Mesoderm Induction II
As we discussed previously, there is strong evidence favoring involvement
of endogenous Vg1 in mesoderm induction. However, testing this hypothesis
has been very difficult due to the difficulties in producing processed VG-1
protein for use in treating animal explants. As discussed previously, RNA
made from chimeric constructs incorporating a cleavage site from BMP has
been used in autoinduction experiments. Although processing is enhanced
by these constructs, the secretion of soluble, processed Vg1 is inefficient
in oocytes injected with BMP4-Vg1 mRNA (Dale et al., 1993). This
brings into question whether induction was mediated by secreted Vg1 in the
autoinduction experiments. Hence, Kessler and Melton (1995) have devised
a means to make soluble, biologically-active Vg1 to apply to animal pole
explants.
The Vg1 was made from an activin ß B-Vg1 hybrid. The activin ß
B element contained both a signal sequence that mediates secretion and a
cleavage site. This facilitated both the processing and secretion of Vg1
protein in oocytes injected with activin ß B-Vg1 mRNA (Fig. 1, Kessler
and Melton, 1995). Supernatant from injected oocytes was tested for its
mesoderm-inducing activity on explanted animal poles. The effects of the
supernatant are illustrated in Fig. 2 (Kessler and Melton, 1995). The explants
elongated, expressed mesoderm-specific markers, and produced notochord,
somitic muscle and neural tissue.
To determine whether the effects of mature Vg1 are mediated by the activin
receptor, two experiments were conducted. First, animal pole explants of
embryos that had been injected with a truncated activin receptor (tAR) were
treated with Vg1-containing supernatant. The truncated receptor blocked
mesoderm induction by mature Vg1. A truncated FGF receptor also blocked
induction by this factor. This suggests that the abilities of these two
receptors to block mesoderm induction in the embryo reflect their effects
on endogenous Vg1 signaling.
Does the activin receptor function as the endogenous Vg1 receptor? To examine
this possibility, a version of the Xenopus activin II receptor that
had an epitope of MYC attached to it (XARmyc) was expressed in Xenopus
oocytes. (This approach is called epitope tagging.) The binding of radioactive
Vg1 or activin to XARmyc was then assessed. The epitope tag enabled the
XARmyc with bound ligand to be immunoprecipitated using an antibody to MYC.
The specificity of binding was assessed by competition using excess unlabeled
ligand. As shown in Fig. 3 (Kessler and Melton, 1995) , activin binds avidly
to the receptor, but Vg1 does not. Furthermore, unlabeled Vg1 fails to compete
for activin binding. These results suggest that (1) the activin receptor
is not a Vg1 receptor and (2) the effects of the truncated activin receptor
on Vg1 signaling are probably mediated through an interaction between the
truncated activin receptor and an endogenous Vg1-specific receptor.
As we have previously discussed, follistatin is an inhibitor of activin.
Previous investigations of the effects of follistatin have utilized mammalian
follistatin. However, Kessler and Melton (1995) have examined the effects
of Xenopus follistatin on both normal development and response of
animal pole explants to activin and Vg1. Injection of follistatin mRNA into
embryos at the two-cell stage perturbed posterior development, but appeared
to enhance anterior dorsal development (Fig. 4, Kessler and Melton, 1995).
Normal expression of dorsal mesodermal markers was detected. Thus, endogenous
induction of dorsal mesoderm is not inhibited, and a role for activin in
dorsal mesoderm induction is not supported by these results. Follistatin
was unable to block the effects of Vg1 on animal pole explants, although
it blocked the effects of activin (Kessler and Melton, 1995, Fig. 5). Once
again, these results cast doubt on the role of activin and support a role
for Vg1 in mesoderm induction. These results may also indicate that follistatin
is involved in patterning of the mesoderm.
Kessler and Melton (1995) propose that the rotation of the cortex at fertilization
triggers localized processing of Vg1 precursor, which imparts on those cells
functionality as the Nieuwkoop center. Mature Vg1 ligand secreted from the
Nieuwkoop center would induce the Spemann organizer. The Spemann organizer,
in turn, would induce neural tissue and also dorsalize lateral mesoderm.
Regionalization of the Mesoderm
The mesoderm is a complex array of tissue-types in three primary domains:
dorsal, ventrolateral, and ventral. It is likely given the large number
of factors that are capable of inducing mesoderm (see Slack, 1994, Table
2) that the regional character of the mesoderm is a result of the combinatorial
action of the endogenous correlates of these factors. We do not have time
to discuss the putative roles of all of these factors. Instead, we will
discuss briefly their diversity and discuss selected factors in some detail.
In addition to Vg1, the messenger RNAs for BMP-2
, BMP-4,
FGF-2, FGF-4, Wnt-11, and noggin are present in the egg. Thus, they
are present during the time when mesoderm induction is initiated. The bone
morphogenetic proteins (BMPs) are TGF-ß-related proteins. BMP-4 induces
ventral mesoderm, suppresses induction of dorsal mesoderm by activin and
inhibits dorsoanterior development of embryos, suggesting that it is a ventralizing
factor. The messengers encoding the BMPs are not localized in the vegetal
hemisphere, which casts some doubt on their role in mesoderm induction.
However, expression of a dominant-negative BMP-4 receptor blocked mesoderm
induction by BMP-4 in isolated animal caps and prevented ventralization
of embryos by BMP-4. Overexpression of the receptor in embryos caused mesoderm
to be dorsalized. These results suggest that BMP-4 may be involved in ventralization
of the mesoderm by counteracting the dorsalizing signals (for review, see
Kessler and Melton, 1994).
The fibroblast growth factors induce ventrolateral mesoderm. Expression
of dominant-negative FGF receptor inhibits mesoderm induction in animal
cap explants and causes defects in trunk and posterior development, while
not affecting anterior development. This result suggests that FGF is necessary,
but not sufficient, for induction of trunk and posterior mesoderm (for review,
see Kessler and Melton, 1994). Noggin mRNA is oogenic, but - like
FGF and BMP mRNA - it is not localized to the vegetal hemisphere. However,
zygotic expression of noggin is interesting because it begins at
the mid-blastula transition (MBT) in the organizer region and later in the
dorsal mid-line of the archenteron roof. This puts it in a position to play
an important role in either dorsalization and/or neural induction (Slack,
1994). It is noteworthy that when ventral marginal zone tissue is exposed
to noggin protein, it induces muscle (Smith et al., 1993). Thus,
noggin is a possible candidate for the third signal in the three
signal model.
The Wnts are a diverse group of proteins related to the transforming int
proteins of mammals and wingless in Drosophila. They were first implicated
in Xenopus development by the demonstration that mouse wnt-1
overexpression could cause induction of ectopic dorsal axial structures
(McMahon and Moon, 1989). This stimulated a search for the Xenopus
homologues and discovery of a number of Xwnt genes. Of these Xwnt-11
is particularly intriguing. It will be discussed in one of the student presentations.
Also to be discussed is Xbra, which encodes a nuclear protein that
is apparently involved in transcriptional regulation of mesodermal fate.
References
Dale, L., G. Matthews, and A. Colman. 1993. Secretion and mesoderm inducing
activity of the TGF-beta-related domain of Xenopus Vg1. EMBO J. 12:
4471-4480.
Kessler, D.S. and D. A. Melton. 1994. Vertebrate embryonic induction: mesodermal
and neural patterning. Science 266: 596-604.
Kessler, D. S. and Melton, D. A. 1995. Induction of dorsal mesoderm by soluble,
mature
Vg1 protein. Development 121: 2155-2164.
McMahon, A.P. and R.T. Moon. 1989. Ectopic expression of the proto-oncogene
int-1 in Xenopus embryos leads to duplication of the embryonic axis.
Cell 58: 1075-.1084.
Slack, J.M.W. 1994. Inducing factors in Xenopus early embryos. Current Biology
4: 116-126.
Smith, W.C., A.K. Knecht, M. Wu and R.M. Harland. 1993. Secreted noggin
protein mimics the Spemann organizer in dorsalizing Xenopus mesoderm.
Nature 361: 547-549.
References for Student Seminars
Cunliffe, V. and J.C. Smith. 1994. Specification of mesodermal pattern
in Xenopus laevis by interactions between Brachyury, noggin
and Xwnt-8.
EMBO J. 13: 349-359.
Ku, M. and D.A. Melton. 1993. Xwnt-11: a maternally expressed Xenopus
wnt gene. Development 119: 1161-1173.
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DEVELOPMENTAL BIOLOGY