N myc; neural, pronephros
N-myc, nucleic acid and polyclonal antisera.
Cloned by Peter Vize. Photos by Peter Vize and Inga Dunkerson
The distribution of N-myc was first examined by immunohistochemistry using a affinity purified polyclonal against human N-myc which was generated by Gerard Evan. This anti-peptide polyclonal was affinity purified using a peptide corresponding to the Xenopus, rather than the human protein (they only differ by 1 aa in this region anyway). It was a little surprizing that the gene product was not observed in the pronephros, as N-myc is expressed in other developing kidneys. Wholemount in situ hybridization revealed that a low level of N-myc mRNA is observed in the pronephros. This pronephric staining is very faint, and transient (stage 20 to 30). Other structures that stained strongly with the antibody, brain, eye, crest, later somites etc. look similar in both antibody and in situ stains. The pronephric stain may have been missed due to its low intensity, or N-myc protein may be particularly unstable in the pronephros. The in situ shows much stronger staining in the crest. In ealy stages RNA is not detected in the heart, but is observed in heart from early-mid 30's. More (=better) pictures to follow
Left, in situ. Right, polyclonal antisera. In both, the stain is observed in the neural tube, eye vesicles and neural crest. Anterior view, dorsal is up.
Left, in situ; Right, polyclonal.
The most obvious stain is in the neural crest (more obvious in situ), eye, brain and otic vesicle. Just behind the posterior branchial crest segment (the strongest crest stain at this stage) faint staining of the pronephric anlage is also observed. Somites also stain.
Left, close up of in situ pattern, stage 24. Note strong crest, and weak pronephric anlage staining (arrow). Right, another stage 24 in situ, anterior view.
- reference: Vize, P.D., Vaughan, A. and Krieg, P.A. (1990). Expression of the N-myc proto-oncogene during the early development of Xenopus laevis. Development 110; 885-896.
- clone; pSPNmycEP, approx 500 bp EcoRI/PstI fragment from 3' UTR of pXNmyc inserted into EcoRI/Pst pSP72 (Promega)
- sequence; 3' UTR has not been sequenced. Sequence of each of the three exons is available seperately. Exon 1. Exon 2. Exon 3. Genbank search
- for antisense probe cut with PstI and transcribe with T7
- DNA probe available from Peter
- anti-peptide antibodies, NSG C-1 and NSG C-2 available from Gerard Evan.
These antisera were affinity purified against a synthetic peptide corresponding to the Xenopus N-myc C-terminus (see Vize et al.,1990 for details). I do not have any peptide left.
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