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To identify cellular interactions that underlie the spatially appropriate transcription of neural genes, we characterized the embryonic development of proopiomelanocortin (POMC) gene expression in Xenopus laevis using in situ hybridization histochemistry. This has led to the establishment of a unique model system for studying how a neuropeptide gene program in four distinct cell groups is set up in pituitary and forebrain. The embryonic onset and patterning of POMC expression was found to be spatially and temporally correlated inside and outside the brain. The first POMC cells in the pituitaryprimordium and diencephalon were juxtaposed near the infundibulum at stage 29/30, indicating they undergo molecular differentiation much earlier than previously reported for this system. By stage 31/32, many more POMC cells appeared in the morphologically undifferentiated pituitaryprimordium and brain. In fact, these cells were seen throughout the presumptive anterior and intermediate lobes of the pituitary and posteriordiencephalon at the same time that the pituitaryprimordium is translocating ventral to diencephalon. By stage 39/40, coordinated morphogenesis produced the adult pattern of POMC cells localized in distinct anterior and intermediate pituitary lobes and two diencephalic nuclei. We propose in light of these findings that embryonic cells in the pituitaryprimordium and brain are simultaneously induced to transcribe the POMC gene, possibly as a result of reciprocal brain-pituitary interactions.
Fig. 1. Diagram (A) showing Xenopus proopiomelanocortin (POMC) gene structure and the molecular probes. Northern
blots (B,C) showing probe specificity. (A) The POMC gene is comprised of three exons with exon 1 encoding the 5'
untranslated region, exon 2 the signal peptide, and exon 3 the prohormone (Martens, 1987). The prohormone is processed
post-translationally at paired basic residues to different neuropeptides (Loh, 1987). Six different Xenopus-speciftc probes
have been used for filter and in situ hybridization. (B,C) Northern blots of total RNA from adult Xenopus brains (Ad) or
stage 47 tadpoles (47) show that the Exon 3 cDNA (B) and oligonucleotide probe 2 (C) hybridize to the same species
(-1.3 kb) of POMC mRNA found by Martens et al. (1985).
Fig. 2. Stage 28: Dark-field and bright-field
photomicrographs showing (A) the onset of POMC
expression, and (B) the relationship of the pituitary
primordium (HP) to the embryonic forebrain (FB).
(A) Coronal section from head (level shown below)
showing the first evidence of POMC mRNA (arrow) in the
posterior end of the translocating pituitary plate at the
juncture of diencephalon and infundibulum ventral to
posterior diencephalon (pDI) and optic vesicles (OV).
(B) Sagittal section through forebrain (anterior is to the
right) showing the anterior-to-posterior extent of the
translocating pituitary plate (HP, arrowheads) between
diencephalon (DI), infundibulum (IF), foregut (FG) and
cement gland (CG). The anterior trailing end is shown
extending to its origin in the stomodeum (S), and the
leading end of the plate does not yet occupy (as in Fig. 5)
the region beneath the juncture of the thicker diencephalic
(long arrow) and thinner infundibular (short arrow) walls.
POMC mRNA was first detected in sections at this level in
pituitary plate at stage 28 (above), and in brain at stage
29/30 (Fig. 3A). Note the plate is connected to brain floor
at attachment sites. Calibration bars are 100um.
Fig. 3. Stage 29/30 and 31/32: High-magnification
photomicrographs of 20,um sections showing POMC
mRNA in pituitary primordium (short arrows) and
posterior diencephalon (pDI, long arrows) using exonic
probe 2 (A,B), but no primary transcript with intronic
probes 4 or 5 (C,D). (A) Posterior coronal section from a
stage 29/30 embryo (the sixth of eight with labeling in
pituitary plate) showing two midline foci of POMC mRNA
in brain adjacent to labeled plate. (B) Similar section at
stage 31/32 showing higher levels of POMC mRNA in
plate and diencephalon. (C,D) Intronic probes 4 and 5
showed no above background labeling of plate (arrows) or
brain at stage 29/30 and 31/32, respectively. Note, these
thicker sections always show an increased background grain
labeling, but also an enhanced signal to noise. Calibration
bar is 40um.
Fig. 4. Stage 31/32: Serial horizontal 12,t/m sections in
bright-field and dark-field from anterior (aFB) to posterior
(pFB) forebrain showing the relationship of POMCexpressing
cells in brain (thin arrows) and pituitary
primordium (thick arrows). (A) Ventrally near the trailing
end at the level of the optic stalk (OT) only the
hypophyseal plate is labeled between diencephalon (DI)
and foregut (FG). (B-D) More dorsally at the leading end
more evidence of POMC mRNA is seen in the midline (B)
and lateral walls of diencephalon (C,D) adjacent to the
labeled plate. Calibration bar is 100 um.
Fig. 5. Stage 33/34: Bright-field and dark-field sagittal view
through forebrain (FB, anterior is to the left) showing the
anterior-to-posterior extent of POMC-expressing cells
within the translocating and elongating pituitary plate. The
plate is now labeled from its trailing end or origin in the
stomodeal ectoderm (S), to its leading end ventral to
forebrain (FB) at the juncture (Ju, arrow) of the thickwalled
diencephalon (Dl) and thin-walled infundibulum
(IF). Note the relationship of the hypophyseal plate to the
expanding foregut (FG), differentiated cement gland (CG)
and notochord (N). Calibration bar is 100um.
Fig. 6. Stage 33/34: Spaced
serial coronal 12j.im sections in
bright-field and dark-field
showing POMC mRNA in the
pituitary plate (thick arrows,
A-F) and posterior
diencephalon (thin arrows,
C-F) before the plate has
condensed rostrocaudally. This
embryo showed label in
sixteen contiguous sections of
plate; sections no. 9 and no.
15 (C,F) were the first and last
to show brain label.
(A) POMC cells are found in
the plate's extreme trailing end
between anterior diencephalon
(aDI) and oral ectoderm at the
level of retina (R) and optic
stalks (OT). (B) More
posteriorly, only plate cells
express POMC between
posterior diencephalon (pDI)
and foregut (FG). (C-E) Brain
POMC cells in midline
posteriorly in the lateral walls
of anterior infundibulum (alF).
(F) POMC cells disappear in
lateral diencephalon, whereas
those in the plate's leading end
become embedded in the
posterior infundibular wall
(pIF). Calibration bar is
Fig. 7. Stage 39/40: Spaced serial
coronal 12 ^m sections in brightfield
and dark-field showing the
adult-like distribution of POMC
cells in brain and pituitary after
the hypophyseal plate has
condensed to half its original
length. This embryo showed label
in twelve contiguous sections;
sections no. 1-8 (A-E) in brain,
no. 4-9 (B-E) in anterior
pituitary, and no. 10-12 (F) in
intermediate lobe. (A,B) Sections
of anterior diencephalon show
POMC cells in the midline
preoptic nucleus (NPO, long
arrows) at the level of optic tract
(C). (C-E) Serial sections of
posterior diencephalon show more
lateral POMC expression in the
ventral infundibular nucleus of the
hypothalamus (NIV, long arrows)
at the level of midbrain (M) and
notochord (N). The
anterior pituitary (short arrows)
can also be seen still attached
anteriorly to the roof of the
pharynx (P) derived from oral
ectoderm (B,C), and more
posteriorly to diencephalon
(D,E). (F) Posterior to anterior
pituitary, the intermediate lobe
(arrow) in the infundibular wall
always shows the most intense
labeling at this and later stages.
Calibration bar is 100um.