Conserved regulatory switches for the transition from natal down to juvenile feather in birds.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We report that extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial-mesenchymal interactions for branching morphogenesis. α-SMA (ACTA2) compartmentalizes dermal papilla stem cells for feather renewal cycling. LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We show that this primary feather transition is largely conserved in chicken (precocial) and zebra finch (altricial) and discuss the possibility that this evolutionary adaptation process started in feathered dinosaurs.

Adaptive patterning of vascular network during avian skin development: Mesenchymal plasticity and dermal vasculogenesis.

A vasculature network supplies blood to feather buds in the developing skin. Does the vasculature network during early skin development form by sequential sprouting from the central vasculature or does local vasculogenesis occur first that then connect with the central vascular tree? Using transgenic Japanese quail Tg(TIE1p.H2B-eYFP), we observe that vascular progenitor cells appear after feather primordia formation. The vasculature then radiates out from each bud and connects with primordial vessels from neighboring buds. Later they connect with the central vasculature. Epithelial-mesenchymal recombination shows local vasculature is patterned by the epithelium, which expresses FGF2 and VEGF. Perturbing noggin expression leads to abnormal vascularization. To study endothelial origin, we compare transcriptomes of TIE1p.H2B-eYFP+ cells collected from the skin and aorta. Endothelial cells from the skin more closely resemble skin dermal cells than those from the aorta. The results show developing chicken skin vasculature is assembled by (1) physiological vasculogenesis from the peripheral tissue, and (2) subsequently connects with the central vasculature. The work implies mesenchymal plasticity and convergent differentiation play significant roles in development, and such processes may be re-activated during adult regeneration. SUMMARY STATEMENT: We show the vasculature network in the chicken skin is assembled using existing feather buds as the template, and endothelia are derived from local bud dermis and central vasculature.

Transition from natal downs to juvenile feathers: conserved regulatory switches in Neoaves.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We discovered that LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial -mesenchymal interactions for branching morphogenesis. ACTA2 compartments dermal papilla stem cells for feather cycling. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We found this primary feather transition largely conserved in chicken (precocious) and zebra finch (altricial) and discussed the possibility that this evolutionary adaptation process started in feathered dinosaurs.

Chicken HOXC8 and HOXC10 genes may play a role in the altered skull morphology associated with the Crest phenotype.

One of the most intriguing traits found in domestic chickens is the Crest phenotype. This trait, characterized by a tuft of elongated feathers sprouted from the head, is found in breeds such as Polish chickens and Silkie chickens. Moreover, some crested chicken breeds also exhibit a protuberance in their anterodorsal skull region. Previous studies have strived to identify the causative factors of this trait. This study aimed to elucidate the role of chicken HOXC8 and HOXC10 in the formation of the Crest phenotype. We explored the effect of ectopic expression of HOXC8 or HOXC10 on the chicken craniofacial morphology using the RCAS retrovirus transformation system. Microcomputed tomography scanning was conducted to measure the 3D structure of the cranial bone of transgenic embryos for geometric morphometric analysis. We found that the ectopic expression of HOXC8 or HOXC10 in chicken heads caused mild morphological changes in the skull compared with the GFP-transgenic control group. Geometric morphometric analysis showed that HOXC8 and HOXC10 transgenic groups expressed a mild upward shape change in the frontal region of the skull compared with the control group, which is similar to what is seen in the crested chicken breeds. In conclusion, this study supports findings in previous studies in which HOX genes play a role in the formation of the altered skull morphology related to the Crest phenotype. It also supports that mutations in HOX genes may contribute to intra- and inter-specific variation in morphological traits in vertebrates.

Regional specific differentiation of integumentary organs: SATB2 is involved in α- and ß-keratin gene cluster switching in the chicken.

BACKGROUND: Animals develop skin regional specificities to best adapt to their environments. Birds are excellent models in which to study the epigenetic mechanisms that facilitate these adaptions. Patients suffering from SATB2 mutations exhibit multiple defects including ectodermal dysplasia-like changes. The preferential expression of SATB2, a chromatin regulator, in feather-forming compared to scale-forming regions, suggests it functions in regional specification of chicken skin appendages by acting on either differentiation or morphogenesis. RESULTS: Retrovirus mediated SATB2 misexpression in developing feathers, beaks, and claws causes epidermal differentiation abnormalities (e.g. knobs, plaques) with few organ morphology alterations. Chicken ß-keratins are encoded in 5 sub-clusters (Claw, Feather, Feather-like, Scale, and Keratinocyte) on Chromosome 25 and a large Feather keratin cluster on Chromosome 27. Type I and II α-keratin clusters are located on Chromosomes 27 and 33, respectively. Transcriptome analyses showed these keratins (1) are often tuned up or down collectively as a sub-cluster, and (2) these changes occur in a temporo-spatial specific manner. CONCLUSIONS: These results suggest an organizing role of SATB2 in cluster-level gene co-regulation during skin regional specification.

Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling.

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental unsolved question. Here, we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration in chickens. Proliferation analyses reveal quiescent, transient-amplifying (TA) and long-term label-retaining dermal cell (LRDC) states. During the growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal TA cells, enriched in the proximal follicle, generate both peripheral pulp, which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and central pulp, which provides nutrition. Entering the resting phase, LRDCs, accompanying collar bulge epidermal label-retaining cells, descend to the apical dermal papilla. In the next cycle, these apical dermal papilla LRDCs are re-activated to become new pulp progenitor TA cells. In the growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare the cyclic topological changes with those of the hair follicle, a convergently evolved follicle configuration. This work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors.

Making region-specific integumentary organs in birds: evolution and modifications.

Birds are the most diversified terrestrial vertebrates due to highly diverse integumentary organs that enable robust adaptability to various eco-spaces. Here we show that this complexity is built upon multi-level regional specifications. Across-the-body (macro-) specification includes the evolution of beaks and feathers as new integumentary organs that are formed with regional specificity. Within-an-organ (micro-) specification involves further modifications of organ shapes. We review recent progress in elucidating the molecular mechanisms underlying feather diversification as an example. (1) ß-Keratin gene clusters are regulated by typical enhancers or high order chromatin looping to achieve macro- and micro-level regional specification, respectively. (2) Multi-level symmetry-breaking of feather branches confers new functional forms. (3) Complex color patterns are produced by combinations of macro-patterning and micro-patterning processes. The integration of these findings provides new insights toward the principle of making a robustly adaptive bio-interface.

Maternal gut microbes shape the early-life assembly of gut microbiota in passerine chicks via nests.

BACKGROUND: Knowledge is growing on how gut microbiota are established, but the effects of maternal symbiotic microbes throughout early microbial successions in birds remain elusive. In this study, we examined the contributions and transmission modes of maternal microbes into the neonatal microbiota of a passerine, the zebra finch (Taeniopygia guttata), based on fostering experiments. RESULTS: Using 16S rRNA amplicon sequencing, we found that zebra finch chicks raised by their biological or foster parents (the society finch Lonchura striata domestica) had gut microbial communities converging with those of the parents that reared them. Moreover, source-tracking models revealed high contribution of zebra finches' oral cavity/crop microbiota to their chicks' early gut microbiota, which were largely replaced by the parental gut microbiota at later stages. The results suggest that oral feeding only affects the early stage of hatchling gut microbial development. CONCLUSIONS: Our study indicates that passerine chicks mainly acquire symbionts through indirect maternal transmission-passive environmental uptake from nests that were smeared with the intestinal and cloacal microbes of parents that raised them. Gut microbial diversity was low in hand-reared chicks, emphasizing the importance of parental care in shaping the gut microbiota. In addition, several probiotics were found in chicks fostered by society finches, which are excellent foster parents for other finches in bird farms and hosts of brood parasitism by zebra finches in aviaries; this finding implies that avian species that can transfer probiotics to chicks may become selectively preferred hosts of brood parasitism in nature. Video Abstract.

Folding Keratin Gene Clusters during Skin Regional Specification.

Regional specification is critical for skin development, regeneration, and evolution. The contribution of epigenetics in this process remains unknown. Here, using avian epidermis, we find two major strategies regulate ß-keratin gene clusters. (1) Over the body, macro-regional specificities (scales, feathers, claws, etc.) established by typical enhancers control five subclusters located within the epidermal differentiation complex on chromosome 25; (2) within a feather, micro-regional specificities are orchestrated by temporospatial chromatin looping of the feather ß-keratin gene cluster on chromosome 27. Analyses suggest a three-factor model for regional specification: competence factors (e.g., AP1) make chromatin accessible, regional specifiers (e.g., Zic1) target specific genome regions, and chromatin regulators (e.g., CTCF and SATBs) establish looping configurations. Gene perturbations disrupt morphogenesis and histo-differentiation. This chicken skin paradigm advances our understanding of how regulation of big gene clusters can set up a two-dimensional body surface map.

Behavioral and brain- transcriptomic synchronization between the two opponents of a fighting pair of the fish Betta splendens.

Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and brain-transcriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing, and biting/striking occurred only during intervals between mouth-locking. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pair showed a strong gene expression correlation, whereas those in D20 fighting pairs showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in a grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in brain-transcriptomic synchronization (PIBS) suggested that this synchronization provides a physiological basis for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 group found genes enriched for ion transport, synaptic function, and learning and memory. Brain-transcriptomic synchronization could be a general phenomenon and may provide a new cornerstone with which to investigate coordinating and sustaining social interactions between two interacting partners of vertebrates.

Feather Evolution from Precocial to Altricial Birds.

Birds are the most abundant terrestrial vertebrates and their diversity is greatly shaped by the feathers. How avian evolution is linked to feather evolution has long been a fascinating question. Numerous excellent studies have shed light on this complex relationship by investigating feather diversity and its underlying molecular mechanisms. However, most have focused on adult domestic birds, and the contribution of feather diversity to environmental adaptation has not been well-studied. In this review, we described bird diversity using the traditional concept of the altricial-precocial spectrum in bird hatchlings. We combined the spectrum with a recently published avian phylogeny to profile the spectrum evolution. We then focused on the discrete diagnostic character of the spectrum, the natal down, and propose a hypothesis for the precocial-to-altricial evolution. For the underlying molecular mechanisms in feather diversity and bird evolution, we reviewed the literature and constructed the known mechanisms for feather tract definition and natal down development. Finally, we suggested some future directions for research on altricial-precocial divergence, which may expand our understanding of the relationship between natal down diversity and bird evolution.

Identification and evolutionary analysis of long non-coding RNAs in zebra finch.

BACKGROUND: Long non-coding RNAs (lncRNAs) are important in various biological processes, but very few studies on lncRNA have been conducted in birds. To identify IncRNAs expressed during feather development, we analyzed single-stranded RNA-seq (ssRNA-seq) data from the anterior and posterior dorsal regions during zebra finch (Taeniopygia guttata) embryonic development. Using published transcriptomic data, we further analyzed the evolutionary conservation of IncRNAs in birds and amniotes. RESULTS: A total of 1,081 lncRNAs, including 965 intergenic lncRNAs (lincRNAs), 59 intronic lncRNAs, and 57 antisense lncRNAs (lncNATs), were identified using our newly developed pipeline. These avian IncRNAs share similar characteristics with lncRNAs in mammals, such as shorter transcript length, lower exon number, lower average expression level and less sequence conservation than mRNAs. However, the proportion of lncRNAs overlapping with transposable elements in birds is much lower than that in mammals. We predicted the functions of IncRNAs based on the enriched functions of co-expressed protein-coding genes. Clusters of lncRNAs associated with natal down development were identified. The sequences and expression levels of candidate lncRNAs that shared conserved sequences among birds were validated by qPCR in both zebra finch and chicken. Finally, we identified three highly conserved lncRNAs that may be associated with natal down development. CONCLUSIONS: Our study provides the first systematical identification of avian lncRNAs using ssRNA-seq analysis and offers a resource of embryonically expressed lncRNAs in zebra finch. We also predicted the biological function of identified lncRNAs.

Regulatory Differences in Natal Down Development between Altricial Zebra Finch and Precocial Chicken.

Birds can be classified into altricial and precocial. The hatchlings of altricial birds are almost naked, whereas those of precocial birds are covered with natal down. This regulatory divergence is thought to reflect environmental adaptation, but the molecular basis of the divergence is unclear. To address this issue, we chose the altricial zebra finch and the precocial chicken as the model animals. We noted that zebra finch hatchlings show natal down growth suppressed anterior dorsal (AD) skin but partially down-covered posterior dorsal (PD) skin. Comparing the transcriptomes of AD and PD skins, we found that the feather growth promoter SHH (sonic hedgehog) was expressed higher in PD skin than in AD skin. Moreover, the data suggested that the FGF (fibroblast growth factor)/Mitogen-activated protein kinase (MAPK) signaling pathway is involved in natal down growth suppression and that FGF16 is a candidate upstream signaling suppressor. Ectopic expression of FGF16 on chicken leg skin showed downregulation of SHH, upregulation of the feather growth suppressor FGF10, and suppression of feather bud elongation, similar to the phenotype found in zebra finch embryonic AD skin. Therefore, we propose that FGF16-related signals suppress natal down elongation and cause the naked AD skin in zebra finch. Our study provides insights into the regulatory divergence in natal down formation between precocial and altricial birds.

Topographical mapping of α- and ß-keratins on developing chicken skin integuments: Functional interaction and evolutionary perspectives.

Avian integumentary organs include feathers, scales, claws, and beaks. They cover the body surface and play various functions to help adapt birds to diverse environments. These keratinized structures are mainly composed of corneous materials made of α-keratins, which exist in all vertebrates, and ß-keratins, which only exist in birds and reptiles. Here, members of the keratin gene families were used to study how gene family evolution contributes to novelty and adaptation, focusing on tissue morphogenesis. Using chicken as a model, we applied RNA-seq and in situ hybridization to map α- and ß-keratin genes in various skin appendages at embryonic developmental stages. The data demonstrate that temporal and spatial α- and ß-keratin expression is involved in establishing the diversity of skin appendage phenotypes. Embryonic feathers express a higher proportion of ß-keratin genes than other skin regions. In feather filament morphogenesis, ß-keratins show intricate complexity in diverse substructures of feather branches. To explore functional interactions, we used a retrovirus transgenic system to ectopically express mutant α- or antisense ß-keratin forms. α- and ß-keratins show mutual dependence and mutations in either keratin type results in disrupted keratin networks and failure to form proper feather branches. Our data suggest that combinations of α- and ß-keratin genes contribute to the morphological and structural diversity of different avian skin appendages, with feather-ß-keratins conferring more possible composites in building intrafeather architecture complexity, setting up a platform of morphological evolution of functional forms in feathers.

Transcriptomic analyses of regenerating adult feathers in chicken.

BACKGROUND: Feathers have diverse forms with hierarchical branching patterns and are an excellent model for studying the development and evolution of morphological traits. The complex structure of feathers allows for various types of morphological changes to occur. The genetic basis of the structural differences between different parts of a feather and between different types of feather is a fundamental question in the study of feather diversity, yet there is only limited relevant information for gene expression during feather development. RESULTS: We conducted transcriptomic analysis of five zones of feather morphologies from two feather types at different times during their regeneration after plucking. The expression profiles of genes associated with the development of feather structure were examined. We compared the gene expression patterns in different types of feathers and different portions of a feather and identified morphotype-specific gene expression patterns. Many candidate genes were identified for growth control, morphogenesis, or the differentiation of specific structures of different feather types. CONCLUSION: This study laid the ground work for studying the evolutionary origin and diversification of feathers as abundant data were produced for the study of feather morphogenesis. It significantly increased our understanding of the complex molecular and cellular events in feather development processes and provided a foundation for future studies on the development of other skin appendages.

Genomic organization, transcriptomic analysis, and functional characterization of avian α- and ß-keratins in diverse feather forms.

Feathers are hallmark avian integument appendages, although they were also present on theropods. They are composed of flexible corneous materials made of α- and ß-keratins, but their genomic organization and their functional roles in feathers have not been well studied. First, we made an exhaustive search of α- and ß-keratin genes in the new chicken genome assembly (Galgal4). Then, using transcriptomic analysis, we studied α- and ß-keratin gene expression patterns in five types of feather epidermis. The expression patterns of ß-keratin genes were different in different feather types, whereas those of α-keratin genes were less variable. In addition, we obtained extensive α- and ß-keratin mRNA in situ hybridization data, showing that α-keratins and ß-keratins are preferentially expressed in different parts of the feather components. Together, our data suggest that feather morphological and structural diversity can largely be attributed to differential combinations of α- and ß-keratin genes in different intrafeather regions and/or feather types from different body parts. The expression profiles provide new insights into the evolutionary origin and diversification of feathers. Finally, functional analysis using mutant chicken keratin forms based on those found in the human α-keratin mutation database led to abnormal phenotypes. This demonstrates that the chicken can be a convenient model for studying the molecular biology of human keratin-based diseases.

Genome-wide patterns of genetic variation in two domestic chickens.

Domestic chickens are excellent models for investigating the genetic basis of phenotypic diversity, as numerous phenotypic changes in physiology, morphology, and behavior in chickens have been artificially selected. Genomic study is required to study genome-wide patterns of DNA variation for dissecting the genetic basis of phenotypic traits. We sequenced the genomes of the Silkie and the Taiwanese native chicken L2 at ∼23- and 25-fold average coverage depth, respectively, using Illumina sequencing. The reads were mapped onto the chicken reference genome (including 5.1% Ns) to 92.32% genome coverage for the two breeds. Using a stringent filter, we identified ∼7.6 million single-nucleotide polymorphisms (SNPs) and 8,839 copy number variations (CNVs) in the mapped regions; 42% of the SNPs have not found in other chickens before. Among the 68,906 SNPs annotated in the chicken sequence assembly, 27,852 were nonsynonymous SNPs located in 13,537 genes. We also identified hundreds of shared and divergent structural and copy number variants in intronic and intergenic regions and in coding regions in the two breeds. Functional enrichments of identified genetic variants were discussed. Radical nsSNP-containing immunity genes were enriched in the QTL regions associated with some economic traits for both breeds. Moreover, genetic changes involved in selective sweeps were detected. From the selective sweeps identified in our two breeds, several genes associated with growth, appetite, and metabolic regulation were identified. Our study provides a framework for genetic and genomic research of domestic chickens and facilitates the domestic chicken as an avian model for genomic, biomedical, and evolutionary studies.

Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses.

BACKGROUND: Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus. RESULTS: We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to N. patriciarum to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative GH contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: ß-glucosidase, endo-1,4-ß-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks. CONCLUSIONS: These GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One ß-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.