Limb Development Research Articles

New insight into the development of synpolydactyly caused by expansion of HOXD13 polyalanine based on weighted gene co-expression network analysis

BackgroundSynpolydactyly (SPD) is mainly caused by mutations of polyalanine expansion (PAE) in the transcription factor gene HOXD13 and the involved cell types and signal pathway are still not clear possible pathways and single-cell expression characteristics of limb bud in HOXD13 PAE mice was analyzed in this study.MethodWe investigated a previous study of a mouse model with SPD and conducted weighted gene co-expression network analysis (WGCNA) using a single-cell RNA sequencing dataset from limb bud cells of SPD mouse model of HOXD13 + 7A heterozygote.ResultsAnalysis of WGCNA revealed that synpolydactyly-associated Hoxd13 PAEs alter the immune response and osteoclast differentiation, and enhance DNA replication. Bmp4, Hand2, Hoxd12, Lnp, Prrx1, Gmnn, and Cdc6 were found to play potentially key roles in synpolydactyly.ConclusionsThese findings evaluated the main genes related to SPD with PAE mutations in HOXD13 and advance our understanding of human limb development.

A novel genetic mouse model of osteoporosis with double heterozygosity of Irx3 and Irx5 characterizes sex-dependent phenotypes in bone homeostasis

Iroquois homeobox gene 3 (Irx3) and Irx5 encode transcription factors that play crucial roles in limb development and bone formation. Previous studies using knockout mice have revealed a role of Irx3 and Irx5 in osteogenesis in young adult mice. However, whether these genes are also essential for bone homeostasis in adulthood and contribute to bone diseases remain poorly understood. Osteoporosis is a disease characterized by lower bone mineral density and disrupted bone microarchitecture, typically occurs in postmenopausal women. Here, we demonstrate that Irx3/5dHet mice with a half-reduction of Irx3 and Irx5 dosage serve as a novel model of osteoporosis. By micro-computed tomography, we found that Irx3/5dHet mice exhibited sex-dependent bone loss patterns. While male Irx3/5dHet mice progressively lost trabecular microstructures with aging, female mutants exhibited lower bone mineral density (BMD) and bone volume fraction (BV/TV) at early adulthood (9–15 weeks old) but without further loss later at 1 year of age. Bone marrow adipocytes are known to be elevated at the expenses of lower osteogenesis in osteoporotic bone marrow. Surprisingly, we found sex-dependent changes in adipogenesis at the age of skeletal maturity that bone marrow adipocytes were reduced in female Irx3/5dHet mice along with deteriorated osteogenesis, while male mice exhibited elevated adipogenesis. In summary, we reported a novel genetic model for osteoporosis-like phenotypes, highlighting sex-dependent bone mineral density and bone marrow adipocyte characteristics.

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

Approximately a quarter of the human genome consists of gene deserts, large regions devoid of genes often located adjacent to developmental genes and thought to contribute to their regulation. However, defining the regulatory functions embedded within these deserts is challenging due to their large size. Here, we explore the cis-regulatory architecture of a gene desert flanking the Shox2 gene, which encodes a transcription factor indispensable for proximal limb, craniofacial, and cardiac pacemaker development. We identify the gene desert as a regulatory hub containing more than 15 distinct enhancers recapitulating anatomical subdomains of Shox2 expression. Ablation of the gene desert leads to embryonic lethality due to Shox2 depletion in the cardiac sinus venosus, caused in part by the loss of a specific distal enhancer. The gene desert is also required for stylopod morphogenesis, mediated via distributed proximal limb enhancers. In summary, our study establishes a multi-layered role of the Shox2 gene desert in orchestrating pleiotropic developmental expression through modular arrangement and coordinated dynamics of tissue-specific enhancers.

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.

8519 Investigating the Role of Protein Inhibitor of Activated STAT1 (Pias1) in Growth Plate Chondrocyte Maturation

Abstract Disclosure: J. Baronas: None. U. Ahmed: None. J.N. Hirschhorn: None. N.E. Renthal: None. Development of growth plate chondrocytes is a complex process that involves a wide array of regulatory elements. Recently our laboratory conducted a genome wide CRISPR knockout (KO) screen of growth plate chondrocyte maturation to discover novel genes and gene networks in the proliferation and maturation of chondrocytes. Among targets uncovered is Protein Inhibitor of Activated STAT 1 (Pias1), an E3 SUMO ligase that plays a multi-faceted role in genetic regulation, driving post-translational modifications, directly binding to transcription factors, and functioning as a DNA-binding protein. While Pias1 has been shown to be involved in the regulation of many cellular processes across multiple tissues, its function in growth plate development and chondrocyte maturation has been previously unstudied. In our genome-wide screen, our laboratory identified that loss of Pias1 led to early maturation of chondrocytes. This project seeks to investigate the role of Pias1 in chondrocyte maturation and differentiation. We first developed a Pias1-KO chondrocyte cell line, observing upregulation of hypertrophic markers Cd200 and Col10a1 in KOs relative to control via qPCR, in addition to proliferative regulators IHH and Pth1R. Through Bulk-RNAseq of the same cell line, we identified additional chondrocyte markers as upregulated in KO, as well as osteoblast marker Sp7. Members of the HoxD family, regulators of limb development, were downregulated. Cell culture data suggests that loss of Pias1 leads to increased cell proliferation and increased matrix production, indicating dysregulation of multiple chondrocyte processes. Immunohistochemistry of micromass culture sections also suggests morphological differences between KO and control, with KO cells significantly larger in diameter. Early SUMO-1 profiling yields limited evidence of decreased global SUMOylation in KO cells, one possible mechanistic explanation for the observed dysregulation. Ongoing studies are focused on identifying specific SUMOylation targets of Pias1 in chondrocytes, analyzing proteomic differences in KO and control, seeking putative DNA-binding sites of Pias1, and observing in vivo murine Pias1-KO growth plates. Our findings suggest that PIAS1 plays a crucial role in the growth plate by prolonging growth and delaying terminal hypertrophy. Our study aims to provide a better understanding of the regulatory pathways involved in growth plate development and related phenotypes such as height, and contribute to the development of novel therapies for growth-related disorders, such as achondroplasia and hypochondroplasia. Presentation: 6/1/2024

Acromesomelic Dysplasia With Homozygosity for a Likely Pathogenic BMPR1B Variant: Postaxial Polydactyly as a Novel Clinical Finding.

Acromesomelic chondrodysplasias are a rare subgroup of the clinically and genetically heterogeneous osteochondrodysplasias that are characterised by abnormalities in the limb development and short stature. Here, we report a 2-year-old boy, offspring of consanguineous parents, with acromesomelic dysplasia and postaxial polydactyly in which exome sequencing identified a novel homozygous missense variant in BMPR1B. The patient showed skeletal malformation of both hands and feet that included complex brachydactyly with the thumbs most severely affected, postaxial polydactyly of both hands, shortened toes as well as a bilateral hypoplasia of the fibula. Whole trio exome sequencing was conducted to identify potential genetic variants in the patient. The analysis identified the biallelic variant NM_001203.3:c.821A > G;p.(Gln274Arg) in BMPR1B, a gene encoding bone morphogenetic protein receptor 1B. The skeletal phenotype can be brought in line with the phenotypes of previously reported cases of BMPR1B-associated chondrodysplasias. However, the postaxial polydactyly described here is a novel clinical finding in a BMPR1B-related case; notably, it has previously been reported in other acromesomelic dysplasia cases caused by homozygous pathogenic variants in GDF5-a gene which encodes for growth differentiation factor 5, a high-affinity ligand to BMPR1B.

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.

An inherited TBX3 alteration in a prenatal case of ulnar-mammary syndrome: Clinical assessment and functional characterization in Drosophila melanogaster.

Ulnar mammary syndrome (UMS) results from heterozygous variants in the TBX3 gene and impacts limb, tooth, hair, apocrine gland, and genitalia development. The expressivity of UMS is highly variable with no established genotype-phenotype correlations. TBX3 belongs to the Tbx gene family, which encodes transcription factors characterized by the presence of a T-box DNA-binding domain. We describe a fetus exhibiting severe upper limb defects and harboring the novel TBX3:c.400 C > T (p.P134S) variant inherited from the mother who remained clinically misdiagnosed until prenatal diagnosis. Literature revision was conducted to uncover the TBX3 clinical and mutational spectrum. Moreover, we generated a Drosophila humanized model for TBX3 to study the developmental consequences of the p.P134S as well as of other variants targeting different regions of the protein. Phenotypic analysis in flies, coupled with in silico modeling on the TBX3 variants, suggested that the c.400 C > T is UMS-causing and impacts TBX3 localization. Comparative analyses of the fly phenotypes caused by the expression of all variants, demonstrated that missense changes in the T-box domain affect more significantly TBX3 activity than variants outside this domain. To improve the clinicians' recognition of UMS, we estimated the frequency of the main clinical features of the disease. Core features often present pre-pubertally include defects of the ulna and/or of ulnar ray, hypoplastic nipples and/or areolas and, less frequently, genitalia anomalies in young males. These results enhance our understanding of the molecular basis and the clinical spectrum of UMS, shedding light on the functional consequences of TBX3 variants in a developmental context.

Elavl1 is dispensable for appendicular skeletal development.

Elavl1/HuR is a RNA binding protein implicated in multiple developmental processes with pleiotropic roles in RNA life cycle. Loss of Elavl1 is incompatible with life with early embryonic loss of Elavl1 in epiblast cells being lethal with defects in placental branching and embryonic tissue growth. Postnatal global deletion of Elavl1/HuR results in lethality with atrophy in multiple tissues mainly due to loss of progenitor cells. However, roles of Elavl1 specifically during embryonic limb development is not well understood. Here we report that deletion of Elavl1 in limb bud mesenchyme in mouse did not reveal any abnormalities during embryonic development with normal development in pre- and postnatal limb skeleton. Analyses of skeletal patterning, morphogenesis and skeletal maturation including skeletal elements in stylopod, zeugopod and autopod during development did not reveal any significant differences between long bones from control and Elavl1 conditional knockout animals. Our study indicates differential dependency and susceptibility to loss of Elavl1 in different stem cell lineages with its functions being dispensable during limb development.

Utilizing Additive Manufacturing for Economical Prosthetic Limb Prototyping: A Guide from Regression Modelling

This study investigated the development of a low-cost prosthetic limb using additive manufacturing techniques, guided by anthropometric data and regression modeling. Anthropometric measurements were collected from 250 healthy young adults to characterize limb dimensions. Statistical analysis revealed the mean, standard deviation, and range for right and left leg and elbow lengths. A regression model was formulated to estimate limb lengths based on these measurements. A robust regression model demonstrated a significant correlation (r = 0.993, r² = 0.986) between right limb measurements and corresponding left limb measurements, indicating that right leg and elbow lengths could accurately predict left side measurements. The derived regression equation (RLL = 2.253 + 0.959 LLL + 0.054 LEL) provides a practical tool for designing customized prosthetics. Utilizing 3D printing technology, these findings can enhance the production of tailored prosthetic limbs, improving comfort and functionality for users. The study recommends further validation across diverse populations, integration into clinical practices, and continued research and development supported by policy and funding initiatives. These steps will advance the accessibility and effectiveness of personalized prosthetic solutions.

Investigation of the potential teratogenic effects of fructose on the embryo using the rat whole embryo culture model

Excessive consumption of fructose-sweetened foods and beverages is a growing concern worldwide. Studies have demonstrated that fructose consumption before and during pregnancy can result in adverse outcomes such as decreased decidualization, increased fetal losses, and low birth weight. The study investigated the teratogenic effects of fructose on rat embryos during organogenesis using whole embryo culture. Within the scope of the study, 4 groups were formed as control, low, medium, and high-dose fructose (HDF) with 10 embryos in each group. The 9.5-day-old rat embryos were cultured with different concentrations of fructose (1, 5 and 10 mM) for 48 h and the possible effects of fructose were examined using morphological scoring, histochemistry, immunofluorescence, and TUNEL methods. According to the analyses, protein synthesis and proliferation were decreased, vascular formation was suppressed, and apoptosis was increased in embryos exposed to fructose, especially at concentrations of 5 mM and above. According to the morphological scoring results, it was determined that heart, hind limb, and somite development were retarded in all experimental groups compared to the control group, developmental retardation increased in direct proportion to fructose concentration, and also significant malformations were observed in all parameters examined in the HDF group. In addition, analysis of yolk sac diameter, head length, crown rump length and somite numbers showed that these parameters were significantly decreased in all experimental groups. End of the study, it was concluded that fructose at concentrations of 1 mM and above may induce embryonic development retardation and other anomalies by decreasing protein synthesis and cell proliferation, suppressing vascular formation, and increasing apoptosis in embryonic tissues.

Cloning of down-regulated genes under cold stress and identification of important genes related to cold tolerance in zebrafish (Danio rerio)

Low-temperature stress poses a significant risk to the survival of both cultivated and wild fish populations. Existing studies have found that the pre-acclimation of fishes to moderate cold stress can stimulate the activation of acclimation pathways, thereby enhancing their tolerance to cold stress. The fitness of fish relies heavily on appropriately controlled transcriptional reactions to environmental changes. Despite previous characterization of gene expression profiles in various fish species during cold acclimation, the specific genes responsible for essential functions in this process remain largely unknown, particularly the down-regulated genes induced by cold acclimation. To investigate the genes involved in cold acclimation, this study employed real-time quantitative PCR (RT-qPCR), molecular cloning, microinjection techniques, and cold stress experiments to determine the genes that play an essential part in cold acclimation. Consequently, 18 genes were discovered to be down-regulated in larval zebrafish experiencing cold stress. All 18 genes successfully detected overexpression in zebrafish at 96 and 126 hpf (fold change ≥3), which declined with the growth of zebrafish. Following microinjection, it was observed that her8a, cyp51, lss, txnipb, and bhlha9 had an adverse impact on the survival rate of zebrafish larvae under cold stress. These genes have been identified to play significant roles in various biological processes. For instance, bhlha9 has been found to be involved in both limb development and temperature sensing and her8a has been implicated in neural development. Additionally, cyp51 and lss have been identified as participants in the cholesterol synthesis pathway. Txnipb has been reported to induce cell apoptosis, thereby potentially influencing the survival rate of zebrafish larvae under cold stress. These findings offered crucial data for the analysis of molecular processes related to cold tolerance and the development of cold-resistant fish breeding.

Spatial Transcriptomics Analysis: Maternal Obesity Impairs Myogenic Cell Migration and Differentiation during Embryonic Limb Development.

Limb muscle is responsible for physical activities and myogenic cell migration during embryogenesis is indispensable for limb muscle formation. Maternal obesity (MO) impairs prenatal skeletal muscle development, but the effects of MO on myogenic cell migration remain to be examined. C57BL/6 mice embryos were collected at E13.5. The GeoMx DSP platform was used to customize five regions along myogenic cell migration routes (myotome, dorsal/ventral limb, limb stroma, limb tip), and data were analyzed by GeomxTools 3.6.0. A total of 2224 genes were down-regulated in the MO group. The GO enrichment analysis showed that MO inhibited migration-related biological processes. The signaling pathways guiding myogenic migration such as hepatocyte growth factor signaling, fibroblast growth factor signaling, Wnt signaling and GTPase signaling were down-regulated in the MO E13.5 limb tip. Correspondingly, the expression levels of genes involved in myogenic cell migration, such as Pax3, Gab1, Pxn, Tln2 and Arpc, were decreased in the MO group, especially in the dorsal and ventral sides of the limb. Additionally, myogenic differentiation-related genes were down-regulated in the MO limb. MO impedes myogenic cell migration and differentiation in the embryonic limb, providing an explanation for the impairment of fetal muscle development and offspring muscle function due to MO.

Splicing is dynamically regulated during limb development

Modifications to highly conserved developmental gene regulatory networks are thought to underlie morphological diversification in evolution and contribute to human congenital malformations. Relationships between gene expression and morphology have been extensively investigated in the limb, where most of the evidence for alterations to gene regulation in development consists of pre-transcriptional mechanisms that affect expression levels, such as epigenetic alterations to regulatory sequences and changes to cis-regulatory elements. Here we report evidence that alternative splicing (AS), a post-transcriptional process that modifies and diversifies mRNA transcripts, is dynamic during limb development in two mammalian species. We evaluated AS patterns in mouse (Mus musculus) and opossum (Monodelphis domestica) across the three key limb developmental stages: the ridge, bud, and paddle. Our data show that splicing patterns are dynamic over developmental time and suggest differences between the two mammalian taxa. Additionally, multiple key limb development genes, including Fgf8, are differentially spliced across the three stages in both species, with expression levels of the conserved splice variants, Fgf8a and Fgf8b, changing across developmental time. Our data demonstrates that AS is a critical mediator of mRNA diversity in limb development and provides an additional mechanism for evolutionary tweaking of gene dosage.

Effects of Conditioning Contractions on Lower-Body Explosive Force Post-Static Stretching.

The present study assessed the impacts of two distinct protocols, static stretching (StS, 4 sets of 30 seconds) and static stretching combined with conditioning contractions (10 repetitive drop jumps) (SC), on neuromuscular response and rate of force development (RFD) in the lower limbs during squat jumps (SJs) at varying initial knee-joint angles (60°,90°,120°). Twelve participants completed three randomized experimental trials (no intervention, StS intervention, and SC intervention). Except for the intervention segments, each trial included standardized warm-ups and SJs at three different angles. Data were collected using a 3-dimensional injury motion capture system, an electromyography (EMG) recording system, and a force platform. The collected EMG data were subjected to amplitude calculations, while force-time data were used for RFD computation. Neither StS nor SC significantly impacted the average or peak EMG amplitudes of the five muscles examined (p>0.05). However, at an initial knee-joint angle of 120°, the StS group demonstrated significantly lower RFD values at three distinct phases (0-50 ms, 50-100 ms, and 0-peakforce) compared to those seen in the SC and control groups (p<0.05). For activities starting with a knee-joint angle of 120°, it is recommended to either avoid StS or combine it with ten repetitive drop jumps to mitigate any potential negative impact on explosiveness.

Heterogeneity of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells in mice.

This study aimed to explore the heterogeneity and gene ontology of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells (CNCs) in mice. The embryos of Wnt1-Cre;R26RmTmG and Pax2-Cre;R26RmTmG at embryonic day (E)8.0-E9.25 were collected for histological observation. We performed immunostaining to compare green fluorescent protein (GFP)-positive CNCs in Pax2-Cre;R26RAi9 and Wnt1-Cre;R26RAi9 mice at E15.5. Single-cell RNA sequencing (scRNA-seq) was used to analyze the first branchial arch GFP-positive CNCs from Wnt1-Cre;R26RmTmG and Pax2-cre;R26RmTmGmice at E10.5. Real time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to validate the differential genes. Wnt1-Cre-marked and Pax2-Cre-marked CNCs migrated from the neural plateto first and second branchial arches and to the first branchial arch, respectively, at E8.0. Although Wnt1-Cre-marked and Pax2-Cre-marked CNCs were found mostly in cranial-facial tissues, the former had higher expression in palate and tongue. The results of scRNA-seq showed that Pax2-Cre-marked CNCs specifically contributed to osteoblast differentiation and ossification, while Wnt1-Cre-marked CNCs participated in limb development, cell migration, and ossification. The q-PCR data also confirmed the results of gene ontology analysis. Pax2-Cre mice are perfect experimental animal models for research on first branchial arch CNCs and derivatives in osteoblast differentiation and ossification.

Sall4 regulates downstream patterning genes during limb regeneration

Many salamanders can completely regenerate a fully functional limb. Limb regeneration is a carefully coordinated process involving several defined stages. One key event during the regeneration process is the patterning of the blastema to inform cells of what they must differentiate into. Although it is known that many genes involved in the initial development of the limb are re-used during regeneration, the exact molecular circuitry involved in this process is not fully understood. Several large-scale transcriptional profiling studies of axolotl limb regeneration have identified many transcription factors that are up-regulated after limb amputation. Sall4 is a transcription factor that has been identified to play essential roles in maintaining cells in an undifferentiated state during development and also plays a unique role in limb development. Inactivation of Sall4 during limb bud development results in defects in anterior-posterior patterning of the limb. Sall4 has been found to be up-regulated during limb regeneration in both Xenopus and salamanders, but to date it function has been untested. We confirmed that Sall4 is up-regulated during limb regeneration in the axolotl using qRT-PCR and identified that it is present in the skin cells and also in cells within the blastema. Using CRISPR technology we microinjected gRNAs specific for Sall4 complexed with cas9 protein into the blastema to specifically knockout Sall4 in blastema cells only. This resulted in limb regenerate defects, including missing digits, fusion of digit elements, and defects in the radius and ulna. This suggests that during regeneration Sall4 may play a similar role in regulating the specification of anterior-proximal skeletal elements.

Positive regulation of Hedgehog signaling via phosphorylation of GLI2/GLI3 by DYRK2 kinase

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

Genomic regions underlying variation in wattles, horns, and supernumerary teats phenotypes in Egyptian goats

Goats play a crucial role in providing humans with various types of valuable products including milk and meat. The underlying genetic mechanisms of important morphological aspects remain largely unknown in goats, highlighting the need for further investigation. A genome-wide association analysis (GWAS) was conducted for three morphological phenotypes in Egyptian goats. All animals were genotyped using the Illumina 65 K SNP BeadChips. Results of GWAS for wattles identified two significant ( P ≤ 1.4 × 10–6, false discovery rate (FDR) ≤ 0.05) single nucleotide polymorphisms (SNPs) on chromosome 10 within a region (72–74 Mb) containing FMN1 and GREM1 genes that are important for limb development and growth. For horns, three significant SNPs were identified on chromosome 1 (119–131 Mb) harbouring candidate genes for embryonic development and tissue differentiation, such as CEP70, DZIP1L, CLDN18, SOX14, and SLC35G2. For supernumerary teats, four significant SNPs located on chromosomes 25 (8.7 Mb), 9 (47.8 Mb), 17 (45.1 Mb), and 28 (6.7 Mb) were identified, harbouring candidate genes involved in morphogenesis and reproductive traits such as EMP2, MDN1, PCDH10, and GHITM. This study identified novel candidate genes alongside previously reported ones in other goat breeds, suggesting their potential as candidate genes for the studied traits in Egyptian goats.

Regulation mechanism of endochondral ossification in Rana zhenhaiensis during metamorphosis based on histomorphology and transcriptome analyses

Endochondral ossification plays a crucial role in the limb development of amphibians. This study explored the ossification sequence in the hindlimb of Rana zhenhaiensis tadpoles and the correlation between thyroid hormones (THs) and endochondral ossification via histomorphology and transcriptional analyses. Our results suggest that ossification of the femur and tibiofibula was initiated during the period of high THs activity (metamorphosis climax). In addition, the results of differentially expressed gene analyses in the hindlimb and tail showed that systemic factors, transcription factors, and locally secreted factors interacted with each other during the metamorphosis climax to regulate the occurrence of endochondral ossification. These results will enrich the morphological data of anurans and provide scientific reference for the evolutionary history of vertebrates.