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eFGF is required for activation of XmyoD expression in the myogenic cell lineage of Xenopus laevis

Department of Biology, University of York, York YO10 5YW, UK

View larger version (48K): [in a new window]   Fig. 1. The normal expression patterns of eFGF and XmyoD showing co-expression in the early mesoderm. Whole-mount in situ hybridisation showing expression of (A) eFGF at stage 10 and (B) Xmyod at stage 10 and (C) Xmyod at stage 10 (+). Expression of XmyoD across the dorsal midline is rapidly excluded as Spemann’s organiser signalling is established.

View larger version (48K): [in a new window]   Fig. 2. Gene expression in animal caps treated with activin or eFGF. RNA was extracted at stages 10, 11, 11.5 and 12 from whole embryos, untreated animal cap explants and animal cap explants treated with eFGF or activin. Total RNA (7 µg) from each stage was analysed by RNAase protection for expression of the marker genes XmyoD, Xbra and the loading control ODC.

View larger version (30K): [in a new window]   Fig. 3. XmyoD induction in eFGF and CHX treated animal caps. (A) RNA was extracted at stage 13 from whole embryos, animal caps in the presence or absence of CHX, and animal caps cultured with eFGF in the presence or absence of CHX. Total RNA (3 µg) was analysed by RNAase protection for the expression of the marker genes XmyoD, Xbra and for the loading control ODC. (B) RNA was extracted at stage 18 from whole embryos, animal caps in the presence or absence of CHX, and animal caps cultured with eFGF in the presence or absence of CHX. Total RNA (10 µg) was analysed by RNAase protection for the expression of the marker gene actin and for the loading control ODC. (C) RNA was extracted at stages 11, 13 and 18 from whole embryos and animal caps cultured in the presence or absence of CHX. Total RNA (10 µg) was analysed by RNAase protection for the marker genes XmyoD, Xbra and the loading control ODC.

View larger version (38K): [in a new window]   Fig. 4. The effect of an eFGF(i) targeted morpholino (eFM) on morphogenic movements and myogenic gene expression. (A) (i) Untreated animal cap controls showing uninduced morphology; (ii) caps from embryos injected with 5 pg of eFGF mRNA showing induced morphology; (iii) caps from embryos co-injected with 5 pg of eFGF mRNA and 80 ng of control morpholino (CM) showing an induced morphology; and (iv) caps from embryos co-injected with 5 pg of eFGF mRNA and 80 ng of eFM showing uninduced morphology. (B) RNA was extracted from whole embryos at stage 13, untreated animal caps, animal caps from embryos injected with 5 pg eFGF mRNA and animal caps from embryos co-injected with 5 pg eFGF mRNA and 80 ng of either CM or eFM. Total RNA (6 µg) was analysed by RNAase protection for expression of the marker genes XmyoD, Xbra and the loading control ODC. (C) RNA was extracted from whole embryos at stage 13, untreated animal caps, animal caps treated with eFGF protein, eFGF treated animal caps injected with 80 ng of CM or eFM. Total RNA (6 µg) was analysed by RNAase protection for expression of the marker genes XmyoD, Xbra and the loading control ODC.

View larger version (75K): [in a new window]   Fig. 5. eFM inhibits XmyoD expression in gastrula but not neurula embryos. (A) (i) RNA was extracted at stages 11 and 14 from whole embryos, embryos injected with 80 ng, and embryos injected with 40, 60 or 80 ng of eFM. Total RNA (10 µg) was assayed by RNAase protection for expression of the mesodermal markers XmyoD and Xsna and for the loading control ODC; (ii) RNA was extracted from whole embryos at stage 13, embryos injected with 80 ng of eFM, embryos injected with 200 pg of ssbFGF, and embryos injected with 80 ng of eFM and 200 pg of ssbFGF. Total RNA (10 µg) was analysed by RNAase protection for expression of the marker genes XmyoD, Xbra and the loading control ODC. (B) Embryos injected unilaterally on the left hand side with 40 ng of eFM were assayed at (i) gastrula stage 10.5 and (ii) neurula stage 14 for XmyoD gene expression by in situ hybridisation. (iii) Gastrula stage 10.5 embryos injected unilaterally on the left-hand side with 40 ng of eFM were also assayed by in situ hybridisation for expression of the mesodermal marker Xsna.

View larger version (38K): [in a new window]   Fig. 6. Activin induces eFGF expression in the presence of CHX. RNA was extracted at stage 11 from whole embryos, untreated animal cap explants, CHX treated animal cap explants and caps treated with activin in the presence or absence of CHX. Total RNA (7 µg) was assayed by RNAase protection for expression of eFGF, the mesodermal marker Xbra and the loading control ODC.

View larger version (6K): [in a new window]   Fig. 7. A pathway for myogenic induction. A molecular pathway depicting the role of eFGF signalling in the induction of XmyoD in response to the mesoderm inducing factor. A maternal factor such as VegT induces the expression of a TGFß family member(s) which act as the endogenous mesoderm inducing factor; this is likely a nodal related factor (Xnr1 and/or Xnr2). We mimic the endogenous mesoderm inducing factor with activin in our experiments and show that it induces the expression of eFGF directly. eFGF protein directly induces the expression of XmyoD, possibly acting through inhibition of a repressor. XmyoD is crucial in the specification of the myogenic cell lineage.