January 1, 1996;
The Xenopus homologue of hepatocyte growth factor-like protein is specifically expressed in the presumptive neural plate during gastrulation.
Using a RT
-PCR approach, we were able to isolate a cDNA encoding the Xenopus homologue of hepatocyte
growth factor-like protein, which we have termed accordingly Xhl
. The deduced Xhl
protein consists of 717 amino acids, contains four putative kringle domains and a serine protease-like domain characteristic for mammalian HGF
-like protein. The mRNA of Xhl
is exclusively expressed in the midline of the prospective neural plate
during the period of neural induction, only. Ectopic expression of Xhl
causes a ''spina bifida''-like phenotype with enlargement of neural tissue
. Activation of Xhl
mRNA transcription can be induced by delayed reaggregation of animal caps and appears to require vertical rather than planar signals from the organizer
. These data suggest that Xhl
is involved in the formation of the embryonic nervous system
[+] show captions
Fig. 1. Nucleotide and deduced amino acid sequence of Xhl. The putative signal sequence is typed in italics and sequences on which primers for F’CR
amplification were based are underlined. Amino acids critical for catalytic activity of serine proteases but exchanged in Xhl are shown in brackets. The
positions of the four putative kringle domains in the a-subunit are indicated. The putative proteolytic cleavage site between amino acid position 486
and 487 is marked by an arrow. The stop codon TAA is indicated by an asterisk.
Fig. 2. Sequence comparison of the deduced protein sequence of Xhl (XHL.PRO) with human HGF-like protein (HGPLPRO) and Xenopus HGF
(XHGF.PRO). Amino acid residues that match the sequence of XHL.PRO are shown in boxes.
Fig. 3. Developmental profile of Xhl mRNA expression. Numbers indicate
the developmental stage, according to Nieuwkoop and Faber
(1967), at which RNA was isolated. The positions of the 28s ribosomal
RNA, 18s ribosomal RNA and Xhl mRNA are indicated. Ethidium
bromide-stained gel was photographed for documentation of RNAloading.
Fig. 4. Northern blot analysis of Xhl mRNA distribution in adult frogs.
1, Brain; 2, kidney; 3, liver; 4, lung; 8pg total RNA were loaded per
lane. Ethidium bromide-stained gel was photographed for documentation
Fig. 5. In situ distribution of Xhl mRNA at stage 12 of development. 1,
whole embryo at stage 12 of development; 2, whole embryo without
prospective neural plate at stage 12; 3, prospective neural plate at stage
12. Position of 28s and 18s ribosomal RNA and the short form of Xhl
mRNA is indicated. Ethidium bromide-stained gel was photographed
for documentation of RNA-loading.
Fig. 6. Whole-mount in situ hybridization showing the endogenous localization of Xhl mRNA during gastrulation and neurulation, respectively. (A)
Dorsal-posterior view of an embryo at late gastrula stage 12. Xhl mRNA is predominantly located to the midline of the neural plate. (B) Dorsal view of
a stage 13 early neurula embryo showing the localization of Xhl mRNA in the midline of the neural plate. Anterior to the top. (C) Same view of a midneurula
stage 15 embryo as in (A) demonstrating that Xhl mRNA is localized in the neural groove of the up-folding neural plate. Space bars are
Fig. 7. Cross-sections of Xhl whole-mount labelled embryos. (a) Section through the posterior part of a stage 12 embryo. Arrows indicate Xhl positive
cells. Xhl mRNA is localized in epithelial and deeper cell layers of the posterior part of the notoplate. (b) Rosin-stained section through a more anterior
part of the same embryo as in (a). Arrows mark epithelial cells expressing Xhl mRNA. Bar indicates the midline of the prospective neural plate. (c)
Section through the posterior part of a stage 13 embryo showing that Xhl mRNA is localized mainly in epithelial cells of the notoplate. Arrow points at
cells of deeper cell-layers expressing Xhl mRNA. Bar marks the region of the notoplate. (d) In more anterior parts of stage 13 embryos Xhl-expressing
cells are exclusively localized in epithelial cells of the notoplate which ate indicated by arrows. (e) Cross-section of a stage 15 embryo showing bottlecells
(arrow) expressing Xhl mRNA. (f) Higher magnification of section shown in (e). Arrow points at neural groove and bottle-cells, respectively.
Since Xhl represents a low-abundant mRNA, only weak signals were observed in cross-sections of whole-mount labelled embryos, Longer staining of
embryos, however, resulted in mom background staining, making it difficult to distinguish specific from unspecific signals. yp, yolk plug; ae, archenteron;
n, notochord; s, somitic mesoderm.
Fig. 8. (A) Northern blot analysis showing that Xhl is not induced by
planar signals. 16, whole embryo at stage 16 of development; Exe,
ectoderm of exogastrulae isolated, when sibling embryos reached stage
16 of development. (B) Northern blot analysis showing that Xhl mRNA
is activated by disaggregation and subsequent reaggregation without
inducer. AC, animal caps kept in culture; Re, cells of disaggregated and
reaggregated animal caps. As a control for neural differentiation in
reaggregated cells of animal caps, the same filter was reprobed with a
neural-specific, putative RNA-binding protein 24-39. The positions of
the 28s and 18s ribosomal RNAs are indicated. Ethidium bromidestained
gels were photographed for documentation of RNA-loading.
Fig. 9. Dorsal view an embryo with 60 pg Xhl-pXeX expression plasmid showing severe malformation of the axis posterior of the brain.
Embryo at the top was injected with pXeX plasmid as a control. (b) Enlargement of Xhl-pXeX injected embryo shown in (a). (c) Anterior view of an
embryo injected with 60 pg Xhl-pXeX showing additional perturbance of head development. (d) Enlargement of Xhl-pXeX injected embryo shown in
(c). Arrows point at tips of split tail.
Fig. 10. (a) Transverse section through the part posterior of the brain region of an embryo injected with Xhl-pXeX expression plasmid. Arrow-head
indicates enlargement and splitting, respectively, of the neural tube (100X magnification). (b) Transverse section through the trunk of an embryo injected
with Xhl-pXeX plasmid. Arrowheads point at neural tissue which has not folded up but appears larger than in control embryos. Tissue dorsal of
neural tissue derives from bent tails (100X magnification). (c) Transverse section through the trunk of a control embryo injected with pXeX. Arrowhead
points at neural tube (100x magnification). n, notochord; m, muscle; t, tail. All sections were stained with eosin.