Southern blot analysis of the Radar gene. Genomic zebrafish C32 or Medaka DNA samples were digested with the restriction enzymes indicated at the top of panels A and B. DNA was transferred to filters and probed with the 383-bp DNA fragment from Z15 cDNA representing the C-terminal region of Radar. Molecular weight standards are indicated on the left of each autoradiogram. (A) Under low stringency conditions, (washes at 37°C, 2 x SSC) several bands appear in each zebrafish lane as well as in the medaka lanes. (B) After high stringency washes (68°C 0.1 x SSC), one band only can be detected in the zebrafish lanes and no band is detected in the medaka lanes. The band detected in the BamHI digest from zebrafish DNA is very weak due to a lower mass of electrophoresed DNA in this lane. (C) Genomic DNA from man, cow, mouse, chick, Xenopus laevis, trout, zebrafish and Drosophila melanogaster was digested with EcoRI, transferred to a filter and probed as described above. In each of the vertebrate species, several bands are detected. No hybridising DNA fragment is detected in Drosophila, the only invertebrate species tested.

Expression of the Radar gene during zebrafish embryogenesis. Whole-mount in situ hybridisation of embryos of different developmental stages. (Anterior is to the left, by convention). (A and B) Dorsal views from embryos at the 90% epiboly (9 hpf) and tailbud (11 hpf) stages. In (A), Radar is expressed in the anterior third of the embryo, at the edges of the neural plate, in two parallel lines of cells starting approximately distal to the future forebrain region. Later (B), the domain of expression has expanded both rostrally and caudally to surround the entire edge of the neural plate. The strongest staining is observed in the future mid- and hindbrain regions. The posterior midline of the embryo is also stained. (C and D) In the 16-somite-stage embryo (14 hpf), Radar is expressed as a salt and pepper pattern along the dorsal side of the embryo with stronger expression in a region posterior to the eye. Staining is also observed in a thin region at the level of the dorsal midline of the embryo and in the posterior part of the spinal cord in a rostro-caudal gradient with higher expression in the tailbud. Fainter staining is detectable in the hatching gland and the hypochord. (C) Left lateral view. A part of the yolk was lost during washing steps. (D) Dorsal view. (E-G) Expression of Radar in the 26-somite-stage embryo (22 hpf). (E) Left lateral view of an entire embryo. Expression persists in the dorsal midline of the embryo. starting rostrally on the sides of the fourth ventricle (tulip-like structure. dorsal view in (G)), continuing just behind the otic vesicle as the roof plate and caudally finishing as the roof plate and the future dorsal and ventral fins. Expression also persists in the hypochord and the hatching gland. New sites of expression include the dorsal quadrant of the eye, exactly opposite to the choroid fissure, the lateral line primordia (see (F). detail of the head region in (E)) and the posterior ventral mesenchyme. In (F), the position of the otic vesicle is indicated by an arrowhead. (H and I) Expression of Radar in posterior mesodermal tissues in wild-type (H) and ntl (I) embryos. The domain of expression in the ntl ventral posterior mesenchyme is smaller than in wild-type embryos. No detection in the region corresponding to the hypochord (above the mesenchyme region) is observed in ntl embryos. Dorsal expression of Radar is maintained in ntl embryos but out of focus because the tail is kinked. Arrowheads in (A), (C) and (E) indicated the level of the sections depicted subsequently.

Serial sections of whole-mount embryos depicted in previous figure. The embryos shown in Fig. 4 were embedded in paraffin and cross-sectioned. Neighbouring sections were either observed with Nomarski optics directly (A, C, E, G, I, K. M) or after staining with haematoxylin and eosin (B, D, F, H, J, K, L, N). Embryos dorsal side oriented up. (A and B) Sections through the head of an embryo at the 90%, epiboly stage. Expression is confined to three to five cells located superficially at the edge of the neural plate thickening. (C and D) Sections through the head of a Ibsomite embryo. Expression is observed as a scattered staining within the epidermis covering the brain and the eyes. Weak expression is seen in the ventral diencephalon. (E and F) Sections through the tailbud region of the same embryo. Expression is detected in the dorsal part of the neural tube which will generate the roof plate and the dorsal fin. (G and H) Sections through the head of a 2bsomite embryo. at the level of the eyes. Radar expression is confined to the dorsal part of the retina. Only the upper half of the sections are shown. (I and J) In the posterior trunk of the same embryo, at the level of the yolk tube, staining is detected in the hypochord (arrowhead), the neural tube and loosely connected cells located between the somites, the hypochord and the gut. These cells may undergo migratory movements. Expression is also detected at three positions within the epidermis surrounding the yolk tube. (K and L) In sections of the anterior trunk from the same embryo, expression is detected only in the roof plate. Only the upper half of the sections is shown. (M and N) Sections at the tail level from the same embryo reveal low levels of expression in the dorsal and ventral fins. Stronger expression is detected in the hypochord and in the median mesenchymal region immediately ventral to it.