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Dorsal-ventral patterning in vertebrate embryos is regulated by members of the TGF-beta family of growth and differentiation factors. In Xenopus the activins and Vg1 are potent dorsal mesoderm inducers while members of the bone morphogenetic protein (BMP) subclass pattern ventralmesoderm and regulate ectodermal cell fates. Receptors for ligands in the TGF-beta superfamily are serine-threonine kinases, but little is known about the components of the signal transduction pathway leading away from these receptors. In Drosophila the decapentaplegic protein (dpp), a homolog of vertebrate BMP-2 and BMP-4, functions in dorsal-ventral axial patterning, and a genetic screen for components involved in signaling by dpp has identified a gene named mothers against decapentaplegic (Mad). Mad encodes a unique, predicted cytoplasmic, protein containing no readily identified functional motifs. This report demonstrates that a gene closely related to Drosophila Mad exists in Xenopus (called XMad) and it exhibits activities consistent with a role in BMP signaling. XMad protein induces ventralmesoderm when overexpressed in isolated animal caps and it ventralizes embryos. Furthermore, XMad rescues phenotypes generated by a signaling-defective, dominant-negative, BMP-2/4 receptor. These results furnish evidence that XMad protein participates in vertebrate embryonic dorsal-ventral patterning by functioning in BMP-2/4 receptor signal transduction.
Fig. 1. Protein sequence comparison of Xenopus Mad with Drosophila Mad. The predicted
Xenopus Mad (XMad) protein sequence is aligned with Drosophila Mad protein (DMad). The
XMad cDNA encodes a predicted protein of 464 amino acids. XMad protein is 75% identical
to DMad at the amino acid level (identical residues are indicated by vertical bars) and 85%
similar when conservative amino acid substitutions are considered (double dots). Note the high
degree of conservation in the N-terminal and C-terminal portions of the proteins. The
GenBank accession number for XMad is U58834.
Fig. 2. Developmental expression of Xenopus Mad. (A) A
developmental northern blot of total embryonic RNA shows that
XMad is maternal and expressed at all stages of early development.
Three transcripts are detected with the sizes (in kb) indicated on the
left. The most abundant mRNA is 3.4 kb, and minor transcripts of
4.3 kb and 3.0 kb are also detected. It is not known whether these
transcripts derive from closely related genes or splicing variants.
Lane numbers correspond to developmental stages (Nieuwkoop and
Faber, 1967): (7, 9) blastula, (11) gastrula, (15, 18) neurula, (26)
tailbud tadpole and (38) swimming tadpole. (B) In situ hybridization
shows that XMad transcripts are uniformly distributed in the early
gastrula (stage 10). Dense staining covers the entire prospective
ectoderm and marginal zone, but vegetal cells (the lighter region) do
not stain efficiently by this procedure (Harland, 1991). A northern
blot on isolated dorsal, ventral, animal and vegetal regions confirmed
the uniformity of XMad expression at early stages (not shown).
(C) A mid-gastrulaembryo (stage 12) split sagitally reveals that
XMad is expressed in the ectoderm and neurectoderm (arrows mark
the ectodermal-mesodermal boundary), and expression in the
underlying mesoderm is greater in the posterior, adjacent to the yolk
plug (YP). This embryo is lightly pigmented and the brown line
anterior to the yolk corresponds to involuted bottle cells. The embryo
is positioned with the anterior to the left and dorsal at the top. (D) At
tailbud tadpole stage 26 XMad expression is high in the central
nervous system and head. (E) A close-up of the head of the embryo
in D, highlighting XMad expression in the brain (b), eye (e) and head
neural crest derivatives (mc, mandibular crest; hc, hyoid crest; abc,
anterior branchial crest; pbc, posterior branchial crest). Expression in
the otic vesicle, between the hyoid crest and anterior branchial crest,
is also visible. Scale bars, 0.1 mm.
Fig. 3. XMad and DMad ventralize Xenopus embryos. Synthetic
mRNAs encoding control (pGem vector) or Mad sequences were
injected into the equatorial region of two dorsal or two ventral
blastomeres at the 4-cell blastula stage, and phenotypes were scored
at tadpole stage 40. Dorsal (A) or ventral (B) injections of control
(pGem vector) mRNA resulted in normal embryos. (C) Dorsal
injection of XMad mRNA caused severe ventralization. (D) Ventral
injection of XMad mRNA caused posterior thickening and a slight
reduction in the tail. The average dorso-anterior index, a measure of
the degree of dorsal and anterior mesodermal patterning (Kao and
Elinson, 1989), was for each group: A, DAI=5 (n=18); B, DAI=5
(n=18); C, DAI=0 (n=18); D, DAI=5 (n=20). (E) Expression of
DMad in dorsal blastomeres ventralized the embryos (DAI=1.9,
n=20), while ventral expression yielded relatively normal embryos
(DAI=5, n=21; not shown). (F) Northern blot analysis of muscle
actin (a dorsal mesodermal marker) and aT1 globin (a ventral
mesodermal marker) gene expression in the control and XMadinjected
embryos shown in A-D. Lanes a-d correspond to embryos in
A-D. Note the loss of muscle actin and the significant increase in
globin expression in embryos injected dorsally with XMad mRNA
(lane c). This indicates that overexpression of XMad protein in the
presumptive dorsal mesoderm respecified its fate to that of ventralmesoderm. Ventral expression of XMad boosted globin expression
slightly (lane d), but dorsal or ventral injections of vector mRNA had
no effect (lanes a and b). Histone H4 mRNA (Perry et al., 1985) was
scored as a control for RNA loading.
Fig. 4. XMad and DMad induce mesoderm in animal caps. The panel
shows an RT-PCR analysis of mesodermal marker gene expression
in animal caps injected with 3.0 ng of mRNA for pGem (C), DMad
(DM), XMad (XM) or Xenopus BMP-4 (B4). The last two lanes
show RT-PCR products from stage-18 embryonic cDNA synthesized
in the presence (emb RT+) or absence (emb RT-) of reverse
transcriptase to control for cDNA synthesis and DNA contamination,
respectively. Note that expression of DMad, XMad and BMP-4
induced each of the ventro-posteriormesoderm markers assayed.
EF1-a was scored as a positive control for cDNA synthesis. Animal
caps were harvested at stage 30 (tadpole) to score aT1 globin
expression, stage 11 (mid-gastrula) to score Xwnt 8 and Xtwist, and
stage 18 (neurula) to score Xhox-3 and Xlhbox6. The eF1-a signal
shown corresponds to that of stage 11 cDNA, but all other cDNA
samples treated with reverse transcriptase were positive.
Fig. 5. XMad rescues dominant-negative BMP2/4 receptor
phenotypes. The panels display phenotypes of whole embryos (A-C)
and isolated ventral marginal zones (VMZs, D-E) expressing the
dominant-negative BMP-2/4 receptor (Graf et al., 1994), alone or in
combination with XMad. 50 pg of each mRNA were injected into the
marginal zone of two ventral blastomeres at the 4-cell stage. VMZs
were explanted at stage 10.5, and embryos and VMZ explants were
scored at stage 40. (A) Expression of the dominant-negative BMP-
2/4 receptor (tBR) from injected mRNA resulted in tadpoles with
secondary axial structures in 33% of the cases (n=18). A typical
secondary axis is indicated by the arrows. (B) Co-expression of
XMad together with tBR resulted in 100% normal embryos (n=18).
(C) Injection of control (pGem) mRNA resulted in 100% normal
embryos (n=21). (D) VMZs expressing tBR elongated and developed
pigmented melanocytes, a neural derivative. (E) VMZs from
embryos co-expressing tBR and XMad were rescued to normalcy
and formed oblong ‘belly pieces’ like control VMZs (F).
(F) Injection of control (pGem) mRNA into VMZs resulted in a
typical wild-type VMZ morphology. (G) A northern blot on RNA
from the VMZs shown in D-F. VMZs expressing tBR (lane d)
developed dorsal mesoderm, as revealed by the expression of muscle
actin, and they lacked ventralmesoderm (red blood) as reflected by
the absence of aT1 globin expression. When XMad was co-expressed
with tBR (lane e) dorsalization of the VMZ was reversed; the explants
lacked muscle and expressed globin, similar to control VMZs (lane f).
Cytoplasmic actin mRNAs cross-hybridize with the muscle actin
probe and migrate as two bands above the muscle-specific message,
providing a positive control for RNA loading in the gel.
Fig. 6. Neural induction by tBR expression in animal cap ectoderm is
inhibited by co-expression of XMad. Lanes 1-4 correspond to an RTPCR
analysis of cDNA from animal caps injected at the 2-cell stage
with 2 ng pGem RNA (lane C),1.0 ng XMad mRNA (lane XM), 50
pg tBR mRNA (lane tBR), and 50 pg tBR mRNA plus 50 pg XMad
mRNA (lane tBR + XM). Caps were cut at stage 8 and harvested at
neurula stage 18. Note the induction of N-CAM and NRP1 (a neuralenriched
ribonucleoprotein; Richter et al., 1990) mRNAs when BMP
signals were blocked by tBR. This effect was suppressed by coexpression
of XMad. Some background expression of NRP1 is
normal. Furthermore, XMad alone did not induce neural tissue (lane
XM). Lanes 5 and 6 are positive and negative controls for RT-PCR
as in Fig. 2. EF1-a is a positive control for cDNA synthesis.
smad1 (SMAD family member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anteriorleft, dorsal up.
