Hox Genes Research Articles

Problems with paralogs: the promise and challenges of gene duplicates in evo-devo research.

Gene duplicates, or paralogs, serve as a major source of new genetic material and comprise seeds for evolutionary innovation. While originally thought to be quickly lost or non-functionalized following duplication, now a vast number of paralogs are known to be retained in a functional state. Daughter paralogs can provide robustness through redundancy, specialize via sub-functionalization, or neo-functionalize to play new roles. Indeed, the duplication and divergence of developmental genes have played a monumental role in the evolution of animal forms (e.g. Hox genes). Still, despite their prevalence and evolutionary importance, the precise detection of gene duplicates in newly sequenced genomes remains technically challenging and often overlooked. This presents an especially pertinent problem for evolutionary developmental biology (evo-devo), where hypothesis testing requires accurate detection of changes in gene expression and function, often in non-traditional model species. Frequently, these analyses rely on molecular reagents designed within coding sequences that may be highly similar in recently duplicated paralogs, leading to cross-reactivity and spurious results. Thus, care is needed to avoid erroneously assigning diverged functions of paralogs to a single gene, and potentially misinterpreting evolutionary history. This perspective aims to overview the prevalence and importance of paralogs and to shed light on the difficulty of their detection and analysis while offering potential solutions.

Precise CRISPR/Cpf1 genome editing system in the Deinococcus radiodurans with superior DNA repair mechanisms

Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.

Persistent DNA Methylation Changes across the First Year of Life and Prenatal Exposure in a Canadian Prospective Birth Study.

Evidence suggests that prenatal air pollution exposure alters DNA methylation (DNAm), which could go on to affect long-term health. It remains unclear whether DNAm alterations present at birth persist through early life. Identifying persistent DNAm changes would provide greater insight into the molecular mechanisms contributing to the association of prenatal air pollution exposure with atopic diseases. This study investigated DNAm differences associated with prenatal nitrogen dioxide () exposure (a surrogate measure of traffic-related air pollution) at birth and 1 y of age and examined their role in atopic disease. We focused on regions showing persistent DNAm differences from birth to 1 y of age and regions uniquely associated with postnatal exposure. Microarrays measured DNAm at birth and at 1 y of age for an atopy-enriched subset of Canadian Health Infant Longitudinal Development (CHILD) study participants. Individual and regional DNAm differences associated with prenatal () were identified, and their persistence at age 1 y were investigated using linear mixed effects models (). Postnatal-specific DNAm differences () were isolated, and their association with in the first year of life was examined. Causal mediation investigated whether DNAm differences mediated associations between and age 1 y atopy or wheeze. Analyses were repeated using biological sex-stratified data. At birth (), 18 regions of DNAm were associated with , with several annotated to HOX genes. Some of these regions were specifically identified in males (), but not females (). The effect of prenatal across CpGs within altered regions persisted at 1 y of age. No significant mediation effects were identified. Sex-stratified analyses identified postnatal-specific DNAm alterations. Regional cord blood DNAm differences associated with prenatal persisted through at least the first year of life in CHILD participants. Some differences may represent sex-specific alterations, but replication in larger cohorts is needed. The early postnatal period remained a sensitive window to DNAm perturbations. https://doi.org/10.1289/EHP13034.

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.

Abstract 5592: HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia

Abstract Although High Mobility Group A1 (HMGA1) chromatin regulators are overexpressed in refractory acute myeloid leukemia (AML) and diverse solid tumors, targetable mechanisms underlying HMGA1 remain elusive. HMGA1 is highly expressed in stem cells and poorly differentiated cancers where it modulates chromatin structure to activate developmental gene networks. In AML arising from JAK2V617F myeloproliferative neoplasms (MPN), HMGA1 is required for leukemogenesis by inducing transcriptional networks that drive aberrant proliferation and cell fate. Like MPN AML, KMT2A-rearranged (KMT2A-r) AML is refractory to therapy and highly lethal. KMT2A-r AML is caused by rearrangements of the KMT2A gene, which encode abnormal fusion proteins that induce pro-leukemogenic genes, including HOX genes. While prior work identified KMT2A fusion partners and protein complexes, this has not led to better therapies for most patients. We therefore sought to examine HMGA1 function in KMT2A-r AML. We hypothesized that: 1) HMGA1 drives plasticity in KMT2A-r AML by altering chromatin state and transcriptional networks, and, 2) Targeting HMGA1 will enhance sensitivity to therapy. In two KMT2A-r AML patient cohorts, HMGA1 is highly overexpressed compared to CD34+ stem and progenitors from healthy controls, suggesting it drives leukemogenesis. To define HMGA1 function, we silenced HMGA1 using CRISPR or short hairpin RNA in KMT2A-r cell lines (MOLM-14, THP-1, MV4-11). Strikingly, silencing HMGA1 disrupts proliferation and clonogenicity in vitro, while prolonging survival in immunosuppressed mice transplanted with KMT2A-r AML cells. Intriguingly, leukemic cells that engraft in the bone marrow from the pool of cells with HMGA1 silencing express higher levels of HMGA1, suggesting that escape from gene silencing and a specific level of HMGA1 is required for leukemic engraftment and expansion. HMGA1 also increases in KMT2A-r AML cells that become resistant to cytarabine (AraC), suggesting that HMGA1 endows AML cells with the capacity to survive and expand after AraC. Because repression of tumor suppressors was recently defined as a mechanism of therapy resistance in AML, we also examined tumor suppressor pathways in both JAK2V617F and KMT2A-r AML. Intriguingly, HMGA1 occupies the promoter region of the CDKN1A locus by chromatin immunoprecipitation and represses its expression in KMT2A-r and JAK2V617F AML cells. To define additional mechanisms underlying HMGA1 in KMT2A-r AML, multi-omics studies are underway. In preliminary work thus far, we also found that HMGA1 induces HOX genes. Together, these findings implicate HMGA1 as a promising therapeutic target and novel epigenetic regulator in KMT2A-r AML by altering chromatin state and downstream gene expression. Citation Format: Bailey E. West, Jung-Hyun Kim, Audrey-Ann Supreme, Iliana Herrera, Zanshé Thompson, Li Z. Luo, Faiza Shaik, Joseph Kim, Hyunsung Woo, Soheil Meshinchi, Rhonda E. Ries, Linda M. Resar. HMGA1 modulates chromatin state and transcriptional networks involved in plasticity in refractory myeloid leukemia [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 5592.

Abstract 1733: SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia

Abstract The SET-NUP214 (S/N) fusion gene, which results from either cryptic t(9:9) (q34;q34) or del(9) (q34.11q34.13), is found in a very narrow spectrum of patients diagnosed with Acute Myeloid Leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL). AML patients with S/N fusion were reported to have relatively poor prognostic outcomes despite receiving myeloablative conditioning. While previously, the fusion has been described to overrepresent HOX cluster genes, the underlying mechanism of gene regulation that contributes to the pathology remains elusive due to the rare incidence of the disease. The current study used the MEGAL (ACC719, DSMZ) cell line, identified as the solo megakaryocytic AML line expressing the S/N-fusion, for determining genome-wide DNA methylation (DNAm) and gene expression using 935K Infinium MethylationEPIC v2.0 BeadChip array and RNA-sequencing, respectively. The differential DNAm and expression data were analyzed and compared to the hematopoietic stem progenitor cells (HSPC) or normal megakaryocytes (MK). The ChIP-sequencing data from the S/N positive T-ALL cell line LOUCY has been used to represent the coincidence of regulatory chromatin marks and differentially methylated CpG (mC) sites. We observed n=1934 genes upregulated and n=2396 downregulated in MEGAL, compared to both HSPC and MK. The direction of expression changes between MEGAL and HSPC and MEGAL and MK showed a high correlation (r=0.92, p< 0.01). Per the previous literature, we observed an overrepresentation of HOX genes (n=19) in MEGAL. Based on DNAm profiling, we noticed a propensity of hypermethylation in MEGAL across the promoters (93%±0.6), bodies (93%±0.8), and intergenic (85%±0.3) regions, compared to HSPC/MK. Next, we integrated the mC and expression at the promoters and identified that the majority (75%±3) of the genes belong to the hyper-down (n=2563 against HPSC; n=1907 against MK) or hyper-up (n=965 against HPSC; n=923 against MK) clusters. We further investigated the HOXB (HOXB7, HOXB8, HOXB9) and HOXC (HOXC10, HOXC12, HOXC13, HOXC13-AS, HOXC4, HOXC5, HOC6, HOXC8, HOXC9, HOXC-AS1, HOXC-AS2, and HOXC-AS3) cluster genes, where hypermethylated CpGs mainly were concentrated at the CpG islands and overlapped with active promoter (H3K4me3, H3K4me1) signatures or enhancer-like (CTCF bound) elements. We also observed frequent overlaps between mC and H3K36me3 across the observed genes. The differentially expressed genes in this AML subtype were enriched for the hippo-signaling pathway, calcium signaling, focal adhesion, and PI3-Akt pathways. Collectively, our data showed that S/N favors genome-wide hypermethylation, which, in conjunction with the cis-regulatory elements, induces overrepresentation of the HOX cluster genes. These novel epigenetic vulnerabilities are worth pursuing for investigating the cellular function and pathophysiology of the disease. Citation Format: Samrat Roy Choudhury, Akhilesh Kaushal. SET-NUP214 rearranges the DNA-methylation landscape to upregulate the HOX-gene cluster in acute myeloid leukemia [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 1733.

Abstract 294: The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte

Abstract Homeobox genes are regulatory genes that encode nuclear proteins acting as transcription factors during normal development and differentiation. One such gene, HOXB7, plays a role in various developmental processes, including hematopoietic differentiation, lymphoid development, and mammary gland development. However, the role of HOX genes in breast cancer development remains largely unexplored. Our previous studies revealed that HOXB7 expression was significantly elevated in primary cancer and distant metastasis. HOXB7 overexpression promoted cell proliferation in SKBR3 breast cancer cells, leading to robustly vascularized xenografts in immunodeficient mice. Furthermore, HOXB7 enhanced tumor growth through TGF beta signaling and recruitment of macrophages, indicating the role of HOXB7 in the crosstalk of stromal components. We found evidence that HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes. By screening secreted factors in adipocytes induced by TCM of MCF-7-HOXB7 cells, it was also found that CCL5 is highly expressed in HOXB7-overexpressing MCF-7 cells. The CCR5 inhibitor, maraviroc, sensitized MCF-7-HOXB7 cells compared to control cells. These findings suggest that blocking the interaction between CCL5 and CCR5 signaling significantly inhibits ER+ breast cancer with HOXB7 overexpression. We aim to further investigate if HOXB7 overexpression in ER+ breast cancer cells promotes tumor cell growth through increased expression and signaling of CCL5/CCR5 in crosstalk with adipocytes and the role of CCL5 in breast cancer cell growth and migration. Citation Format: Ahone Gina Akume, Kideok Jin. The HOXB7 protein promotes breast cancer cell growth through activation of the CCL5/CCR5 pathway in the crosstalk of adipocyte [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 294.

Abstract 4885: Integrating mutational profiles and transcriptional data with ASTUTE to elucidate the key molecular functions involved in the pathogenesis of cancer

Abstract Cancer is a highly heterogeneous disease characterized by genomic and phenotypic changes that differ among tumor subtypes. A comprehensive understanding of the molecular heterogeneity is crucial for identifying molecular biomarkers and developing targeted therapies. While recent years have seen significant progress in genomic studies and advancements in next-generation sequencing technologies, elucidate the impact of individual genomic alterations on the transcriptome landscape of cancer cells remains essential. This knowledge is crucial for developing personalized therapies. To integrate mutational profiles and transcriptional data, we have developed a novel computational framework named ASTUTE (Association of SomaTic mUtaTions to Expression). ASTUTE establishes associations between somatic mutations and gene expression profiles, quantifying the results as fold changes and comparing the effects of individual mutations. In particular, ASTUTE leverages LASSO regularized regression for feature selection, identifying the most relevant mutated genes that exhibit a strong association with expression levels. Our goal is to create a valuable resource for identifying diagnostic markers and advancing the development of targeted therapies in cancer. To this end, we applied ASTUTE to two published bulk RNA-seq datasets of adult acute myeloid leukemia (AML) patients from the Beat AML program and the TCGA study to verify the capacity of our approach to identify genes whose expression correlates with the presence of specific somatic mutations. The first dataset comprises 585 samples, while the second one includes 173 specimens, both of them contain mutations and RNA-seq data. We executed ASTUTE on the two datasets independently, considering all genes for RNA-seq data and the alterations in the top 10 most mutated genes. We considered the associations consistently discovered in both cohorts. Our analysis revealed a strong association between NPM1 mutations and the expression of HOX genes. Specifically, HOXB6, HOXB5, HOXB3, HOXA5, HOXB2, and HOXA10 exhibited a log2 fold change higher than 1.5. The upregulation of these genes has been reported in association with NPM1 mutations in AML adult patients. Additionally, ASTUTE identified HOXA6, HOXA7, HOXA4, and HOXA9, whose expression has been described to be upregulated in the presence of NPM1 mutations in AML pediatric samples. Finally, we identified a robust relationship between NPM1 mutations and the expression of PBX3, MEIS, and ITM2A. The first two genes are upregulated in the presence of NPM1 mutations, while the third one is downregulated. Our analysis showcases ASTUTE's capability to identify potential markers and underscores the possibility to apply ASTUTE to different tumors datasets, with the aim to better characterize cancer heterogeneity and develop targeted therapies. Citation Format: Valentina Crippa, Diletta Fontana, Ivan Civettini, Luca Mologni, Rocco Piazza, Carlo Gambacorti-Passerini, Daniele Ramazzotti. Integrating mutational profiles and transcriptional data with ASTUTE to elucidate the key molecular functions involved in the pathogenesis of cancer [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 4885.

Evolutionary immunology to explore original antiviral strategies.

Over the past 25 years, the field of evolutionary developmental biology (evo-devo) has used genomics and genetics to gain insight on the developmental mechanisms underlying the evolution of morphological diversity of animals. Evo-devo exploits the key insight that conserved toolkits of development (e.g. Hox genes) are used in animals to produce genetic novelties that provide adaptation to a new environment. Like development, immunity is forged by interactions with the environment, namely the microbial world. Yet, when it comes to the study of immune defence mechanisms in invertebrates, interest primarily focuses on evolutionarily conserved molecules also present in humans. Here, focusing on antiviral immunity, we argue that immune genes not conserved in humans represent an unexplored resource for the discovery of new antiviral strategies. We review recent findings on the cGAS-STING pathway and explain how cyclic dinucleotides produced by cGAS-like receptors may be used to investigate the portfolio of antiviral genes in a broad range of species. This will set the stage for evo-immuno approaches, exploiting the investment in antiviral defences made by metazoans over hundreds of millions of years of evolution. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

A signal-switchable photoelectrochemical biosensor for ultrasensitive detection of long non-coding RNA in cancer cells

Long non-coding RNA (LncRNA) as an emerging tumor biomarker plays a key factor in the early diagnosis of cancer. Herein, an innovative signal-switchable photoelectrochemical (PEC) biosensor based on ZrO2@CuO bimetallic oxides and T7 Exo-assisted signal amplification is reported for the ultrasensitive and selective detection of lncRNA (HOX gene antisense intergenic RNA, HOTAIR) in cancer cells. Firstly, MOFs-derived TiO2 nanodisks as an excellent photoactive material show an anodic background signal. When target lncRNA exists, the abundant auxiliary DNA1 is freed from T7 Exo-assisted cycle signal amplification, and then competitively hybridizes with auxiliary DNA2 on the electrode. Subsequently, bimetallic MOFs-derived ZrO2@CuO octahedra with a high specific surface area and porous structure are introduced into TiO2 nanodisks-modified biosensor, which appears a cathodic photocurrent and achieves a switchable signal. The developed signal-switchable PEC biosensor shows ultrasensitive detection of lncRNA HOTAIR with a detection limit of 0.12 fM, and can eliminate the false interference. Importantly, the established PEC biosensor has good correlation with RT-qPCR analysis (P < 0.05) for the quantification of lncRNA HOTAIR in cancer cells, which has great potential application for biomarker detection in the early diagnosis of cancer.

Gene-expression patterns during regeneration of the multi-organ complex after evisceration in the sea cucumber Eupentacta quinquesemita

Many species of sea cucumbers (Echinodermata, Holothuroidea) perform evisceration, i.e., ejection of internal organs including digestive tracts when responding to environmental stresses. After evisceration, they also show a high regenerative capacity, in which all the eviscerated organs regenerate. Especially in anterior evisceration species, the oral complex structure consisting of multiple organs, including the digestive tract and central nervous system, are reconstructed. The detailed developmental mechanisms underlying the process remains unclear, and therefore, in this study, focusing on Eupentacta quinquesemita, gene expression analyses in the regenerating tissues were carried out. For the formation of the gut tube, genes involved in mesenchymal-epithelial transition were upregulated consistently with the histological changes. Upregulation of Hox and Parahox genes along the anterior-posterior axis was observed, implying the involvement of these genes in the spatial differentiation of the digestive tract. In addition, the expression of otx, six and pax, i.e., transcription factors patterning anterior nervous tissues, was upregulated during the regeneration of the central nervous system. Taken together, these results suggest that conserved genes are co-opted to the internal organ regeneration after evisceration in sea cucumbers.

Drug Repurposing and Personalized Treatment Strategies for Bipolar Disorder Using Transcriptomic.

The present study combined transcriptomic data and computational techniques based on gene expression signatures to identify novel bioactive compounds or FDA-approved drugs for the management of Bipolar Disorder (BD). Five transcriptomic datasets, comprising a total of 165 blood samples from BD case-control, were selected from the Gene Expression Omnibus repository (GEO). The number of subjects varied from 6 to 60, with a mean age ranging from 35 to 48, with a gender variation between them. Most of the patients were on pharmacological treatment. Master Regulator Analysis (MRA) and Gene Set Enrichment Analysis (GSEA) were performed to identify statistically significant genes between BD and HC and their association with the mood states of BD. Additionally, existing molecules with the potential to reverse the transcriptomic profiles of disease-altered regulons in BD were identified using the LINCS and cMap databases. MRA identified 59 potential MRs candidates modulating the regulatory units enriched with genes altered in BD, while the GSEA identified 134 enriched genes, and a total of 982 regulons had their activation state determined. Both analyses showed genes exclusively associated with mania, depression, or euthymia, and some genes were common between the three mood states. We identified bioactive compounds and licensed drug candidates, including antihypertensives and antineoplastics, as promising candidates for treating BD. Nevertheless, experimental validation is essential to authenticate these findings in subsequent studies. Although preliminary, our data provides some insights regarding the biological patterns of BD into distinct mood states and potential therapeutic targets. The combined transcriptomic and bioinformatics strategy offers a route to advance drug discovery and personalized medicine by tapping into gene expression information.

Morphological variation, modularity and integration in the scapula and humerus of Lissotriton newts.

The modular organization of tetrapod paired limbs and girdles, influenced by the expression of Hox genes is one of the primary driving forces of the evolution of animal locomotion. The increased morphological diversification of the paired limbs is correlated with reduced between-limb covariation, while correlation within the elements is usually higher than between the elements. The tailed amphibians, such as Lissotriton newts, have a biphasic lifestyle with aquatic and terrestrial environments imposing different constraints on limb skeleton. By employing the methods of computerized microtomography and 3D geometric morphometrics, we explored the pattern of morphological variation, disparity, modularity and morphological integration in the proximal parts of the anterior limbs of six species of Eurasian small bodied newts. Although the species significantly differ in limb shape, there is a great overlap in morphology of scapula and humerus, and there are no differences in morphological disparity. For the scapula, the shape differences related to the duration of the aquatic period are in length, depth and curvature. The shape of the humerus is not affected by the length of aquatic period, and shape differences between the species are related to robustness of the body. The length of aquatic period has statistically supported phylogenetic signal. The scapula and humerus are structures of varying modularity. For the humerus, the strongest support on the phylogenetic level was for the capitulum/shaft hypothesis, which can also be interpreted as functional modularity. For the scapula, the greatest support was for the antero-posterior hypothesis of modularity in case of Lissotriton vulgaris, which can be explained by different functional roles and muscle insertion patterns, while there was no phylogenetic modularity. The modularity patterns seem to correspond with the general tetrapod pattern, with modularity being more pronounced in the distal structure. The future research should include more salamandrid taxa with different habitat preferences and both adult and larval stages, in order to explore how size, phylogeny and ecology affect the morphology and covariation patterns of limbs.

Global gene expression profile of proliferative verrucous leukoplakia and its underlying biological disease mechanisms

BackgroundProliferative verrucous leukoplakia (PVL) is a rare and enigmatic oral potentially malignant disorder which almost invariably results in oral squamous cell carcinoma (OSCC). The aims of this project were to use transcriptome profiling to characterise PVL gene expression patterns for biomarker identification and gain insight into the molecular aetiopathogenesis of PVL. MethodsForty-three oral cavity mucosal biopsies from 32 patients with oral lesions clinically compatible with either PVL or non-PVL conventional oral leukoplakia (OLK) underwent transcriptome profiling by RNA sequencing. Data was analysed by hierarchical clustering, differential gene expression, functional enrichment and network analysis, sparse partial least squares discriminant analysis sPLS-DA, and immune cell phenotypic estimation. ResultsWe found 464 genes significantly differentially expressed at least 2-fold between PVL and non-PVL OLK (193 up and 271 down). HOX genes, including HOXA1 and HOXB7, keratin-associated proteins (KRTAPs) and olfactory receptor G proteins (OR) were significantly upregulated in PVL. Other upregulated genes in PVL included FOS, WNT16 and IFNA1. Pathway analysis showed that there was a significant downregulation of connective tissue signalling in PVL. Classifying multivariate models based upon 22 genes discriminated PVL from non-PVL OLK. Bioinformatic profiling showed that immune cell profiles in PVL and OLK were similar except that fibroblast markers were reduced in PVL. ConclusionThese results demonstrate that PVL and conventional OLK are molecularly distinct with upregulation of many cancer-associated genes. They provide insight into the pathogenesis of PVL and show that biomarker based molecular diagnostics is feasible to discriminate and inform diagnosis and management.

Heat-Resistant Inbred Lines Coordinate the Heat Response Gene Expression Remarkably in Maize (Zea mays L.).

High temperatures are increasingly becoming a prominent environmental factor accelerating the adverse influence on the growth and development of maize (Zea mays L.). Therefore, it is critical to identify the key genes and pathways related to heat stress (HS) tolerance in maize. Great challenges have been faced in dissecting genetic mechanisms and uncovering master genes for HS tolerance. Here, Z58D showed more thermotolerance than AF171 at the seedling stage with a lower wilted leaf rate and H2O2 accumulation under HS conditions. Transcriptomic analysis identified 3006 differentially expressed genes (DEGs) in AF171 and 4273 DEGs in Z58D under HS treatments, respectively. Subsequently, GO enrichment analysis showed that commonly upregulated genes in AF171 and Z58D were significantly enriched in the following biological processes, including protein folding, response to heat, response to temperature stimulus and response to hydrogen peroxide. Moreover, the comparison between the two inbred lines under HS showed that response to heat and response to temperature stimulus were significantly over-represented for the 1234 upregulated genes in Z58D. Furthermore, more commonly upregulated genes exhibited higher expression levels in Z58D than AF171. In addition, maize inbred CIMBL55 was verified to be more tolerant than B73, and more commonly upregulated genes also showed higher expression levels in CIMBL55 than B73 under HS. These consistent results indicate that heat-resistant inbred lines may coordinate the remarkable expression of genes in order to recover from HS. Additionally, 35 DEGs were conserved among five inbred lines via comparative transcriptomic analysis. Most of them were more pronounced in Z58D than AF171 at the expression levels. These candidate genes may confer thermotolerance in maize.

Adaptive tail-length evolution in deer mice is associated with differential Hoxd13 expression in early development

Variation in the size and number of axial segments underlies much of the diversity in animal body plans. Here we investigate the evolutionary, genetic and developmental mechanisms driving tail-length differences between forest and prairie ecotypes of deer mice (Peromyscus maniculatus). We first show that long-tailed forest mice perform better in an arboreal locomotion assay, consistent with tails being important for balance during climbing. We then identify six genomic regions that contribute to differences in tail length, three of which associate with caudal vertebra length and the other three with vertebra number. For all six loci, the forest allele increases tail length, indicative of the cumulative effect of natural selection. Two of the genomic regions associated with variation in vertebra number contain Hox gene clusters. Of those, we find an allele-specific decrease in Hoxd13 expression in the embryonic tail bud of long-tailed forest mice, consistent with its role in axial elongation. Additionally, we find that forest embryos have more presomitic mesoderm than prairie embryos and that this correlates with an increase in the number of neuromesodermal progenitors, which are modulated by Hox13 paralogues. Together, these results suggest a role for Hoxd13 in the development of natural variation in adaptive morphology on a microevolutionary timescale.

Evidence for the Involvement of Gene Regulation of Inflammatory Molecules in the Accumulation of Intracellular Cholesterol: The Mechanism of Foam Cell Formation in Atherosclerosis.

The relationship between the cellular pro-inflammatory response and intracellular lipid accumulation in atherosclerosis is not sufficiently studied. Transcriptomic analysis is one way to establish such a relationship. Previously, we identified 10 potential key genes (IL-15, CXCL8, PERK, IL-7, IL-7R, DUSP1, TIGIT, F2RL1, TSPYL2, and ANXA1) involved in cholesterol accumulation in macrophages. It should be noted that all these genes do not directly participate in cholesterol metabolism, but encode molecules related to inflammation. In this study, we conducted a knock-down of the 10 identified key genes using siRNA to determine their possible role in cholesterol accumulation in macrophages. To assess cholesterol accumulation, human monocyte-derived macrophages (MDM) were incubated with atherogenic LDL from patients with atherosclerosis. Cholesterol content was assessed by the enzymatic method. Differentially expressed genes were identified with DESeq2 analysis. Master genes were determined by the functional analysis. We found that only 5 out of 10 genes (IL-15, PERK, IL-7, IL-7R, ANXA1) can affect intracellular lipid accumulation. Knock-down of the IL-15, PERK, and ANXA1 genes prevented lipid accumulation, while knock-down of the IL-7 and IL-7R genes led to increased intracellular lipid accumulation during incubation of MDM with atherogenic LDL. Seventeen overexpressed genes and 189 underexpressed genes were obtained in the DGE analysis, which allowed us to discover 20 upregulated and 86 downregulated metabolic pathways, a number of which are associated with chronic inflammation and insulin signaling. We also elucidated 13 master regulators of cholesterol accumulation that are immune response-associated genes. Thus, it was discovered that 5 inflammation-related master regulators may be involved in lipid accumulation in macrophages. Therefore, the pro-inflammatory response of macrophages may trigger foam cell formation rather than the other way around, where intracellular lipid accumulation causes an inflammatory response, as previously assumed.

Embryonic spatio-temporal expression pattern of Folded gastrulation (Fog) suggests roles in multiple morphogenetic events and regulation by AbdA.

In Drosophila, the signaling pathway activated by the ligand Folded gastrulation (Fog) is amongst the few known G-protein coupled receptor (GPCR) pathways to regulate cell shape change with a well characterized role in gastrulation. However, an understanding of the spectrum of morphogenetic events regulated by Fog signaling is still lacking. Here, we present an analysis of the expression pattern and regulation of fog using a genome engineered Fog::sfGFP line. We show that Fog expression is widespread and in tissues previously not associated with the signaling pathway including germ cells, trachea and amnioserosa. In the CNS, we find that the ligand is expressed in multiple types of glia indicating a prominent role in the development of these cells. Consistent with this, we have identified three intronic enhancers whose expression in the CNS overlaps with Fog::sfGFP. Further, we show that enhancer-1, (fog intenh-1) located proximal to the coding exon is responsive to AbdA. Supporting this, we find that fog expression is downregulated in abdA mutants. Together, our study highlights the broad scope of Fog-GPCR signaling during embryogenesis and identifies Hox gene AbdA as a novel regulator of fog expression.

Polycomb repression of Hox genes involves spatial feedback but not domain compaction or phase transition.

Polycomb group proteins have a critical role in silencing transcription during development. It is commonly proposed that Polycomb-dependent changes in genome folding, which compact chromatin, contribute directly to repression by blocking the binding of activating complexes. Recently, it has also been argued that liquid-liquid demixing of Polycomb proteins facilitates this compaction and repression by phase-separating target genes into a membraneless compartment. To test these models, we used Optical Reconstruction of Chromatin Architecture to trace the Hoxa gene cluster, a canonical Polycomb target, in thousands of single cells. Across multiple cell types, we find that Polycomb-bound chromatin frequently explores decompact states and partial mixing with neighboring chromatin, while remaining uniformly repressed, challenging the repression-by-compaction or phase-separation models. Using polymer simulations, we show that these observed flexible ensembles can be explained by 'spatial feedback'-transient contacts that contribute to the propagation of the epigenetic state (epigenetic memory), without inducing a globular organization.

Phenotypic divergence between broiler and layer chicken lines is regulated at the molecular level during development

BackgroundUnderstanding the molecular underpinnings of phenotypic variations is critical for enhancing poultry breeding programs. The Brazilian broiler (TT) and laying hen (CC) lines exhibit striking differences in body weight, growth potential, and muscle mass. Our work aimed to compare the global transcriptome of wing and pectoral tissues during the early development (days 2.5 to 3.5) of these chicken lines, unveiling disparities in gene expression and regulation.ResultsDifferent and bona-fide transcriptomic profiles were identified for the compared lines. A similar number of up- and downregulated differentially expressed genes (DEGs) were identified, considering the broiler line as a reference. Upregulated DEGs displayed an enrichment of protease-encoding genes, whereas downregulated DEGs exhibited a prevalence of receptors and ligands. Gene Ontology analysis revealed that upregulated DEGs were mainly associated with hormone response, mitotic cell cycle, and different metabolic and biosynthetic processes. In contrast, downregulated DEGs were primarily linked to communication, signal transduction, cell differentiation, and nervous system development. Regulatory networks were constructed for the mitotic cell cycle and cell differentiation biological processes, as their contrasting roles may impact the development of distinct postnatal traits. Within the mitotic cell cycle network, key upregulated DEGs included CCND1 and HSP90, with central regulators being NF-κB subunits (RELA and REL) and NFATC2. The cell differentiation network comprises numerous DEGs encoding transcription factors (e.g., HOX genes), receptors, ligands, and histones, while the main regulatory hubs are CREB, AR and epigenetic modifiers. Clustering analyses highlighted PIK3CD as a central player within the differentiation network.ConclusionsOur study revealed distinct developmental transcriptomes between Brazilian broiler and layer lines. The gene expression profile of broiler embryos seems to favour increased cell proliferation and delayed differentiation, which may contribute to the subsequent enlargement of pectoral tissues during foetal and postnatal development. Our findings pave the way for future functional studies and improvement of targeted traits of economic interest in poultry.