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Development
1999 Apr 01;1267:1467-82. doi: 10.1242/dev.126.7.1467.
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derrière: a TGF-beta family member required for posterior development in Xenopus.
Sun BI
,
Bush SM
,
Collins-Racie LA
,
LaVallie ER
,
DiBlasio-Smith EA
,
Wolfman NM
,
McCoy JM
,
Sive HL
.
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TGF-beta signaling plays a key role in induction of the Xenopus mesoderm and endoderm. Using a yeast-based selection scheme, we isolated derrière, a novel TGF-beta family member that is closely related to Vg1 and that is required for normal mesodermal patterning, particularly in posterior regions of the embryo. Unlike Vg1, derrière is expressed zygotically, with RNA localized to the future endoderm and mesoderm by late blastula, and to the posteriormesoderm by mid-gastrula. The derrière expression pattern appears to be identical to the zygotic expression domain of VegT (Xombi, Brat, Antipodean), and can be activated by VegT as well as fibroblast growth factor (FGF). In turn, derrière activates expression of itself, VegT and eFGF, suggesting that a regulatory loop exists between these genes. derrière is a potent mesoderm and endoderm inducer, acting in a dose-dependent fashion. When misexpressed ventrally, derrière induces a secondary axis lacking a head, an effect that is due to dorsalization of the ventral marginal zone. When misexpressed dorsally, derrière suppresses head formation. derrière can also posteriorize neurectoderm, but appears to do so indirectly. Together, these data suggest that derrière expression is compatible only with posterior fates. In order to assess the in vivo function of derrière, we constructed a dominant interfering Derrière protein (Cm-Derrière), which preferentially blocks Derrière activity relative to that of other TGFbeta family members. Cm-derrière expression in embryos leads to posterior truncation, including defects in blastopore lip formation, gastrulation and neural tube closure. Normal expression of anterior and hindbrain markers is observed; however, paraxial mesodermal gene expression is ablated. This phenotype can be rescued by wild-type derrière and by VegT. Our findings indicate that derrière plays a crucial role in mesodermal patterning and development of posterior regions in Xenopus.
Fig. 1. Derrière protein sequence
and alignments. Amino acid
residues are shown. Numbers at
left indicate amino acid
positions. (A) Derrière protein
sequence. Arrow indicates signal
sequence cleavage site predicted
by von Heijne algorithm.
Maturation cleavage signal
(RAKR) is boxed. These four
amino acids are substituted with
GVDG in Cm-Derrière. The
mature region of Derrière protein
is underlined. The seven
cysteines within the mature
region are underscored with solid
bars. (B) Alignment of Derrière
protein sequence to Xenopus Vg1
(Vg1), zebrafish Vg1 (zVg1),
chicken Vg1 (CVg1) and
incomplete newt Vg1 (nVg1).
Consensus amino acid residues
in proteins are shaded. Sequence
gaps introduced for optimal
protein alignment are indicated
by dashes. The percentage
identities between full-length
Derrière and other Vg1-related
proteins are 56% (xVg1), 51%
(cVg1) and 50% (zVg1), with
mature region identities of 79%
(xVg1), 73% (cVg1) and 76%
(zVg1).
Fig. 2. Temporal and spatial expression patterns of derrière.
(A) Northern analysis of Xenopus embryos. One embryo equivalent
per lane was analyzed for derrière RNA (top row) at various
embryonic stages shown. Ethidium-bromide-stained 28S rRNA is a
loading control (bottom row). Lanes as marked. (B) Whole-mount in
situ hybridization analysis of derrière expression. Embryo
orientations are indicated by vegetal (Vg), dorsal (D) and posterior
(P). Bl, blastocoel. Purple staining represents derrière expression.
(a) Stage 9, late blastula; (b) stage 10.5, early gastrula; (c) stage 11.5,
mid-gastrula; (d) stage 12.5, late gastrula; (e) stage 14, early neurula;
(f) a sagittal section of a stage 11.5embryo. White arrow indicates
superficial layer, white arrowhead indicates deep cells and black
arrowhead indicates the anterior limit of archenteron.
Fig. 3. derrière can be induced by known mesoderm inducers in
animal caps. (A) Experimental scheme. Stage 8.5 animal caps of
uninjected embryos were cultured with purified proteins until stage
11.5 (mid gastrula). Alternatively, embryos were injected at the
animal pole of one blastomere with test RNA at the 2-cell stage.
Animal caps were removed from stage 8.5 embryos and were
cultured until sibling embryos reached stage 11.5. Animal caps and
whole embryos were harvest for RT-PCR. (B) Induction of derrière
in animal caps. ODC served as a loading control. Lanes as marked;
lanes 1, 5, BSA served as a negative control; lanes 4, 10, whole
embryo controls. See Materials and Methods for amount of proteins
used.
Fig. 4. derrière induces mesodermal and endodermal
markers in animal caps. (A) Experimental scheme.
Wild-type embryos were injected at the animal pole of
one blastomere with test RNA at the 2-cell stage.
Animal caps were removed from stage 8.5 embryos
and were cultured until sibling embryos reached
stages indicated by the triangles. Animal caps and
whole embryos were harvest for RT-PCR or
morphology. (B) Expression of marker genes (see
Results) in animal caps after injection of 400 pg RNA.
Injection of globin served as negative controls. ODC
served as a loading control. VegT has a very similar
expression pattern to derrière. eFGF is expressed
posteriorly and in the notochord. gsc is a prechordal
plate marker, siamois is expressed in the dorsal
mesendoderm. eomesodermin a mesendodermal
marker. Xlim-1 and Xnot are dorsal mesodermal
markers. Xvent-1 is a ventral ectodermal and
mesodermal marker. HoxB9 marks posterior spinal
cord. Xsox17a is an endodermal marker. XK81 is a
ventral ectodermal (epidermal) marker and N-CAM is
a neural-specific marker. Lanes as marked.
(C) Mesodermal markers respond to derrière
induction in a dose-dependent manner. HoxA7 is a
posterior mesodermal and ectodermal marker. At the
time of harvest, Pintallavis weakly marks the dorsal
mesoderm and tailbud. m-actin is a muscle-specific
marker and Xbra is expressed posteriorly and in the
notochord. Lanes as marked. b-globin-capped RNA
was used to make up for differences in amount of test
RNA injected such that all embryos received the same
total amount of RNA. (D) XFD blocks derrièreinduced
cap elongation. (a) 200 pg derrière and 800
pg globin; (b) 200 pg derrière and 800 pg XFD; (c)
800 pg XFD and 200 pg globin.
Fig. 5. derrière suppresses head formation or
induces a posterior secondary axis in whole
embryos. (A) Experimental scheme. Wild-type or
albino embryos were injected with 50 pg derrière
and 80 pg lacZ RNA in the marginal zone of one
blastomere at 2-cell stage, either dorsally or
ventrally. Albino embryos were harvested at stage
24 (tailbud) for in situ hybridization and wild-type
embryo were harvested at stage 35 (hatching) for
morphological analysis. (B) Dorsal misexpression
of derrière results in microcephaly. In all panels
anterior (A) is to the left and posterior (P) is to the
right. Light blue indicates lineage tracer b-gal.
(a,b) Wild-type embryo injected with derrière
RNA (a) and globin RNA (b). (c-h) Albino
embryos processed for in situ hybridization (see
Results). Purple staining represents probes as
indicated on the left. m-actin is a muscle-specific
marker, otx2 marks the forebrain, XCG marks the
cement gland, en-2 marks the midbrain/hindbrain
junction and Krox20 marks rhombomeres 3 and 5
in the hindbrain. (c-e) White arrow and arrowhead
indicate otx2forebrain and eye staining,
respectively. Black arrowhead indicates anterior
limit of HoxB9 staining. (f-h) White arrow
indicates en-2 staining, white arrowhead indicates
XCG staining, bracket indicates krox20 staining.
(C) Ventral misexpression of derrière results in a
posterior secondary axis. In all panels, anterior (A)
is to the left and posterior (P) is to the right.
(a,b) Wild-type embryos. White arrow indicates a
secondary axis. (c-j) Albino embryos processed for
in situ hybridization (see Results). Markers as for
B, except for N-CAM, which is a general neural
marker. Arrow indicates secondary axis. (i,j) Arrow
indicates en-2 staining, bracket indicates krox20hindbrain staining.
Fig. 6. derrière increases dorsal character of the ventral marginal
zone. (A) Experimental scheme. 25 pg derrière RNA was injected
into the marginal zone of both ventral blastomeres of 4-cell stage
wild-type embryos. The VMZ was dissected at stage 10.25 and
cultured until harvest for RT-PCR at stage 17. (B) Dorsalization of
the VMZ by derrière. Lanes as marked.
Fig. 7. Derrière posteriorizes isolated neurectoderm indirectly.
(A) Experimental scheme. Anterior dorsal ectoderm (aDE, indicated
by the cut marks, see Materials and Methods) was isolated from
stage 11 and 11.5 (mid-gastrula) embryos. Explants were cultured in
saline alone or with added factors until harvest for RT-PCR at stage
22 (tailbud). (B) Expression of marker genes in aDE explants (see
Results). BSA-treated explants served as negative controls. See
Materials and Methods for amount of protein used. HoxA7 and
Xcad3 are expressed in posterior mesoderm and ectoderm, m-actin is
a muscle marker, XCG is a cement gland marker, en-2 marks the
midbrain/hindbrain junction and Krox20 is a hindbrain marker. Lanes
as marked.
Fig. 8. Specificity of Cm-derrière.
(A) Rationale for dominant negative activity.
Solid bars and open bars indicate the prepro
region and the mature region of Derrière
protein, respectively. Gray boxes represent
mutated maturation cleavage signal. The
link between two open boxes represents the
disulfide bond of a dimer. (B) Experimental
scheme for C-E. Wild-type embryos were
injected at the animal pole in one cell at the
2-cell stage with test RNA. Animal caps
were removed from stage 8.5 (mid-blastula)
embryos and were cultured until sibling
embryos reached stage 20 (late neurula).
Animal caps and control whole embryos
were harvested for RT-PCR (C,D) and for
morphological analysis (E). (C) Ratio of
derrière: Cm-derrière at which marker
expressions are inhibited in animal caps.
Injection of globin alone served as a
negative control. ODC was used as a loading
control. Test RNA is indicated at the top. wt,
wild-type derrière; Cm, Cmderrière.
endodermin (Edd) is an endodermal
marker. Lanes as marked; lane 7, uninjected
whole embryo control. globin RNA was
injected at 1 ng; In derrière and Cm-derrière
injections 20 pg of derrière RNA was
injected with Cm-derrière RNA to make up
the indicated ratio of derrière: Cm-derrière;
and globin RNA making the total mass
injected equal to 1 ng. (D) Effects of Cmderrière
on other TGF-b family members:
molecular assay. Injection of Cm-derrière
alone served as a negative control. Test RNA
is indicated at the top and the presence or
absence of ten-fold mass excess of Cmderrière
is indicated by + or – sign. Lanes as
marked; lane 16, uninjected whole embryo
control. derrière, BVg1, Xnr1, Xnr2, Xnr3
and Xnr4 RNAs were injected at 50 pg and
activin RNA was injected at 5 pg.
(E) Effects of Cm-derrière on other TGF-b
family members: animal cap elongation
assay. Cm-derrière was used at a 10:1 ratio
to co-injected TGF-bs, shown above the
panels. derrière, BVg1, Xnr1, Xnr2, Xnr3
and Xnr4 RNAs were injected at 50 pg and
activin RNA was injected at 5 pg.
Fig. 9. Phenotype and in situ hybridization analysis of
Cm-derrière-injected embryos and rescue of Cmderrière
by wild-type derrière and VegT.
(A) Experimental scheme. Wild-type or albino embryos
were injected with test and lacZ RNA in the marginal
zone. At stages indicated below, albino embryos were
harvested for in situ hybridization and wild-type embryo
were harvested for morphological analysis. (B) Effects
of Cm-derrière expression on whole embryos. Light
blue indicates lineage tracer b-gal and purple represents
specific RNA expression. (a,b) Vegetal (Vg) views of
stage 11-11.5 embryos injected at 4-cell stage with 250
pg Cm-derrière (a) and globin (b) into each of the two
ventral blastomeres. Black arrowhead: extent of
blastopore formation. (c,d) Posterior (P) views of stage
17 embryos injected at 4-cell stage with 250 pg Cmderrière
(c) and globin (d) into each of the two ventral
blastomeres. D, dorsal; Y, yolk cells. (e,f) Lateral views
of stage 35 embryos injected at 2-cell stage with 500 pg
Cm-derrière (e) and globin (f) into both blastomeres. A,
anterior; P, posterior. (g-j) Albino embryos processed for
in situ hybridization. (g) XCG (arrowhead) en (arrow)
and krox20 (bracket) probes; dorsal view of a stage 20embryo injected with 500 pg Cm-derrière. (h) XCG, en
and krox20 probes; head-on view of a stage 20embryo
injected with 500 pg globin. (i,j) m-actin probe; lateral
views of stage 26 embryos injected with 500 pg Cmderrière
(i) and globin (j). (k,l) Rescue experiments.
(k) 50 pg derrière and 500 pg Cm-derrière co-injected;
(l) 50 pg VegT and 500 pg Cm-derrière co-injected.
Fig. 10. Model for derrière activity. At the mid-blastula transition, we speculate that maternal transcription factors (such as VegT) and possibly
secreted factors activate derrière expression in the presumptive mesoderm and endoderm (wide hatching). During early gastrula, derrière
expression is maintained in the mesoderm by a positive feed-back loop that includes FGFs (such as eFGF), Xbra and zygotic VegT (close
hatching). derrière expression in the endoderm is not maintained presumably because such a feed-back loop cannot be established there. During
early gastrula stages, derrière may activate mesendodermal fates in both the future head region and more posteriorly. In particular, derrière may
play a role in activating posterolateral (paraxial) mesodermal fates at this time, before somite formation. By mid to late gastrula, derrière is
excluded from anteriormesoderm and from the dorsal midline (close hatching), and continues to promote posterolateral fates. An, animal pole;
Vg, vegetal pole; A, anterior; P, posterior; meso, mesodermal fates; endo, endodermal fates.