Expression patterns of Xenopus FGF receptor-like 1/nou-darake in early Xenopus development resemble those of planarian nou-darake and Xenopus FGF8.

Fibroblast growth factors (FGFs) mediate many cell-to-cell signaling events during early development. Nou-darake (ndk), a gene encoding an FGF receptor (FGFR)-like molecule, was found to be highly and specifically expressed in the head region of the planarian Dugesia japonica, and its functional analyses provided strong molecular evidence for the existence of a brain-inducing circuit based on the FGF signaling pathway. To analyze the role of ndk during vertebrate development, we isolated the Xenopus ortholog of ndk, the vertebrate FGFR-like 1 gene (XFGFRL1). Expression of XFGFRL1/Xndk was first detected in the anterior region at the late gastrula stage and dramatically increased at the early neurula stage in an overall anterior mesendodermal region, including the prechordal plate, paraxial mesoderm, anterior endoderm, and archenteron roof. This anterior expression pattern resembles that of ndk in planarians, suggesting that the expression of FGFRL1/ndk is conserved in evolution between these two distantly diverged organisms. During the tail bud stages, XFGFRL1/Xndk expression was detected in multiple regions, including the forebrain, eyes, midbrain-hindbrain boundary, otic vesicles, visceral arches, and somites. In many of these regions, XFGFRL1/Xndk was coexpressed with XFGF8, indicating that XFGFRL1/Xndk is a member of the XFGF8 synexpression group, which includes sprouty, sef, and isthmin.     

Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Xenopus tropicalis transgenic lines and their use in the study of embryonic induction.

For over a century, amphibian embryos have been a source of significant insight into developmental mechanisms, including fundamental discoveries about the process of induction. The recently developed transgenesis for Xenopus offers new approaches to these poorly understood processes, particularly when undertaken in the quickly maturing species Xenopus tropicalis, which greatly facilitates establishment of permanent transgenic lines. Several X. tropicalis transgenic lines have now been generated, and experiments demonstrating the value of these lines to study induction in embryonic tissue recombinants and explants are presented here. A revised protocol for transgenesis in X. tropicalis resulting in a significant increase in the percentage of transgenic animals that reach adulthood is presented, as well as improvements in tadpole and froglet husbandry, which have facilitated the raising of large numbers of adults. Working transgenic populations have been rapidly expanded, and some transgenes have been bred to homozygosity. Established lines include those bearing the promoter regions of Pax-6, Otx-2, Rx, and EF1alpha coupled to fluorescent reporter genes. Multireporter lines combining, in a single animal, up to three gene promoters coupled to different fluorescent reporters have also been established. The value of X. tropicalis transgenic lines for the study of induction is demonstrated by showing activation of Pax-6 by noggin treatment of Pax-6/GFP transgenic animal caps, illustrating how reporter lines allow a rapid, in vivo assay for an inductive response. An experiment showing lens induction in gamma-crystallin/GFP transgenic lens ectoderm when it is recombined with mouse optic vesicle demonstrates conservation of inducing signals from amphibians and mammals. It also shows how the warmer culture temperatures tolerated by X. tropicalis embryos can be used in assays of factors produced by mammalian cells and tissues. The many applications of transgenic reporter lines and other lines designed to target gene expression in particular tissues promise to bring significant new insights to the classic issues first defined in amphibian systems.     

Development of the amygdalohypothalamic projection in the mouse embryonic forebrain

The amygdalohypothalamic projection, a major component of the stria terminalis, is involved in the conduction of emotional and olfactory information integrated in the amygdala to the hypothalamus to elicit emotional reactions. Despite the extensive studies on functional aspects of the amygdaloid complex, developmental mechanisms of the amygdala and related structures are still poorly understood. To investigate the development of the amygdalohypothalamic projection in the mouse embryonic brain, carbocyanine dye was applied to the amygdala to label the growing axons anterogradely and to the hypothalamus to label the amygdaloid neurons retrogradely. The initial outgrowth of the stria terminalis was found to be as early as E11.5. The pathway crossed in a saddle over the internal capsule, another prominent connection in the developing forebrain of the mammalian embryo. Bipolar immature neurons were distributed along the stria terminalis at the telencephalo-diencephalic boundary, and the internal capsule was also surrounded by these cells. These cells expressed immunoreactivities to calretinin and the lot-1 antigen which has been shown to be involved in guidance of the developing lateral olfactory tract. Ultrastructural analysis revealed an adherens-like junction between the stria terminalis and the apposed cells, implying contact-mediated guidance. These results suggest that, in the development of the stria terminalis, the axonal outgrowth is guided by a mechanism similar to that of the developing lateral olfactory tract, a major amygdalopetal connection.     

胰岛素增强子结合蛋白1在小鼠胚胎心的时空分布

目的 观察转录因子胰岛素增强子结合蛋白1（ISL1）在小鼠胚胎心的表达与心、第二生心区及前肠内胚层的发育。 方法 胚龄8～13d小鼠胚胎心共18个,连续石蜡切片,用抗心肌肌球蛋白重链（MHC）、抗ISL1、抗增殖细胞核抗原（PCNA）和抗α-平滑肌肌动蛋白(α-SMA)抗体进行免疫组织化学染色、免疫荧光染色和Western blotting检测。 结果 胚龄9d,ISL1阳性心前体细胞进入流出道远端。胚龄10d,ISL1阳性细胞延伸入流出道近端及静脉窦心肌。胚龄11～12d,心内ISL1表达量逐渐增多并达高峰,动脉端ISL1阳性细胞分布于流出道远端壁、心包内主肺动脉壁及主肺动脉隔,静脉端ISL1阳性细胞主要限于窦房结和静脉瓣。动脉端前肠内胚层细胞索增至最长,周围前生心区ISL1阳性细胞密度也达高峰,并且明显多于后生心区。胚龄13d,心内及第二生心区ISL1阳性细胞显著减少,内胚层细胞索趋于消失。 结论 ISL1阳性细胞在小鼠胚胎心的表达主要集中在胚龄9～13d,其表达模式与第二生心区及前肠内胚层的发育密切相关。     

Expression of botulinum toxin binding sites in Xenopus oocytes.

The binding of iodinated botulinum toxin type B to nerve membranes was studied by using rat and mouse preparations. The toxin was examined both in the single-chain and in the proteolytically processed dichain form, and binding sites both in the spinal cord and in various brain regions were assayed. Rat and mouse brains possessed specific binding sites for botulinum toxin type B. The average Kd values for the various rat and mouse membrane preparations examined were 4.2 +/- 0.7 nM and 3.7 +/- 0.9 nM, respectively. The average Bmax values for the same tissue preparations were 7.3 +/- 0.7 pmol/mg of protein and 7.5 +/- 1.9 pmol/mg protein, respectively. The binding of botulinum toxin type B to rat brain membranes was not antagonized by a polyclonal antibody against the cytosolic domain of synaptotagmin 1 or by a monoclonal antibody directed against the luminal domain of synaptotagmin 1. In addition, these antibodies did not protect the mouse phrenic nerve-hemidiaphragm from toxin-induced neuromuscular blockade. Extraction of whole-brain mRNA and injection into Xenopus oocytes led to expression of binding sites for botulinum toxin. Extraction and injection of cerebellar mRNA led to expression of a higher density of binding sites. The number of binding sites was not diminished when oocytes were pretreated with antibodies against the cytosolic and luminal domains of synaptotagmin 1. These findings are likely to aid in the isolation, characterization, and reconstitution of toxin binding sites.     

Expression of the mouse FGF-1 and FGF-1.A mRNAs during embryonic development and in the aging heart

The mouse Fgf-1 gene contains at least four upstream promoters that are alternatively spliced to the first protein coding exon, giving rise to different Fgf-1 mRNA variants (1.A, 1.B, 1.C, and 1.G), each expressed in a tissue specific manner. Only the Fgf-1.A promoter contains TATA and CAAT consensus sequences, and its corresponding mRNA is mainly expressed in the mouse heart. In situ hybridization with an Fgf-1.A-specific cRNA probe showed the 1.A message to be very low in embryonic heart, tongue, spinal cord and smooth muscle of the small intestine. Moderate 1.A mRNA levels were revealed in head mesenchyme, paraxial mesoderm, in the embryonic inferior cunniculus, hyppocampus, and thymus. These results suggest a limited role of the Fgf-1.A mRNA variant during development.     

Docking protein SNT1 is a critical mediator of fibroblast growth factor signaling during Xenopus embryonic development.

The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from several growth factor receptors to the mitogen-activated protein (MAP) kinase signaling cascade, but its biological function during development is not well characterized. Here, we show that the Xenopus homolog of mammalian SNT1/FRS-2 (XSNT1) plays a critical role in the appropriate formation of mesoderm-derived tissue during embryogenesis. XSNT1 has an expression pattern that is quite similar to the fibroblast growth factor receptor-1 (FGFR1) during Xenopus development. Ectopic expression of XSNT1 markedly enhanced the embryonic defects induced by an activated FGF receptor, and increased the MAP kinase activity as well as the expression of a mesodermal marker in response to FGF receptor signaling. A loss-of-function study using antisense XSNT1 morpholino oligonucleotides (XSNT-AS) shows severe malformation of trunk and posterior structures. Moreover, XSNT-AS disrupts muscle and notochord formation, and inhibits FGFR-induced MAP kinase activation. In ectodermal explants, XSNT-AS blocks FGFR-mediated induction of mesoderm and the accompanying elongation movements. Our results indicate that XSNT1 is a critical mediator of FGF signaling and is required for early Xenopus development.     

Expression of nerve growth factor in astrocytes of the hippocampal CA1 area following transient forebrain ischemia.

We have examined by immunoassay and immunohistochemistry, the expression of nerve growth factor in the rat hippocampus, one to 28 days after transient forebrain ischemia. In the CA1 area, the overall level of nerve growth factor expression remained constant over the first three days of reperfusion while it increased by about 45% of control levels after longer postischemic periods. In contrast, a slight decrease in nerve growth factor levels, which was most prominent at three days postlesion, was observed in the other hippocampal regions. Immunohistochemical analysis of the distribution of nerve growth factor showed that its expression was up-regulated in astrocytes but not in microglia of the postischemic CA1 region and that the intensity and temporal profile of the changes in nerve growth factor immunostaining in these cells, was consistent with that observed in the immunoassay. Interestingly, the regulation of the nerve growth factor expression in reactive astrocytes of the postischemic CA1 area closely parallels that of kainate receptor subunits GluR5-7, raising the possibility of a cause-effect relationship. These results indicate that after ischemia nerve growth factor expression is up-regulated in reactive astrocytes suggesting that these cells may contribute to rescuing damaged neurons by means of increasing nerve growth factor production.     

Expression of cVg1 mRNA during chicken embryonic development

Using degenerated PCR-primers to identify known and novel BMPs that are expressed in the developing chicken heart, we identified not only BMP2, -4, and -7 mRNA, but also the TGFbeta superfamily member cVg1. The expression pattern of cVg1 mRNA was determined during chicken development from HH4 to HH44. In early developmental stages, cVg1 mRNA is expressed in the primitive streak, paraxial mesoderm, developing somites, and developing neural tube. Subsequently, cVg1 mRNA is expressed in the developing central and peripheral nervous system, retina, auditory vesicle, notochord, lung alveoli, and olfactory mucosa. In the heart, cVg1 is initially expressed through the linear heart tube, but becomes restricted to the forming chamber myocardium, in an expression domain similar to that of atrial natriuretic factor (ANF) mRNA.     

Myogenic waves and myogenic programs during Xenopus embryonic myogenesis

Although Xenopus is a key model organism in developmental biology, little is known about the myotome formation in this species. Here, we assessed the expression of myogenic regulatory factors of the Myod family (MRFs) during embryonic development and revealed distinct MRF programs. RESULTS: The expression pattern of each MRF during embryonic development highlights three successive myogenic waves. We showed that a first median and lateral myogenesis initiates before dermomyotome formation: the median cell population expresses Myf5, Myod, and Mrf4, whereas the lateral one expresses Myod, moderate levels of Myogenin and Mrf4. The second wave of myoblasts arising from the dermomyotome is characterized by the full MRF program expression, with high levels of Myogenin. The third wave is revealed by Myf5 expression in the myotome and could contribute to the formation of plurinucleated fibers at larval stages. Furthermore, Myf5- or Myod-expressing anlagen are identified in craniofacial myogenesis. CONCLUSIONS: The first median and lateral myogenesis and their associated MRF programs have probably disappeared in mammals. However, some aspects of Xenopus myogenesis have been conserved such as the development of somitic muscles by successive myogenic waves and the existence of Myf5-dependent and -independent lineages.