Toshiaki Okada

FGF8 signaling patterns the telencephalic midline by regulating putative key factors of midline development

FGF8 has been reported to act as a primary regulator of neocortical patterning along the anteroposterior (AP) axis in the mouse telencephalon, and disruption of FGF signaling causes distortion of molecular arealization along the AP axis. Since hypoplasia of midline structures is observed in Fgf8 mutant mice, FGF8 is also postulated to be involved in telencephalic midline development. In this study we analyzed the role of FGF8 in midline development by means of gain-of-function and loss-of-function experiments. The results showed that FGF8 up-regulates the expression of transcription factor (TF) genes, including putative key factors involved in midline development. Although FGF8 had been thought to act downstream of SHH signaling, ectopic FGF8 up-regulates the expression of midline TF genes in Shh null mice, suggesting that FGF signaling acts as an upstream positive regulator of midline TFs during midline development independently of SHH.

Ptch1-mediated dosage-dependent action of Shh signaling regulates neural progenitor development at late gestational stages

Sonic hedgehog (Shh) signaling regulates cell differentiation and proliferation during brain development. However, the role of Shh in neurogenesis during late gestation (embryonic day 13.5-18.5) remains unclear. Herein, we used a genetic approach and in utero electroporation to investigate the role of mouse Shh and patched homolog 1 (Ptch1), the putative receptor for Shh. Proliferating cortical intermediate (basal) progenitor cells (IPCs) were severely reduced in Shh mutant mice, suggesting that endogenous Shh signaling could play an essential role in cortical IPC development. During cortical neurogenesis, strong upregulation of Shh signaling enhanced the transition from ventricular zone (VZ) progenitors to ventralized IPCs, while low levels of signaling enhanced the generation and proliferation of cortical IPCs in the subventricular zone. The effects of Shh upregulation in this study were consistent with a phenotype of conditional loss of function of Ptch1, and the phenotype of a hypomorphic allele of Ptch1, respectively. These data indicated that endogenous Ptch1 mediates the broad effects of Shh on the transition from VZ progenitors to IPCs and activation of proliferation of the IPCs in the cortex during late gestational stages.

ORIGIN OF THE GONAD IN THE JUVENILE OF A SOLITARY ASCIDIAN, CIONA INTESTINALIS

In the just‐metamorphosed juveniles of Ciona intestinalis, a round mass of tissue debris derived from the resorbed tadpole tail is situated in the broad space enclosed by the peritoneal membrane and the epidermis around the ventral side of the esophagus. In living juveniles, the origin of the gonad rudiment was traced back to the mass of tissue debris. Electron microscopically, the round mass was a clump of irregular‐shaped phagocytotic cells engulfing degenerated cell fragments. On the surface of the cell clump, a small number of singly occurring round cells were found and identified as primordial germ cells on the basis of morphological continuity to obvious germ cells in later stages. Presence of nuage around the nucleus characterized the germ cells. In a few days the germ cells assembled to form a solid slender body (gonad rudiment) together with smaller somatic cells. The gonad rudiment left the space around the esophagus, moving into the narrow mesenteric space connecting the stomach and intestine on the fourth day after metamorphosis. It gradually increased in size by proliferation of the germ cells and somatic cells. The solid gonad rudiment changed into an oval vesicle with an eccentrically located cavity on about the seventh day after metamorphosis. The vesicle comprised a thinner wall made of a simple epithelium without germ cells and a thicker wall containing germ cells and somatic cells.

Differentiation of the gonad rudiment into ovary and testis in the solitary ascidian, Ciona intestinalis

In the early juveniles of Ciona intestinalis, primordial germ cells arise on the degenerated mass of the resorbed tadpole tail, and assemble to form a discrete gonad rudiment. The present study elucidated the morphological sequences during differentiation of the gonad rudiment into the testis and ovary. In 11‐ to 12‐day juveniles, the gonad rudiment, an elongate sac, divided into the testicular and ovarian rudiments. The testicular rudiment separated as a round vesicle from the thickened wall of the elongate sac. The original sac, after separation of the round vesicle, developed into the ovary. In the testicular rudiment, germ cells formed a continuous central mass without association of somatic cells, while in the ovarian rudiment, each germ cell was associated with somatic cells within the epithelium composing the wall of the rudiment. In 13‐ to 15‐day juveniles the testicular rudiment changed into branched tubes ending in club‐shaped follicles. Cells characterized by many flattened cisternae of rough endoplasmic reticulum (distal cells) constituted the distal wall of each follicle. Spermatogenic cells were freely present in the follicular lumen, but the largest spermatogonia were in contact with the distal cells. Both in the testicular and ovarian rudiments, germ cells entered meiosis in 18‐day juveniles. A novel body (periesophageal body) was found just beneath the ventral margin of the esophageal opening. It comprised irregular follicles made up of one cell type whose cytoplasm, filled with round vesicles and Golgi complexes, was suggestive of an endocrine function. Fragments derived from the periesophageal body were present around the developing ovary.

127 Expression of a calcium channel alpha1-subunit during neuronal and muscular differentiation of ascidian embryos

Masashi Umemiya To investigate dopaminergic modulation in the striatum, we recorded excitatory postsynaptic currents, evoked by local stimulation, from medium spiny neurons in postnatal rat brain slices. Recording were made using the whole-cell patch clamp technique under voltage-clamp condition. Incubation of slices in 1OmM dopamine resulted in a highly variable decrease in the amplitude of EPSCs. The variability of dopamine’s effect on EPSCs is consistent with activation of different receptor subtypes with potentially opposing effects. Incubation of slices in 5 mM SKF 386393, a Di-type dopamine receptor antagonist, resulted in potentiation of the EPSC. On the other hand, 5 mM SKF 386393 had no effect on currents activated by application of glutamate or kainate. However, because of the limitation of slow application of agonists, our results leave possibility that the effect of Di receptor activation on the EPSC is mediated via postsynaptic glutamate receptor modulation.

132 TuNaII: A putative sodium channel gene specifically expressed in ascidian motoneuron lineage

In the previous study, we reported that PC12 rat pheochromocytoma cells treated with nerve growth factor, a model for mammalian sympathetic neurons, had some characteristics of cold sensitive neurons. The resting membrane potential of PC12 cells was depolarized and amplitude of macroscopic inward currents elicited by brief depolarizing step pulses was increased by cooling from normal body temperature of rats. Here we analyzed voltage dependence of the thermosensitive comgonent of the membrane current by applying an identical ramp command voltage pulse at different temperature using whole-cell patch-clamp configuration. The cold-activated current was obtained by subtracting current produced by the ramp command pulse at 370c from that at 31% or 33%. Although there was no remarkable change in current below -3OmV by cooling, an inward current having a peak near 0 mV and an outward current above +lO m\’ were observed in the coldactivated current. The inward current decreased during superfusion with tetrodotoxin (TTX, SxlO~‘M) but the outward current had not TTX sensitivity. These findings indicate that PC12 cell has two component of thermosensitive membrane currents, one is TTX sensitive and the other is insensitive.