[Show abstract][Hide abstract] ABSTRACT: Because the molecular mechanisms of morphogenesis of the hepatic cord and sinus are unclear, we investigated the involvement of bone morphogenetic protein (BMP4) in hepatic sinusoid morphogenesis.
We used embryonic chicken livers, which develop rapidly, as our model, and investigated expression of BMP-related genes. BMP4 activity was manipulated by overexpressing BMP4 and its antagonist, noggin.
During hepatic cord morphogenesis, BMP4 and its receptors are expressed in both peri-sinusoidal cells and hepatoblasts as the sinusoids form, whereas noggin is expressed transiently in peri-sinusoidal cells at early stages. Suppression of BMP activity with noggin overexpression disrupted normal hepatic sinusoid structure, leading to liver congestion, failure of fibronectin deposition, and markedly reduced numbers of peri-sinusoidal cells. However, overexpression of BMP did not change sinusoidal morphology but increased endothelial cell number. Noggin overexpression resulted in disrupted cord organization, and dilated sinusoidal space, eventually leading to increased apoptosis and failed hepatocyte differentiation.
Our results show that proper BMP signaling mediates peri-sinusoidal cell-hepatoblast interactions during development; this is essential for hepatic cord organization among hepatoblasts, endothelium, and presumptive hepatic stellate cells.
[Show abstract][Hide abstract] ABSTRACT: 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 β-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 β-keratin genes in various skin appendages at embryonic developmental stages. The data demonstrate that temporal and spatial α- and β-keratin expression is involved in establishing the diversity of skin appendage phenotypes. Embryonic feathers express a higher proportion of β-keratin genes than other skin regions. In feather filament morphogenesis, β-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 β-keratin forms. α- and β-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 β-keratin genes contribute to the morphological and structural diversity of different avian skin appendages, with feather-β-keratins conferring more possible composites in building intrafeather architecture complexity, setting up a platform of morphological evolution of functional forms in feathers.
Proceedings of the National Academy of Sciences 11/2015; DOI:10.1073/pnas.1520566112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1966-6) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: The midline pattern of sternal ossification characteristic of the Cretaceous enantiornithine birds is unique among the Ornithodira, the group containing birds, non-avian dinosaurs and pterosaurs. This has been suggested to indicate that Enantiornithes is not the sister group of Ornithuromorpha, the clade that includes living birds and their close relatives, which would imply rampant convergence in many non-sternal features between enantiornithines and ornithuromorphs. However, detailed comparisons reveal greater similarity between neornithine (i.e. crown group bird) and enantiornithine modes of sternal ossification than previously recognized. Furthermore, a new subadult enantiornithine specimen demonstrates that sternal ossification followed a more typically ornithodiran pattern in basal members of the clade. This new specimen, referable to the Pengornithidae, indicates that the unique ossification pattern observed in other juvenile enantiornithines is derived within Enantiornithes. A similar but clearly distinct pattern appears to have evolved in parallel in the ornithuromorph lineage. The atypical mode of sternal ossification in some derived enantiornithines should be regarded as an autapomorphic condition rather than an indication that enantiornithines are not close relatives of ornithuromorphs. Based on what is known about molecular mechanisms for morphogenesis and the possible selective advantages, the parallel shifts to midline ossification that took place in derived enantiornithines and living neognathous birds appear to have been related to the development of a large ventral keel, which is only present in ornithuromorphs and enantiornithines. Midline ossification can serve to medially reinforce the sternum at a relatively early ontogenetic stage, which would have been especially beneficial during the protracted development of the super-precocial Cretaceous enantiornithines.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Wnt10b is a signaling protein regulating skin development and homeostasis, and the expression of Wnt10b is restricted to epidermal keratinocytes in embryonic and postnatal skin. Recent studies indicate an elevated expression of Wnt10b in skin tumors. However, how Wnt10b regulates skin tumorigenesis remains largely unknown. Here we report that continuous expression of Wnt10b mediates transformation of epidermal keratinocytes through activating genes involved in EGF/MAPK signaling pathways. We first established a prolonged Wnt10b overexpression system in JB6P- cells to represent the elevated Wnt10b expression level in skin keratinocytes. Through expression assays and observations under phase-contrast microscopy, prolonged expression of Wnt10b activated Wnt/β-catenin pathway and induced morphological changes of cells showing longer protrusions and multilayer growth, indicating early-stage cell transformation. Wnt10b also increased cellular proliferation and migration according to BrdU incorporation and cell mobility assays. Furthermore, multi-doses of AdWnt10b treatment to JB6P- cells induced colony formation, stronger invasive ability in transwell system, and anchorage-independent growth in agar gel. In molecular level, AdWnt10b treatment induced increased transcriptional expressions of Egf, downstream Mapk pathway factors, and MMPs. Administration of Wnt antagonist DKK1 blocked the tumor promotion process induced by Wnt10b. Taken together, these findings clearly demonstrate that Wnt10b promotes epidermal keratinocyte transformation through induced Egf pathway.
[Show abstract][Hide abstract] ABSTRACT: The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.
[Show abstract][Hide abstract] ABSTRACT: Hair follicles undergo cyclic behavior through regression (catagen), rest (telogen) and regeneration (anagen) during postnatal life. The hair cycle transition is strictly regulated by the autonomous and extrinsic molecular environment. However, whether there is a switch controlling catagen-telogen transition remains largely unknown. Here we show that hair follicles cycle from catagen to the next anagen without transitioning through a morphologically typical telogen after Gsdma3 mutation. This leaves an ESLS (epithelial strand-like structure) during the time period corresponding to telogen phase in WT mice. Molecularly, Wnt10b is upregulated in Gsdma3 mutant mice. Restoration of Gsdma3 expression in AE (alopecia and excoriation) mouse skin rescues hair follicle telogen entry and significantly decreases the Wnt10b-mediated Wnt/β-catenin signaling pathway. Overexpression of Wnt10b inhibits telogen entry by increasing epithelial strand cell proliferation. Subsequently, hair follicles with a Gsdma3 mutation enter the second anagen simultaneously as WT mice. Hair follicles cannot enter the second anagen with ectopic WT Gsdma3 overexpression. A luciferase reporter assay proves Gsdma3 directly suppresses Wnt signaling. Our findings suggest Gsdma3 plays an important role in catagen-telogen transition by balancing the Wnt signaling pathway, and that morphologically typical telogen is not essential for the initiation of a new hair cycle.Journal of Investigative Dermatology accepted article preview online, 10 April 2015. doi:10.1038/jid.2015.147.
[Show abstract][Hide abstract] ABSTRACT: Background: Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. Results: We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. Conclusion: We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.
[Show abstract][Hide abstract] ABSTRACT: Tendons and ligaments exhibit limited regenerative capacity following injury, with damaged tissue being replaced by a fibrotic scar. The physiological role of scar tissue is complex and has been studied extensively. In this study we demonstrate that rat tendons contain a unique subpopulation of cells exhibiting stem cell characteristics, including clonogenicity, multipotency, and self-renewal capacity. Additionally, these putative stem cells expressed markers consistent with neural crest stem cells. Using immunofluorescent labeling, we identified P75+ (p75 neurotrophin receptor) cells in the perivascular regions of native rat tendon. Importantly, P75+ cells were frequently localized near vascular cells expressing α-smooth muscle actin (SMA+) and increased in number within the peritenon after injury. Ultrastructural analysis showed that perivascular cells detached from vessels in response to injury, migrated into the interstitial space and deposited extracellular matrix (ECM). Characterization of P75+ cells isolated from the scar tissue indicated that this population also expressed the neural crest stem cell markers vimentin, Sox 10, and Snail. In conclusion, our results suggest that neural crest-like stem cells of perivascular origin reside within the rat peritenon and give rise to scar-forming stromal cells following tendon injury.
Stem Cells and Development 11/2014; 24(7). DOI:10.1089/scd.2014.0036 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulation of adult stem cells (SCs) is fundamental for organ maintenance and tissue regeneration. On the body surface, different ectodermal organs exhibit distinctive modes of regeneration and the dynamics of their SC homeostasis remain to be unraveled. A slow cycling characteristic has been used to identify SCs in hair follicles and sweat glands; however, whether a quiescent population exists in continuously growing nails remains unknown. Using an in vivo label retaining cells (LRCs) system, we detected an unreported population of quiescent cells within the basal layer of the nail proximal fold, organized in a ring-like configuration around the nail root. These nail LRCs express the hair stem cell marker, keratin 15 (K15), and lineage tracing show that these K15-derived cells can contribute to both the nail structure and peri-nail epidermis, and more toward the latter. Thus, this stem cell population is bifunctional. Upon nail plucking injury, the homeostasis is tilted with these SCs dominantly delivering progeny to the nail matrix and differentiated nail plate, demonstrating their plasticity to adapt to wounding stimuli. Moreover, in vivo engraftment experiments established that transplanted nail LRCs can actively participate in functional nail regeneration. Transcriptional profiling of isolated nail LRCs revealed bone morphogenetic protein signaling favors nail differentiation over epidermal fate. Taken together, we have found a previously unidentified ring-configured population of bifunctional SCs, located at the interface between the nail appendage organ and adjacent epidermis, which physiologically display coordinated homeostatic dynamics but are capable of rediverting stem cell flow in response to injury.
Proceedings of the National Academy of Sciences 10/2014; 111(42). DOI:10.1073/pnas.1318848111 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ectodermal appendages such as feathers, hair, mammary glands, salivary glands, and sweat glands form branches, allowing much-increased surface for functional differentiation and secretion. Here, the principles of branching morphogenesis are exemplified by the mammary gland and feathers.
[Show abstract][Hide abstract] ABSTRACT: Feathers are hallmark avian integument appendages, although they were also present on theropods. They are composed of flexible corneous materials made of α- and β-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 β-keratin genes in the new chicken genome assembly (Galgal4). Then, using transcriptomic analysis, we studied α- and β-keratin gene expression patterns in five types of feather epidermis. The expression patterns of β-keratin genes were different in different feather types, while those of α-keratin genes were less variable. In addition, we obtained extensive α- and β-keratin mRNA in situ hybridization data, showing that α-keratins and β-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 β-keratin genes in different intra-feather 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.
[Show abstract][Hide abstract] ABSTRACT: Feathers regenerate from stem cells localized in a region of the follicle indicated as the bulge of the collar. Stem cells are slow cycling cells and some of these cells can be identified after labeling experiments using 5-bromo-deoxyuridine to detect label retaining cells (5BrdU LRCs). The present electron microscopic analysis of 5BrdU LRCs has described the ultrastructural characteristics of small cells present in the bulge region of the follicle in regenerating feathers of chickens that include stem cells. Labeled feather stem cells are smaller than 10 lm in average diameter, possess large nuclei with high nuclear/cytoplasmic ratio, and contain evenly distributed ribosomes, sparse bundles of intermediate filaments, scarce or no endoplasmic reticulum, and few mitochondria. The nuclei are mainly euchromatic with a variable amount of heterochromatin clumps and the nucleoli show developed granular and fibrillar components. These features indicate that feather stem cells are transcriptionally active cells for ribosomal and proteins synthesis. The cell surface of feather stem cells often shows small and irregular folds resembling microvilli in contact with the surrounding cells. The latter characteristics may be related to the exchange of molecules and/or with the migration of stem cells among other epithelial cells of the collar epithelium.
Journal of Morphology 07/2014; 275(7):768-74. DOI:10.1002/jmor.20257 · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lizard skin can produce scales during embryonic development, tail regeneration, and wound healing; however, underlying molecular signaling and extracellular matrix protein expression remains unknown. We mapped cell proliferation, signaling and extracellular matrix proteins in regenerating and developing lizard scales in different body regions with different wound severity. Following lizard tail autotomy (self-amputation), de novo scales regenerate from regenerating tail blastema. Despite topological differences between embryonic and adult scale formation, asymmetric cell proliferation produces the newly formed outer scale surface. Regionally different responses to wounding were observed; open wounds induced better scale regeneration from tail skin than trunk skin. Molecular studies suggest NCAM enriched dermal regions exhibit higher cell proliferation associated with scale growth. β-catenin may be involved in epidermal scale differentiation. Dynamic tenascin-C expression suggests its involvement in regeneration. We conclude that different skin regions exhibit different competence for de novo scale formation. While cellular and morphogenetic paths differ during development and regeneration of lizard scale formation, they share general proliferation patterns, epithelial-mesenchymal interactions and similar molecular modules composed of adhesion and extracellular matrix molecules.
[Show abstract][Hide abstract] ABSTRACT: Avian feathers have robust growth and regeneration capability. To evaluate the contribution of signaling molecules and pathways in these processes, we profiled gene expression in the feather follicle using an absolute quantification approach. We identified hundreds of genes that mark specific components of the feather follicle: the dermal papillae (DP) which controls feather regeneration and axis formation, the pulp mesenchyme (Pp) which is derived from DP cells and nourishes the feather follicle, and the ramogenic zone epithelium (Erz) where a feather starts to branch. The feather DP is enriched in BMP/TGF-β signaling molecules and inhibitors for Wnt signaling including Dkk2/Frzb. Wnt ligands are mainly expressed in the feather epithelium and pulp. We find that while Wnt signaling is required for the maintenance of DP marker gene expression and feather regeneration, excessive Wnt signaling delays regeneration and reduces pulp formation. Manipulating Dkk2/Frzb expression by lentiviral-mediated overexpression, shRNA-knockdown, or by antibody neutralization resulted in dual feather axes formation. Our results suggest that the Wnt signaling in the proximal feather follicle is fine-tuned to accommodate feather regeneration and axis formation.
[Show abstract][Hide abstract] ABSTRACT: The hair follicle (HF) regeneration paradigm provides a unique opportunity for studying the collective behavior of stem cells in living animals. Activation of HF stem cells depends on the core inhibitory BMP and activating WNT signals operating within the HF microenvironment. Additionally, HFs receive multilayered signaling inputs from the extrafollicular macroenvironment, which includes dermis, adipocytes, neighboring HFs, hormones, and external stimuli. These activators/inhibitors are integrated across multiple stem-cell niches to produce dynamic hair growth patterns. Because of their pigmentation, these patterns can be easily studied on live shaved animals. Comparing to autonomous regeneration of one HF, populations of HFs display coupled decision making, allowing for more robust and adaptable regenerative behavior to occur collectively. The generic cellular automata model used to simulate coordinated HF cycling here can be extended to study population-level behavior of other complex biological systems made of cycling elements.
Cold Spring Harbor Perspectives in Medicine 01/2014; 4(1). DOI:10.1101/cshperspect.a015198 · 9.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent progress in epigenetics reveals dynamic chromatin interactions in the nucleus during development, regeneration, reprogramming, and in disease. Higher-order chromatin organization is manifested as changes in the topological distribution of eu-/heterochromatin and in nuclear morphology. We are now able to gain new knowledge about these changes at the genomic level.