxa10b, Hoxb2a, Hoxc5a, Hoxd3a, Hoxa9a, and Hoxb1a, as positive regulators and Hoxc11b, as a negative regulator for thrombocyte development.

Is HOXA5 a Novel Prognostic Biomarker for Uterine Corpus Endometrioid Adenocarcinoma?

Endometrial cancer (EC) is one of the most pervasive malignancies in females worldwide. HOXA5 is a member of the homeobox (HOX) family and encodes the HOXA5 protein. HOXA5 is associated with various cancers; however, its association with EC remains unclear. This study aimed to determine the association between HOXA5 gene expression and the prognosis of endometrioid adenocarcinoma, a subtype of EC (EAEC). Microarray data of HOXA5 were collected from the Gene Expression Omnibus datasets, consisting of 79 samples from GSE17025 and 20 samples from GSE29981. RNA-sequencing, clinical, and survival data on EC were obtained from The Cancer Genome Atlas cohort. Survival analysis revealed that HOXA5 overexpression was associated with poor overall survival in patients with EAEC (p = 0.044, HR = 1.832, 95% CI = 1.006-3.334). Cox regression analysis revealed that HOXA5 was an independent risk factor for poor prognosis in EAEC. The overexpression of HOXA5 was associated with a higher histological grade of EAEC, and it was also associated with TP53 mutation or the high copy number of EC. Our findings suggest the potential of HOXA5 as a novel biomarker for predicting poor survival outcomes in patients with EAEC.