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We have examined the role of FGFs in neural induction and posteriorization of the central nervous system (CNS). Using embryos micro-injected with dominant negative FGF receptor RNA (XFD), we show that a patterned CNS can still develop following inhibition of FGF signaling. The most severely affected embryos developed with strong posterior defects. In these embryos, head development and expression of a marker of forebrain and midbrain, and of markers of the hindbrain, occurred relatively normally. However, the expression levels of a posterior marker, Hoxb-9, were considerably reduced compared to those in control embryos. The results support the idea that FGFs are involved in inducing posterior development, but they suggest that other signals are also necessary for antero-posterior patterning of the primary body axis.
Fig. 1. Overexpression of dominant negative FGF receptors
inhibits FGF signaling and causes posterior defects. (A). Morphology
of XFD embryos. (Top) Control stage 46 embryo. (Bottom)
XFD mRNA injected embryos grown to stage 46. Left phenotype
of the most severely affected embryos; middle, a less severely
affected phenotype with a shortened, split axis starting from the
trunk region; right, mildly affecred embryo with a shortened, split
tall. (B). An/mal caps from XFD mRNA-InJected embryos do not
respond to bFGF. (Top) Animal caps from control embryos: (left)
induced to form mesoderm-containing vesicles by 60 nglml
recombinant Xenopus bFGF;(right) uninduced controls which formed
spheroids of atypical epidermis. (Bottom) Animal caps from XFD
mRNA injecredembryos rreatedwith 60nglml bFGF. Mosrexplants
failed to be induced. Only one animal cap is induced to the same
extent as the caps from the control embryos (arrow), IC). Brachyury
expression /s reduced In XFD mRNA-injected embryos. Whole
mount in siru hybridization of mid-gastrula stage embryos with an
xbra anti-sense probe: (top) conrrol embryos show a ring of strong
expression around rhe blastopore; (bottom) XFD mRNA-injected
embryos show enlarged blastopores and reduced expression of
xbra transcnpts.
Fig. 2. Splitting of the midbrain in a severely affected XFD mANA-injected embryo.
CLSM images (see Materials and Methods) of stage 46 tadpoles following whole-mount
immunofluorescence using an anti-neural antibody (Xen-l). (AI Brain of a control tadpole;
(B) CNS of an XFD mRNA~injected embryo. Abbreviations: n, olfactory organ; t, telencephalon;
d, diencephalon; m, midbrain; r, hindbrain; s, spinal cord
Fig. 3. Localization of injected RNA by lineage
labeling. B-galactosidase e;o..pression (light blue)
and whole-mount in situ hybridization with
antisense Hoxb-3 probe (purple) in embryos
coinjected with B-galactosidase and HA VNOT
RNAs (AI or B-galactosidase and XFD RNAs (B).
Embryos are shown with anterior to the top
Injected RNA can be seen along the CNS and in
epidermis in both A and B. The arrows in B
Indicate domains of double staining where there
is Hoxb-3 expression in cells containing lineage
label. Double staimng is also visible in A
Fig. 4. Expression of antero-posterior markers in XFD mRNA injected embryos. Wholemount
in Situ hybridization analysis of.-IA,C,E,II stage 20 control embryos; IB.D,F,JI severely
affected XFD embryos cultured untIl control embryos reached stage 20. Each embryo is
positioned wIth the open blastopore in the centre. bordered by the posteriorly spM CNS. (GI
Stage 28 control embryo; IHI XFD embryo, stage 28_ IA,BJ ot\--2 eApression In the fore-brain
and mid-brain regions; IC) en-2 expression at the midbramlhrndbrain border: (D) embryos
hybfldized wIth two probes, en-2 and the spina! cord marker. Ho-.;b-9. Strong stripes of en-2
e\pression are seen near the anterior end of the embryos (top) and very weak Ho\b-9
e\press1on is vIsible in the posterior half of the CNS; IE,F) HOAb-3 e:\pression in the hindbrain
region, (GJ Ho),b-4 e>.pression In the posterior hindbrain region and som/res of a control embryo;
(HI weak Hoxb-4 expression IS visible In rhe CNS of an XFD embryo (arrowhead), and som/ric
e\preSSlon is reduced to very low levels: (I,JI HOltb-9 e-\pression /n the spinal cord region. In
the XFD embryos, it ISmuch weaker than in controls. Arrowheads po,m to the borders of Hoxb-
9 expression on one side of the CNS In an XFD embryo. IA-F,H-JI Anterior IS to the top; (GI
anrenor is to the left.
