Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Differentiation
1993 Mar 01;523:195-200. doi: 10.1111/j.1432-0436.1993.tb00631.x.
Show Gene links
Show Anatomy links
Proopiomelanocortin gene expression as a neural marker during the embryonic development of Xenopus laevis.
Heideveld M
,
Ayoubi TA
,
van de Wiel MH
,
Martens GJ
,
Durston AJ
.
???displayArticle.abstract???
Proopomelanocortin (POMC) is the precursor protein for a number of peptide hormones and neuropeptides, and the POMC gene is transcriptionally very active in the pars intermedia of the pituitary of the amphibian Xenopus laevis (Xenopus). We analysed the expression of this gene during Xenopus embryogenesis, in order to examine whether it can function as a (novel) neural marker. We investigated the spatio-temporal distribution of POMC mRNA, using a single-stranded probe that corresponds to the 3' untranslated region of Xenopus POMC gene B mRNA. Gene transcripts were first detected at stage 25 of development via RNase protection assays. In situ hybridization analysis performed at stage 46 showed clearly that these transcripts are localised in a region representing the future pars intermedia of the pituitary. Experiments using Xenopus explants indicate that the POMC gene can be used successfully as an indirect marker in studies on neural induction: in the absence of interactions with mesoderm, ectoderm fails to express the POMC gene, whereas POMC transcripts are readily detectable in conjugates of ectoderm and mesoderm. Artificial application of two different signals, which are likely to be relevant for neural differentiation (namely retinoic acid and the activation of protein kinase C via phorbol ester), was not effective in evoking POMC gene expression in cultured ectoderm explants. However, retinoic acid treatment of conjugates of Xenopus ectoderm and mesoderm successfully prevented POMC expression. We conclude that POMC gene expression can be used as an indirect marker for anterior neural differentiation in Xenopus.
Fig. 1. Schematic representation of
the Xenopus proopiomelanocortin
(POMC) gene. A 1.1 kb fragment
from the Xenopus POMC gene B
cDNA clone pXP20 [14], cloned
downstream of the SP6 RNA polymerase
promoter, was used to produce
a synthetic RNA fragment of
284 bp (that corresponds to part of
the 3' untranslated region) for RNase
protection assays. Only 28 nucleotides
remain unprotected during
these assays. The same cDNA clone
was labeled by random primed incorporation
of digoxigenin-labeled deoxyuridine
triphosphate using a
DNA labeling kit, for in situ hybridization
experiments
Fig. 2. Spatio-temporal aspects of POMC expression. Arrowheads
indicate the position of the 256 nucleotide protected probe; in
some lanes input-probe (284 nucleotides) can be seen as an upper
band. A. Timing of POMC gene expression as judged by RNase
protection analysis. RNA was extracted from different developmental
stages and examined for the presence of POMC gene B
transcripts. These were first detected at stage 25 of development.
No transcripts were detected when total oocyte RNA (ooc) or
tRNA was assayed using the same probe. B. Localization of POMC
gepe expression. Stage 42/43 embryos were dissected into component
parts, pooled and used for RNA extraction. For analysis,
5 pg total RNA of head, back, belly and tail samples were used.
The gene appears to be transcribed only in the head region; RNase
protection analysis of total stage 42 material is shown as a control.
The integrity of the RNA from the dissected samples was checked
by RNase protection assay using a 5s RNA probe [29]. We conclude
that, from stage 25 onwards, POMC transcripts can be detected
in the developing Xenopus embryo ; its expression is restricted
to the head region
Fig. 3. In situ hybridization using
a Xenopus POMC cDNA probe.
Stage 46 larvae were examined as
described in Methods: experiments
were performed with the
POMC (A and C; C is a higher
magnification of A) or pBR 328
probe (B). Immunodetection of
digoxigenin-labeled DNA reveals
only POMC transcripts. These
are highly localized in a region
representing the future pars intermedia
of the pituitary. This is in
line with the results of the dissection
experiment described above
(see Fig. 2B), where the POMC
transcripts were shown to be expressed
only in the head. Several
anatomical structures are indicated
in B
Fig. 4. Signals mediating POMC expression. Arrowheads indicate
the position of the 256 nucleotide protected probe; in some lanes
input-probe (284 nucleotides) can be seen as an upper band. A.
The influence of light conditions on POMC gene expression during
development. Embroys were raised in total darkness from stage
10 until stage 40 and compared with embryos that had been reared
in a daily lightldark cycle. 5s RNA serves as a internal control
for the amounts of RNA that were used in this experiment. The
figure shows, that total darkness (dark) results in overexpression
of POMC mRNA when compared to control embryos (normal).
Ooc, oocyte RNA. B. POMC gene expression as a molecular
marker for neural induction. Explants containing both ectoderm
and mesoderm (EM) were made from early gastrulae and cultured
until control embryos had reached stage 42. These explants showed
neural differentiation and expressed the POMC gene. However,
ectoderm explants ( E ) , in which neural induction does not occur,
failed to express the POMC gene. The expression of the POMC
gene is thus consistent with that expected for a neural marker.
Again, 5s RNA is shown for a comparison. Moreover, analysis
of XIF6 expression, known to be a general neural marker [27],
was used to control for the effectiveness of the 12-0-tetradecanoylphorbol-
acetate (TPA) treatment to induce neural tissue. Neither
artificial activation of protein kinase C (E TPA; known to result
in neural differentiation: see the XIF6 expression), nor retinoic
acid-treatment (which suppresses the development of anterior neural
tissue; E RA), could induce POMC gene expression in ectoderm
explants. Apparently, these signals can not substitute for the endogenous
signals that are transmitted from the mesoderm to the ectoderm
during neural induction. 42, expression in 10 Fg total RNA
from stage 42 material. When ectoderm-mesoderm recombinates
were treated with retinoic acid (EM RA), although expressing the
general neural marker, they failed to show expression of the POMC
gene
