Gamer LW and Wright CV (1995), Autonomous endodermal determination in Xenopus:...

XB-ART-19320

Dev Biol 1995 Sep 01;1711:240-51. doi: 10.1006/dbio.1995.1275.

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Autonomous endodermal determination in Xenopus: regulation of expression of the pancreatic gene XlHbox 8.

Gamer LW , Wright CV .

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In neural plate stage Xenopus embryos, XlHbox 8 expression marks anterior endodermal cells fated to develop into pancreas/duodenum, and expression continues in adult pancreas in exocrine duct, acinar, and islet cells. Here, XlHbox 8 is used as a marker in experiments addressing the mechanisms of early endodermal patterning, particularly with respect to the role of specific polypeptide growth factors. When mesoderm-free vegetal explants (VEs) from early blastula stage embryos are cultured in isolation, XlHbox 8 expression develops autonomously in the dorsal region, strongly suggesting that endodermal region-specific determination occurs before MBT. Data from microinjection experiments using RNA encoding the activin and FGF dominant negative receptors and growth factor treatments of isolated VEs suggest that activin positively regulates XlHbox 8 expression, whereas bFGF is a potent negative regulator. Moreover, bFGF induces mesodermal marker expression in VEs. This suggests that the early endodermal determination state is plastic and that elevated levels of bFGF may convert vegetal (endodermal) cells into mesoderm. We propose a model for XlHbox 8 regulation in which an early signal from the Nieuwkoop center (whose eventual fate is endoderm) predisposes dorsovegetal cells for autonomous XlHbox 8 expression, in an area of high local activin (or activin-like) ligand concentration, and low relative concentrations of bFGF.

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Species referenced: Xenopus
Genes referenced: actl6a bmp4 fgf2 fgfr1 gcg gdf1 ins nog pdx1 sst.1 tbxt yes1
???displayArticle.antibodies??? Gcg Ab2 Gut Ab1 Ins Ab2 Pdx1 Ab1 Pdx1 Ab2

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	FIG. 1. Developmental expression of XlHbox 8. (A) RNase protection analysis of total RNA hom whole Xenopus embryos of stages 9 (late blastula), 10 !initial gastrula}, ll (mid-gastrula!, 12.5 /initial neural plate!, 14 (neural plate), 16 (neural fold), 19jneural tube), 25 (tailbudJ, and 35 (tadpole). The upper bands represent the protected 225-bp XlHbox 8 fragment, with I 5 embryo-equivalents of total RNA per reaction. The lower bands represent protected fragments for EF-1 a, using one embryo-equivalent of RNA per reaction. EF- 1 a in this and subsequent figures is a control for RNA loading and integrity. {EI Northern analysis of RNA from stage lO, 15, 25, and 35 Xenopus embryos. 3 .ug of poly(A)+ RNA was loaded per lane. The same filter was stripped and reprobed with EF-1 a (lower band!. The locations of 285 and ISS rRNAs are marked.
	FIG. 2. Colocalization of XlHbox 8 and islet hormones in adult frog pancreas. Sections of frog pancreas were immunostained with XlHbox 8 antibody, detected using alkaline phosphatase·linked secondary antibody {A, C, E), and then immunostained with hormone antibodies detected with fluorescently labeled secondary antibody (B, D, F). Arrowheads indicate representative cells coexpressing XlHbox 8 and hormone. (A, Bl XlHbox 8 :md insulin colocalization in an islet cell cluster. (C, Dl XlHbox 8 and glucagon colocalization near the pancreatic duct. IE, F) XlHbox 8 and somatostatin co\ocalization in an endocrine cell cluster. 500 cells positive for each hormone were scored for nuclear XlHbox 8. Bar, 50 .urn.
	FIG. 3. Expression of XlHbox 8 in embryos treated with agents that alter embryonic patterning. Input RNA for all hybridization reactions was 15 embryos for XlHbox 8 and 1 embryo for E.F-1 a. (AJ Embryos were ventralized by UV irradiation be.fore first cleavage and assayed with untreated siblings /WE} at stages 14, 25, and 35 for XlHbox 8 RNA by RNase protection. DAis of embryos used in this experiment ranged from 0-l. jB) Embryos were dorsalized by incubating 32- to 64-cell embryos in 0.2 M LiCl for 3 min IDAI 6- 7), 5 min (DAI 8), or 7 min [DAllO). Treated embryos and controls were assayed at stage 24. !CI Embryos were injected at the 1-cell stage with BMP-4/DVR-4 RNA. XlHbox 8 was assayed at stage 24. As a control for the efficiency of the BMP-4 effect, 5 injected and 5 uninjected (WE.) embryos were assayed for Xhox 3 at stage 13. Xhox 3 mRNA is upregulated in embryos overexpressing BMP-4 (Jones et a!, 1992}. (DJ Embryos were injected at the 1-ceJl stage with 10 or 100 pg of Xwnt-8 RNA and assayed at stage 24.
	FIG. 4. RNase protection analysis of XIHbox 8 expression in embryos injected with the dominant negative activin or FGF receptor RNAs. (AI Two-cell embryos were injected into each cell with 2 ng of dominant negative activin receptor RNA jDNActRI and whole embryos (\\'E) processed at sibling stages 11, 14, and 25. As a control for the proper phenotype, two injected and two control uninjected embryos were assayed for expression of Xbra. [B} Embryos were injected at the 2-celJ stage with 2 x 2 ng of dominant negative FGF receptor IDNFCFR) mRNA and processed at sibling stages 14, 22, and 25. As a control for the proper phenotype, one injected or one control embryo was assayed for expression of muscle-specific actin {ms-actin). In [A) and (B), RNA from 15 whole embryos IWE) was used in each X}Hbox 8 hybridization.
	FIG. 5. Autonomous expression of XlHbox 8 in vegetal pole explants (VEs). [A) Embryos were dissected into animal cap (AC] and vegetal pole lVE) pieces at stage 8 and assayed with whole embryos (WE} for XlHbox 8 RNA at stage 2.5. Thirty-five animal caps or VEs or 15 WEs were used per hybridization. (B) VE.s were isolated from stage 8 embryos. Total RNA from 2.5 explants was isolated when siblings reached stages 14, 25, and 35 and used in RNase protection analysis. (C) VEs were explanted from stage 8 embryos and cultured until siblings reached stage 25. Twenty-five explants were assayed for muscle-specific actin, Xbra, or XlHbox 8 expression using RNase protection analysis. For comparison o{ relat ive levels of actin expression, RNA from 5, l, or 0.2 whole embryos was assayed. Cytoskeletal actin [lower band) shows the relative level of RNA per sample. For comparison of relative levels of Xbra and X!Hbox 8, RNA from 5 and IS embryos, respectively, was assayed. {D) Whole-mount immunostaining of VEs for XJHbox 8. VEs were isolated from stage 8 embryos and immunostained at sibling stage 35. We infer that XlHbox 8 is expressed in an area in the dorsal side (d) of the e}._"Plants (bracketed} based on the data shown in E. Scale bar, 300 JliTI. (E) At the 4-cell stage embryos were marked on the ventral side. At stage 8, VEs were dissected into dorsal or ventral halves, cultured until siblings reached stage 35, and immunostained with XIHbox 8 antibody. Dorsal VEs expressed XlHbox 8 protein over much of their surface, whereas ventral VEs remained unstained (n = 30 dorsal-ventral explants). Endogenous melanin pigmentation is visible in some ventral explants.
	FIG. o. Growth factor effects on vegetal pole explants /YEs). For XlHbox B hybridizations, 2$ vegetal explants (YEs) or 15 whole embryos (WEs) were used. Two VEs or 1 WE were used in EF-1 a hybridiz.ations. Growth fact<Jr was present for the entire culture period except in (DI. (A) Fertilized embryos were irradiated with UV light for 45 sec (resulting in sibling DAI l l or 30 sec I resulting in sibling DAJ 2- 3), and VEs were isolated at stage 8. The explants were cultured alone or with 20 ng/ml activin, or SO ng/ml bFGF, until sibling stage 25. (B) VEs were isolated at stage 8 and treated with increasing doses of bFGF (1, 15, 50 ng/mll. Explants were assayed at sibling stage 25. ICI VEs were isolated at stage 8. In Janes 1 and 2, explants were cultured alone and collected when siblings reached stage 25 or 35. In lane 3, VEs were treated with SO ngfml FGF at stage 23 and then collected at stage 35 for RNase protection analysis. IDl VEs were isolated at stage 8 and either cultured alone (lane 1) or with growth factors. In Jane 2 VEs were cultured with 20 ng/m] activin alone, in lane 3 with 50 ng{ml bFGF alone, and in lane 4 with 2G ngjm\ activin and SG ng/ml bFGF. VEs wete assayed at sibling stage 25. (EJ VEs were isolated at stage 8 and treated with growth factors (activin 50 ng/ml and FGF 50 ng/ml}. Twenty-five YEs were assayed for Xbw at stages 11 and 24 and for XlHbox Bat stage 24. Ten stage 11 or 24 embryos were used as positive controls (lane 1). In a few experiments, a very small amount of residual Xbra transcript was detected in untreated VEs, which is probably due to expression by cells at the marginalfendodermal boundary. However, in these cases, activin completely removes Xbra from these VEs {lane 3]. (F) VEs were dissected from unjnjected embryos and from DNActR- or DNFGFR-injected embryos 14 ng total, same as in Fig. 4] at stage 8-9 and assayed at stage 25 for XlHbox 8.
	FIG. 7. Vegetal pole explants overexpressing Xwnt-8 have elevated levels of XIHbox 8. IAJ Embryos were injected at the 1-cell stage with LO or lOO pg of Xwnt-8 RNA. At stage 8, 25 VEs were isolated from control and injected embryos, culture<l until sibling stage 25, and then assayed for XlHbox 8. IBl One-cell nonalbino embryos were injected with 100 pg of Xwnt-8 mRNA and VEs were isolated at stage 8. Injected VEs were fixed at stage 35 for whole-mount immunostaining with XlHhox 8 antibody. Xwnt-8-injected VEs expressed more XlHbox 8 protein over a broader domain than controls (compare with C). ICI Control, uninjected VEs immunostained with XlHbox 8 antibody. Asterisks indicate local areas of extracellular nonspecific background separate from the nuclear XlHbox 8 staining. Scale bar, 300 #ill.
	FIG. 8. Animals caps treated with activin express XIHbox 8. Animal caps were isolated from stage 8 embryos and cultured in 0.75x NAM alone or in 0.75x NAM with 20 or 50 ng/ml activin or 50 ngfml bFGF. Fifty animal caps from each treatment were collected when siblings reached stage 35 and processed for RNase protection analysis. The arrowhead indicates the very low levels of XlHbox 8 RNA induced by 20 ng/ml activin. Two animal caps were used in EF-1 a hybridizations .