Frank D and Harland RM (1992), Localized expression of a Xenopus POU gene depe...

XB-ART-23701

Development 1992 Jun 01;1152:439-48.

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Localized expression of a Xenopus POU gene depends on cell-autonomous transcriptional activation and induction-dependent inactivation.

Frank D , Harland RM .

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We have cloned a cDNA encoding a Xenopus POU domain protein, XLPOU91, which is expressed at high levels in gastrula embryos. XLPOU91 transcription initiates at the midblastula transition, and declines to low levels by late neurula stages. In early neurula embryos, XLPOU91 transcripts are enriched 35-fold in the most ventroposterior versus anterior regions. Initial transcriptional activation of the gene is cell autonomous; the gene is activated in dissociated gastrula stage embryos as well as in animal cap explants. Cell-cell communication is needed for proper temporal down-regulation of XLPOU91 expression in late neurula embryos; cell dissociation during blastula stages or removal of explants from the embryo prevents normal transcriptional shunt down. Explants treated with peptide growth factors (PGFs) mimic the normal temporal and spatial shut down in whole embryos. This negative regulatory pathway may be important for determining cell fate or maintaining an inducible state in the ventroposterior region of the embryo.

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Species referenced: Xenopus laevis
Genes referenced: actc1 actl6a fgf2 fn1 krt12.4 myod1 pou5f3

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	Fig. 1. Comparison of the amino acid sequence of XLPOU91 to POU genes of the five representative families (1-5). The amino acid sequence of XLPOU91 is presented in the sixth line of the figure. Amino acids that are identical to XLPOU91 are shaded. The histidine residue in the basic lysine/arginine-rich region is starred.
	Fig. 2. (A) Expression of XLPOU91 mRNA during Xenopus development. Total RNA was isolated from oocytes and developing embryos through stage 42 (lanes 1-15). One-half embryo equivalent of RNA was electrophoresed on a formaldehyde gel and blotted onto a nylon membrane for northern analysis. The filter was then hybridized sequentially with complementary eDNA probes of XLPOU91 and fibronectin (see Materials and methods). (B) Expression of XLPOU91 mRNA in all three germ layers in blastula and neurula embryos. Stage 9 blastulae were dissected into animal pole, marginal wne and vegetal pole regions (lanes 1-4) and lysed immediately. Stage 15 neurulae were dissected into dorsal and ventral halves (see Materials and methods) and the yolky endoderm cells were scooped out of the midposterior region of the ventral half using a hairloop and eyebrow knife, the remainder of the embryo was collected as meso-ectoderm (lanes 5-7), and tissues were lysed immediately. Total RNA was isolated as a pool from ten whole embryos and dissected regions. Two embryo equivalents of control (C) RNA were loaded per well at stage 9 (lane 1). Four marginal zone (MZ) and six animal (AC) and vegetal (V) pole explant equivalents of RNA were loaded per well (lanes 2-4). One embryo equivalent of control (C) RNA and one meso-ectoderm (ME) and six endoderm (EN) equivalents of explant RNA were loaded per well (lanes 5-7). The stage 9 filter was hybridized sequentially with complementary DNA probes of XLPOU91 and GS-17 (the zygotic GS- 17 transcript is expressed ubiquitously throughout mid-late blastula stage embryos) and the stage 15 filter was hybridized sequentially with eDNA probes of XLPOU91, XMyoD, and EFl a- (see Materials and methods).
	Fig. 3. (A) Local expression of XLPOU91 in early neurula stage embryos. Stage 14-15 embryos were dissected into six pieces along the dorsoventral and anteroposterior axis as described in the text. Total RNA was isolated as a pool from ten dissected embryos in two separate experiments. One-half embryo equivalent of control RNA was loaded per well for northern analysis (lane 1). An equivalent of RNA from one dissected region was loaded per well: DA, dorsoanterior (lane 2); DM, dorsomiddle (lane 3); DP, dorsoposterior (lane 4); VA, ventroanterior (lane 5); VM, ventromiddle (lane 6); and VP, ventroposterior (lane 7). The filter was sequentially hybridized with cDNA probes for XLPOU91, XMyoD, and EF1 a (see Materials and methods). (B) Schematic lateral view of dissected neurulae in A. Relative levels of XLPOU91 mRNA in each region are shown (see Materials and methods). (C) Expression of XLPOU91 in UV-ventralized gastrulae and neurulae. Embryos were ventralized by UV irradiation (see Materials and methods). Total RNA was isolated from pools of ten normal and ventralized embryos at each stage. Two embryo equivalents of RNA were loaded per well for northern analysis: controls, stage 11 (lane 1), stage 12 (lane 2), stage 13 (lane 3) and stage 14 (lane 4); ventralized, stage 11 (lane 5), stage 12 (lane 6), stage 13 (lane 7) and stage 14 (lane 8). The filter was hybridized sequentially with the cDNA probes of XLP0U91 and fibronectin (see Materials and methods).
	Fig. 4. Albino embryos were examined by whole-mount hybridization using digoxigenin-labeled XLPOU91 antisense and sense probes (see Materials and methods). (A) Ventral view of a late neurula embryo hybridized with an antisense probe. (B) Lateral view of a late neurula embryo hybridized with a sense probe. Both embryos are oriented anterior-posterior from left to right.
	Fig. 5. (A) Autonomous expression of XLPOU91 in CMFM-treated gastrula stage embryos. Embryos were treated with CMFM (see Materials and methods) as described below. Total RNA was isolated from a pool of ten embryos from each treatment, and two embryo equivalents were loaded per well for northern analysis. Lane 1: controls grown in l/3xMR until stage 11. Lane 2: embryos grown in CMFM from the 4 to 8-cell stage until stage 11. Lane 3: embryos grown in l/3xMR until stage 7, and in CMFM from stages 7-11. Lane 4: embryos grown in CMFM from the 4 to 8-cell stage until stage 7, and in 1/3XMR from stages 7-11. Filters were sequentially hybridized with the cDNA probes for XLPOU91, XMyoD, and EF1 a (see Materials and methods). (B) Effect of PGFs on XLPOU91 expression in gastrula and neurula animal cap explants. Animal caps were dissected from embryos at stage 8. Explants were incubated with the appropriate PGF until stage 11 (see Materials and methods). Total RNA was isolated from pools of five explants at stages 11 and 18. One embryo equivalent of control whole embryo RNA was loaded per well for northern analysis and two and a half to four animal cap explants were loaded per well. Lane 1: Stage 11 embryo. Lane 2: Stage 11 animal cap. Lane 3: Stage 11 animal cap, PIF 1:1. Lane 4: Stage 11 animal cap, PEF 1:10. Lane 5: Stage 11 animal cap, medium bFGF (200 ng/ml). Lane 6: Stage 11 animal cap, low bFGF (40 ng/ml). Lane 7: Stage 18 embryo. Lane 8: Stage 18 animal cap. Lane 9: Stage 18 animal cap, PIF 1:1. Lane 10: Stage 18 animal cap, PIF 1:10. Lane 11: Stage 18 animal cap, high bFGF (800 ng/ml). Lane 12: Stage 18 animal cap, medium bFGF (200 ng/ml). Lane 13: Stage 18 animal cap, low bFGF (40 ng/ml). The filter was sequentially hybridized with the cDNA probes for XLPOU91, muscle-specific cardiac actin, £75-cytokeratin and EFltx (see Materials and methods).
	Fig. 6. Inhibition of XLPOU91 down-regulation by dissociation of embryos. Embryos were transiently treated with CMFM as described in the text. Total RNA was isolated from pools of ten embryos for each indicated treatment at stage 20, and two embryonic equivalents of RNA were loaded per well for northern analysis. At each tune point of CMFM treatment was paired with a control group. Lane 1: Control in 1/3xMR. Lane 2: CMFM treatment stages 4 to 11.5, 1/3 x MR stages 11.5 to 20. Lane 3: Control in 1/3XMR. Lane 4: CMFM treatment stages 6 to 11.5, l/3xMR stages 11.5 to 20. Lane 5: Control in 1/3XMR. Lane 6: CMFM treatment stages 7 to 11.5, 1/3 x MR stages 11.5 to 20. Filters were sequentially hybridized with the cDNA probes for XLPOU91, XMyoD, cytokeratin and EFla (see Materials and methods).