Neural differentiation in cleavage‐arrested ascidian blastomeres induced by a proteolytic enzyme.

Abstract

SUMMARY1. As previously reported, ectodermal a4-2 blastomeres isolated from 8-cell embryos of the ascidian, Halocynthia roretzi or aurantium, and cultured under conditions of cleavage arrest always differentiated into an epidermal phenotype, showing long-lasting Ca2+-dependent action potentials and/or tunic on the cell surface. a4-2 blastomeres contacted by a chordamesodermal blastomere, A4-1, differentiated into a neural phenotype, characterized by fast Na+-dependent spikes. Differentiation to a similar neural phenotype occurred when isolated a42 blastomeres from H. aurantium embryos were treated with > 0 003 % subtilisin for 60 min at the 32-cell stage of the control embryo. Comparisons between induction by cell contact and induction by proteolytic enzymes were made and showed them to be similar in several respects.2. When the serine protease, subtilisin, was used as the neural inducer, neural competence of a4-2 blastomeres, measured as the percentage frequency of the induction of Na+ spikes, increased after the 32-cell stage and decreased during the gastrula stage. The time course of the neural competence was the same as that for contact with the A4-1 blastomere.3. The neural competence of four different ectodermal blastomeres isolated from the 16-cell embryo was also examined using subtilisin as a neural inducer, and by contact with the A41 blastomere from the 8-cell embryo. The competence was higher in anterior blastomeres than in posterior blastomeres for both types of induction. This regional difference in neural competence along the antero-posterior axis paralleled that expected from neural cell lineage during normal development, i.e. blastomeres with more cells of neural lineage among their derivatives showed higher competence.4. Streptomyces subtilisin inhibitor, SSI (0 1%), a specific protease inhibitor for subtilisin-type serine proteases, significantly suppressed (50%) neural induction of the ectodermal blastomere, a4-2, by contact wtih the chordamesodermal blastomere, A4-1.

Neural induction suppresses early expression of the inward‐rectifier K+ channel in the ascidian blastomere.

“…Previous work in our laboratory (Okado & Takahashi, 1990b) has shown that neural differentiation was induced by cell contact with the blastomere, A4-1, which contains the presumptive notochordal area, only if contact occurred before the late gastrula stage; the same critical period existed when neural differentiation was induced by protease (Okado & Takahashi, 1990b, 1993.…”

“…5A) and as large as that in protease-induced neurally differentiating cells. If a4-2 blastomeres are contacted with A4-1 blastomeres or treated with proteases after the late gastrula stage, neural induction does not occur and a4-2 blastomeres differentiate into epidermal-type cells (Okado & Takahashi, 1990b;1993). When the blastomere was treated with subtilisin at 15 h. later than the sensitive period for neural induction, the cell differentiated into an epidermal-type cell and exhibited the same level of inward-rectifier K+ channel current as that of cells not treated with protease (U in Fig.…”

“…In this solution, denuded embryos were dissociated into single cells with a fine glass needle.The anterior ectodermal blastomere (designated 'a4-2') that contains the presumptive neural region (Conklin, 1905;Nishida & Satoh, 1983) was isolated. In our experiments, treatment with a proteolytic enzyme (Okado & Takahashi, 1993) was used to induce neural differentiation. At the 64-cell stage, the isolated cleavage-arrested a4-2 blastomeres were incubated for 30 min in sea water containing 0-1 % subtilisin and 2-0 ,ag/ml cytochalasin B.…”

Subfamily-Specific Posttranscriptional Mechanism Underlies K+ Channel Expression in a Developing Neuronal Blastomere

“…Isolation, cleavage arrest, neural induction, and culture of a4.2 cells were performed as previously described (Okamura et al, 1994). C ells were neurally induced either by treatment with 0.2% subtilisin for 1 hr or by cell-contact with A4.1 cell (Okado and Takahashi, 1993). Electrical recording was performed in the two-electrode voltage-clamp configuration using AxoC lamp2B (Axon Currents were elicited by membrane depolarization pulses ranging from Ϫ50 to ϩ80 mV in 10 mV increments after a 50 msec prepulse at Ϫ120 mV.…”

Ion Channel Development, Spontaneous Activity, and Activity-Dependent Development in Nerve and Muscle Cells

“…The disappearance of the Na ϩ current is very gradual and follows a complex time course, with a transient peak of density as the larval tail elongates (243). In a further elegant series of experiments, this group developed an in vitro two-cell neural induction system, in which individual cleavage-arrested neural-and notochord-lineage cells are placed into physical contact and subsequently separated to study the exact developmental timing of induction, as well as the timing of competence in each cell (454,455,457). With the use of this system, it was shown that neural induction triggered the expression of a neural-type Na ϩ channel whose biophysical properties were distinct from the Na ϩ channel expressed in the egg (461,462).…”