RESUMEN
Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10, which triggers molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5), and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4, zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing 1 allele of Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre-mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation.
Asunto(s)
Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior , Proteínas con Homeodominio LIM , Factores de Transcripción , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ratones , Miembro Posterior/embriología , Miembro Posterior/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Esbozos de los Miembros/metabolismo , Esbozos de los Miembros/embriología , Ratones Noqueados , Embrión de Mamíferos/metabolismo , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismoRESUMEN
The synovial cavity and its fluid are essential for joint function and lubrication, but their developmental biology remains largely obscure. Here, we analyzed E12.5 to E18.5 mouse embryo hindlimbs and discovered that cavitation initiates around E15.0 with emergence of multiple, discrete, µm-wide tissue discontinuities we term microcavities in interzone, evolving into a single joint-wide cavity within 12â h in knees and within 72-84â h in interphalangeal joints. The microcavities were circumscribed by cells as revealed by mTmG imaging and exhibited a carbohydrate and protein content based on infrared spectral imaging at micro and nanoscale. Accounting for differing cavitation kinetics, we found that the growing femur and tibia anlagen progressively flexed at the knee over time, with peak angulation around E15.5 exactly when the full knee cavity consolidated; however, interphalangeal joint geometry changed minimally over time. Indeed, cavitating knee interzone cells were elongated along the flexion angle axis and displayed oblong nuclei, but these traits were marginal in interphalangeal cells. Conditional Gdf5Cre-driven ablation of Has2 - responsible for production of the joint fluid component hyaluronic acid (HA) - delayed the cavitation process. Our data reveal that cavitation is a stepwise process, brought about by sequential action of microcavities, skeletal flexion and elongation, and HA accumulation. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Miembro Posterior , Articulaciones , Animales , Embrión de Mamíferos , Miembro Posterior/embriología , Articulaciones/embriología , RatonesRESUMEN
Thyroid hormone (T3) receptors (TRs) mediate T3 effects on vertebrate development. We have studied Xenopus tropicalis metamorphosis as a model for postembryonic human development and demonstrated that TRα knockout induces precocious hind limb development. To reveal the molecular pathways regulated by TRα during limb development, we performed chromatin immunoprecipitation- and RNA-sequencing on the hind limb of premetamorphic wild type and TRα knockout tadpoles, and identified over 700 TR-bound genes upregulated by T3 treatment in wild type but not TRα knockout tadpoles. Interestingly, most of these genes were expressed at higher levels in the hind limb of premetamorphic TRα knockout tadpoles than stage-matched wild-type tadpoles, suggesting their derepression upon TRα knockout. Bioinformatic analyses revealed that these genes were highly enriched with cell cycle and Wingless/Integrated (Wnt) signaling-related genes. Furthermore, cell cycle and Wnt signaling pathways were also highly enriched among genes bound by TR in wild type but not TRα knockout hind limb. These findings suggest that direct binding of TRα to target genes related to cell cycle and Wnt pathways is important for limb development: first preventing precocious hind limb formation by repressing these pathways as unliganded TR before metamorphosis and later promoting hind limb development during metamorphosis by mediating T3 activation of these pathways.
Asunto(s)
Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Metamorfosis Biológica , Organogénesis , Receptores alfa de Hormona Tiroidea/metabolismo , Vía de Señalización Wnt , Animales , Femenino , Masculino , Receptores alfa de Hormona Tiroidea/genética , Xenopus laevisRESUMEN
The chick limb bud has plasticity to reconstruct a normal skeletal pattern after a part of mesenchymal mass is excised to make a hole in its early stage of development. To understand the details of hole closure and re-establishment of normal limb axes to reconstruct a normal limb skeleton, we focused on cellular and molecular changes during hole repair and limb restoration. We excised a cube-shaped mass of mesenchymal cells from the medial region of chick hindlimb bud (stage 23) and observed the following morphogenesis. The hole had closed by 15 âh after excision, followed by restoration of the limb bud morphology, and the cartilage pattern was largely restored by 48 âh. Lineage analysis of the mesenchymal cells showed that cells at the anterior and posterior margins of the hole were adjoined at the hole closure site, whereas cells at the proximal and distal margins were not. To investigate cell polarity during hole repair, we analyzed intracellular positioning of the Golgi apparatus relative to the nuclei. We found that the Golgi apparatus tended to be directed toward the hole among cells at the anterior and posterior margins but not among cells at identical positions in normal limb buds or cells at the proximal and distal hole margins. In the manipulated limb buds, the frequency of cell proliferation was maintained compared with the control side. Tbx3 expression, which was usually restricted to anterior and posterior margins of the limb bud, was temporarily expanded medially and then reverted to a normal pattern as limb reconstruction proceeded, with Tbx3 negative cells reappearing in the medial regions of the limb buds. Thus, mesenchymal hole closure and limb reconstruction are mainly mediated by cells at the anterior and posterior hole margins. These results suggest that adjustment of cellular properties along the anteroposterior axis is crucial to restore limb damage and reconstruct normal skeletal patterns.
Asunto(s)
Tipificación del Cuerpo/fisiología , Esbozos de los Miembros/citología , Esbozos de los Miembros/embriología , Células Madre Mesenquimatosas/metabolismo , Mesodermo/citología , Mesodermo/embriología , Esqueleto/embriología , Animales , Proteínas Aviares/metabolismo , Núcleo Celular/metabolismo , Polaridad Celular/fisiología , Proliferación Celular/fisiología , Embrión de Pollo , Extremidades/embriología , Aparato de Golgi/metabolismo , Miembro Posterior/embriología , Transducción de Señal/fisiología , Esqueleto/citología , Esqueleto/metabolismo , Proteínas de Dominio T Box/metabolismoRESUMEN
Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción Paired Box/genética , Acetilación , Animales , Cromatina/química , Cromatina/metabolismo , Tejido Conectivo/crecimiento & desarrollo , Tejido Conectivo/metabolismo , Embrión de Mamíferos , Epigénesis Genética , Miembro Posterior/citología , Miembro Posterior/embriología , Miembro Posterior/metabolismo , Esbozos de los Miembros/citología , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Ratones , Modelos Genéticos , Factores de Transcripción Paired Box/metabolismo , Eliminación de SecuenciaRESUMEN
SMAD4 regulates gene expression in response to BMP and TGFß signal transduction, and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously downregulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in upregulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are upregulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH- and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.
Asunto(s)
Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Hedgehog/metabolismo , Esbozos de los Miembros/embriología , Transducción de Señal , Proteína Smad4/metabolismo , Animales , Proteínas Morfogenéticas Óseas/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Miembro Posterior/embriología , Ratones , Ratones Transgénicos , Proteína Smad4/genéticaRESUMEN
In vertebrates, 5'-Hoxd genes (Hoxd9), which are expressed in the hindlimb bud mesenchyme, participate in limb growth and patterning in early embryonic development. In the present study, We investigated the mechanisms by which ATRA regulates cultured E12.5 rat embryo hindlimb bud mesenchymal cells (rEHBMCs). Following exposure to ATRA over 24 h, mRNA and protein expression levels of HoxD9 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), and western blotting. Flow cytometry was used to detect apoptosis. ATRA inhibited the condensation and proliferation, and promoted the apoptosis rate of the rEHBMCs in a dose-dependent manner. Sox9 and Col2a1 in rEHBMCs were downregulated by ATRA in a dose-dependent manner at both mRNA and protein levels. Similarly, HoxD9 was downregulated by ATRA in a dose-dependent manner, in parallel with the cartilage-specific molecules Sox9 and Col2a1. Both qPCR and western blotting showed that both Shh and Gli3 were downregulated. Overexpression of HoxD9 reversed the effects of ATRA. These results demonstrate that ATRA suppresses chondrogenesis in rEHBMCs by inhibiting the expression of HoxD9 and its downstream protein targets, including Sox9 and Col2a1. This effect may also be correlated with inhibition of the Shh-Gli3 signaling pathway.
Asunto(s)
Condrogénesis/efectos de los fármacos , Miembro Posterior , Proteínas de Homeodominio/genética , Proteínas de Neoplasias/genética , Tretinoina/farmacología , Animales , Células Cultivadas , Pie Equinovaro , Embrión de Mamíferos/efectos de los fármacos , Miembro Posterior/efectos de los fármacos , Miembro Posterior/embriología , Proteínas de Homeodominio/metabolismo , Proteínas de Neoplasias/metabolismo , RatasRESUMEN
Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb-reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in Chalcides and limb reduction in Scelotes). The genus Brachymeles, a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake-like species Brachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos of B. lukbani in early stages of development, on the other hand, show a truncated but well-developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole-mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb of B. lukabni. Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well-developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well-developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well-developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that "limbless" lizards that possess these muscles may have or have had at least transient developing limbs, as we find in B. lukbani. Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation called cryptomelia. The results of this work add to our growing understanding of clade-specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes.
Asunto(s)
Desarrollo Embrionario/fisiología , Miembro Anterior/anatomía & histología , Miembro Posterior/anatomía & histología , Lagartos/anatomía & histología , Animales , Miembro Anterior/embriología , Miembro Posterior/embriología , FilogeniaRESUMEN
The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1R206H/+;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1R206H mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1R206H mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Articulaciones/embriología , Miositis Osificante/embriología , Miositis Osificante/metabolismo , Dedos del Pie/embriología , Animales , Tipificación del Cuerpo , Condrogénesis , Modelos Animales de Enfermedad , Miembro Anterior/anomalías , Miembro Anterior/embriología , Factor 5 de Diferenciación de Crecimiento/metabolismo , Placa de Crecimiento/embriología , Miembro Posterior/anomalías , Miembro Posterior/embriología , Articulaciones/anomalías , Articulaciones/metabolismo , Ratones , Osteogénesis , Transducción de Señal , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Células Madre/fisiología , Dedos del Pie/anomalíasRESUMEN
L-arginine/NOS/NO signaling pathway plays a critical role in controlling variety of vascular diseases. However, whether NOS inhibition by L-NAME suppresses late embryonic development is undefined. The aim of this study is to determine whether NOS inhibition by L-NAME is critical for late embryonic rat hind limb development. The pregnant rat at E13.5 administrated L-NAME by consecutive intraperitoneal injection. The embryos been harvested from E16.5 to E 20.5. Hematoxylin and Eosin Staining, Immunofluorescence and Immunohistochemistry performed to determine hind limb Vasculogenesis, HUVEC culture, Adenoviral PFKFB3 infection, Real time PCR and western blot were performed to determine whether L-arginine/NOS/NO pathway controlling late embryonic hind limb development through PFKFB3 mediated angiogenetic pathway. NOS inhibition by L-NAME resulting in late embryonic hind limb developmental defects characterized by severe hemorrhage. The in vivo studies showed that NOS inhibition strongly suppressed hind limb angiogenetic remodeling by impairing differentiation of endothelial cells and smooth muscle cells, and extracellular matrix synthesis. For underlie mechanism, our studies indicated that L-NAME treatment dramatically suppresses PFKFB3 expression in hematopoietic progenitor cells, tubulogenetic endothelial cells and smooth muscle cells. Knockdown of PFKFB3 dramatically inhibits the expression of angiogenetic genes, as well as tubulogenesis and extracellular matrix related genes. Taken together, our data in this study demonstrated that L-arginine-eNOS-NO pathway is important for rat hind limb development during late embryonic stage. This could be both a useful animal model and a promising therapeutic treatment for defects of late embryonic developmental hind limbs.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Miembro Posterior/embriología , NG-Nitroarginina Metil Éster/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Fosfofructoquinasa-2/metabolismo , Animales , Femenino , Miembro Posterior/irrigación sanguínea , Embarazo , Ratas , Flujo Sanguíneo Regional/efectos de los fármacosRESUMEN
Disruption of the minor spliceosome due to mutations in RNU4ATAC is linked to primordial dwarfism in microcephalic osteodysplastic primordial dwarfism type 1, Roifman syndrome, and Lowry-Wood syndrome. Similarly, primordial dwarfism in domesticated animals is linked to positive selection in minor spliceosome components. Despite being vital for limb development and size regulation, its role remains unexplored. Here, we disrupt minor spliceosome function in the developing mouse limb by ablating one of its essential components, U11 small nuclear RNA, which resulted in micromelia. Notably, earlier loss of U11 corresponded to increased severity. We find that limb size is reduced owing to elevated minor intron retention in minor intron-containing genes that regulate cell cycle. As a result, limb progenitor cells experience delayed prometaphase-to-metaphase transition and prolonged S-phase. Moreover, we observed death of rapidly dividing, distally located progenitors. Despite cell cycle defects and cell death, the spatial expression of key limb patterning genes was maintained. Overall, we show that the minor spliceosome is required for limb development via size control potentially shared in disease and domestication.
Asunto(s)
Enanismo/genética , Extremidades/embriología , Retardo del Crecimiento Fetal/genética , Microcefalia/genética , Osteocondrodisplasias/genética , ARN Nuclear Pequeño/metabolismo , Animales , Tipificación del Cuerpo/genética , Ciclo Celular/genética , Femenino , Miembro Anterior/embriología , Miembro Anterior/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Miembro Posterior/ultraestructura , Intrones/genética , Masculino , Ratones Endogámicos C57BL , Mutación/genética , ARN Nuclear Pequeño/genética , Células Madre/metabolismoRESUMEN
Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms.
Asunto(s)
Embrión de Pollo/embriología , Pollos/genética , Condrogénesis/genética , Metilación de ADN , Inestabilidad Genómica , Miembro Posterior/metabolismo , Huesos de la Pierna/embriología , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Diferenciación Celular/genética , Expresión Génica , Miembro Posterior/embriologíaRESUMEN
Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5' Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5' Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb.
Asunto(s)
Embrión de Mamíferos/embriología , Geminina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Animales , Embrión de Mamíferos/citología , Geminina/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Miembro Posterior/citología , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Factores de Transcripción/genéticaRESUMEN
The coordinated spatial and temporal regulation of gene expression in the murine hindlimb determines the identity of mesenchymal progenitors and the development of diversity of musculoskeletal tissues they form. Hindlimb development has historically been studied with lineage tracing of individual genes selected a priori, or at the bulk tissue level, which does not allow for the determination of single cell transcriptional programs yielding mature cell types and tissues. To identify the cellular trajectories of lineage specification during limb bud development, we used single cell mRNA sequencing (scRNA-seq) to profile the developing murine hindlimb between embryonic days (E)11.5-E18.5. We found cell type heterogeneity at all time points, and the expected cell types that form the mouse hindlimb. In addition, we used RNA fluorescence in situ hybridization (FISH) to examine the spatial locations of cell types and cell trajectories to understand the ancestral continuum of cell maturation. This data provides a resource for the transcriptional program of hindlimb development that will support future studies of musculoskeletal development and generate hypotheses for tissue regeneration.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Miembro Posterior/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Animales , Diferenciación Celular , Linaje de la Célula , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Miembro Posterior/metabolismo , Hibridación Fluorescente in Situ , Masculino , Ratones , Análisis de Secuencia de ARNRESUMEN
The tetrapod limb has long served as a paradigm to study vertebrate pattern formation. During limb morphogenesis, a number of distinct tissue types are patterned and subsequently must be integrated to form coherent functional units. For example, the musculoskeletal apparatus of the limb requires the coordinated development of the skeletal elements, connective tissues, muscles and nerves. Here, using light-sheet microscopy and 3D-reconstructions, we concomitantly follow the developmental emergence of nerve and muscle patterns in chicken wings and legs, two appendages with highly specialized locomotor outputs. Despite a comparable flexor/extensor-arrangement of their embryonic muscles, wings and legs show a rotated innervation pattern for their three main motor nerve branches. To test the functional implications of these distinct neuromuscular topologies, we challenge their ability to adapt and connect to an experimentally altered skeletal pattern in the distal limb, the autopod. Our results show that, unlike autopod muscle groups, motor nerves are unable to fully adjust to a changed peripheral organisation, potentially constrained by their original projection routes. As the autopod has undergone substantial morphological diversifications over the course of tetrapod evolution, our results have implications for the coordinated modification of the distal limb musculoskeletal apparatus, as well as for our understanding of the varying degrees of motor functionality associated with human hand and foot malformations.
Asunto(s)
Miembro Posterior/embriología , Alas de Animales/embriología , Animales , Embrión de Pollo , Pollos , Extremidades/embriología , Músculos/embriología , Sistema Nervioso/embriología , Organogénesis/fisiologíaRESUMEN
During skeletal development, limb progenitors become specified as chondrocytes and subsequently differentiate into specialized cartilage compartments. We previously showed that the arginine dimethyl transferase, PRMT5, is essential for regulating the specification of progenitor cells into chondrocytes within early limb buds. Here, we report that PRMT5 regulates the survival of a separate progenitor domain that gives rise to the patella. Independent of its role in knee development, PRMT5 regulates several distinct types of chondrocyte differentiation within the long bones. Chondrocytes lacking PRMT5 have a striking blockage in hypertrophic chondrocyte differentiation and are marked by abnormal gene expression. PRMT5 remains important for articular cartilage and hypertrophic cell identity during adult stages, indicating an ongoing role in homeostasis of these tissues. We conclude that PRMT5 is required for distinct steps of early and late chondrogenic specialization and is thus a critical component of multiple aspects of long bone development and maintenance.
Asunto(s)
Cartílago/metabolismo , Rótula/embriología , Proteína-Arginina N-Metiltransferasas/metabolismo , Animales , Desarrollo Óseo , Huesos/metabolismo , Cartílago/embriología , Cartílago Articular/citología , Diferenciación Celular/genética , Condrocitos/metabolismo , Condrogénesis/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Esbozos de los Miembros , Masculino , Ratones , Proteína-Arginina N-Metiltransferasas/genética , Células Madre/citologíaRESUMEN
Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectoderm-derived surface epithelium. Here, we report means for induction of an LB-like mesenchymal/epithelial complex tissues from murine pluripotent stem cells (PSCs) in vitro. The LB-like tissues selectively differentiate into forelimb- or hindlimb-type mesenchymes, depending on a concentration of retinoic acid. Comparative transcriptome analysis reveals that the LB-like tissues show similar gene expression pattern to that seen in LBs. We also show that manipulating BMP signaling enables us to induce a thickened epithelial structure similar to the apical ectodermal ridge. Finally, we demonstrate that the induced tissues can contribute to endogenous digit tissue after transplantation. This PSC technology offers a first step for creating an artificial limb bud in culture and might open the door to inducing other mesenchymal/epithelial complex tissues from PSCs.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Esbozos de los Miembros/embriología , Células Madre Embrionarias de Ratones/fisiología , Ingeniería de Tejidos/métodos , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Ectodermo/citología , Ectodermo/metabolismo , Embrión de Mamíferos , Desarrollo Embrionario , Epitelio/metabolismo , Femenino , Miembro Anterior/embriología , Miembro Anterior/trasplante , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/fisiología , Miembro Posterior/embriología , Miembro Posterior/trasplante , Esbozos de los Miembros/trasplante , Masculino , Ratones , Células Madre Embrionarias de Ratones/trasplante , Transducción de Señal/fisiologíaRESUMEN
The tetrapod limb is a stunning example of evolutionary diversity, with dramatic variation not only among distantly related species, but also between the serially homologous forelimbs (FLs) and hindlimbs (HLs) within species. Despite this variation, highly conserved genetic and developmental programs underlie limb development and identity in all tetrapods, raising the question of how limb diversification is generated from a conserved toolkit. In some breeds of domestic pigeon, shifts in the expression of two conserved limb identity transcription factors, PITX1 and TBX5, are associated with the formation of feathered HLs with partial FL identity. To determine how modulation of PITX1 and TBX5 expression affects downstream gene expression, we compared the transcriptomes of embryonic limb buds from pigeons with scaled and feathered HLs. We identified a set of differentially expressed genes enriched for genes encoding transcription factors, extracellular matrix proteins, and components of developmental signaling pathways with important roles in limb development. A subset of the genes that distinguish scaled and feathered HLs are also differentially expressed between FL and scaled HL buds in pigeons, pinpointing a set of gene expression changes downstream of PITX1 and TBX5 in the partial transformation from HL to FL identity. We extended our analyses by comparing pigeon limb bud transcriptomes to chicken, anole lizard, and mammalian datasets to identify deeply conserved PITX1- and TBX5-responsive components of the limb identity program. Our analyses reveal a suite of predominantly low-level gene expression changes that are conserved across amniotes to regulate the identity of morphologically distinct limbs.
Asunto(s)
Tipificación del Cuerpo/genética , Pie/embriología , Miembro Posterior/embriología , Animales , Columbidae/genética , Extremidades/embriología , Plumas , Pie/fisiología , Miembro Anterior/embriología , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Esbozos de los Miembros/metabolismo , Morfogénesis/genética , Organogénesis/genética , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Transducción de Señal , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismoRESUMEN
Tissue mechanics play a crucial role in organ development. They rely on the properties of cells and the extracellular matrix (ECM), but the relative physical contribution of cells and ECM to morphogenesis is poorly understood. Here, we have analyzed the behavior of the peripodial epithelium (PE) of the Drosophila leg disc in the light of the dynamics of its cellular and ECM components. The PE undergoes successive changes during leg development, including elongation, opening and removal to free the leg. During elongation, we found that the ECM and cell layer are progressively uncoupled. Concomitantly, the tension, mainly borne by the ECM at first, builds up in the cell monolayer. Then, each layer of the peripodial epithelium is removed by an independent mechanism: while the ECM layer withdraws following local proteolysis, cellular monolayer withdrawal is independent of ECM degradation and is driven by myosin II-dependent contraction. These results reveal a surprising physical and functional cell-matrix uncoupling in a monolayer epithelium under tension during development.This article has an associated 'The people behind the papers' interview.
Asunto(s)
Drosophila melanogaster/embriología , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Matriz Extracelular/fisiología , Miembro Posterior/embriología , Morfogénesis/fisiología , Animales , Animales Modificados Genéticamente , Membrana Basal/embriología , Membrana Basal/crecimiento & desarrollo , Fenómenos Biomecánicos , Tipificación del Cuerpo/fisiología , Comunicación Celular/fisiología , Proliferación Celular , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero , Miembro Posterior/crecimiento & desarrollo , Miosina Tipo II/fisiología , Proteolisis , Tensión SuperficialRESUMEN
Embryos from aquatic birds are the primary models for the study of flipper development. While some staging of early embryogenesis in duck have been studied, characterization of the stages of the entire embryonic development period in water birds has not been described. This study aimed to establish a comparison of complete morphological development staging for ducks (Anas platyrhynchos) and geese (Anser cygnoides) with the embryonic staging system by Hamburger and Hamilton (HH) for the chicken (Gallus gallus). Our results show that morphological development in the chicken, duck, and goose are similar in the early stages. The major differences occurred after stage 27 of embryonic development, where the beak shape in ducks and geese was wider and longer than in chickens. In addition, the second and third interdigital webs of the hind limb of the chicken were found to be degraded from stage 31, and eventually vanished at stage 35; however, they were retained in ducks and geese. Rapid physical development occurred in the mid-to-late stages in ducks and geese. To our best knowledge, this is the first description of complete embryonic development for the duck and goose. Establishment of an embryonic staging system for duck and goose provides new models for the study of waterfowl development.