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Prenotochord cell sorting is regarded as one of the first cell sorting events in early chordate development. We recently demonstrated that this sorting event occurs in vitro, although the mediator of this activity remains unidentified. Herein, we report the isolation of a full-length cDNA clone of Axial protocadherin (AXPC), the homologue of human protocadherin-1 (PCD1). AXPC encodes a transmembrane protein (AXPC) that is expressed exclusively in the notochord at the neurula stage and in the pronephros, somites, heart, optic vesicle, otic vesicle, and distinct parts of the brain at the tailbud stage. Cell dissociation and reaggregation assays and in vivo microinjection experiments demonstrated that cells overexpressing a membrane-tethered form of AXPC (MT-AXPC) acquired the same adhesive properties as prenotochord cells. Moreover, microinjection of either mRNA encoding the dominant negative form of AXPC (DN-AXPC) or morpholino oligonucleotides interferes with the sorting activity of prenotochord cells and normal axis formation. This study suggests that AXPC is necessary and sufficient for prenotochord cell sorting in the gastrulating embryo, and may also mediate sorting events later in development.
FIG. 2. Expression of AXPC. (A) Temporal expression analyzed by RT-PCR. Low levels of AXPC expression were detected before gastrulation. Expression levels increased at stage 10, with high levels of expression being maintained by stage 38. (B) AXPC was expressed exclusively in the dorsal marginal zone at stage 10. V, ventral marginal zone. D, dorsal marginal zone. L, lateral marginal zone. no, notochord. so, somite. (C) Whole-mount in situ hybridization analysis at late neurula (stage 18). AXPC is expressed in the notochord region at stage 18. Ant., anterior. Post., posterior. (C) Section of (C) clearly shows the AXPC expression in notochord. (D) At stage 28, AXPC was expressed strongly in the pronephros region (pn) and weakly in the somites (so) and the heart (white arrowhead). (D) Enlarged view of (D) shows the expression of AXPC in the optic vesicle (op), otic vesicle (ot), and some parts of central nervous system (white asterisks). (E) AXPC expression in activin-treated animal caps. Animal caps were treated with 0 500 ng/ml of activin for 1 h, then cultured for 5 h. High AXPC expression was detected in 50 or 100 ng/ml activin-treated animal caps, and low levels of AXPC expression were detected in 10 or 500 ng/ml activin-treated animal caps. Note: Concentrations of 50 100 ng/ml are known to
most effectively induce notochord in this animal cap assay (Asashima et al., 1990). (F) AXPC expressions in activin-treated dissociation-and-reaggregation animal caps (Fig. 3A, Method 1). Lanes 1 4 indicate AXPC expression in the reaggregate treated with activin for 1 h, reaggregated, and cultured for 5 h. Very high expression levels of AXPC were detected in the reaggregates treated
with 1 ng/ml activin. No expression was detected in the other samples. Note: A concentration of 1 ng/ml induces notochord only in this dissociated and reaggregated animal cap assay (Kuroda et al., 1999). Lanes 5 9 show AXPC expression in dissociated cells with 1 ng/ml activin-treatment. Low AXPC expression was detected at 30 (lane 6) and 45 min (lane 7) after the initiation of activin treatment, and high AXPC expression was detected at 60 (lane 8) and 120 min (lane 9) after the initiation of activin treatment.
