Actinotrichia collagens and their role in fin formation

The skeleton of zebrafish fins consists of lepidotrichia and actinotrichia. Actinotrichia are fibrils located at the tip of each lepidotrichia and play a morphogenetic role in fin formation. Actinotrichia are formed by collagens associated with non-collagen components. The non-collagen components of actinotrichia (actinodins) have been shown to play a critical role in fin to limb transition. The present study has focused on the collagens that form actinotrichia and their role in fin formation. We have found actinotrichia are formed by Collagen I plus a novel form of Collagen II, encoded by the col2a1b gene. This second copy of the collagen II gene is only found in fishes and is the only Collagen type II expressed in fins. Both col1a1a and col2a1b were found in actinotrichia forming cells. Significantly, they also expressed the lysyl hydroxylase 1 (lh1) gene, which encodes an enzyme involved in the post-translational processing of collagens. Morpholino knockdown in zebrafish embryos demonstrated that the two collagens and lh1 are essential for actinotrichia and fin fold morphogenesis. The col1a1 dominant mutant chihuahua showed aberrant phenotypes in both actinotrichia and lepidotrichia during fin development and regeneration. These pieces of evidences support that actinotrichia are composed of Collagens I and II, which are post-translationally processed by Lh1, and that the correct expression and assembling of these collagens is essential for fin formation. The unique collagen composition of actinotrichia may play a role in fin skeleton morphogenesis.     

Critical Early Roles for col27a1a and col27a1b in Zebrafish Notochord Morphogenesis,Vertebral Mineralization and Post-embryonic Axial Growth

Background

Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development.

Methodology/Principal Findings

We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically.

Conclusions/Significance

Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.     

Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration

The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.     

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.     

Cx43-Dependent Skeletal Phenotypes Are Mediated by Interactions between the Hapln1a-ECM and Sema3d during Fin Regeneration

Skeletal development is a tightly regulated process and requires proper communication between the cells for efficient exchange of information. Analysis of fin length mutants has revealed that the gap junction protein Connexin43 (Cx43) coordinates cell proliferation (growth) and joint formation (patterning) during zebrafish caudal fin regeneration. Previous studies have shown that the extra cellular matrix (ECM) protein Hyaluronan and Proteoglycan Link Protein1a (Hapln1a) is molecularly and functionally downstream of Cx43, and that hapln1a knockdown leads to reduction of the glycosaminoglycan hyaluronan. Here we find that the proteoglycan aggrecan is similarly reduced following Hapln1a knockdown. Notably, we demonstrate that both hyaluronan and aggrecan are required for growth and patterning. Moreover, we provide evidence that the Hapln1a-ECM stabilizes the secreted growth factor Semaphorin3d (Sema3d), which has been independently shown to mediate Cx43 dependent phenotypes during regeneration. Double knockdown of hapln1a and sema3d reveal synergistic interactions. Further, hapln1a knockdown phenotypes were rescued by Sema3d overexpression. Therefore, Hapln1a maintains the composition of specific components of the ECM, which appears to be required for the stabilization of at least one growth factor, Sema3d. We propose that the Hapln1a dependent ECM provides the required conditions for Sema3d stabilization and function. Interactions between the ECM and signaling molecules are complex and our study demonstrates the requirement for components of the Hapln1a-ECM for Sema3d signal transduction.     

Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish

Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.     

Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these results suggest that microporous electrospun scaffolds pre-seeded with fibroblasts promote greater wound-healing than acellular scaffolds.     

Collagen IX is required for the integrity of collagen II fibrils and the regulation of vascular plexus formation in Zebrafish caudal fins

Capillary plexuses form during both vasculogenesis and angiogenesis and are remodeled into mature vessel types and patterns which are delicately orchestrated with the sizes and shapes of other tissues and organs. We isolated a zebrafish mutation named prp (for persistent plexus) that causes persistent formation of vascular plexuses in the caudal fins and consequent mispatterning of bony fin rays and the fin shape. Detailed analyses revealed that the prp mutation causes a significant reduction in the size and dramatic structural defects in collagen II-rich extracellular matrices called actinotrichia of both embryonic finfolds and adult fins. prp was mapped to chromosome 19 and found to encode the zebrafish collagen9α1 (col9α1) gene which is abundantly expressed in developing finfolds. A point mutation resulting in a leucine-to-histidine change was detected in the thrombospondin domain of the col9α1 gene in prp. Morpholino-mediated knockdown of col9α1 phenocopied the prp small-finfold phenotype in wild-type embryos, and an injection of plasmids containing the col9α1 cDNA into prp embryos locally restored the finfold size. Furthermore, we found that osteoblasts in prp mutants were mispatterned apparently following the abnormal vascular plexus pattern, demonstrating that blood vessels play an important role in the patterning of bony rays in zebrafish caudal fins.     

Expression patterns of dnmt3aa,dnmt3ab,and dnmt4 during development and fin regeneration in zebrafish

Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72 h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72 h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells.     

Structural and mechanical properties of the proliferative zone of the developing murine growth plate cartilage assessed by atomic force microscopy

The growth plate (GP) is a dynamic tissue driving bone elongation through chondrocyte proliferation, hypertrophy and matrix production. The extracellular matrix (ECM) is the major determinant of GP biomechanical properties and assumed to play a pivotal role for chondrocyte geometry and arrangement, thereby guiding proper growth plate morphogenesis and bone elongation. To elucidate the relationship between morphology and biomechanics during cartilage morphogenesis, we have investigated age-dependent structural and elastic properties of the proliferative zone of the murine GP by atomic force microscopy (AFM) from the embryonic stage to adulthood. We observed a progressive cell flattening and arrangement into columns from embryonic day 13.5 until postnatal week 2, correlating with an increasing collagen density and ECM stiffness, followed by a nearly constant cell shape, collagen density and ECM stiffness from week 2 to 4 months. At all ages, we found marked differences in the density and organization of the collagen network between the intracolumnar matrix, and the intercolumnar matrix, associated with a roughly two-fold higher stiffness of the intracolumnar matrix compared to the intercolumnar matrix. This difference in local ECM stiffness may force the cells to arrange in a columnar structure upon cell division and drive bone elongation during embryonic and juvenile development.     

Zebrafish type XVII collagen: Gene structures,expression profiles,and morpholino “knock-down” phenotypes

The human COL17A1 gene encodes type XVII collagen (also known as the 180-kDa bullous pemphigoid antigen), an integral component of hemidesmosomes, attachment complexes providing integrity to the dermal–epidermal junction. Zebrafish, a useful model system to study skin development, displays fully developed hemidesmosomes at approximately 5 days post-fertilization (dpf). We have identified two COL17A1 orthologues in the zebrafish genome, col17a1a and col17a1b, which are expressed in the skin and the neural system, respectively. The proteins coded by these genes have structural module organizations homologous to the human type XVII collagen. “Knock-down” of the expression of col17a1a with a specific morpholino targeting the 5′ UTR of the gene resulted in a blistering phenotype and in perturbations in the basement membrane zone. “Knock-down” of col17a1b expression resulted in ablation or in marked reduction of neuromasts in the lateral line. Thus, zebrafish has two COL17A1 orthologues which may have evolved tissue-specific functions during vertebrate development. Collectively, zebrafish provides a model system to study the molecular aspects of skin development and offers insights into the corresponding human diseases.     

Clonal Analysis of kit ligand a Functional Expression Reveals Lineage-Specific Competence to Promote Melanocyte Rescue in the Mutant Regenerating Caudal Fin

The study of regeneration in an in vivo vertebrate system has the potential to reveal targetable genes and pathways that could improve our ability to heal and repair damaged tissue. We have developed a system for clonal labeling of discrete cell lineages and independently inducing gene expression under control of the heat shock promoter in the zebrafish caudal fin. Consequently we are able to test the affects of overexpressing a single gene in the context of regeneration within each of the nine different cell lineage classes that comprise the caudal fin. This can test which lineage is necessary or sufficient to provide gene function. As a first example to demonstrate this approach, we explored which lineages were competent to functionally express the kit ligand a protein as assessed by the local complementation of the mutation in the sparse-like (kitlga ^tc244b) background. We show that dermal fibroblast expression of kit ligand a robustly supports the rescue of melanocytes in the regenerating caudal fin. kit ligand a expression from skin and osteoblasts results in more modest and variable rescue of melanocytes, while lateral line expression was unable to complement the mutation.     

Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction

The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BM functions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that aberrant structure and function of the COL4A1 protein may also be a significant factor in modulating immunity.     

Extracellular matrix of adipogenically differentiated mesenchymal stem cells reveals a network of collagen filaments,mostly interwoven by hexagonal structural units

Extracellular matrix (ECM) is the non-cellular component of tissues, which not only provides biological shelter but also takes part in the cellular decisions for diverse functions. Every tissue has an ECM with unique composition and topology that governs the process of determination, differentiation, proliferation, migration and regeneration of cells. Little is known about the structural organization of matrix especially of MSC-derived adipogenic ECM. Here, we particularly focus on the composition and architecture of the fat ECM to understand the cellular behavior on functional bases. Thus, mesenchymal stem cells (MSC) were adipogenically differentiated, then, were transferred to adipogenic propagation medium, whereas they started the release of lipid droplets leaving bare network of ECM. Microarray analysis was performed, to indentify the molecular machinery of matrix. Adipogenesis was verified by Oil Red O staining of lipid droplets and by qPCR of adipogenic marker genes PPARG and FABP4. Antibody staining demonstrated the presence of collagen type I, II and IV filaments, while alkaline phosphatase activity verified the ossified nature of these filaments. In the adipogenic matrix, the hexagonal structures were abundant followed by octagonal structures, whereas they interwoven in a crisscross manner. Regarding molecular machinery of adipogenic ECM, the bioinformatics analysis revealed the upregulated expression of COL4A1, ITGA7, ITGA7, SDC2, ICAM3, ADAMTS9, TIMP4, GPC1, GPC4 and downregulated expression of COL14A1, ADAMTS5, TIMP2, TIMP3, BGN, LAMA3, ITGA2, ITGA4, ITGB1, ITGB8, CLDN11. Moreover, genes associated with integrins, glycoproteins, laminins, fibronectins, cadherins, selectins and linked signaling pathways were found. Knowledge of the interactive-language between cells and matrix could be beneficial for the artificial designing of biomaterials and bioscaffolds.     

Defective collagen VI α6 chain expression in the skeletal muscle of patients with collagen VI-related myopathies

Collagen VI is a non-fibrillar collagen present in the extracellular matrix (ECM) as a complex polymer; the mainly expressed form is composed of α1, α2 and α3 chains; mutations in genes encoding these chains cause myopathies known as Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM). The collagen VI α6 chain is a recently identified component of the ECM of the human skeletal muscle. Here we report that the α6 chain was dramatically reduced in skeletal muscle and muscle cell cultures of genetically characterized UCMD, BM and MM patients, independently of the clinical phenotype, the gene involved and the effect of the mutation on the expression of the “classical” α1α2α3 heterotrimer. By contrast, the collagen VI α6 chain was normally expressed or increased in the muscle of patients affected by other forms of muscular dystrophy, the overexpression matching with areas of increased fibrosis. In vitro treatment with TGF-β1, a potent collagen inducer, promoted the collagen VI α6 chain deposition in the ECM of normal muscle cells, whereas, in cultures derived from collagen VI-related myopathy patients, the collagen VI α6 chain failed to develop a network outside the cells and accumulated in the endoplasmic reticulum. The defect of the α6 chain points to a contribution to the pathogenesis of collagen VI-related disorders.     

Engineering cell alignment in vitro

Cell alignment plays a critical role in various cell behaviors including cytoskeleton reorganization, membrane protein relocation, nucleus gene expression, and ECM remodeling. Cell alignment is also known to exert significant effects on tissue regeneration (e.g., neuron) and modulate mechanical properties of tissues including skeleton, cardiac muscle and tendon. Therefore, it is essential to engineer cell alignment in vitro for biomechanics, cell biology, tissue engineering and regenerative medicine applications. With advances in nano- and micro-scale technologies, a variety of approaches have been developed to engineer cell alignment in vitro, including mechanical loading, topographical patterning, and surface chemical treatment. In this review, we first present alignments of various cell types and their functionality in different tissues in vivo including muscle and nerve tissues. Then, we provide an overview of recent approaches for engineering cell alignment in vitro. Finally, concluding remarks and perspectives are addressed for future improvement of engineering cell alignment.     

Comparative Expression Profiling Reveals an Essential Role for Raldh2 in Epimorphic Regeneration

Zebrafish have the remarkable ability to regenerate body parts including the heart and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage larvae also possess the ability to regenerate the caudal fin. A comparative microarray analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart, and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of retinoic acid, as one of the most highly induced genes across the three regeneration platforms. In situ localization and functional studies indicate that raldh2 expression is critical for the formation of wound epithelium and blastema. Patterning during regenerative outgrowth was considered to be the primary function of retinoic acid signaling; however, our results suggest that it is also required for early stages of tissue regeneration. Expression of raldh2 is regulated by Wnt and fibroblast growth factor/ERK signaling.