Kintner CR and Melton DA (1987), Expression of Xenopus N-CAM RNA in ectoderm is ...

XB-ART-28253

Development 1987 Mar 01;993:311-25.

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Expression of Xenopus N-CAM RNA in ectoderm is an early response to neural induction.

Kintner CR , Melton DA .

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We have isolated Xenopus laevis N-CAM cDNA clones and used these to study the expression of N-CAM RNA during neural induction. The results show that the first marked increase in N-CAM RNA levels occurs during gastrulation when mesoderm comes in contact with ectoderm and induces neural development. In situ hybridization results show that the early expression of N-CAM RNA is localized to the neural plate and its later expression is confined to the neural tube. Induction experiments with explanted germ layers show that N-CAM RNA is not expressed in ectoderm unless there is contact with inducing tissue. Together these results suggest an approach to studying how ectoderm is committed to form neural rather than epidermal tissue. Specifically, the data suggest that neural commitment is marked and perhaps mediated by the transcriptional activation of genes, like N-CAM, in the neural ectoderm.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actc1 actl6a krt12.4 mt-tr ncam1 ptprf tbx2 trna

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	Fig. 1. Restriction maps for the Nl and N5 Xenopus N-CAM cDNAs. The Pvull/Pstl fragment of Nl marked by "probe" was used for RNase protection assays. This fragment is likely to be representative for all N-CAM transcripts because a labelled RNA probe synthesized from this fragment in vitro hybridizes on a Northern blot to the same four transcripts detected by the EcoRl fragments of the Nl and N5 cDNAs. Moreover, the sequence of this fragment reveals that it contains one of the immunoglobulin-like repeats known from the sequence of chicken N-CAM to be present in the extracellular domain which is shared by all N-CAM polypeptides (Hemperly et al. 1986). The sites for £coRl (R), Hindlll (H), Pvull (Pv), Pstl (P) and Kpnl (K) are denoted.
	Fig. 2. Transcripts detected on Northern analysis by the Nl cDNA. 2-5/ig of poly(A)+ RNA from eggs (E), blastula (stage 9), early neural plate (stage 14), neurula (stage 18), or early tadpole (stage 22) were electrophoresed in a formaldehyde agarose gel, transferred to Gene Screen and hybridized with radiolabelled Nl cDNA. An autoradiogram obtained by a 3-day exposure to preflashed X-Omat film is shown. An identical transcript pattern was detected when the N5 cDNA (Fig. 1) was used as the probe. The molecular weight markers (M) on the left refer to the size in kilobases of fragments produced in a Hindlll digest of A DNA.
	Fig. 3. In situ hybridization of N-CAM to the neural tube of a stage-22 embryo. A RNA probe synthesized from the N5 N-CAM cDNA was hybridized to a cross section of a stage-22 albino embryo. Albino embryos were used to avoid pigment granules which are indistinguishable from autoradiographic grains. Panel A shows a tissue section from the trunk region photographed under phase-contrast optics. The neural tube (nt), myotomes (m), notochord («) and endoderm (e) are denoted. Panel B shows the same section under dark-field optics where autoradiographic grains appear white. The neural tube shows strong hybridization with the N-CAM probe. Panels C-E show a series of near adjacent transverse sections from a stage-20 albino embryo. Panel C shows a section photographed under phase-contrast optics with specific regions of the embryo denoted as in panel A. Panel D shows hybridization with an epidermal keratin Xek3 probe. Panel E shows hybridization with a.muscle actin, AC 100, probe. Hybridization is to epidermis in panel D and to the myotomes in panel E.
	Fig. 4. /n 5«M hybridization of a N-CAM probe to the neural plate. Transverse sections from a stage-16 albino embryo were hybridized with RNA probes synthesized from either the epidermal keratin Xek3 cDNA (A,B) or the N5 N-CAM cDNA (C-F). In each case a phase-contrast photograph (A,C,E) and dark-field photograph (B,D,F) is shown. B shows hybridization of an epidermal keratin Xek3 probe to a section from a more posterior region of the embryo. Note that hybridization occurs to surrounding ectoderm (se), but not to the neural plate (np) as labelled in the phase-contrast image shown in panel A. D shows hybridization of the N5 N-CAM probe to a section from a similar region of the embryo. Note hybridization to the neural plate (np), but not to surrounding ectoderm (se). F shows hybridization of the N5 N-CAM probe to sections from more anterior regions of a stage-16 embryo. Note that hybridization is in the neural plate, but not surrounding ectoderm. In addition, hybridization is concentrated in the medial, deep regions of the neural plate. Low or no hybridization is detected to the superficial ectoderm (arrows) or to lateral neural plate (arrowheads).
	Fig. 5. /n situ hybridizations of Nl N-CAM, muscle-specific and epidermal specific probes to a neural-plate-stage embryo. A series of transverse section from an early neural plate (stage 14) albino embryo were hybridized with RNA probes synthesized from the Nl N-CAM cDNA (B), the epidermal keratin Xek3 cDNA (C) and the muscle actin cDNA (D). A shows a section from this series photographed under phase-contrast optics. The dorsal midline is denoted with an arrow while the boundary between the neural plate and surrounding epidermis is denoted with an arrowhead. Note that N-CAM hybridization is concentrated toward the dorsal midline (arrow) and appears to be absent from lateral regions of the neural plate (arrowhead). Xek3 hybridization is to superficial ectoderm and stops at the boundary between the neural plate and surrounding ectoderm. Muscle actin hybridizes to the mesoderm. This dorsal mesoderm underlies the region of ectoderm expressing N-CAM RNA.
	Fig. 6. Expression of N-CAM RNA in early development. RNA samples prepared from eggs, stage 8, 10, 12, 14 and 16 embryos were assayed by RNase protection for N-CAM and EF-lcr RNA as described in Materials and Methods. The results were quantified by parallel assays performed using known quantities of sense N-CAM and EF-la- RNA synthesized in vitro. Note that EF-lar RNA increases and marks the MBT whereas NCAM RNA first increases during gastrulation, between stages 10 and 12. The maternal level of N-CAM RNA (present in eggs through stage 10) is approximately 0-01pg/embryo.
	Fig. 7. Induction of N-CAM RNA in recombinates and exogastrula. Ectoderm from the animal pole (A) and endoderm from the vegetal pole (V) were isolated from a stage-8 blastula as diagrammed and cultured alone or in combination (A/V). After 18 h in culture, RNA samples were prepared and assayed by RNase protection with probes for EF-la, NCAM, or the epidermal keratin Xek3 RNA (Epi.Ker.). The EF-la-probe is used to control for cell viability and number (see text). Lane N shows the protected probe obtained from RNA samples of normal st 22 embryos while lane C shows the probe protected by a negative control, tRNA. Note that N-CAM RNA is not expressed in the animal (A) or vegetal (V) fragment when these are cultured separately, but is in the A/V recombinate. Xek3 RNA is not detected in the V sample, but is in both the A sample and the A/V recombinate. Exogastrula were generated as described in Materials and methods and grown to the equivalent of stage 22. RNA samples were prepared from the total exogastrula (E) or from exogastrula divided into an ectodermal half (AE) and a mesodermal/endodermal half (VE) as shown. Samples were assayed as above. Note that N-CAM RNA is present at similar levels in exogastrula (E) and normal embryos (N). This N-CAM expression is concentrated in the ectodermal half (AE) of the exogastrula.
	Fig. 8. Localized expression of N-CAM RNA in recombinates. The ectodermal and endodermal portion of stage-8 albino blastula were recombined as shown in Fig. 7 and cultured for 18 h. A series of transverse sections were hybridized with a Nl N-CAM probe (A,B), an epidermal keratin Xek3 probe (C,D) or a muscle actin probe (E,F). In each case a phase-contrast photograph (A,C,E) and dark-field photograph (B,D,F) is shown. White arrows point to NCAM hybridization in B, epidermal keratin Xek3 hybridization in D, and muscle actin hybridization in F. Note the regions hybridized with these three probes do not overlap.
	Fig. 9. A series of longitudinal sections from albino exogastrula were hybridized with the epidermal keratin Xek3 probe (B), the Nl N-CAM probe (C), and the muscle actin probe (D). A shows a photograph under brightfield optics of one section from this series. The ectodermal sack (ec) is to the left of the photograph and the mesoderm/endoderm (m/en) portion is to the right. Note that the epidermal keratin Xek3 probe hybridizes to the ectodermal sack (arrow in B) and that the muscle actin probe hybridizes to a block of tissue in the mesodermal half (arrow in D). The N-CAM probe hybridizes to the junction between ectoderm and mesoderm (arrow in C).