Dev Growth Differ
March 1, 2008;
VegT, eFGF and Xbra cause overall posteriorization while Xwnt8 causes eye-level restricted posteriorization in synergy with chordin in early Xenopus development.
We examined several candidate posterior
/mesodermal inducing molecules using permanent blastula
-type embryos (PBEs) as an assay system. Candidate molecules were injected individually or in combination with the organizer
mRNA. Injection of chordin
alone resulted in a white hemispherical neural tissue
surrounded by a large circular cement gland, together with anterior
neural gene expression and thus the development of the anterior
-most parts of the embryo
, without mesodermal tissues. When VegT
mRNAs were injected into a different blastomere
of the chordin
-injected PBEs, the embryos elongated and formed eye
and pigment cells, and expressed mesodermal and posterior
neural genes. These embryos formed the full spectrum of the anteroposterior embryonic axis. In contrast, injection of CSKA-Xwnt8
DNA into PBEs injected with chordin
resulted in eye
formation and expression of En2
, a midbrain/hindbrain marker, and Xnot
, a notochord
marker, but neither elongation, muscle
formation nor more posterior
gene expression. Injection of chordin
and posteriorizing molecules into the same cell did not result in elongation of the embryo
. Thus, by using PBEs as the host test system we show that (i) overall anteroposterior neural development, mesoderm
) formation, together with embryo
elongation can occur through the synergistic effect(s) of the organizer
, and each of the ''verall posteriorizing molecules''eFGF, VegT
; (ii) Xwnt8
-mediated posteriorization is restricted to the eye
level and is independent of mesoderm
formation; and (iii) proper anteroposterior patterning requires a separation of the dorsalizing and posteriorizing gene expression domains.
Dev Growth Differ
[+] show captions
Fig. 1. Schematic representation of the experimental procedure.
(Left) Permanent blastula-type embryos (PBEs) were made by
ablating vegetal cytoplasm and egg surface so that more than
60% of the egg surface was deleted. (Middle) At the 4-cell stage,
one of the candidate posteriorizing molecules (VegT, eFGF, or
Xbra mRNAs or CSKA-Xwnt8 DNA construct) was injected into
the vegetal region of a single blastomere. (Right) At the 8-cell
stage, chordin mRNA was injected into a vegetal blastomere
opposite the injection site of the candidate posteriorizing molecules.
Fig. 2. Reverse transcription–polymerase chain reaction (RT–
PCR) analysis of experimental embryos. The PCR products from
four permanent blastula-type embryos (PBEs) or one control
embryo (stage 24) were loaded in each lane. PCR primer pairs
and cycling conditions are shown in Table 1. See also Materials
and methods. Lane 1, a normal embryo at stage 24. Lane 2, naive
PBEs without injection were expressed Krox20, but not dorsal/
neural and endomesodermal genes. Lane 3, PBEs injected with
100 pg chordin mRNA. Lane 4, PBEs injected with 1 pg eFGF
mRNA. Lane 5, PBEs injected with 100 pg chordin and 1 pg
eFGF (separate injection, see Fig. 1). Lane 6, PBEs injected
with 50 pg CSKA-Xwnt8 DNA. Lane 7, PBEs injected with
100 pg chordin and 50 pg CSKA-Xwnt8 (separate injection).
Fig. 3. Morphology of permanent blastula-type embryos (PBEs) injected with chordin or posteriorizing candidate molecules. (A)
Naive PBEs without injection. (B) PBEs injected with 100 pg chordin. A white neural area is surrounded by a circular cement gland.
(C) PBEs injected with 1 pg eFGF. Note the accumulation of cells (white arrowheads). (D) PBEs injected with 50 pg CSKA-Xwnt8
DNA. Note the cell accumulation on the right side of the embryos. (E) Fluorescent image showing that chordin-injected blastomeres
developed into white neural cells. FDA (green) injected with chordin mRNA at the 8-cell stage was localized in neural cells. (F) eFGF
mRNA expressing domain as shown by coinjection of RDA (magenta). All embryos are at stage 24. Bar in A for A–D, 1 mm. Bar in E
for E and F, 0.5 mm. Broken lines in E and F indicate the outline of the embryo.
Fig. 4. Injection of eFGF, VegT and Xbra triggered endogenous
expression of these molecules and Xwnt8 expression, whereas
CSKA-Xwnt8 did not induce eFGF, VegT or Xbra. Permanent
blastula-type embryos (PBEs) were injected into one
blastomere with VegT, eFGF, Xbra mRNA or CSKA-Xwnt8 DNA
at the 4-cell stage and assayed at stage 11. The PCR products
from four PBEs or one control embryo were loaded in each lane.
See also Materials and methods. Lane 1, a normal embryo.
Lane 2, naive PBEs without injection. Lane 3, PBEs injected with
6 pg VegT mRNA. Lane 4, PBEs injected with 50 pg CSKAXwnt8
DNA. Lane 5, PBEs injected with 1 pg eFGF mRNA. Lane
6, PBEs injected with 400 pg Xbra mRNA. Lane 7, PBEs
injected with 100 pg chordin mRNA.
Fig. 5. Overall posteriorization by VegT, eFGF, Xbra mRNA
and eye-level restricted posteriorization by CSKA-Xwnt8 DNA.
(A) PBE/chd/eFGFs at stage 24 (B) A PBE/chd/eFGF at stage
40. cg, cement gland; ey, eye; me, melanocytes; tf, tail fin. (C,
D) A PBE/chd/eFGFs at stage 24. Chordin mRNA was injected
together with a lineage marker, FDA (green) while eFGF was
injected together with RDA (magenta). (E) A PBE/chd/VegT at
stage 40. (F) A PBE/chd/Xbra at stage 40. Embryos shown in B,
E and F twitched, suggesting the presence of motor neurons
and skeletal muscle. (G) PBE/chd/CSKA-Xwnt8s at stage 24.
These embryos formed smaller cement glands compared with
PBE/chds (Fig. 3B). (H) A PBE/chd/CSKA-Xwnt8 at stage 40.
This embryo did not elongate, but formed a large eye (ey) and
a cement gland (cg). Bars in A, B, and E–H, 1 mm; bars in C
and D, 0.5 mm.
Fig. 6. PBE/chd/eFGF formed eye and notochord while PBE/
chd/CSKA-Xwnt8 formed eye but not notochord. (A, B) Sections
of a PBE/chd/eFGF shown in Figure 5(B). (A) A transverse
section at the eye level. (B) Posterior region forming a
notochord. (C) A section of PBE/chd/CSKA-Xwnt8 shown in
Figure 5(H). (D) An eye of a control embryo. le, lens; ne,
neuroepithelial cells; no, notochord; re, retinal-pigmented
epithelium. Bar in A for A, C, and E, 0.1 mm. Bar in B, 0.2 mm.
Fig. 7. Anteroposteriorly organized gene expression in PBE/chd/eFGFs. (A–C) In situ hybridization of PBE/chd/eFGFs. Top: normal
embryo at stage 24. Bottom: PBE/chd/eFGFs at stage 24. Anterior to the left. (A) NCAM (B) Krox20 (C) HoxB9. (D and E)
Immunohistochemical staining using 12/101 (muscle marker, D) and MZ15 (notochord marker, E) antibody. Top: normal embryo at
stage 40. Bottom: PBE/chd/eFGFs at stage 40. White arrowheads mark the anterior end of the stain while black arrowheads mark the
posterior end. Bars: 1 mm.
Fig. 8. Co-injection of chordin and posteriorizing molecules
into the same blastomere did not result in elongation of the
embryos and proper anteroposterior patterning. (A) PBE/
chd + eFGFs at stage 24. These embryos did not elongate and
had semicircular cement glands. (B) A PBE/chd + eFGF at
stage 40. A small eye remnant was seen. (C) PBE/chd + CSKAXwnt8s
at stage 24. These embryos resembled PBE/
chd + eFGFs. (D) A PBE/chd + CSKA-Xwnt8 at stage 40. Eye
structure was absent. cg, cement gland; ey, eye remnant. (E)
Reverse transcription–polymerase chain reaction (RT–PCR)
analysis for coinjection experiments. The PCR products from
four permanent blastula-type embryos (PBEs) or one control
embryo were loaded in each lane. See also Materials and
methods. Lane 1, a normal embryo at stage 24. Lane 2, PBEs
injected with the mixture of 100 pg chordin and 1 pg eFGF into
one blastomere at the 8-cell stage. Lane 3, PBEs injected with
the mixture of 100 pg chordin and 50 pg CSKA-Xwnt8 into one
blastomere at the 8-cell stage. In spite of their non-elongated
morphology, PBE/chd + eFGFs expressed notochord and
muscle markers. Bars in A–D, 1 mm.
Fig. 9. Proposed model of anteroposterior patterning in
Xenopus embryos. (A) After gene expression commences (after
mid-blastula transition [MBT]), the Xenopus embryo is divided
into three domains, as shown by previous tissue transplantation
experiments; competent animal domain (green), dorsalizing
organizer domain (the Spemann-Mangold organizer) (magenta)
and posteriorizing vegetal domain (cyan–light-blue gradient).
Tissue transplantation experiments revealed whole vegetal
hemisphere (stage 10) Xenopus embryos have overall
posteriorizing activity; however, this activity could not be
attributed to zygotic Xwnt8, because Xwnt8 did not exhibit
overall posteriorizing activity. The molecule responsible for the
overall posteriorizing activity in the non-dorsal marginal zone
may be VegT, eFGF and Xbra; however, these molecules are
absent in the vegetal pole. Unknown molecules in the gastrula
vegetal pole are most likely responsible for the overall
posteriorizing activity. (B) Posteriorization at the neurula stage.
At this stage, Xwnt8 is expressed in the mid-anterior neural fold,
and may be involved in the eye and midbrain level
posteriorization. The expression domain of eFGF, VegT and
Xbra suggests that these molecules are responsible for overall