ABSTRACT
By comparison with skeletal or cardiac developmental programs, little is known regarding the specific factors that promote specification and differentiation of smooth muscle cells from pluripotent cells. We have analyzed the developmental expression of a subset of smooth muscle genes during Xenopus early development and showed that similar to mammals and avians, Xenopus smooth muscle myosin heavy chain (SM-MHC) is a highly specific marker of smooth muscle differentiation. Embryonic cells from animal pole explants of Xenopus blastula can be induced by basic fibroblast growth factor, Wnt, and bone morphogenetic protein signals to adopt the smooth muscle pathway. Explants from early embryos that contain neural crest cells can also differentiate into cells expressing smooth muscle genes. We examined the interplay of several transcription factors, that is SRF, myocardin, and GATA6, that induce the expression of SM-MHC in animal cap cells and found that myocardin-dependent expression of smooth muscle genes in animal cap cells is synergized by SRF but is strongly antagonized by GATA6.
Subject(s)
Blastula/embryology , Gene Expression Regulation, Developmental/physiology , Muscle, Smooth/metabolism , Myosin Heavy Chains/biosynthesis , Transcription Factors/metabolism , Animals , Blastula/cytology , Bone Morphogenetic Proteins/metabolism , Fibroblast Growth Factor 2/metabolism , Muscle, Smooth/cytology , Wnt Proteins/metabolism , Xenopus laevisABSTRACT
OBJECTIVE: Clinically, the effect of chronic hypoxia (CH) in the pulmonary circulation alternates between phases of pulmonary artery hypertension (CH-PAHT) and normoxic normotensive remission (N-RE). Little information is available on the role of calcium-sensitive potassium channels (BK(Ca)) in both CH-PAHT and N-RE phases. In the present study, we investigated the effects of both CH and N-RE on BK(Ca) channels activity and their consequences on hypoxic pulmonary vasoconstriction (HPV). METHODS: Using isolated ring preparation, the patch-clamp technique, RT-PCR and Western immunoblotting, we examined the role of the BK(Ca) channel in normoxic, CH-PAHT and N-RE rat pulmonary artery smooth muscle cells (PASMCs). RESULTS: In intrapulmonary arterial rings, acute hypoxia induced contraction in control vessels, relaxation in the N-RE rats, and had no effect in CH-PAHT. The hypoxia-induced relaxation in the N-RE rat pulmonary arteries was abolished by iberiotoxin (IbTx), a specific BK(Ca) blocker. The IbTx-sensitive whole-cell K(Ca) channel current was reduced in CH-PAHT and increased in N-RE rat PASMCs. The BK(Ca) channel conductance and voltage sensitivity were not altered in CH and N-RE rat PASMCs, whereas its calcium sensitivity was decreased and increased in CH and N-RE rat PASMCs, respectively. Results of RT-PCR and Western blot analysis revealed a decrease in the mRNA and protein of the BK(Ca) alpha-subunit in CH, whereas no change at protein level was observed in the N-RE. CONCLUSION: In rat PASMCs, CH and N-RE are associated with a down- and up-regulation of BK(Ca) activity, respectively, mainly due to modifications of its Ca(2+) sensitivity. This could explain the acute hypoxic pulmonary constriction and relaxation observed in CH and N-RE rats, respectively.
Subject(s)
Hypertension, Pulmonary/physiopathology , Muscle, Smooth, Vascular/physiopathology , Potassium Channels, Calcium-Activated/physiology , Pulmonary Artery/physiopathology , Pulmonary Disease, Chronic Obstructive/physiopathology , Animals , Blotting, Western/methods , Calcium/metabolism , Hypertension, Pulmonary/metabolism , Hypoxia , In Vitro Techniques , Male , Muscle, Smooth, Vascular/metabolism , Patch-Clamp Techniques , Peptides/pharmacology , Potassium Channel Blockers/pharmacology , Potassium Channels, Calcium-Activated/analysis , Potassium Channels, Calcium-Activated/genetics , Pulmonary Artery/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , RNA, Messenger/analysis , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Vasodilation/drug effectsABSTRACT
The goal of this laboratory course is to introduce vertebrate developmental biology to undergraduate students, emphasizing both classical and contemporary aspects of this field. During the course, the students combine the use of living Xenopus laevis material with active tutorial participation, with the aim of illustrating how the fertilized egg can generate the diversity of cell types and complexity of pattern seen only a few days later in the embryo. Special emphasis is given to the observation and manipulation of living material. The laboratory course includes a comprehensive analysis of both oogenesis and early development and is divided into two overlapping parts that combine tutorial and practical approaches. The first part is devoted to oogenesis; oocytes are sorted out, allowed to mature in vitro and observed in histological section. In the second part, students perform an in vitro fertilization of Xenopus eggs and a mesoderm and neural induction assay of animal cap explants. Successful induction of the explants is confirmed by morphological, histological and molecular analyses. Finally, the students observe and comment on selected slides to illustrate the organization of the body plan of the amphibian embryo at an early stage of organogenesis.
