RESUMO
BACKGROUND: During primitive hematopoiesis in Xenopus, cebpa and spib expressing myeloid cells emerge from the anterior ventral blood island. Primitive myeloid cells migrate throughout the embryo and are critical for immunity, healing, and development. Although definitive hematopoiesis has been studied extensively, molecular mechanisms leading to the migration of primitive myelocytes remain poorly understood. We hypothesized these cells have specific extracellular matrix modifying and cell motility gene expression. RESULTS: In situ hybridization screens of transcripts expressed in Xenopus foregut mesendoderm at stage 23 identified seven genes with restricted expression in primitive myeloid cells: destrin; coronin actin binding protein, 1a; formin-like 1; ADAM metallopeptidase domain 28; cathepsin S; tissue inhibitor of metalloproteinase-1; and protein tyrosine phosphatase nonreceptor 6. A detailed in situ hybridization analysis revealed these genes are initially expressed in the aVBI but become dispersed throughout the embryo as the primitive myeloid cells become migratory, similar to known myeloid markers. Morpholino-mediated loss-of-function and mRNA-mediated gain-of-function studies revealed the identified genes are downstream of Spib.a and Cebpa, key transcriptional regulators of the myeloid lineage. CONCLUSIONS: We have identified genes specifically expressed in migratory primitive myeloid progenitors, providing tools to study how different gene networks operate in these primitive myelocytes during development and immunity.
Assuntos
Linhagem da Célula/genética , Movimento Celular/genética , Células Mieloides/citologia , Xenopus laevis/genética , Animais , Destrina/genética , Destrina/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Células Mieloides/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2(+/-)) similar to those found in the majority of HPAH patients. We show that Bmpr2(+/-) PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2(+/-) PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2(+/-) PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2(+/-) PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Hipertensão Pulmonar Primária Familiar/genética , Transporte Proteico/genética , Quinases da Família src/genética , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Cavéolas/metabolismo , Cavéolas/patologia , Caveolina 1/genética , Endocitose/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Hipertensão Pulmonar Primária Familiar/fisiopatologia , Mutação da Fase de Leitura , Heterozigoto , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Pirimidinas/administração & dosagem , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
The liver, pancreas, and lungs are induced from endoderm progenitors by a series of dynamic growth factor signals from the mesoderm, but how the temporal-spatial activity of these signals is controlled is poorly understood. We have identified an extracellular regulatory loop required for robust bone morphogenetic protein (BMP) signaling in the Xenopus foregut. We show that BMP signaling is required to maintain foregut progenitors and induce expression of the secreted frizzled related protein Sizzled (Szl) and the extracellular metalloprotease Tolloid-like 1 (Tll1). Szl negatively regulates Tll activity to control deposition of a fibronectin (FN) matrix between the mesoderm and endoderm, which is required to maintain BMP signaling. Foregut-specific Szl depletion results in a loss of the FN matrix and failure to maintain robust pSmad1 levels, causing a loss of foregut gene expression and organ agenesis. These results have implications for BMP signaling in diverse contexts and the differentiation of foregut tissue from stem cells.