RESUMO
OBJECTIVE: Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH. METHODS: SMC-specific Bmpr2-/- mice (BKOSMC) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKOSMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism. RESULTS: BKOSMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (ß-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (ß-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension. CONCLUSIONS: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Humanos , Camundongos , beta-Arrestina 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hipertensão Pulmonar/metabolismo , Hipóxia/complicações , Hipóxia/genética , Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/metabolismo , RNA/metabolismoRESUMO
Zebrafish robustly regenerate fins, including their characteristic bony ray skeleton. Amputation activates intra-ray fibroblasts and dedifferentiates osteoblasts that migrate under a wound epidermis to establish an organized blastema. Coordinated proliferation and re-differentiation across lineages then sustains progressive outgrowth. We generate a single cell transcriptome dataset to characterize regenerative outgrowth and explore coordinated cell behaviors. We computationally identify sub-clusters representing most regenerative fin cell lineages, and define markers of osteoblasts, intra- and inter-ray fibroblasts and growth-promoting distal blastema cells. A pseudotemporal trajectory and in vivo photoconvertible lineage tracing indicate distal blastemal mesenchyme restores both intra- and inter-ray fibroblasts. Gene expression profiles across this trajectory suggest elevated protein production in the blastemal mesenchyme state. O-propargyl-puromycin incorporation and small molecule inhibition identify insulin growth factor receptor (IGFR)/mechanistic target of rapamycin kinase (mTOR)-dependent elevated bulk translation in blastemal mesenchyme and differentiating osteoblasts. We test candidate cooperating differentiation factors identified from the osteoblast trajectory, finding IGFR/mTOR signaling expedites glucocorticoid-promoted osteoblast differentiation in vitro. Concordantly, mTOR inhibition slows but does not prevent fin regenerative outgrowth in vivo. IGFR/mTOR may elevate translation in both fibroblast- and osteoblast-lineage cells during the outgrowth phase as a tempo-coordinating rheostat.
Assuntos
Transdução de Sinais , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Diferenciação Celular , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Receptores de Somatomedina/metabolismo , Nadadeiras de Animais/metabolismoRESUMO
Organs stop growing to achieve a characteristic size and shape in scale with the body of an animal. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency.
Assuntos
Nadadeiras de Animais/fisiologia , Expressão Ectópica do Gene/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Nadadeiras de Animais/anatomia & histologia , Animais , Sistemas CRISPR-Cas , Calcineurina/metabolismo , Proliferação de Células , Expressão Ectópica do Gene/genética , Éter , Canais de Potássio Éter-A-Go-Go/genética , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Tamanho do Órgão , Regeneração/fisiologia , Transdução de Sinais/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Caudal fin symmetry characterizes teleosts and likely contributes to their evolutionary success. However, the coordinated development and patterning of skeletal elements establishing external symmetry remains incompletely understood. We explore the spatiotemporal emergence of caudal skeletal elements in zebrafish to consider evolutionary and developmental origins of caudal fin symmetry. RESULTS: Transgenic reporters and skeletal staining reveal that the hypural diastema-defining gap between hypurals 2 and 3 forms early and separates progenitors of two plates of connective tissue. Two sets of central principal rays (CPRs) synchronously, sequentially, and symmetrically emerge around the diastema. The two dorsal- and ventral-most rays (peripheral principal rays, PPRs) arise independently and earlier than adjacent CPRs. Muscle and tendon markers reveal that different muscles attach to CPR and PPR sets. CONCLUSIONS: We propose that caudal fin symmetry originates from a central organizer that establishes the hypural diastema and bidirectionally patterns surrounding tissue into two plates of connective tissue and two mirrored sets of CPRs. Further, two peripheral organizers unidirectionally specify PPRs, forming a symmetric "composite" fin derived from three fields. Distinct CPR and PPR ontogenies may represent developmental modules conferring ray identities, muscle connections, and biomechanical properties. Our model contextualizes mechanistic studies of teleost fin morphological variation.
Assuntos
Diastema , Peixe-Zebra , Nadadeiras de Animais/anatomia & histologia , Animais , Animais Geneticamente Modificados , Evolução Biológica , Peixe-Zebra/anatomia & histologiaRESUMO
Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent 'tethering' to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.
Assuntos
Nadadeiras de Animais/embriologia , Células Epidérmicas/fisiologia , Epiderme/embriologia , Proteínas Hedgehog/fisiologia , Morfogênese , Proteínas de Peixe-Zebra/fisiologia , Nadadeiras de Animais/citologia , Nadadeiras de Animais/metabolismo , Animais , Benzamidas/farmacologia , Movimento Celular , Epiderme/metabolismo , Receptor Patched-2/metabolismo , Quinazolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/fisiologia , Peixe-ZebraRESUMO
Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.
Assuntos
Biologia Molecular/métodos , RNA/isolamento & purificação , Coloração e Rotulagem/métodos , Tiouracila/metabolismo , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Encéfalo/embriologia , Encéfalo/metabolismo , Quimera , Perfilação da Expressão Gênica , Camundongos , Transgenes/genéticaRESUMO
Zebrafish innately regenerate amputated fins by mechanisms that expand and precisely position injury-induced progenitor cells to re-form tissue of the original size and pattern. For example, cell signaling networks direct osteoblast progenitors (pObs) to rebuild thin cylindrical bony rays with a stereotypical branched morphology. Hedgehog/Smoothened (Hh/Smo) signaling has been variably proposed to stimulate overall fin regenerative outgrowth or promote ray branching. Using a photoconvertible patched2 reporter, we resolve active Hh/Smo output to a narrow distal regenerate zone comprising pObs and adjacent motile basal epidermal cells. This Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which nevertheless functions atypically to support bone maturation. Using BMS-833923, a uniquely effective Smo inhibitor, and high-resolution imaging, we show that Shha/Smo is functionally dedicated to ray branching during fin regeneration. Hh/Smo activation enables transiently divided clusters of Shha-expressing epidermis to escort pObs into similarly split groups. This co-movement likely depends on epidermal cellular protrusions that directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. Progressively separated pObs pools then continue regenerating independently to collectively re-form a now branched skeletal structure.
Assuntos
Regeneração Óssea , Comunicação Celular , Células Epidérmicas , Proteínas Hedgehog/metabolismo , Osteoblastos/citologia , Regeneração , Células-Tronco/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Nadadeiras de Animais/efeitos dos fármacos , Nadadeiras de Animais/fisiologia , Animais , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Benzamidas/farmacologia , Regeneração Óssea/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Quinazolinas/farmacologia , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated and stratified valves. Wnt signaling and its canonical effector ß-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/ß-catenin signaling by chemically inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Furthermore, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/ß-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/ß-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects.
Assuntos
Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Organogênese , Via de Sinalização Wnt , Animais , Proteína Axina/metabolismo , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Proliferação de Células/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Coxins Endocárdicos/citologia , Coxins Endocárdicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Valvas Cardíacas/efeitos dos fármacos , Camundongos Transgênicos , Valva Mitral/efeitos dos fármacos , Valva Mitral/embriologia , Valva Mitral/metabolismo , Miocárdio/metabolismo , Organogênese/efeitos dos fármacos , Organogênese/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Trimethylation of lysine 27 on histone 3 (H3K27me3) by the Polycomb repressive complex 2 (PRC2) contributes to localized and inherited transcriptional repression. Kdm6b (Jmjd3) is a H3K27me3 demethylase that can relieve repression-associated H3K27me3 marks, thereby supporting activation of previously silenced genes. Kdm6b is proposed to contribute to early developmental cell fate specification, cardiovascular differentiation, and/or later steps of organogenesis, including endochondral bone formation and lung development. We pursued loss-of-function studies in zebrafish to define the conserved developmental roles of Kdm6b. kdm6ba and kdm6bb homozygous deficient zebrafish are each viable and fertile. However, loss of both kdm6ba and kdm6bb shows Kdm6b proteins share redundant and pleiotropic roles in organogenesis without impacting initial cell fate specification. In the developing heart, co-expressed Kdm6b proteins promote cardiomyocyte proliferation coupled with the initial stages of cardiac trabeculation. While newly formed trabecular cardiomyocytes display a striking transient decrease in bulk cellular H3K27me3 levels, this demethylation is independent of collective Kdm6b. Our results indicate a restricted and likely locus-specific role for Kdm6b demethylases during heart ventricle maturation rather than initial cardiogenesis.
Assuntos
Ventrículos do Coração/crescimento & desenvolvimento , Histona Desmetilases/genética , Histona Desmetilases com o Domínio Jumonji/genética , Miócitos Cardíacos/metabolismo , Organogênese/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Histona Desmetilases/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Metilação , Miócitos Cardíacos/citologia , Organogênese/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Identifying coronary artery progenitors and their developmental pathways could inspire novel regenerative treatments for heart disease. Multiple sources of coronary vessels have been proposed, including the sinus venosus (SV), endocardium and proepicardium, but their relative contributions to the coronary circulation and the molecular mechanisms regulating their development are poorly understood. We created an ApjCreER mouse line as a lineage-tracing tool to map SV-derived vessels onto the heart and compared the resulting lineage pattern with endocardial and proepicardial contributions to the coronary circulation. The data showed a striking compartmentalization to coronary development. ApjCreER-traced vessels contributed to a large number of arteries, capillaries and veins on the dorsal and lateral sides of the heart. By contrast, untraced vessels predominated in the midline of the ventral aspect and ventricular septum, which are vessel populations primarily derived from the endocardium. The proepicardium gave rise to a smaller fraction of vessels spaced relatively uniformly throughout the ventricular walls. Dorsal (SV-derived) and ventral (endocardial-derived) coronary vessels developed in response to different growth signals. The absence of VEGFC, which is expressed in the epicardium, dramatically inhibited dorsal and lateral coronary growth but left vessels on the ventral side unaffected. We propose that complementary SV-derived and endocardial-derived migratory routes unite to form the coronary vasculature and that the former requires VEGFC, revealing its role as a tissue-specific mediator of blood endothelial development.
Assuntos
Linhagem da Célula/fisiologia , Vasos Coronários/embriologia , Átrios do Coração/embriologia , Neovascularização Fisiológica/fisiologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/fisiologia , Vasos Coronários/citologia , Átrios do Coração/citologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Mutantes , Microscopia de FluorescênciaRESUMO
The formation of intricately organized aortic and pulmonic valves from primitive endocardial cushions of the outflow tract is a remarkable accomplishment of embryonic development. While not always initially pathologic, developmental semilunar valve (SLV) defects, including bicuspid aortic valve, frequently progress to a disease state in adults requiring valve replacement surgery. Disrupted embryonic growth, differentiation, and patterning events that "trigger" SLV disease are coordinated by gene expression changes in endocardial, myocardial, and cushion mesenchymal cells. We explored roles of chromatin regulation in valve gene regulatory networks by conditional inactivation of the Brg1-associated factor (BAF) chromatin remodeling complex in the endocardial lineage. Endocardial Brg1-deficient mouse embryos develop thickened and disorganized SLV cusps that frequently become bicuspid and myxomatous, including in surviving adults. These SLV disease-like phenotypes originate from deficient endocardial-to-mesenchymal transformation (EMT) in the proximal outflow tract (pOFT) cushions. The missing cells are replaced by compensating neural crest or other non-EMT-derived mesenchyme. However, these cells are incompetent to fully pattern the valve interstitium into distinct regions with specialized extracellular matrices. Transcriptomics reveal genes that may promote growth and patterning of SLVs and/or serve as disease-state biomarkers. Mechanistic studies of SLV disease genes should distinguish between disease origins and progression; the latter may reflect secondary responses to a disrupted developmental system.
Assuntos
Valva Aórtica/embriologia , DNA Helicases/fisiologia , Endocárdio/embriologia , Doenças das Valvas Cardíacas/etiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Fatores de Transcrição NFATC/fisiologiaRESUMO
Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1), and a cell cycle inhibitor, p21(cip1), to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNA-binding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.
Assuntos
DNA Helicases/genética , Células-Tronco Multipotentes/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Sistema Cardiovascular/citologia , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/metabolismo , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/irrigação sanguínea , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Camundongos , Microscopia de Fluorescência , Mutação , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/metabolismo , Proteínas Nucleares/metabolismo , Gravidez , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Understanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults. RESULTS: Using a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages. CONCLUSIONS: We conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.
Assuntos
Perfilação da Expressão Gênica/métodos , Neurogênese , Neurônios/citologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Neuropeptídeos/genética , Análise de Sequência de RNA/métodos , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Adult zebrafish fins regenerate to their original size regardless of damage extent, providing a tractable model of organ size and scale control. Gain-of-function of voltage-gated K + channels expressed in fibroblast-lineage blastema cells promotes excessive fin outgrowth, leading to a long-finned phenotype. Similarly, inhibition of the Ca 2+ -dependent phosphatase calcineurin during regeneration causes dramatic fin overgrowth. However, Ca 2+ fluxes and their potential origins from dynamic membrane voltages have not been explored or linked to fin size restoration. We used fibroblast-lineage GCaMP imaging of regenerating adult fins to identify dynamic and heterogeneous Ca 2+ transients in distal blastema cells. Membrane depolarization of isolated regenerating fin fibroblasts triggered Ca 2+ spikes dependent on voltage-gated Ca 2+ channel activity. Single cell transcriptomics identified the voltage-gated Ca 2+ channels cacna1c (L-type channel), cacna1ba (N-type), and cacna1g (T-type) as candidate mediators of fibroblast-lineage Ca 2+ signaling. Small molecule inhibition revealed L- and/or N-type voltage-gated Ca 2+ channels act during regenerative outgrowth to restore fins to their original scale. Strikingly, cacna1g homozygous mutant zebrafish regenerated extraordinarily long fins due to prolonged outgrowth. The regenerated fins far exceeded their original length but with otherwise normal ray skeletons. Therefore, cacna1g mutants uniquely provide a genetic loss-of-function long-finned model that decouples developmental and regenerative fin outgrowth. Live GCaMP imaging of regenerating fins showed T-type Cacna1g channels enable Ca 2+ dynamics in distal fibroblast-lineage blastemal mesenchyme during the outgrowth phase. We conclude "bioelectricity" for fin size control likely entirely reflects voltage-modulated Ca 2+ dynamics in fibroblast-lineage blastemal cells that specifically and steadily decelerates outgrowth at a rate tuned to restore the original fin size.
RESUMO
Unlike humans, some vertebrate animals are able to completely regenerate damaged appendages and other organs. For example, adult zebrafish will regenerate the complex structure of an amputated caudal fin to a degree that the original and replacement fins are indistinguishable. The blastema, a mass of cells that uniquely forms following appendage amputation in regenerating animals, is the major source of regenerated tissue. However, the cell lineage(s) that contribute to the blastema and their ultimate contribution(s) to the regenerated fin have not been definitively characterized. It has been suggested that cells near the amputation site dedifferentiate forming multipotent progenitors that populate the blastema and then give rise to multiple cell types of the regenerated fin. Other studies propose that blastema cells are non-uniform populations that remain restricted in their potential to contribute to different cell lineages. We tested these models by using inducible Cre-lox technology to generate adult zebrafish with distinct, isolated groups of genetically labeled cells within the caudal fin. We then tracked populations of several cell types over the entire course of fin regeneration in individual animals. We found no evidence for the existence of multipotent progenitors. Instead, multiple cell types, including epidermal cells, intra-ray fibroblasts, and osteoblasts, contribute to the newly regenerated tissue while remaining highly restricted with respect to their developmental identity. Our studies further demonstrate that the regenerating fin consists of many repeating blastema "units" dedicated to each fin ray. These blastemas each have an organized structure of lineage restricted, dedifferentiated cells that cooperate to regenerate the caudal fin.
Assuntos
Nadadeiras de Animais/citologia , Nadadeiras de Animais/fisiologia , Desdiferenciação Celular , Regeneração , Peixe-Zebra/fisiologia , Animais , Movimento Celular , Células Epidérmicas , Fibroblastos/citologia , Integrases/genética , Células-Tronco Multipotentes/citologia , Osteoblastos/citologiaRESUMO
Developing myocardial cells respond to signals from the endocardial layer to form a network of trabeculae that characterize the ventricles of the vertebrate heart. Abnormal myocardial trabeculation results in specific cardiomyopathies in humans and yet trabecular development is poorly understood. We show that trabeculation requires Brg1, a chromatin remodeling protein, to repress ADAMTS1 expression in the endocardium that overlies the developing trabeculae. Repression of ADAMTS1, a secreted matrix metalloproteinase, allows the establishment of an extracellular environment in the cardiac jelly that supports trabecular growth. Later during embryogenesis, ADAMTS1 expression initiates in the endocardium to degrade the cardiac jelly and prevent excessive trabeculation. Thus, the composition of cardiac jelly essential for myocardial morphogenesis is dynamically controlled by ADAMTS1 and its chromatin-based transcriptional regulation. Modification of the intervening microenvironment provides a mechanism by which chromatin regulation within one tissue layer coordinates the morphogenesis of an adjacent layer.
Assuntos
Proteínas ADAM/metabolismo , DNA Helicases/metabolismo , Endocárdio/metabolismo , Coração/embriologia , Morfogênese , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS1 , Animais , Linhagem Celular , DNA Helicases/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endotélio/citologia , Endotélio/metabolismo , Eritropoese , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/embriologia , Humanos , Camundongos , Neovascularização Fisiológica , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Saco Vitelino/irrigação sanguíneaRESUMO
Glycogen synthase kinase-3beta (GSK-3beta) has integral roles in a variety of biological processes, including development, diabetes, and the progression of Alzheimer's disease. As such, a thorough understanding of GSK-3beta function will have a broad impact on human biology and therapeutics. Because GSK-3beta interacts with many different pathways, its specific developmental roles remain unclear. We have discovered a genetic requirement for GSK-3beta in midline development. Homozygous null mice display cleft palate, incomplete fusion of the ribs at the midline and bifid sternum as well as delayed sternal ossification. Using a chemically regulated allele of GSK-3beta (ref. 6), we have defined requirements for GSK-3beta activity during discrete temporal windows in palatogenesis and skeletogenesis. The rapamycin-dependent allele of GSK-3beta produces GSK-3beta fused to a tag, FRB* (FKBP/rapamycin binding), resulting in a rapidly destabilized chimaeric protein. In the absence of drug, GSK-3beta(FRB)*(/FRB)* mutants appear phenotypically identical to GSK-3beta-/- mutants. In the presence of drug, GSK-3betaFRB* is rapidly stabilized, restoring protein levels and activity. Using this system, mutant phenotypes were rescued by restoring endogenous GSK-3beta activity during two distinct periods in gestation. This technology provides a powerful tool for defining windows of protein function during development.
Assuntos
Fissura Palatina/enzimologia , Fissura Palatina/patologia , Quinase 3 da Glicogênio Sintase/deficiência , Quinase 3 da Glicogênio Sintase/metabolismo , Alelos , Animais , Fissura Palatina/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Camundongos , Mutação/genética , Fenótipo , Sirolimo/farmacologiaRESUMO
Fraser Syndrome is a rare, multisystemic autosomal recessive disorder characterized by disrupted epithelial-mesenchymal associations upon loss of Fraser Complex genes. Disease manifestation and affected organs are highly variable. Digit malformations such as syndactyly are common but of unclear developmental origins. We explored if zebrafish fraser extracellular matrix complex subunit 1 (fras1) mutants model Fraser Syndrome-associated appendicular skeleton patterning defects. Approximately 10% of fras1 mutants survive to adulthood, displaying striking and varied fin abnormalities, including endochondral bone fusions, ectopic cartilage, and disrupted caudal fin symmetry. The fins of surviving fras1 mutants frequently have fewer and unbranched bony rays. fras1 mutant fins regenerate to their original size but with exacerbated ray branching and fin symmetry defects. Single cell RNA-Seq analysis, in situ hybridizations, and antibody staining show specific Fraser complex expression in the basal epidermis during regenerative outgrowth. Fras1 and Fraser Complex component Frem2 accumulate along the basal side of distal-most basal epidermal cells. Greatly reduced and mislocalized Frem2 accompanies loss of Fras1 in fras1 mutants. The Sonic hedgehog signaling between distal basal epidermis and adjacent mesenchymal pre-osteoblasts that promotes ray branching persists upon Fraser Complex loss. However, fras1 mutant regenerating fins exhibit extensive sub-epidermal blistering associated with a disorganized basal epidermis and adjacent pre-osteoblasts. We propose Fraser Complex-supported tissue layer adhesion enables robust integrated tissue morphogenesis involving the basal epidermis and osteoblasts. Further, we establish zebrafish fin development and regeneration as an accessible model to explore mechanisms of Fraser Syndrome-associated digit defects and Fraser Complex function at epithelial-mesenchymal interfaces.
RESUMO
Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.
Assuntos
Valvas Cardíacas/embriologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Endocárdio/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/embriologia , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Modelos Cardiovasculares , Gravidez , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Across species, hematopoietic stem and progenitor cells (HSPCs) arise during embryogenesis from a specialized arterial population, termed hemogenic endothelium. Here, we describe a mechanistic role for the epigenetic regulator, Enhancer of zeste homolog-1 (Ezh1), in vertebrate HSPC production via regulation of hemogenic commitment. Loss of ezh1 in zebrafish embryos favored acquisition of hemogenic (gata2b) and HSPC (runx1) fate at the expense of the arterial program (ephrinb2a, dll4). In contrast, ezh1 overexpression blocked hematopoietic progression via maintenance of arterial gene expression. The related Polycomb group subunit, Ezh2, functioned in a non-redundant, sequential manner, whereby inhibition had no impact on arterial identity, but was capable of blocking ezh1-knockdown-associated HSPC expansion. Single-cell RNA sequencing across ezh1 genotypes revealed a dropout of ezh1+/- cells among arterial endothelium associated with positive regulation of gene transcription. Exploitation of Ezh1/2 modulation has potential functional relevance for improving in vitro HSPC differentiation from induced pluripotent stem cell sources.