Compartmental structure of the otic placode: junctional permeability and clonal restriction

E Vazquez, E Geijo, T Schimmang, F Giraldez
1996 International Journal of Developmental Biology  
The otic placode is induced in the head ectoderm by signals which are believed to arise from the neighbouring mesoderm and the neural tube. Specification of the otic domain stans very early in development. but the otic primordium is fully committed at the placode stage. when the first evidence of a morphological structure associated with the ear is apparent. The purpose of this work was to study whether the otic placode forms a developmental compartment. This may be defined as structural and
more » ... ctional domain which is isolated from the surrounding and originally equivalent territory. and that is developmentally committed. Operationally. it can be identified by demonstrating boundaries and restricted communication between the proposed compartment and its surroundings. The present experiments were carried out in order to study: 1) the occurrence of limits to intercellular communication via gap junctions between otic and extra-otic ectoderm. 2) the presence of clonal restriction within the otic placode. The results show that the otic placode forms an uniform compartment which is limited by the otic ridge. This forms a functional boundary between the otic placode and the surrounding ectoderm regarding cell-cell communication and clonal expansion. Experiments were carried out on chick embryos from stage 11 to 14 (Hamburger & Hamilton. 1951). For dye injection. embryos were dissected. the heart primordium removed and immobilised on a Sylgard coated well and viewed under a binocular microscope. Microinjection was carried out following Fraser et al.(1990) and Martinez et al.(1991). Pipettes were pulled from borosolicate glass. tips filled with the corresponding dye solution and backfilled with 1 M LiC!. Biocytin (Sigma. 30mglml) and Iysinafed rhodamine dextran (LRD) 1Ox10' mw. Molecular Probes. 100mglml were micro-iontophoretically injected using a conventional microelectrode amplifier with 0.5-1 nAiSOO ms current pulses for 2-3 min for biocytin injections or 30-45 see for LRD. Electrode resistance was between 70 and 150 Mohm. Cells with membrane potentials lower than -15mV were discarded and those used for this work were typically about -40 mV. Specimens were fixed for two-three hours with 4% paraformaldehyde in 0.1 M phosphate buffer. after 20 min for biocytin injections. For clonal studies with LRD injections. the otic region was explanted and cultured for 36 hours on collagen gels with M-199IHank's medium and 5% fetal calf serum added. Explants were transversal sections of the embryo containing the neural tube and the otic placodes. Embryos were usually dissected further and flat mounted to be examined under epifluorescence microscopy and bright field illumination. In situ mRNA hybridisation was performed following the melhod described by Nieto et al.(1996) and whole mount immunodetection. The cek-8 probe was a kind gift of David Wilkinson and the HNK-I/Leu-7 antibody was from Becklon and Dikinson. Microinjections of biocytin were carried out on the edges and within the body of the otic placode. Injections performed at the bottom of the otic placode were typically oval or round shaped. expanded several cell diameters and showed a high dilution of the dye. In contrast. injections at the otic ridge showed sharp edges. expanded only few cell diameters and were intensely stained (Fig. 1) . Diffusion of the dye out of the ridge was limited both towards the ectoderm and towards the placode. and aiso laterally within the axis of the ridge. Similar results were obtained using Lucifer yellow as a dye. We examined a total of eleven ridge injections and eighteen body injections. The internal aspect of the otic placode is in close apposition to the neural tube. Biocytin injections on cells at the neural tube or the otic placode. however. never expanded into each other territory. A B OV ect. Figure 1. Restricted junctionalpermeability at the otic ridge. A, Squematic diagram of the experiment and the possibilities expected for the diffusion of biocytin into neighbouring cens. e, Whole-mount of a stage 13 chick embryo. Two cells located at the otic ridge, a and b were injected with biocytin for 3 and 1 minute, respectively. OV= otic vesicle, or= otic ridge. Exp.27296E12. Magnification=100x
pmid:9087785 fatcat:em6fjmey3naqniyng4ta5aeb6u