Beck CW et al. (1998), Involvement of NF-kappaB associated proteins in...

XB-ART-15336

Int J Dev Biol 1998 Jan 01;421:67-77.

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Involvement of NF-kappaB associated proteins in FGF-mediated mesoderm induction.

Beck CW , Sutherland DJ , Woodland HR .

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In this report, we have used mRNA injection to study the action of mutants of XrelA, a Xenopus homolog of the RelA (p65) component of NF-kappaB, on the induction of mesoderm in Xenopus embryos. A region of the rel homology domain of XrelA was deleted to create XrelA deltaSP, which retains the dimerization and activation domains, but no longer binds to DNA. We also made an analogous derivative of mammalian NF-kappaB1 (p50). We show that both constructs have dominant inhibitory activity. When message encoding either is injected into eggs or oocytes, DNA binding of rel family members is suppressed, as is transactivation of a kappaB-dependent promoter in embryos. Expression of XrelA deltaSP in animal caps blocks the induction of mesoderm by bFGF. In addition, this mutant prevents elongation movements generated by activin, but has little effect on posterior dorsal cytodifferentiation, which in marked contrast is blocked by inhibition of the FGF signal transduction pathway between the receptor and MAP kinase. The specificity of the XrelA deltaSP effect on FGF signaling is shown by rescue of mesodermal marker expression when XrelA deltaSP is co-expressed with a specific rel inhibitor. The target of these dominant negative constructs seems to be neither XrelA itself, nor p50, but rather some other molecule with which XrelA, rather than NF-kappaB1, heterodimerizes. We show that XrelA deltaSP blocks FGF induction of mesoderm downstream of MAP kinase and Xbra expression. Thus it prevents the maintenance of Xbra expression by inhibiting its autoregulation by embryonic FGF (eFGF). We suggest that XrelA deltaSP differs from other reported inhibitors of FGF signaling because it inhibits only gastrula stage FGF signaling and not the maternally programmed signaling at the blastula stage. Our results therefore suggest that zygotic FGF action is required for cell movements rather than dorsal differentiation.

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Species referenced: Xenopus
Genes referenced: abl1 arhgef7 bmp4 fgf2 fgf4 lipa mapk1 nfkb1 rel rela tbx2 tbxt

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	Fig. 1. Schematic diagram of rei deletion constructs.
	Fig. 2.1nhibition of XreiA and p50 function by mutants with dominant negative activity. (A) Each of the DNA bmdmg defic1ent delet1on constructs was tested for abl/1ty to mh1b1t DNA bmdmg of XreiA.l222. which conrams the w1ld-rype Xre/A ref homology domam. and of p50. The appropnate consrrucrs were co-translated m oocyres. and DNA bmdmg v1sua/1sed using gel mobility shift assays The rauo of dommanr negat1ve mRNA to w1/d-type RNA was 20:1 (4 ng 200 pgJ m each case. (B) The same delet1on constructs were also tested for the abli1ty to mh1b1t transactiVatlon by w1ld-type Xre/A from an reporter construct lpLC2R) dnven by the HIV L TR. DNA and RNA as shown were tn}ected mto 2-ce/1 embryos and CAT actiVIty measured at stage 11. (B') The product in B was quantified with respect to total soluble protein.
	Fig. 3. Effects of rei deletion constructs on embryos. (A. A', A H) Comparison of phenorypes at stage 25. IAI Representative embryo from a batch injected with 1 n9 XrelAjSP mRNA showing reduction in posreflor structures. IA') More severely affected embryo from same batch showmg alt's split dorsally around an e'-posed yoll<: plug. (A'" Uninjecred control sibling embryo. IB) Comparison of control stage 40 embryo (upper) wirh sibling embryo In- Jecred with Ing XreIA...1SP mRNA (lower). Note the complete lack of tail formatIOn In rhe lower embryo. IB'I Com- Pdnson of tYPical embryo at stage 38 (upper) wIth 2 embryos from batches Injected with 0.5 ng XFO mRNA (lower). IC and C'lln Situ hyb"d,zations comparing e ~press,on of Xbra at stage 10 In an unlnjected embryo (0 and In a sibling embryo bllarerally injected wIth , ngXrelA.J.SPmRNA IC'). Note the reduction In Xbra e>l.pression to a small arc ID and D'iln situ hybridizations for the expression of the notochord marker collagen II. fD) Control embryo at stage 32 showing the normal development of the notochord and weaker staimng in the somites. 10') Distribution of dorsal a)(lal tissues around the e>l.wsed yolk plug in an cleared embryo Inlecred with 1 ng XrelAlSP RNA Note that a notochord IS present only on one side of the plug. while the other side of the plug has only umdentlf,ed loosely staining tissue (possIbly either disorganized somite or notochord'.
	Fig. 4 A.E: Effect of XrelA and p50 dominant negative expression on the induction of morphological movements in animal caps. Embryos were uOinJecredlAI, or InJecred with 1ngofmRNA encodmg the dommant negarlves XrelA1SP222 IBI. XrefA1222lCl, p50-1SPIDI or X,elA1SPIEI ar the two cell stage. Animal caps were Isolated at stage 8 5 and cultured In Barth's medium alone. or containmg either 1 .\ WIF or 50 ng/ml bFGF. Normal morphogenetic movements of the tissue, scored at control stage 17, are seen in response ro both facrors with the exception of caps taken from embryos e)(pressing XreIAlSP. Inhibition of FGFand activin mduced e:tr.tension movemenrs by XrelA1SP IShighly reproducible. Bar, 250 um
	Fig. 5. Differential effects of XrelADSP on expression of mesodermal marker genes induced by FGF and activin. Embryos were injected with XrelA.JSP mRNA as shown and animal caps were excised at stage 8.5 before treatment with mesoderm mducmg factors. fAI Expression of markergenes at stage 11. (81 Expression of muscle and neural specific genes at stage 17. (CI Rescue of the effects of XrelA.JSP. Co-mjectJon of 500 pg of XrelA.lSP mRNA with 1ng of mRNA encoding h.-a. a specific inhibitor of NF-",.Bproteins. fully rescues the induction of Xbra by bFGF.
	Fig. 6. Animal caps from embryos injected with XreiA. XrelA.J222 or p50JSP were excised and treated 8S in Fig. 5 and analyzed for Xbra expression at stage 11. Wild rype XrelA IAI equally reC1uces induction of Xbr8 by borh aCClvln and FGF. The dommanr negatives p50.'JSP 181 and XrelA.J222 ICI have no discernible effect on mductlon of Xbra by either facror
	Fig. 7, XrelAjSP does not inhibit the induction of mesoderm by BMP-4. Animal caps were injected with I ng BMP-4 mANA alone or in combination with 2 ng Xre/AJSP mANA, as indicated. (AI RT-PCR analysis of marker expression showing that XrefAJSP has IIrrteor no effect on the mductlon of ventral and intermediate mesodermal markers by BMP-4. (8- D) Histology of animal caps at stage 40. (8) uninjected caps contain only atvpical epidermis (ae), IC) caps from embryos mjected with BMP-4 mRNA develop vesicles containing mesenchyme (mc). (D) caps from embryos injected with BMP-4 and XreJAJSP develop as C. Bar in 0, 100 ,urn and applies to panels B-D
	Fig. 8 (AI. Assay for the activation of MAP kinase in response to FGF treatment of animal caps. Lanes 1 and 2 are positive and negative controls respectively for the detection of active MAP kinase and correspond to extracts from matured (1) and immature (2) stage Vloocytes Active MAP kinase IS vIsible as a 42 kOa band. Caps were assayed for MAP kinase specific phosphorylation of myelin basic protein after 20min contact with 50 ng/ml bFGF according to Mason et al., 1996. The lanes in 3 are extracts from uninjected animal caps, MAP kinase is activated by FGF. Caps from embryos injected with 400 pg of XFD mRNA (4) do not show an activation of MAP kinase in response to FGF. In contrast, Injection of 2 ng XrelA..15P does not affect the activation of MAP kinase by FGF in animal caps (5). fBI RT.PCR assay for the induction of Xbra and XeFGF by exogenous Brachyury. 600 pg mRNA from the mouse Brachyury gene was injected into 2 cell Xenopus embryos with and without 1 ng XrelA..1SP mRNA Animal caps were isolated at stage 9 and assayed for Induction of Xbra and XeFGF at stage 11.
	Fig. 9. Model showing possible points of action of XFD and Xrel~P in animal cap inductions. This is simplrfied Since it is clear that activin and FGF do more than induce Xbra. and the latter does more than Induce XeFGF.