We previously showed that FGF was capable of inducing Xenopus gastrula ectoderm cells in culture to express position-specific neural markers along the anteroposterior axis in a dose-dependent manner. However, conflicting results have been obtained concerning involvement of FGF signaling in the anterior neural induction in vivo using the same dominant-negative construct of Xenopus FGF receptor type-1 (delta XFGFR-1 or XFD). We explored this issue by employing a similar construct of receptor type-4a (XFGFR-4a) in addition, since expression of XFGFR-4a was seen to peak between gastrula and neurula stages, when the neural induction and patterning take place, whereas expression of XFGFR-1 had not a distinct peak during that period. Further, these two FGFRs are most distantly related in amino acid sequence in the Xenopus FGFR family. When we injected mRNA of a dominant-negative version of XFGFR-4a (delta XFGFR-4a) into eight animal pole blastomeres at 32-cell stage, anterior defects including loss of normal structure in telencephalon and eye regions became prominent as examined morphologically or by in situ hybridization. Overexpression of delta XFGFR-1 appeared far less effective than that of delta XFGFR-4a. Requirement of FGF signaling in ectoderm for anterior neural development was further confirmed in culture: when ectoderm cells that were overexpressing delta XFGFR-4a were cocultured with intact organizer cells from either early or late gastrula embryos, expression of anterior and posterior neural markers was inhibited, respectively. We also showed that autonomous neuralization of the anterior-type observed in ectoderm cells that were subjected to prolonged dissociation was strongly suppressed by delta XFGFR-4a, but not as much by delta XFGFR-1. It is thus indicated that FGF signaling in ectoderm, mainly through XFGFR-4, is required for the anterior neural induction by organizer. We may reconcile our data to the current "neural default model," which features the central roles of BMP4 signaling in ectoderm and BMP4 antagonists from organizer, simply postulating that the neural default pathway in ectoderm includes constitutive FGF signaling step.

SUMMARY1. The action potential and the membrane current of the mouse oocyte were analysed by current-clamp and voltage-clamp techniques and they were compared with those of other animal oocytes.2. The matured and unfertilized oocyte of the mouse in standard medium with 6 mM-K showed the resting potential of -23 1 + 2-9 mV. The resting potential was relatively large in the medium with 20 mM-Ca or 10 mM-Mn, being -357 + 2-6 mV and further increased to -469 + 48 mV with replacement of Na in the medium by choline.3. At the cessation of large hyperpolarization below -90 mV in standard medium, a regenerative potential was often elicited in the form of an off-response. The off-response depended upon the external concentration of Ca. In 20 mM-Ca medium it was constantly observed with hyperpolarization below -60 mV. Its critical level was -40 mV and its overshoot was +15 mV.4. The time and potential-dependent inward current was observed both in standard and 20 mM-Ca media under voltage-clamp condition. In 20 mM-Ca medium the inward current was observed by depolarization beyond -40 mV and showed its maximum at -15 mV. It was greatly reduced by replacing the external Ca with Mn but retained by substituting Sr or Ba for Ca. The selectivity ratios among these alkali earth cations were Ca: Sr: Ba = 1-0: 1-4:0-7.5. The current-voltage relation in Ca and Na-deficient and 10 mm-Mn medium was linear from -200 to +25 mV. The hyperpolarization below -200 mV revealed an inward-going rectification. The depolarization above +50 mV under voltage-clamp condition induced the outward surge current with activation and inactivation processes.6. In contrast to the mouse oocyte, the matured and unfertilized oocyte H. OKAMOTO AND OTHERS of the sea urchin showed a large resting potential of -70 mV in 30 Ca ASW and the depolarization beyond -40 mV elicited an action potential with an overshoot of 20 mV. The action potential showed a notch in the rising phase and lasted about 1 to 2 sec.7. Under the voltage-clamp condition both Ca inward current and the outward surge current were observed in the sea urchin oocyte membrane just as in the mouse oocyte membrane.8. The selectivity ratios among alkali earth cations, Ca: Sr:Ba, for 'Ca channels' of the oocyte membranes were 10:14:0-7 in the mouse, 1*0:1-7:1*1 in the tunicate and 1-0:0-7:0-5 in the sea urchin. When the current density through Ca channels are revised in terms of the respective critical levels for Ca channels, the revised selectivity sequences become Ca > Sr > Ba, being common to all three species.

SUMMARY1. Ionic currents of the egg membrane of a certain tunicate, Halocynthia roretzi Drashe, were studied by the voltage-clamp technique.2. The membrane depolarization beyond -55 mV in standard artificial sea water induced mainly transient inward current and slight outward currents, when the holding potential was kept at -90 mV.3. The transient inward current was composed of two components; the major one showed a faster time course, a more negative critical level of about -55 mV, and a reversal potential around + 60 mV and the minor one showed a slower time course, a less negative critical level of -10 mV, and no definite reversal potential.4. The major component became maximum at about -25 mV with the peak time of 6-9 msec at 15°C, and the maximum currents ranged from 0-5 to 15 x10-5 A/cm2.5. The major component of the inward current was abolished by the replacement of Na with choline or Tris or Cs ions, while it was almost unaltered by the replacement with Li. The minor component was independent of Na concentration in the external solution.6. This major component showed the activation and inactivation identical with those of Na current of other excitable membranes. A conditioning depolarization over -90 mV inactivated the Na current and the half inactivation of the major inward current was obtained by a conditioning pulse to -56 mV, when the pulse duration was 400 msec and the temperature was at 150 C. 6HARUMASA OKAMOTO AND OTHERS 7. The time course of the Na current was formulated with m and h parameters in the following equations: i=a gia(Em -ENa)m h . gNa(Eme-tENa)M2 Ie-Tm)2e-t/7h; M = o+ (m,m-o)(O)e-tIrm), h = h, + (ho -ho)e-tITh. 8. The kinetic parameters Tm and Th of egg Na current were calculated and compared with those of the squid axon. The potential dependence of Tm and Th was almost identical with that of the axon, but the absolute values of both Tm and Th were ten-to twentyfold larger than those of the axon in any range of the membrane potential.9. The temperature dependence of the kinetic parameters Tm, Th and of the chord conductance 9Na was studied. The Q10's for Tm and Th were both 4 0, while the Q10 for 9Na was 2-0 in the temperature range from 5 to 200 C.10. The outward and inward rectifying conductances of egg membrane were remarkably activated at the potential level above + 100 mV and below -70 mV respectively in standard artificial sea water. Both increased currents were subsequently subject to inactivation.11. It was suggested that Na, Ca, K inward rectifying and K outward rectifying conductances all exist separately in the egg cell membrane and the Na current was essentially identical with that through the Na channel in other excitable membranes.

Neural crest is formed at the boundary of epidermal and neural ectoderm. To understand the molecular mechanism of neural crest formation, we focused on the transcriptional regulation of the Slug gene. In the upstream sequence of the chicken Slug gene, we have identified potential binding sites for transcription factors, such as Lef / Tcf and Smad1. Transgenic mouse embryos carrying the chicken Slug promoter-reporter gene showed a crest-specific activation of the reporter, suggesting the isolated sequence included the cis-regulatory elements to receive Slug-inducing signals in the mouse neural crest. While these potential cis-regulatory elements could be recognized and activated by corresponding transcription factors, such as Lef1 and Smad1, Wnt-Lef-β-catenin signal failed to induce endogenous Slug expression in quail neural plate tissue prepared from forebrain and midbrain levels. In contrast, Slug expression and subsequent epithelialmesenchymal transition were effectively induced by BMP4. Consistently, while we could detect phosphorylation of Smad1 in the ectoderm including the neural plate and the neural fold region, the activation of a reporter gene for a detection of canonical Wnt signal activation was below the level of detection at the forebrain and midbrain levels. These observations indicated that in the anterior ectoderm BMP signal has a predominant role for Slug expression.

The dysferlin gene is defective in Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). Dysferlin is a sarcolemmal protein that is implicated in calcium-dependent membrane repair. Affixin (beta-parvin) is a novel, integrin-linked kinase-binding protein that is involved in the linkage between integrin and the cytoskeleton. Here we show that affixin is a dysferlin binding protein that colocalizes with dysferlin at the sarcolemma of normal human skeletal muscle. The immunoreactivity of affixin was reduced in sarcolemma of MM and LGMD2B muscles, although the total amount of the affixin protein was normal. Altered immunoreactivity of affixin was also observed in other muscle diseases including LGMD1C, where both affixin and dysferlin showed quite similar changes with a reduction of sarcolemmal staining with or without cytoplasmic accumulations. Colocalization of dysferlin and affixin was confirmed by immunofluorescence analysis using dysferlin-expressing C2 myoblasts. Wild-type and mutant dysferlin colocalized with endogenous affixin. The interaction of dysferlin and affixin was confirmed by immunoprecipitation study using normal human and mouse skeletal muscles. Using immunoprecipitation with deletion mutants of dysferlin, we have identified that C-terminal region of dysferlin is an apparent binding site for affixin. We also found N-terminal calponin homology domain of affixin as a binding site for dysferlin. Our results suggest that affixin may participate in membrane repair with dysferlin.