AGIDB: a versatile database for genotype imputation and variant decoding across species.

The high cost of large-scale, high-coverage whole-genome sequencing has limited its application in genomics and genetics research. The common approach has been to impute whole-genome sequence variants obtained from a few individuals for a larger population of interest individually genotyped using SNP chip. An alternative involves low-coverage whole-genome sequencing (lcWGS) of all individuals in the larger population, followed by imputation to sequence resolution. To overcome limitations of processing lcWGS data and meeting specific genotype imputation requirements, we developed AGIDB (https://agidb.pro), a website comprising tools and database with an unprecedented sample size and comprehensive variant decoding for animals. AGIDB integrates whole-genome sequencing and chip data from 17 360 and 174 945 individuals, respectively, across 89 species to identify over one billion variants, totaling a massive 688.57 TB of processed data. AGIDB focuses on integrating multiple genotype imputation scenarios. It also provides user-friendly searching and data analysis modules that enable comprehensive annotation of genetic variants for specific populations. To meet a wide range of research requirements, AGIDB offers downloadable reference panels for each species in addition to its extensive dataset, variant decoding and utility tools. We hope that AGIDB will become a key foundational resource in genetics and breeding, providing robust support to researchers.

Potential therapeutic effects of milk-derived exosomes on intestinal diseases.

Exosomes are extracellular vesicles with the diameter of 30 ~ 150 nm, and are widely involved in intercellular communication, disease diagnosis and drug delivery carriers for targeted disease therapy. Therapeutic application of exosomes as drug carriers is limited due to the lack of sources and methods for obtaining adequate exosomes. Milk contains abundant exosomes, several studies have shown that milk-derived exosomes play crucial roles in preventing and treating intestinal diseases. In this review, we summarized the biogenesis, secretion and structure, current novel methods used for the extraction and identification of exosomes, as well as discussed the role of milk-derived exosomes in treating intestinal diseases, such as inflammatory bowel disease, necrotizing enterocolitis, colorectal cancer, and intestinal ischemia and reperfusion injury by regulating intestinal immune homeostasis, restoring gut microbiota composition and improving intestinal structure and integrity, alleviating conditions such as oxidative stress, cell apoptosis and inflammation, and reducing mitochondrial reactive oxygen species (ROS) and lysosome accumulation in both humans and animals. In addition, we discussed future prospects for the standardization of milk exosome production platform to obtain higher concentration and purity, and complete exosomes derived from milk. Several in vivo clinical studies are needed to establish milk-derived exosomes as an effective and efficient drug delivery system, and promote its application in the treatment of various diseases in both humans and animals.

The genomic characteristics affect phenotypic diversity from the perspective of genetic improvement of economic traits.

The genetic improvement of economic traits suggests that chicken is an excellent model for exploring the genetic changes and molecular mechanisms underlying phenotypic diversity and artificial selection. Here, the sequencing data including 477 samples from 25 breeds worldwide were used to reveal the genomic patterns of chicken domestication. We analyzed 7.4 Tb clean data with 14.8× per individual to identify 23,504,766 SNPs, 3,289,782 InDels, and 27,027 SVs. The diversity analysis indicates that high-intensity artificial selection would accelerate population differentiation. We also found that the human-driven traits are controlled by polygenes and major genes, such as the primary candidates SOX5 and IGF1 for body size, and NEDD4 for sperm storage capacity. Our findings provide an important reference for understanding how genomic patterns shape phenotypes in livestock.

Revealing the impacts on shaping scutate scales in goose skin.

Feather follicles and scales are two types of skin appendages distributed on different parts of avian skin. The morphogenesis and development of scales in waterfowl remain largely unknown. Here, we used H&E staining, ISH and RNA sequencing to reveal the morphological and molecular variations at the early development of scutate scales in goose shank skin. Transcriptome analysis produced 1824 differentially expressed genes (DEGs) regulating the induction of scales and further enriched gene function in cell adhesion and Wnt signaling pathway, etc. A total of 8 candidate genes (ALDOC, CSRP2, KRT15, KRT75, LGALS1, S100A6, OGN and SFRP2) were further detected by RT-qPCR to show upregulated (6 genes) and downregulated (2 genes) from pre-placodal to placode stage during the induction of goose scales. The localization of 7 candidate genes (ALDOC, CSRP2, CD109, KRT15, KRT75, S100A6, and OGN) by ISH suggests the potential roles for dermal and epidermal development during the induction of scutate scales. The dynamic molecular changes and specific gene expression patterns revealed in this report provide general knowledge of scale development in waterfowl as well as skin appendage diversity.

HOXC10 intronic duplication is associated with unsealed skull and crest in crested chicken with cerebral hernia.

The genetic basis and developmental mechanism of unsealed skull in crested chicken with cerebral hernia remain unclear. Here, a genomic region including six HOXC genes was mapped by bulked segregant analysis (BSA) in a crested chicken resource population. A 195-bp intronic tandem duplication was further confirmed in the HOXC10 gene. HOXC genes, particularly HOXC10, were expressed ectopically in fetal skin and meningeal tissues of crested chicken with cerebral hernia, indicating its impact on the cranial mesenchymal tissues that drive the development of scalp skin, frontal bone, and meninges. The restricted expansion of frontal bone progenitors labeled with anti-RUNX2 antibody in the supraorbital mesenchyme of the fetal head implied abnormal migration, which contributed to the formation of the unsealed skull. This study suggests that HOXC genes were potent drivers for the abnormalities of the head crest and unsealed skull observed in crested chicken with cerebral hernia.

Elevated EDAR signalling promotes mammary gland tumourigenesis with squamous metaplasia.

Ectodysplasin A receptor (EDAR) is a death receptor in the Tumour Necrosis Factor Receptor (TNFR) superfamily with roles in the development of hair follicles, teeth and cutaneous glands. Here we report that human Oestrogen Receptor (ER) negative breast carcinomas which display squamous differentiation express EDAR strongly. Using a mouse model with a high Edar copy number, we show that elevated EDAR signalling results in a high incidence of mammary tumours in breeding female mice. These tumours resemble the EDAR-high human tumours in that they are characterised by a lack of oestrogen receptor expression, contain extensive squamous metaplasia, and display strong ß-catenin transcriptional activity. In the mouse model, all of the tumours carry somatic deletions of the third exon of the CTNNB1 gene that encodes ß-catenin. Deletion of this exon yields unconstrained ß-catenin signalling activity. We also demonstrate that ß-catenin activity is required for transformed cell growth, showing that increased EDAR signalling creates an environment in which ß-catenin activity can readily promote tumourigenesis. Together, this work identifies a novel death receptor oncogene in breast cancer, whose mechanism of transformation is based on the interaction between the WNT and Ectodysplasin A (EDA) pathways.

Transcriptomic and metabolomic insights into the variety of sperm storage in oviduct of egg layers.

In birds, the sperm storage tubules (SST) are dispersed in uterovaginal junction (UVJ) and highly correlated with differential capacity of sperm storage (SS) in and among species with unspecified mechanisms. Here, the SS duration of 252 egg layer breeders was evaluated in 5 rounds with 3 phenotypic traits to screen high- and low-SS individuals, respectively, followed with transcriptome of UVJ tissues and metabolome of serum (high-SS vs. low-SS) to decipher the candidate genes and biochemical markers correlated with differential SS capacity. Histological characterization suggested slightly higher density of SST in UVJ (high-SS vs. low-SS). Transcriptome analyses identified 596 differentially expressed genes (336 upregulated vs. 260 downregulated), which were mainly enriched in gene ontology terms of homeostasis, steroid and lipid metabolism and hormone activity, and 12 significant pathways (P < 0.05) represented by calcium, steroid, and lipid metabolism. Immunohistochemical staining of GNAQ, ST6GAL1, ADFP, and PCNA showed similar distribution in UVJ tissues between 2 groups. Several candidates (HSD11B2, DIO2, AQP3, GNAQ, NANS, ST6GAL1) combined with 4 (11ß-prostaglandin F2α, prostaglandin B1, 7α-hydroxytestosterone, and N-acetylneuraminic acid) of 40 differential metabolites enriched in serum metabolome were considered as regulators and biomarkers of SS duration in egg layer breeders. The integrated transcriptome and metabolome analyses of chicken breeder hens will provide novel insights for exploration and improvement of differential SS capacity in birds.

Transcriptome analysis and identification of age-associated fertility decreased genes in hen uterovaginal junction.

Avian sperm storage tubules (SSTs), which are located in the uterovaginal junction (UVJ) of the oviduct, are primary sperm storage sites after mating or artificial insemination. The mechanism underlying reduced sperm storage efficiency of SSTs which is highly correlated with decreased fertility rates in aged laying breeders remains largely unclear. Here, comparative transcriptomic analysis between the aged and young White Leghorn hens (120 vs. 30 wk) was applied to identify gene expression changes of UVJs containing SSTs. Bioinformatics analysis revealed 567 upregulated and 1998 downregulated differentially expressed genes. Gene ontology analysis was highly enriched in terms of immune system, cell adhesion, and cytoskeleton proteins. Kyoto Encyclopedia of Genes and Genomes analysis revealed 5 significant (P < 0.05) pathways including inositol phosphate and glycerophospholipid metabolism. ß-Galactosidase staining of chicken UVJ sections suggested increased cell senescence via aging. Oil Red O staining and immunohistochemistry detection of ADFP both confirmed distribution of lipid droplets in SST cells with increased intensity in aged breeders. The lipid synthesis and metabolism-related genes represented by TFAP2 and PLD1 were differentially expressed in aged laying breeders. The upregulation of IL15 and downregulation of a large number of immune-related genes in aged breeders indicate altered immune homeostasis in UVJs and SSTs. The increased accumulation of lipids, and altered immunity homeostasis, combined with other factors (TJP1, MYL9, AFDN, and RPL13, etc.) are potentially dominant effectors to decrease the sperm storage efficiency and egg fertility in aged laying breeders.

Deciphering the Forebrain Disorder in a Chicken Model of Cerebral Hernia.

Cerebral hernia in crested chicken has been characterized as the protrusion of cerebral hemispheres into the unsealed skull for hundreds of years, since Charles Darwin. The development of deformed forebrain (telencephalon) of cerebral hernia remains largely unknown. Here, the unsealed frontal skull combined with misplaced sphenoid bone was observed and potentially associated with brain protuberance. The shifted pallidum, elongated hippocampus, expanded mesopallium and nidopallium, and reduced hyperpallium were observed in seven regions of the malformed telencephalon. The neurons were detected with nuclear pyknosis and decreased density. Astrocytes showed uneven distribution and disordered protuberances in hyperpallium and hippocampus. Transcriptome analyses of chicken telencephalon (cerebral hernia vs. control) revealed 547 differentially expressed genes (DEGs), mainly related to nervous system development, and immune system processes, including astrocyte marker gene GFAP, and neuron and astrocyte developmental gene S100A6. The upregulation of GFAP and S100A6 genes in abnormal telencephalon was correlated with reduced DNA methylation levels in the promoter regions. The morphological, cellular, and molecular variations in the shape, regional specification, and cellular states of malformed telencephalon potentially participate in brain plasticity and previously reported behavior changes. Chickens with cerebral hernia might be an interesting and valuable disease model to further explore the recognition, diagnosis, and therapy of cerebral hernia development of crested chickens and other species.

Comparative investigation of coarse and fine wool sheep skin indicates the early regulators for skin and wool diversity.

The hair follicle is an excellent mini-system illustrating the mechanisms governing organogenesis and regeneration. Although the general mechanisms modulating skin and hair follicle development are widely studied in mouse and chicken models, the delicate network regulating skin and hair diversity remains largely unclear. Sheep is an additional model to address the various wool characteristics observed in nature. The coarse and fine wool sheep with diverse fibers were examined to show differences in the primary wool follicle size and skin thickness. The molecular dynamics in skin staged at the primary wool follicle induction between two sheep lines were investigated by RNA-sequencing analyses to generate 1994 differentially expressed genes revealing marker genes for epithelium (6 genes), dermal condensate (38 genes) and dermal fibroblast (58 genes) highly correlated with skin and wool follicle morphological differences. The DEGs were enriched in GO terms represented by epithelial cell migration and differentiation, regulation of hair follicle development and ectodermal placode formation, and KEGG pathways typified by WNT and Hedgehog signaling pathways governing the differences of skin structure. The qPCR detection of 9 genes confirmed the similar expression tendency with RNA-sequencing profiles. This comparative study of coarse and fine wool sheep skin reveals the presence of skin and wool follicle differences at primary wool follicle induction stage, and indicates the potential effectors (APCDD1, FGF20, DKK1, IGFBP3 and SFRP4) regulating the skin compartments during the early morphogenesis of primary wool follicles to shape the variable wool fiber thickness in later developmental stages.

Transcriptome analysis and identification of genes associated with chicken sperm storage duration.

The sperm storage tubules located in the mucosal folds of the uterovaginal junction (UVJ) are the primary site of sperm storage in chicken hens after natural mating or artificial insemination (AI). The short-term sperm storage (24 h after mating or AI) in hens was highly associated with immunity and pH-related pathway genes. However, the underlying mechanism of longer duration of sperm storage in female birds remains largely unclear. In the present study, transcriptome analysis was applied to uncover the dynamic gene expression changes in chicken UVJ tissues at two time points (day 3 and day 9) after AI. A total of 574 differentially expressed genes (DEG) were enriched, including 266 upregulated and 308 downregulated DEG. The validation of 5 DEG using quantitative PCR showed a similar expression tendency with RNA sequencing results. The gene ontology terms of DEG were highly enriched in heparin binding (9 genes including COMP, CTGF, and IMPG2), glycosaminoglycan binding (10 genes including PCOLCE, POSTN, and RSPO3), and response to estradiol and ion transport (AREG, RAMP3, SFRP1, and SSTR1). Kyoto encyclopedia of genes and genomes pathway-enrichment analyses of DEG revealed 10 significant pathways (P < 0.05) represented by calcium signaling pathway (7 genes including CACNA1G, PDE1C, PDGFRB, and SLC8A1) and glycosaminoglycan biosynthesis (B3GNT7, CSGALNACT1, GLCE, and ST3GAL1). Protein-protein interaction network of DEG established the connection-regulating epithelial cell or cell-matrix adhesion and migration. The enriched pathways and genes were highly correlated with temporary sperm storage in and possibly sequential sperm release from chicken UVJ overtime after AI. Of these, HIP1, PDE1C, and calcium-related genes were the most interesting candidates associated with sperm storage duration. This report provided a global gene expression profile of the chicken UVJ regarding the capacity of sperm storage overtime after AI. The outcome of this study will contribute to further understanding of the long-term sperm maintenance in avian females and eventually improving the duration of fertile egg performance by selected chicken breeding.

Exploration of key regulators driving primary feather follicle induction in goose skin.

The primary feather follicles are universal skin appendages widely distributed in the skin of feathered birds. The morphogenesis and development of the primary feather follicles in goose skin remain largely unknown. Here, the induction of primary feather follicles in goose embryonic skin (pre-induction vs induction) was investigated by de novo transcriptome analyses to reveal 409 differentially expressed genes (DEGs). The DEGs were characterized to potentially regulate the de novo formation of feather follicle primordia consisting of placode (4 genes) and dermal condensate (12 genes), and the thickening of epidermis (5 genes) and dermal fibroblasts (17 genes), respectively. Further analyses enriched DEGs into GO terms represented as cell adhesion and KEGG pathways including Wnt and Hedgehog signaling pathways that are highly correlated with cell communication and molecular regulation. Six selected Wnt pathway genes were detected by qPCR with up-regulation in goose skin during the induction of primary feather follicles. The localization of WNT16, SFRP1 and FRZB by in situ hybridization showed weak expression in the primary feather primordia, whereas FZD1, LEF1 and DKK1 were expressed initially in the inter-follicular skin and feather follicle primordia, then mainly restricted in the feather primordia. The spatial-temporal expression patterns indicate that Wnt pathway genes DKK1, FZD1 and LEF1 are the important regulators functioned in the induction of primary feather follicle in goose skin. The dynamic molecular changes and specific gene expression patterns revealed in this report provide the general knowledge of primary feather follicle and skin development in waterfowl, and contribute to further understand the diversity of hair and feather development beyond the mouse and chicken models.

Expression patterns of three JAK-STAT pathway genes in feather follicle development during chicken embryogenesis.

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) (JAK-STAT) pathway is shown to restrain the hair follicles in catagen and telogen and prevent anagen reentry in murine hair follicle cycling. The early roles of JAK-STAT pathway genes in skin development remain uncharacterized in mouse and chicken models. Here, we revealed the expression patterns of three JAK-STAT pathway genes (JAK1, JAK2, and TYK2) in chicken embryonic skin at E6-E10 stages which are key to feather follicle morphogenesis. Multiple sequence alignment of the three genes from chicken and other species all showed a closely related homology with birds like quail and goose. Whole mount in situ hybridization (WISH) revealed weak expression of JAK1, JAK2, and TYK2 in chicken skin at E6 and E7, and followed with the focally restricted signals in the feather follicles of neck and body skin located dorsally at E8 for JAK1, E9 for TYK2 and E10 for JAK2 gene. All three genes displayed stronger expression in feather follicles of neck skin than that of body skin. The expression levels of JAK1 and TYK2 were much stronger than those of JAK2. Quantitative real-time PCR (qRT-PCR) analysis revealed the increased expression tendency for JAK2 both in the neck and body skin from E6 to E10, and the much stronger expression in neck and body skin at later stages (E8-E10) than earlier stages (E6 and E7) for JAK1 and TYK2. Overall, these findings suggest that JAK1 and TYK2, not JAK2 are important to specify the feather follicle primordia, and to arrange the proximal-distal axis of feather follicles, respectively, during the morphogenesis of feather follicles in embryonic chicken skin.

Transcriptome Reveals Long Non-coding RNAs and mRNAs Involved in Primary Wool Follicle Induction in Carpet Sheep Fetal Skin.

Murine primary hair follicle induction is driven by the communication between the mesenchyme and epithelium and mostly governed by signaling pathways including wingless-related integration site (WNT), ectodysplasin A receptor (EDAR), bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), as observed in genetically modified mouse models. Sheep skin may serve as a valuable system for hair research owing to the co-existence of sweat glands with wool follicles in trunk skin and asynchronized wool follicle growth pattern similar to that of human head hair follicles. However, the mechanisms underlying wool follicle development remain largely unknown. To understand how long non-coding RNAs (lncRNAs) and mRNAs function in primary wool follicle induction in carpet wool sheep, we conducted high-throughput RNA sequencing and revealed globally altered lncRNAs (36 upregulated and 26 downregulated), mRNAs (228 elevated and 225 decreased), and 80 differentially expressed novel transcripts. Several key signals in WNT (WNT2B and WNT16), BMP (BMP3, BMP4, and BMP7), EDAR (EDAR and EDARADD), and FGF (FGFR2 and FGF20) pathways, and a series of lncRNAs, including XLOC_539599, XLOC_556463, XLOC_015081, XLOC_1285606, XLOC_297809, and XLOC_764219, were shown to be potentially important for primary wool follicle induction. GO and KEGG analyses of differentially expressed mRNAs and potential targets of altered lncRNAs were both significantly enriched in morphogenesis biological processes and transforming growth factor-ß, Hedgehog, and PI3K-Akt signaling, as well as focal adhesion and extracellular matrix-receptor interactions. The prediction of mRNA-mRNA and lncRNA-mRNA interaction networks further revealed transcripts potentially involved in primary wool follicle induction. The expression patterns of mRNAs and lncRNAs of interest were validated by qRT-PCR. The localization of XLOC_297809 and XLOC_764219 both in placodes and dermal condensations was detected by in situ hybridization, indicating important roles of lncRNAs in primary wool follicle induction and skin development. This is the first report elucidating the gene network of lncRNAs and mRNAs associated with primary wool follicle early development in carpet wool sheep and will shed new light on selective wool sheep breeding.

Defining Key Genes Regulating Morphogenesis of Apocrine Sweat Gland in Sheepskin.

The apocrine sweat gland is a unique skin appendage in humans compared to mouse and chicken models. The absence of apocrine sweat glands in chicken and murine skin largely restrains further understanding of the complexity of human skin biology and skin diseases, like hircismus. Sheep may serve as an additional system for skin appendage investigation owing to the distributions and histological similarities between the apocrine sweat glands of sheep trunk skin and human armpit skin. To understand the molecular mechanisms underlying morphogenesis of apocrine sweat glands in sheepskin, transcriptome analyses were conducted to reveal 1631 differentially expressed genes that were mainly enriched in three functional groups (cellular component, molecular function and biological process), particularly in gland, epithelial, hair follicle and skin development. There were 7 Gene Ontology (GO) terms enriched in epithelial cell migration and morphogenesis of branching epithelium that were potentially correlated with the wool follicle peg elongation. An additional 5 GO terms were enriched in gland morphogenesis (20 genes), gland development (42 genes), salivary gland morphogenesis and development (8 genes), branching involved in salivary gland morphogenesis (6 genes) and mammary gland epithelial cell differentiation (4 genes). The enriched gland-related genes and two Kyoto Encyclopedia of Genes and Genomes pathway genes (WNT and TGF-ß) were potentially involved in the induction of apocrine sweat glands. Genes named BMPR1A, BMP7, SMAD4, TGFB3, WIF1, and WNT10B were selected to validate transcript expression by qRT-PCR. Immunohistochemistry was performed to localize markers for hair follicle (SOX2), skin fibroblast (PDGFRB), stem cells (SOX9) and BMP signaling (SMAD5) in sheepskin. SOX2 and PDGFRB were absent in apocrine sweat glands. SOX9 and SMAD5 were both observed in precursor cells of apocrine sweat glands and later in gland ducts. These results combined with the upregulation of BMP signaling genes indicate that apocrine sweat glands were originated from outer root sheath of primary wool follicle and positively regulated by BMP signaling. This report established the primary network regulating early development of apocrine sweat glands in sheepskin and will facilitate the further understanding of histology and pathology of apocrine sweat glands in human and companion animal skin.

Cryptic patterning of avian skin confers a developmental facility for loss of neck feathering.

Vertebrate skin is characterized by its patterned array of appendages, whether feathers, hairs, or scales. In avian skin the distribution of feathers occurs on two distinct spatial levels. Grouping of feathers within discrete tracts, with bare skin lying between the tracts, is termed the macropattern, while the smaller scale periodic spacing between individual feathers is referred to as the micropattern. The degree of integration between the patterning mechanisms that operate on these two scales during development and the mechanisms underlying the remarkable evolvability of skin macropatterns are unknown. A striking example of macropattern variation is the convergent loss of neck feathering in multiple species, a trait associated with heat tolerance in both wild and domestic birds. In chicken, a mutation called Naked neck is characterized by a reduction of body feathering and completely bare neck. Here we perform genetic fine mapping of the causative region and identify a large insertion associated with the Naked neck trait. A strong candidate gene in the critical interval, BMP12/GDF7, displays markedly elevated expression in Naked neck embryonic skin due to a cis-regulatory effect of the causative mutation. BMP family members inhibit embryonic feather formation by acting in a reaction-diffusion mechanism, and we find that selective production of retinoic acid by neck skin potentiates BMP signaling, making neck skin more sensitive than body skin to suppression of feather development. This selective production of retinoic acid by neck skin constitutes a cryptic pattern as its effects on feathering are not revealed until gross BMP levels are altered. This developmental modularity of neck and body skin allows simple quantitative changes in BMP levels to produce a sparsely feathered or bare neck while maintaining robust feather patterning on the body.

Enhanced ectodysplasin-A receptor (EDAR) signaling alters multiple fiber characteristics to produce the East Asian hair form.

Hair morphology differs dramatically between human populations: people of East Asian ancestry typically have a coarse hair texture, with individual fibers being straight, of large diameter, and cylindrical when compared to hair of European or African origin. Ectodysplasin-A receptor (EDAR) is a cell surface receptor of the tumor necrosis factor receptor (TNFR) family involved in the development of hair follicles, teeth, and sweat glands. Analyses of genome-wide polymorphism data from multiple human populations suggest that EDAR experienced strong positive selection in East Asians. It is likely that a nonsynonymous SNP in EDAR, rs3827760, was the direct target of selection as the derived p.Val370Ala variant is seen at high frequencies in populations of East Asian and Native American origin but is essentially absent from European and African populations. Here we demonstrate that the derived EDAR370A common in East Asia has a more potent signaling output than the ancestral EDAR370 V in vitro. We show that elevation of Edar activity in transgenic mice converts their hair phenotype to the typical East Asian morphology. The coat texture becomes coarse, with straightening and thickening of individual hairs and conversion of fiber cross-sectional profile to a circular form. These thick hair fibers are produced by enlarged hair follicles, which in turn develop from enlarged embryonic organ primordia. This work shows that the multiple differences in hair form between East Asian and other human populations can be explained by the simplest of genetic alterations.

The Edar subfamily in feather placode formation.

A subgroup of the TNF receptor family, composed of Edar, Troy and Xedar, are implicated in the development of ectodermal appendages, such as hair follicles, teeth and sweat glands. We have isolated chicken orthologues of these three receptors and analysed their roles in early feather development. Conservation of protein sequences between mammalian and avian proteins is variable, with avian Edar showing the greatest degree of sequence identity. cXedar differs from its mammalian orthologue in that it contains an intracellular death domain. All three receptors are expressed during early feather morphogenesis and dominant negative forms of each receptor impair the epithelial contribution to feather bud morphogenesis, while the dermal contribution appears unaffected. Hyperactivation of each receptor leads to more widespread assumption of placode fate, though in different regions of the skin. Receptor signaling converges on NF-kappaB, and inhibiting this transcription factor alters feather bud number and size in a stage-specific manner. Our findings illustrate the roles of these three receptors during avian skin morphogenesis and also suggest that activators of feather placode fate undergo mutual regulation to reach a decision on skin appendage location and size.

The identification of lymphocyte-like cells and lymphoid-related genes in amphioxus indicates the twilight for the emergence of adaptive immune system.

To seek evidence of a primitive adaptive immune system (AIS) before vertebrate, we examined whether lymphocytes or lymphocyte-like cells and the related molecules participating in the lymphocyte function existed in amphioxus. Anatomical analysis by electron microscopy revealed the presence of lymphocyte-like cells in gills, and these cells underwent morphological changes in response to microbial pathogens that are reminiscent of those of mammalian lymphocytes executing immune response to microbial challenge. In addition, a systematic comparative analysis of our cDNA database of amphioxus identified a large number of genes whose vertebrate counterparts are involved in lymphocyte function. Among these genes, several genes were found to be expressed in the vicinity of the lymphocyte-like cells by in situ hybridization and up-regulated after exposure to microbial pathogens. Our findings in the amphioxus indicate the twilight for the emergence of AIS before the invertebrate-vertebrate transition during evolution.

Generation of the primary hair follicle pattern.

Hair follicles are spaced apart from one another at regular intervals through the skin. Although follicles are predominantly epidermal structures, classical tissue recombination experiments indicated that the underlying dermis defines their location during development. Although many molecules involved in hair follicle formation have been identified, the molecular interactions that determine the emergent property of pattern formation have remained elusive. We have used embryonic skin cultures to dissect signaling responses and patterning outcomes as the skin spatially organizes itself. We find that ectodysplasin receptor (Edar)-bone morphogenetic protein (BMP) signaling and transcriptional interactions are central to generation of the primary hair follicle pattern, with restriction of responsiveness, rather than localization of an inducing ligand, being the key driver in this process. The crux of this patterning mechanism is rapid Edar-positive feedback in the epidermis coupled with induction of dermal BMP4/7. The BMPs in turn repress epidermal Edar and hence follicle fate. Edar activation also induces connective tissue growth factor, an inhibitor of BMP signaling, allowing BMP action only at a distance from their site of synthesis. Consistent with this model, transgenic hyperactivation of Edar signaling leads to widespread overproduction of hair follicles. This Edar-BMP activation-inhibition mechanism appears to operate alongside a labile prepattern, suggesting that Edar-mediated stabilization of beta-catenin active foci is a key event in determining definitive follicle locations.