RESUMO
Modulation of bone morphogenetic protein (BMP) activity is essential to the progression of limb development in the mouse embryo. Genetic disruption of BMP signaling at various stages of limb development causes defects ranging from complete limb agenesis to oligodactyly, polydactyly, webbing, and chondrodysplasia. To probe the state of BMP signaling in early limb buds, we designed two sets of primers to measure both spatially and quantitatively the transcription of nine key genes indicative of canonical BMP activity. One set is used to generate digoxigenin (DIG)-labeled antisense RNA probes for whole-mount mRNA in situ hybridization, while the second set is used for SYBR® Green-based quantitative PCR on limb bud cDNA. Here we describe step-by-step protocols for both methods around this specific set of genes.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
BACKGROUND: The endogenous ability to dedifferentiate, re-pattern, and re-differentiate adult cells to repair or replace damaged or missing structures is exclusive to only a few tetrapod species. The Mexican axolotl is one example of these species, having the capacity to regenerate multiple adult structures including their limbs by generating a group of progenitor cells, known as the blastema, which acquire pattern and differentiate into the missing tissues. The formation of a limb regenerate is dependent on cells in the connective tissues that retain memory of their original position in the limb, and use this information to generate the pattern of the missing structure. Observations from recent and historic studies suggest that blastema cells vary in their potential to pattern distal structures during the regeneration process; some cells are plastic and can be reprogrammed to obtain new positional information while others are stable. Our previous studies showed that positional information has temporal and spatial components of variation; early bud (EB) and apical late bud (LB) blastema cells are plastic while basal-LB cells are stable. To identify the potential cellular and molecular basis of this variation, we compared these three cell populations using histological and transcriptional approaches. RESULTS: Histologically, the basal-LB sample showed greater tissue organization than the EB and apical-LB samples. We also observed that cell proliferation was more abundant in EB and apical-LB tissue when compared to basal-LB and mature stump tissue. Lastly, we found that genes associated with cellular differentiation were expressed more highly in the basal-LB samples. CONCLUSIONS: Our results characterize histological and transcriptional differences between EB and apical-LB tissue compared to basal-LB tissue. Combined with our results from a previous study, we hypothesize that the stability of positional information is associated with tissue organization, cell proliferation, and pathways of cellular differentiation.
Assuntos
Ambystoma mexicanum/embriologia , Plasticidade Celular/genética , Extremidades/embriologia , Botões de Extremidades/embriologia , Regeneração/genética , Ambystoma mexicanum/genética , Animais , Diferenciação Celular/genética , Plasticidade Celular/fisiologia , Proliferação de Células/genética , Botões de Extremidades/fisiologia , Regeneração/fisiologia , Transdução de Sinais/genéticaRESUMO
We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Botões de Extremidades/efeitos dos fármacos , Tecido Nervoso/citologia , Ambystoma mexicanum , Animais , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Botões de Extremidades/citologia , Botões de Extremidades/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Tecido Nervoso/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteínas Smad/metabolismoRESUMO
Amphibians can regenerate missing body parts, including limbs. The regulation of collagen has been considered to be important in limb regeneration. Collagen deposition is suppressed during limb regeneration, so we investigated collagen deposition and apical epithelial cap (AEC) formation during axolotl limb regeneration. The accessory limb model (ALM) has been developed as an alternative model for studying limb regeneration. Using this model, we investigated the relationship between nerves, epidermis, and collagen deposition. We found that Sp-9, an AEC marker gene, was upregulated by direct interaction between nerves and epidermis. However, collagen deposition hindered this interaction, and resulted in the failure of limb regeneration. During wound healing, an increase in deposition of collagen caused a decrease in the blastema induction rate in ALM. Wound healing and limb regeneration are alternate processes.
Assuntos
Ambystoma mexicanum/fisiologia , Colágeno/fisiologia , Regeneração/fisiologia , Animais , Extremidades/inervação , Extremidades/fisiologia , Botões de Extremidades/fisiologia , Pele/inervação , Fenômenos Fisiológicos da PeleRESUMO
This paper analyzes the influence of infrared radiation (IR) on regeneration, after autotomy of limb buds of Neohelice granulata and consequently the time molt. Eyestalks were ablated to synchronize the start of molt. Afterward, animals were autotomized of five pereopods and divided into control and irradiated groups. The irradiated group was treated for 30 min daily until molt. Limb buds from five animals of days 4, 16 and 20 were collected and histological sections were made from them. These sections were photographed and chitin and epithelium content measured. Another group was made, and after 15 days limb buds were extracted to analyze mitochondrial enzymatic activity from complex I and II. The irradiated group showed a significant reduction in molt time (19.38+/-1.22 days) compared with the control group (32.69+/-1.57 days) and also a significant increase in mitochondrial complex I (388.9+/-27.94%) and II (175.63+/-7.66%) in the irradiated group when compared with the control group (100+/-17.90; 100+/-7.82, respectively). However, these effects were not accompanied by histological alterations in relation to chitin and epithelium. This way, it was possible to demonstrate that IR increases complex I and II activity, reduces the time molt and consequently increases the appendage regeneration rate.
Assuntos
Crustáceos/efeitos da radiação , Raios Infravermelhos , Muda , Regeneração , Animais , Crustáceos/fisiologia , Botões de Extremidades/crescimento & desenvolvimento , Botões de Extremidades/fisiologia , Botões de Extremidades/efeitos da radiaçãoRESUMO
Antecedentes: la tosferina es una enfermedad respiratoria aguda de una alta transmisibilidad sobre todo en ámbitos intrafamiliares o intradomiciliarios.En febrero de 2008, fue informado un brote de tosferina con casos confirmados por nexo epidemiológico, algunos de los cuales no presentaban vacunación certificada y se carecía de claridad sobre sus características clínico-epidemiológicas para determinar el comportamiento de la enfermedad.Materiales y Métodos:estudio descriptivo de corte transversal.Se estableció la definición de caso confirmado por laboratorio y confirmado por nexo epidemiológico; además, se realizó búsqueda activa comunitaria e institucional, barrido de vacunación y monitoreo rápido de coberturas de vacunación y establecimiento de medidas quimio-profilácticas.Resultados:se confirmó un caso por laboratorio y 4 por nexo epidemiológico.Período de incubación =16,2 días; tasa de ataque total para las dos viviendas implicadas 33 por ciento (5/15).De las 52 casas visitadas, 42 fueron efectivas, no se presentó ausentismo de niños en las instituciones visitadas y se evidenció 100 por ciento de cobertura para DPT.De 24170 registros revisados, 3227 tenían diagnóstico compatible, 17 fueron clasificados como probables de tosferina, 7 investigados.Conclusiones:existen dificultades en la captación de casos de tosferina, subnotificación al SIVIGILA y dificultades en el diagnóstico por laboratorio para el aislamiento de la bacteria; se hace necesario reforzar la vigilancia del evento por parte de todas instituciones de salud y fortalecer la capacidad del laboratorio de salud pública...
Assuntos
Humanos , Botões de Extremidades/imunologia , CoquelucheRESUMO
Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. In the present study, we have utilized the ALM to identity the buttonhead-like zinc-finger transcription factor, Sp9, as being involved in the formation of the regeneration epithelium. Sp9 expression is induced in basal keratinocytes of the apical blastema epithelium in a pattern that is comparable to its expression in developing limb buds, and it thus is an important marker for dedifferentiation of the epidermis. Induction of Sp9 expression is nerve-dependent, and we have identified KGF as an endogenous nerve factor that induces expression of Sp9 in the regeneration epithelium.
Assuntos
Ambystoma mexicanum/fisiologia , Células Epidérmicas , Epiderme/fisiologia , Botões de Extremidades/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Regeneração , Cicatrização , Animais , Metaloproteinase 9 da Matriz/metabolismo , Modelos AnimaisRESUMO
Molecular mechanisms patterning the tetrapod limb, including anterior-posterior axis determination involving Sonic hedgehog (Shh), have received much attention, particularly in amniotes. Anterior-posterior patterning in urodele amphibians differs radically from that of amniotes in that it shows a pronounced anterior-to-posterior sequence of digit development. In contrast, amniotes develop their digits almost simultaneously with a slight posterior-to-anterior polarity. Here we use cyclopamine, an inhibitor of the Hedgehog signaling pathway, to investigate the role of Shh in anterior-posterior patterning in the urodele limb. Inhibition of Shh signal transduction affects digit number long before their morphological appearance. In accordance with the apparently derived order of digit development in urodeles, exposure reproducibly removes digits in a posterior-to-anterior sequence, the inverse of their developmental sequence. This pattern of digit loss mimics the order of digit loss in natural variation. We suggest that variation in Shh expression and/or signal transmission may explain natural variation in digit number in urodeles.
Assuntos
Padronização Corporal , Extremidades/embriologia , Proteínas Hedgehog/fisiologia , Transdução de Sinais , Ambystoma mexicanum , Animais , Proteínas Hedgehog/antagonistas & inibidores , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Fenótipo , Filogenia , Alcaloides de Veratrum/farmacologiaRESUMO
Topological and histological analyses of Mabuya mabouya embryos at different developmental stages showed an extraembryonic membrane sequence as follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula stages; mesodermal split and formation of an exocoelom in the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to form a chorioallantoic membrane at pharyngula stages; complete extension of the allantois into the exocoelom between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac placenta; chorioplacenta; allantoplacenta. All placentas are highly specialized for nutrient absorption from early developmental stages. The bistratified extraembryonic ectoderm possesses an external layer with cuboidal cells and a microvillar surface around the whole yolk sac, which absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of development. The dorsal absorptive plaque will constitute the placentome and paraplacentome during allantoplacental development. At late gastrula-early neurula stages some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and seem to have a specialized histotrophic and/or metabolic role. The extraembryonic mesoderm does not ingress into the yolk sac and neither an isolated yolk mass nor a yolk cleft are formed. This derived pattern of development may be related to the drastic reduction of the egg size and obligatory placentotrophy from early developmental stages. Our results show new specialized placentotrophic structures and a novel arrangement of extraembryonic membrane morphogenesis for Squamata.
Assuntos
Desenvolvimento Embrionário , Lagartos/embriologia , Animais , Ectoderma/citologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/citologia , Endoderma/citologia , Epitélio/anatomia & histologia , Feminino , Gástrula/citologia , Botões de Extremidades/anatomia & histologia , Botões de Extremidades/citologia , Botões de Extremidades/crescimento & desenvolvimento , Lagartos/anatomia & histologia , Mesoderma/citologia , Morfogênese , Mucosa/anatomia & histologia , Mucosa/citologia , Oviductos/anatomia & histologia , Óvulo/citologia , Gravidez , América do Sul , Útero/anatomia & histologia , Útero/citologiaRESUMO
During limb skeletal muscle formation, committed muscle cells proliferate and differentiate in the presence of extracellular signals that stimulate or repress each process. Proteoglycans are extracellular matrix organizers and modulators of growth factor activities, regulating muscle differentiation in vitro. Previously, we characterized proteoglycan expression during early limb muscle formation and showed a spatiotemporal relation between the onset of myogenesis and the expression of decorin, an important muscle extracellular matrix component and potent regulator of TGF-beta activity. To evaluate decorin's role during in vivo differentiation in committed muscle cells, we grafted wild type and decorin-null myoblasts onto chick limb buds. The absence of decorin enhanced the migration and distribution of myoblasts in the limb, correlating with the inhibition of skeletal muscle differentiation. Both phenotypes were reverted by de novo decorin expression. In vitro, we determined that both decorin core protein and its glycosaminoglycan chain were required to reverse the migration phenotype. Results presented here suggest that the enhanced migration observed in decorin-null myoblasts may not be dependent on chemotactic growth factor signaling nor the differentiation status of the cells. Decorin may be involved in the establishment and/or coordination of a critical myoblast density, through inhibition of migration, that permits normal muscle differentiation during embryonic myogenesis.
Assuntos
Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Proteoglicanas/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Movimento Celular , Transplante de Células , Embrião de Galinha , Técnicas de Cocultura , Decorina , Proteínas da Matriz Extracelular , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos , Botões de Extremidades/citologia , Botões de Extremidades/crescimento & desenvolvimento , Camundongos , Músculo Esquelético/citologia , Mioblastos Esqueléticos/citologia , Miogenina/metabolismo , Retroviridae/genética , Fator de Crescimento Transformador beta/metabolismo , Transplante Heterólogo , beta-Galactosidase/metabolismoRESUMO
After arriving at the limb bud, migrating myogenic precursor cells express transcription factors responsible for the induction of terminal skeletal muscle differentiation. One such factor is myogenin, a member of the basic helix-loop-helix family, known to activate the expression of muscle-specific genes. The extracellular signals involved in activating the myogenic program in the muscle precursor cells that reach the limb in vivo are not known. However, in vitro, it has been shown that proteoglycans, macromolecules composed of a core protein and glycosaminoglycan chains, modulate the triggering of myogenin activity. To understand the role of proteoglycans during limb muscle development, we assessed the synthesis of proteoglycans in limb bud explants at 10.5 days post coitum, when migrating cells arrive, evaluated the expression and nature of these macromolecules during in vivo early limb bud formation, and determined the colocalization of myoblasts expressing myogenin with specific proteoglycans. We found that the expression of myogenin was temporally and spatially coincident with the expression of syndecan-3 and decorin, two essential proteoglycans in the modulation of skeletal muscle differentiation. This article is the first report of myogenic activation and proteoglycan expression during limb muscle formation.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteoglicanas de Heparan Sulfato/genética , Músculo Esquelético/embriologia , Animais , Linhagem da Célula/fisiologia , Proteoglicanas de Sulfatos de Condroitina/análise , Decorina , Proteínas da Matriz Extracelular , Feminino , Proteoglicanas de Heparan Sulfato/análise , Botões de Extremidades/citologia , Botões de Extremidades/embriologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Camundongos , Músculo Esquelético/química , Músculo Esquelético/citologia , Gravidez , Proteoglicanas/análise , Proteoglicanas/genética , RNA Mensageiro/análise , Células-Tronco/química , Células-Tronco/fisiologia , Sindecana-3Assuntos
Cartilagem/citologia , Cartilagem/fisiologia , Diferenciação Celular/efeitos dos fármacos , Proteoglicanas/biossíntese , Fator de Crescimento Transformador beta/farmacologia , Animais , Cartilagem/efeitos dos fármacos , Células Cultivadas , Cinética , Botões de Extremidades , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , CamundongosRESUMO
An antibody raised against the recombinant Xenopus laevis Hoxb-7 protein (López and Carrasco [1992] Mech. Dev. 36:153-164) recognizes the 30 kDa translation product of the Hoxb-7 gene in X. laevis and the cognate nuclear protein in chicken embryos. The X. laevis Hoxb-7 protein was expressed maternally and zygotically. Treatment of X. laevis and chicken embryos with either all-trans retinoic acid (RA) or the retinoid antagonist Ro 41-5253 (Ro; Apfel et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:7129-7133) during early development induced malformations of the neural tube and complementary changes in the expression domain of the homeoprotein Hoxb-7. Treatment of X. laevis embryos with retinoic acid during gastrulation induced an anterior shift of the Hoxb-7 expression domain and was correlated with an enlargement of rhombomere r7. In addition to a reduction in rhombomere numbers and of forebrain size, various malformations involving all three germ layers were observed. Treatment of X. laevis embryos with the antagonist Ro before or during gastrulation caused a progressive reduction of the Hoxb-7 domain and also dose-dependent malformations of all three germ layers. RA or Ro treatment of chicken embryos from the beginning of gastrulation caused changes of the Hoxb-7 expression domain very similar to those observed in X. laevis. In particular, either a dose-dependent loss of the Hoxb-7 protein in the neural tube or an ectopic expression in the forebrain region was observed. The results of this study indicate that endogenous retinoids regulate the spatial expression of homeobox-containing genes in vertebrates.