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???displayArticle.abstract??? Tissue distribution and localization of RNAs from the Xeb1 homoeo-box-containing gene were monitored with Northern blots and in situ hybridization. Xeb1 transcript distribution in larval stage embryos was established by blotting RNAs extracted from microdissected embryos. Those transcripts are restricted to a limited number of embryonic regions such as the dorsal trunk. The tissue/cell localization of Xeb1 transcripts was then monitored at several embryonic stages with in situ hybridization methods using [35S]RNA probes. These homoeo-box transcripts accumulated in a progressive and dynamic fashion. First localized in late gastrulae, they are distributed along the neural tube and in caudal mesoderm at later stages. By the swimming tadpole stage the spatial distribution of the homoeo-box transcripts is limited to specific regions of the central nervous system. Adult spinal cord shows the signal in specific neurons in the ventrolateral field of the gray matter.
FIG. 1. Restriction map of the genomic recombinant clone and the pcXeb 1 cDNA clone. The Xebl cDNA was isolated from a gtl0 phage library of stage 12 Xenopus laevis embryos. The black box indicates the region homologous to the Drosophila homoeo-box (Carrasco et al., 1984). The direction of transcription is from left to right (arrow). The arrows in both directions in the lower diagram indicate the 900~bp BgiII-EcoRI fragment which was used to produce single-stranded RNA probes.
FIG. 2. Northern blots of RNAs extracted from dissected embryos. Poly(A)* RNA from 200 dissections of stage 41 tadpoles was loaded in each gel (1.2% formaldehyde-agarose) lane and eventually was transferred to GeneScreen membranes (New England Nuclear). Hybridization was performed at high stringency with a âP-nick-translated 1.4-kb Xebl fragment (>107 cpm/pg). Transcript sizes, indicated in kilobases, were estimated with rRNA standards (Carrasco et ah, 1984). (a) Planes of dissection shown in embryo diagram. Lane w = whole embryo (10 pg RNA-equivalent to yield from approx 50 embryos); 1 = head (5 Fg); 2 = dorsal (5 fig); 3 = ventral (3 Kg); (-) = 20 pg poly(A)) RNA from whole embryos. The same filter was washed and rehybridized with a cytoplasmic actin probe. (b) Planes of dissection shown in embryo diagram. Lane w = whole embryo (8 wg RNA-from approx 50 embryos); 1 = anterior head region (6 pg); 2 = posterior head region (2.5 pg); 3 = trunk (5 fig); 4 = tail (5 fig). Also included is the rehybridization of the filter with a mixture of muscle specific (m) and cytoplasmic (c) actin cDNA probes. Two-week autoradiographic exposure.
FIG. 3. In situ hybridization localization of Xebl transcript accumulation in gastrulae [st. 12 (Nieuwkoop and Faber, 1956)]. (a) Darkfield photo of in situ hybridization of a sagittal section of a stage 12 gastrula. A 900-bp [%]UTP-labeled RNA probe complimentary to the Xebl coding strand transcript was employed. This section shows both the dorsal and ventral regions around the blastopore (bl). The bars mark the edges of the blastopore. Label can be observed in the dorsal lip (dl) region, but not in the ventral lip (vl) region. 17X magnification of a 40-day exposure. (h) Higher magnification view (66X) of (a), showing label in a limited region of the dorsal lip (IO-day exposure). (c) Section hybridized with the opposite strand (sense probe). No signal could be detected in the dl cells after 40 days exposure. 66x magnification.
Fig.4 Xeb1 transcript distribution in neurula and tailbud stages
Fig. 5 Accumulation of Xenb1 transcripts in late tailbud stage
FIG. 6. Restriction of Xebl transcripts to the cervical spinal cord of the swimming tadpole. In silu hybridization (using same probes as Fig. 3) of longitudinal sections of stage 41 larvae. (a) Hybridized with sense RNA strand (control); (b, c) hybridized with antisense strand. (c) Higher magnification (80X) of spinal cord region of (b). Exposed for 40 days. R = rostral; br = brain; y = yolk-laden endoderm; SC = spinal cord; IV = 4th ventricle of brain; hbr = hindbrain; m = mesoderm; n = notochord; sm = somites; p = pharynx.
FIG. 7. Transcript accumulation in the adult spinal cord. Darkfield photographs of transverse sections of the adult spinal cord. (a) and (b) Are at low magnification (9X), lshowing the entire spinal cord. (c) and (d) Are at higher magnification (47x), showing a detail focused on the left half of the gray matter. Tissue was dissected from an adult female and fixed in methanol, as described for embryos. Likewise, in situ hybridization was carried out in the same way as described for embryos. (a) and (c) Represent controls stained with Giemsa and photographed in darkfield to show cellular details of the sections. In the ventral horn the large motoneurons (m) can be identified. The rest of the gray matter shows groups of neurons (n, with pointers) with different shapes. (b) Shows a section adjacent to (a) hybridized with the Xeb1 antisense probe in the same way as was performed in embryo sections, and exposed for 40 days. Brackets show the region which is magnified in (d). (d) shows the bracketed region, enlarged from (b), of half of the spinal cord gray matter. Groups of neurons (n, pointers) display signal in the lateral field [compare (d) with (e) to localize the cell position in the same half of the spinal cord). (e) Represents a section through the medulla oblongata hybridized concomitantly with the section shown in (a). No signal can be observed in the section.
