December 15, 1997;
FGF-8 is associated with anteroposterior patterning and limb regeneration in Xenopus.
has attracted attention particularly because of its importance for limb
development in the chick and mouse, although it also has a number of earlier expression domains in these species. We have now cloned an FGF-8
homologue from Xenopus in which it is easier to do functional studies on early development. There is no maternal expression, while zygotic expression is highest in the gastrula
is expressed as a ring around the blastopore
and subsequently in the tail bud
. There are several domains in the head
including the hatching gland
, the branchial clefts, and the midbrain
border. At later stages there is a prominent band of expression in the limb bud epidermis
. Although there is no morphological apical ridge
, this band of expression suggests that the Xenopus limb bud
contains a cryptic region with a similar ability to stimulate mesenchymal outgrowth. The mesoderm
-inducing activity of XFGF-8
is somewhat lower than that of other FGFs, while the posteriorizing activity is similar. These differences are probably due to the different receptor specificity. The posterior
expression and high posteriorizing activity suggest that XFGF-8
contributes to the patterning of the anterior
axis by FGF family members during gastrulation. In contrast to the amniotes, Xenopus limb
buds can regenerate following damage. We show that regeneration is correlated with the reexpression of XFGF-8
in the distal epidermis
, suggesting that this ability is critical for successful limb
[+] show captions
FIG. 3. Spatial expression of XFGF-8 during early development. Detection by whole-mount in situ hybridization of XFGF-8 DIG-labeled antisense RNA. (A) Vegetal view of a stage 10 embryo. XFGF-8 expression comes up in a ring around the future blastopore. (B) Vegetal view of a stage 11 embryo. Expression around blastopore broadens. (C and D) Dorsal and anterior view, respectively, of a stage 14 embryo. XFGF-8 transcribed in two patches in the future midbrain/hindbrain junction (black arrowheads), the prospective hatching gland region (white arrow), and an epidermal crescent outside the neural plate area (black arrow). Periblastoporal expression becomes localized to the posterior. (E) Side view of stage 20 embryo. Transcription in future hatching gland is increased and the expression previously seen in a crescent outside the neural plate territory has now moved to a lateral epidermal fold. (F, G, and I) Stage 33 embryos. (F and G) While expression in the hatching gland and frontal plate (white arrow) and the midbrain/hindbrain junction (black arrowhead) persists, new expression domains are detected in the forebrain (fb), the ear vesicle (e), the notochord, and the pronephros (*). The lateral epidermal fold expression is now seen in the branchial clefts (bc). In the tail, transcripts are confined to the tail bud (tb). (H) Close-up of stage 38 tail bud. (I) Sections of whole-mount in situs. (I and K) Parasagittal sections through the head showing the expression at the midbrain/ hindbrain junction (black arrowhead) and labeling in the forebrain adjacent to the frontal plate (fb). (L) Parasagittal section through the tail bud. Transcripts are detected in the posterior wall (pw) of the neuroenteric canal and the chordoneural hinge (cnh).
FIG. 4. Whole-mount in situ hybridization of XFGF-8 to early limb bud stages. (A and B) Ventral and distal view, respectively, of a stage 50 limb bud. XFGF-8 is expressed along the dorsoventral boundary in a broad and fuzzy band. (C and D) Longitudinal sections through a stage 50 limb bud. Phase contrast (C) and bright field (D) picture of the same section. XFGF-8 transcripts are confined to ectodermal cells. Note that no morphological AER is visible at this stage. (E) Dorsal view of an early stage 52 limb bud. XFGF-8 transcripts are concentrated in the anterior ridge. (F) Ventral side of a tilted stage 56 limb. XFGF-8 expression confined to connective tissue on the ventral and dorsal side of the digits.
Regeneration in manipulated limb buds. In all panels the right (operated) leg is shown on the right side. (A, C, and E) Tip removal at stage 50. (B, D, and F) Removal of the epidermis at stage 48/49. (A and B) Specimens fixed right after the operation. (A) An XFGF-8 in situ shows a small spot of persisting expression. (B) A section of a limb bud where the epidermis was removed completely. (C, D) After 10 days the right limb bud has regulated for the loss
FIG. 6. Overexpression of XFGF-8. (A) Overexpression phenotype with three different doses of RNA. From top to bottom: Uninjected, 20, 50, and 100 pg of XFGF-8 mRNA, respectively. (A) Note enlarged proctodeum region. In situ hybridization with En-2 (B) and otx-2 (C), showing dosage-dependent loss of anterior structures. No ectopically formed midbrain structure is detected with either of the markers.