RESUMO
Skin can be mechanically stimulated to grow through a clinical procedure called tissue expansion (TE). Using a porcine TE model, we determined that expansion promptly activates transcription of SFRP2 in skin and we revealed that in the epidermis, this protein is secreted by Langerhans cells (LCs). Similar to well-known mechanosensitive genes, the increase in SFRP2 expression was proportional to the magnitude of tension, showing a spike at the apex of the expanded skin. This implies that SFRP2 might be a newly discovered effector of mechanotransduction pathways. In addition, we found that acute stretching induces accumulation of b-catenin in the nuclei of basal keratinocytes (KCs) and LCs, indicating Wnt signalling activation, followed by cell proliferation. Moreover, TE-activated LCs proliferate and migrate into the suprabasal layer of skin, suggesting that LCs rebuild their steady network within the growing epidermis. We demonstrated that in vitro hrSFRP2 treatment on KCs inhibits Wnt/b-catenin signalling and stimulates KC differentiation. In parallel, we observed an accumulation of KRT10 in vivo in the expanded skin, pointing to TE-induced, SFRP2-augmented KC maturation. Overall, our results reveal that a network of LCs delivers SFRP2 across the epidermis to fine-tune Wnt/b-catenin signalling to restore epidermal homeostasis disrupted by TE.
Assuntos
Células de Langerhans , beta Catenina , Animais , Epiderme/metabolismo , Mecanotransdução Celular , Suínos , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Human disorders of the post-squalene cholesterol biosynthesis pathway frequently result in skeletal abnormalities, yet our understanding of the mechanisms involved is limited. In a forward-genetic approach, we have found that a late-onset skeletal mutant, named kolibernu7 , is the result of a cis-acting regulatory mutation leading to loss of methylsterol monooxygenase 1 (msmo1) expression within pre-hypertrophic chondrocytes. Generated msmo1nu81 knockdown mutation resulted in lethality at larval stage. We demonstrated that this is a result of both cholesterol deprivation and sterol intermediate accumulation by creating a mutation eliminating activity of Lanosterol synthase (Lss). Our results indicate that double lssnu60;msmo1nu81 and single lssnu60 mutants survive significantly longer than msmo1nu81 homozygotes. Liver-specific restoration of either Msmo1 or Lss in corresponding mutant backgrounds suppresses larval lethality. Rescued mutants develop dramatic skeletal abnormalities, with a loss of Msmo1 activity resulting in a more-severe patterning defect of a near-complete loss of hypertrophic chondrocytes marked by col10a1a expression. Our analysis suggests that hypertrophic chondrocytes depend on endogenous cholesterol synthesis, and blocking C4 demethylation exacerbates the cholesterol deficiency phenotype. Our findings offer new insight into the genetic control of bone development and provide new zebrafish models for human disorders of the cholesterol biosynthesis pathway.
Assuntos
Doenças do Desenvolvimento Ósseo/metabolismo , Osso e Ossos/metabolismo , Colesterol/biossíntese , Condrócitos/metabolismo , Fígado/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/patologia , Osso e Ossos/patologia , Condrócitos/patologia , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Modelos Animais de Doenças , Predisposição Genética para Doença , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutação , Fenótipo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
We have developed four experimental models of mechanical stimulation applied to the back skin using tissue expansion (TE) procedure performed on minipigs. The technique is used by plastic surgeons for decades, to amend the congenital or accidental skin defects, though underlying changes in epidermis are not well understood. We found that the initial stretching increased proliferation of basal keratinocytes leading to elongation of the basal layer, and increased cellular density. The increased number of the rete ridges, suggests that they absorbed the impact of excessive proliferation, preserving layered organization of epidermis. We found ß1 integrin to be a very sensitive responder to stimulation instigated by TE procedure, able to dynamically relocate to adjust the basal cell against external force. Repeated mechanical stimulation with a seven-day interval generated healthy tissue without detrimental effects. Given the similarities between the structure of the porcine and human epidermis, we speculate that a similar mechanism functions in human skin. A better understanding of the underlying process could help improve medical care and outcomes for patients undergoing surgical reconstruction.
Assuntos
Células Epidérmicas/metabolismo , Epiderme/crescimento & desenvolvimento , Estresse Mecânico , Animais , Humanos , SuínosRESUMO
Fibroblast growth factor (FGF) signaling is essential for many developmental processes and plays a pivotal role in skeletal homeostasis, regeneration and wound healing. FGF signals through one of five tyrosine kinase receptors: Fgfr1a, -1b, -2, -3, -4. To characterize the expression of zebrafish fgfr3 from the larval stage to adulthood, we used RNAscope in situ hybridization on paraffin sections of the zebrafish head. Our study revealed spatial and temporal distribution of fgfr3 transcript in chondrocytes of the head cartilages, osteoblasts involved in bone formation, ventricular zone of the brain, undifferentiated mesenchymal cells of the skin, and lens epithelium of the eye. In general, the expression pattern of zebrafish fgfr3 is similar to the expression observed in higher vertebrates.
Assuntos
Encéfalo/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Encéfalo/citologia , Cartilagem/citologia , Células Cultivadas , Condrócitos/citologia , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Tissue expansion is a common technique in reconstructive surgery used to grow skin in vivo for correction of large defects. Despite its popularity, there is a lack of quantitative understanding of how stretch leads to growth of new skin. This has resulted in several arbitrary expansion protocols that rely on the surgeon's personal training and experience rather than on accurate predictive models. For example, choosing between slow or rapid expansion, or small or large inflation volumes remains controversial. Here we explore four tissue expansion protocols by systematically varying the inflation volume and the protocol duration in a porcine model. The quantitative analysis combines three-dimensional photography, isogeometric kinematics, and finite growth theory. Strikingly, all four protocols generate similar peak stretches, but different growth patterns: Smaller filling volumes of 30â¯ml per inflation did not result in notable expander-induced growth neither for the short nor for the long protocol; larger filling volumes of 60â¯ml per inflation trigger skin adaptation, with larger expander-induced growth in regions of larger stretch, and more expander-induced growth for the 14-day compared to the 10-day expansion protocol. Our results suggest that expander-induced growth is not triggered by the local stretch alone. While stretch is clearly a driver for growth, the local stretch at a given point is not enough to predict the expander-induced growth at that location. From a clinical perspective, our study suggests that longer expansion protocols are needed to ensure sufficient growth of sizable skin patches.
Assuntos
Simulação por Computador , Expansão de TecidoRESUMO
BACKGROUND: The relative effects of skin growth and stretch during tissue expansion have not been studied. The authors use novel analytic techniques that allow calculation of these factors at any point of a skin patch. OBJECTIVE: The authors sought to determine how stretch and growth change with different expansion rates and to correlate these values with histologic and cellular changes in skin. MATERIALS AND METHODS: Two minipigs were implanted with a total of 5 tissue expanders under tattooed skin grids. One pig was expanded over 35 days and the second over 15 days. Isogeometric analysis allowed calculation of growth and stretch. Expanders with similar total deformation were compared between protocols. Regression analysis determined predictive effects of stretch and growth on histologic data from the second animal. RESULTS: Deformation was more attributable to stretch in rapid than in slow expansion (1.40 vs1.12, p < .001). Growth was higher in slow expansion than in rapid (1.52 vs 1.07, p < .001). Both growth and stretch predicted epidermal thickness, dermal thinning, and keratinocyte proliferation. Growth predicted vascularity. CONCLUSION: Isogeometric analysis allows determination of precise surface area changes for correlation to microscopic-level data. Using the model, the authors identified that skin deformation in rapid expansion is more attributable to stretch.
Assuntos
Pele/crescimento & desenvolvimento , Expansão de Tecido , Animais , Proliferação de Células , Epiderme/anatomia & histologia , Epiderme/crescimento & desenvolvimento , Queratinócitos/citologia , Modelos Animais , Pele/anatomia & histologia , Pele/irrigação sanguínea , Suínos , Porco MiniaturaRESUMO
Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.
Assuntos
Suturas Cranianas/citologia , Osso Frontal/citologia , Regulação da Expressão Gênica no Desenvolvimento , Osso Occipital/citologia , Osteogênese/genética , Osso Parietal/citologia , Animais , Colágeno/genética , Colágeno/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Suturas Cranianas/crescimento & desenvolvimento , Suturas Cranianas/metabolismo , Osso Frontal/crescimento & desenvolvimento , Osso Frontal/metabolismo , Humanos , Osso Occipital/crescimento & desenvolvimento , Osso Occipital/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Osso Parietal/crescimento & desenvolvimento , Osso Parietal/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição Twist/genética , Fatores de Transcrição Twist/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The Wnt/Planar Cell Polarity (PCP) pathway controls cell morphology and behavior during animal development. Several zebrafish mutants were identified as having perturbed Wnt/PCP signaling. Many of these mutants have defects in craniofacial formation. To better understand the role that Wnt/PCP plays in craniofacial development we set out to identify which of the mutants, known to be associated with the Wnt/PCP pathway, perturb head cartilage formation by disrupting chondrocyte morphology. Here we demonstrate that while vang-like 2 (vangl2), wnt11 and scribbled (scrib) mutants have severe craniofacial morphogenesis defects they do not display the chondrocyte stacking and intercalation problems seen in glypican 4 (gpc4) and wnt5b mutants. The function of Gpc4 or Wnt5b appears to be important for chondrocyte organization, as the neural crest in both mutants is specified, undergoes migration, and differentiates into the same number of cells to compose the craniofacial cartilage elements. We demonstrate that Gpc4 activity is required cell autonomously in the chondrocytes and that the phenotype of single heterozygous mutants is slightly enhanced in embryos double heterozygous for wnt5b and gpc4. This data suggests a novel mechanism for Wnt5b and Gpc4 regulation of chondrocyte behavior that is independent of the core Wnt/PCP molecules and differs from their collaborative action of controlling cell movements during gastrulation.
Assuntos
Condrócitos/metabolismo , Condrogênese/genética , Glipicanas/genética , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Região Branquial/embriologia , Região Branquial/metabolismo , Contagem de Células , Movimento Celular/genética , Tamanho Celular , Condrócitos/citologia , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Glipicanas/deficiência , Mutação , Crista Neural/embriologia , Crista Neural/metabolismo , Fenótipo , Proteínas Wnt/deficiência , Via de Sinalização Wnt/genética , Proteína Wnt-5a , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiênciaRESUMO
Multiple developmental processes require tightly controlled Wnt signaling, and its misregulation leads to congenital abnormalities and diseases. Glypicans are extracellular proteins that modulate the Wnt pathway. In addition to interacting with Wnts, these glycosophosphotidylinositol (GPI)-anchored, heparan-sulfate proteoglycans bind ligands of several other signaling pathways in both vertebrates and invertebrates. In Drosophila, Notum, a secreted α/ß-hydrolase, antagonizes the signaling of the prototypical Wnt Wingless (Wg), by releasing glypicans from the cell surface. Studies of mammalian Notum indicate promiscuous target specificity in cell culture, but the role of Notum in vertebrate development has not been studied. Our work shows that zebrafish Notum 1a, an ortholog of mammalian Notum, contributes to a self-regulatory loop that restricts Wnt/ß-catenin signaling. Notum 1a does not interact with Glypican 4, an essential component of the Wnt/planar cell polarity (PCP) pathway. Our results suggest a surprising specific role of Notum in the developing vertebrate embryo.
Assuntos
Hidrolases/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , beta Catenina/metabolismo , Animais , Padronização Corporal , Polaridade Celular , Desenvolvimento Embrionário , Glipicanas/metabolismo , Hidrolases/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Delta-like ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR-27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets.
Assuntos
Artérias/embriologia , Endotélio Vascular/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/fisiologia , Neovascularização Fisiológica , Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Aorta/citologia , Aorta/metabolismo , Artérias/metabolismo , Biomarcadores/metabolismo , Northern Blotting , Western Blotting , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Diferenciação Celular , Movimento Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endotélio Vascular/metabolismo , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases , Pseudópodes/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
The heparan sulfate proteoglycan Glypican 4 (Gpc4) is part of the Wnt/planar cell polarity pathway, which is required for convergence and extension during zebrafish gastrulation. To observe Glypican 4-deficient phenotypes at later stages, we rescued gpc4(-/-) (knypek) homozygotes and raised them for more than one year. Adult mutants showed diverse cranial malformations of both dermal and endochondral bones, ranging from shortening of the rostral-most skull to loss of the symplectic. Additionally, the adult palatoquadrate cartilage was disorganized, with abnormal chondrocyte orientation. To understand how the palatoquadrate cartilage normally develops, we examined a juvenile series of wild type and mutant specimens. This identified two novel domains of elongated chondrocytes in the larval palatoquadrate, which normally form prior to endochondral ossification. In contrast, gpc4(-/-) larvae never form these domains, suggesting a failure of chondrocyte orientation, though not differentiation. Our findings implicate Gpc4 in the regulation of zebrafish cartilage and bone morphogenesis.
Assuntos
Padronização Corporal , Cartilagem , Ossos Faciais , Glipicanas/metabolismo , Crânio , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Cartilagem/anormalidades , Cartilagem/anatomia & histologia , Cartilagem/crescimento & desenvolvimento , Ossos Faciais/anormalidades , Ossos Faciais/anatomia & histologia , Ossos Faciais/crescimento & desenvolvimento , Técnicas de Silenciamento de Genes , Glipicanas/genética , Humanos , Fenótipo , Crânio/anormalidades , Crânio/anatomia & histologia , Crânio/crescimento & desenvolvimento , Peixe-Zebra/anormalidades , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
The cerebellum, which forms from anterior hindbrain, coordinates motor movements and balance. Sensory input from the periphery is relayed and modulated by cerebellar interneurons, which are organized in layers. The mechanisms that specify the different neurons of the cerebellum and direct its layered organization remain poorly understood. Drawing from investigations of spinal cord, we hypothesized that the embryonic cerebellum is patterned on the dorsoventral axis by opposing morphogens. We tested this using zebrafish. Here we show that expression of olig2, which encodes a bHLH transcription factor, marks a distinct subset of neurons with similarities to eurydendroid neurons, the principal efferent neurons of the teleost cerebellum. In combination with other markers, olig2 reveals a dorsoventral organization of cerebellar neurons in embryos. Disruption of Hedgehog signaling, which patterns the ventral neural tube, produced a two-fold increase in the number of olig2(+) neurons. By contrast, olig2(+) neurons did not develop in embryos deficient for Wnt signaling, which patterns dorsal neural tube, nor did they develop in embryos deficient for both Hedgehog and Wnt signaling. Our data indicate that Hedgehog and Wnt work in opposition across the dorsoventral axis of the cerebellum to regulate formation of olig2(+) neurons. Specifically, we propose that Hedgehog limits the range of Wnt signaling, which is necessary for olig2(+) neuron development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cerebelo/embriologia , Proteínas Hedgehog/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Calbindina 2 , Cerebelo/citologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Morfogênese , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Fator de Transcrição 2 de Oligodendrócitos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Receptor Smoothened , Alcaloides de Veratrum/metabolismo , Proteínas Wnt/genética , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression.
Assuntos
Melanoma/patologia , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Blástula/transplante , Linhagem Celular Tumoral , Embrião não Mamífero , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Melanócitos/metabolismo , Melanócitos/patologia , Proteínas de Membrana/antagonistas & inibidores , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Oligonucleotídeos Antissenso/farmacologia , Transplante Heterólogo , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
Delta-Notch signaling is essential for somitogenesis in vertebrate embryos. In a search for genes that control somite formation in zebrafish we have identified two paralogues encoding proteins related to Nrarp (Notch regulated ankyrin repeat protein). Zebrafish nrarp-a and-b encode small proteins with two ankyrin repeat domains. Here, we report the expression patterns of both genes in normal and mutant embryos.
Assuntos
Regulação da Expressão Gênica , Proteínas/genética , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Mutação , Biossíntese de Proteínas , Peixe-Zebra/metabolismo , Proteínas de Peixe-ZebraRESUMO
We describe here simple methods for producing transgenic zebrafish reporter lines using BAC clones. The use of BAC clones facilitates creation of useful transgenics as the large amounts of genomic DNA they contain increase the likelihood that reporter gene expression will be properly regulated. Combined with recent advances in live embryo image analysis, this strategy has the potential to greatly advance the investigation of neural cell behavior during development.