Hayes WP and Loh YP (1990), Correlated onset and patterning of proopiomelan...

XB-ART-25496

Development 1990 Nov 01;1103:747-57.

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Correlated onset and patterning of proopiomelanocortin gene expression in embryonic Xenopus brain and pituitary.

Hayes WP , Loh YP .

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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 pituitary primordium 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 pituitary primordium and brain. In fact, these cells were seen throughout the presumptive anterior and intermediate lobes of the pituitary and posterior diencephalon at the same time that the pituitary primordium 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 pituitary primordium and brain are simultaneously induced to transcribe the POMC gene, possibly as a result of reciprocal brain-pituitary interactions.

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Species referenced: Xenopus laevis
Genes referenced: gtf2a1l pdia2 pomc tbx2

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	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 diencephalon continue 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 100um.
	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 morphologically differentiated 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.