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Abstract
The Runt domain gene AML1 is essential for definitive hematopoiesis during murine embryogenesis. We have isolated Xaml, a Xenopus AML1 homologue in order to investigate the patterning mechanisms responsible for the generation of hematopoietic precursors. Xaml is expressed early in the developing ventralblood island in a pattern that anticipates that of later globin. Analysis of globin and Xaml expression in explants, in embryos with perturbed dorsal ventral patterning, and by lineage tracing indicates that the formation of the ventralblood island is more complex than previously thought and involves contributions from both dorsal and ventral tissues. A truncated Xaml protein interferes with primitive hematopoiesis. Based on these results, we propose that Runt domain proteins function in the specification of hematopoietic stem cells in vertebrate embryos.
Fig. 1. XAML, CBFa2 and XRD sequence alignment. The predicted amino acid sequence for
XAML (GenBank accesion number AF035446) is approximately 90% identical to CBFa2.
Insertion/deletions and regions of the proteins which cannot be aligned are lower case. The darker
shading indicates a high pair-mean score (obtained by the Macaw sequence alignment program).
Fig. 2. Expression of Xaml in Xenopus
embryogenesis. Xaml (A-G,I) and a-
globin (H) expression patterns, in all
panels anterior is to the right. Bracket
in D-F, 400 mm. Arrows indicate
Xaml-expressing cells (C,I). (A) RTPCR
of Xaml expression at different
embryonic stages. (B) Dorsal view of
Xaml expression in neuroblasts at
stage 17. (C) Sagittal section through
anterior endomesoderm of a stage 14embryo, Xaml is in cells of the
endomesoderm which make direct
contact with the ectodermal layer, Bar,
20 mm. (D) Ventral view of a stage 14embryo. (E) Ventral view of embryo
shown in B; note that Xaml expression
has expanded posteriorly. (F) Detail of
EA Xaml expression at stage 22. At
this stage Xaml expression refines into
a V-shape, but some Xaml cells remain
anterior to the VBI (right arrow).
(G) Ventral Xaml expression in a stage
26 embryo extends to the proctodeum
(arrow). (H) a-globin expression in a
stage 26embryo, has not yet reached
the proctodeum (arrow) demonstrating
that Xaml expression precedes
a-globin in the VBI. (I) Transverse
section of a stage 26embryo at approximately 30% embryonic length. Xaml expression is seen in isolated cells of the lateral plate mesoderm,
top panel is a high magnification view of the region boxed in the lower panel. Abbreviations: N, notochord, nt, neural tube, o, olfactory placode.
Fig. 3. Schematic representation of gastrulation in Xenopus. Presumptive EA Xamlexpressing
cells are colored magenta. The embryo shown on the left is a stage 10
embryo; the brackets indicate the region of the embryo excised in explant
experiments. The middle embryo shows a mid-gastrulation stage embryo. The
embryo on the right is at a similar stage to the earliest that we detect Xaml by wholemount
in situ hybridization. Abbreviations: A, archenteron, B, blastocoel cavity; BC,
bottle cells; D, dorsal; V, ventral. (Adapted from Keller, 1991).
Fig. 4. Xaml in situ hybridization in dorsal and ventral explants.
(A) Dorsal explant at control stage 15-17 showing positive Xaml
staining. The position of Xaml expression is in a ventral location
relative to the head folds, similar to the position of EA Xaml in a
whole embryo. (B) Ventral explant at control stage 15-17 with lack
of Xaml staining. (C) Dorsal explant at control stage 25-27 with lack
of Xaml staining. (D) Ventral explant at control stage 25-27; note
intense Xaml staining at the âanteriorâ tip (pointing down) of the
explant, this location of staining is similar to that seen with an
a-globin probe (data not shown).
Fig. 5. Comparison of the effects of UV ventralization and LiCl on
Xaml (A-D) expression and a-globin (D-G) expression. Arrowheads
indicate untreated controls in F and G. The bracket indicates the
region of a DAI=8 embryo where Xaml is increased but a-globin is
absent (A,D,E). (A) LiCl-treated embryos at control stage 27
following in situ for Xaml. The top two embryos are DAI=8 the
bottom two embryos are DAI=9. (B)Ventral view of an embryo
probed for Xaml expression after UV treatment (left embryo); the
arrow indicates the region of the UV embryo where Xaml expression
is missing. A wild-type control embryo is shown on the right.
(C) Dorsal view of embryos shown in B (see text). (D) LiCl embryos
at control stage 27 (DAI=8) following whole-mount in situ
hybridization for Xaml (right embryo) or a-globin (left embryo).
(E) LiCl-treated embryos at control stage 27 following aT1 globin in
situ. The top two embryos have DAI=8; the bottom two embryos
have DAI 9. (F) a-globin expression in UV ventralized embryos at
control stage 26 (G) a-globin expression in UV ventralized embryos
at control stage 28-29. Abbreviations: o, olfactory placode; WT,
untreated control.
Fig. 6. a-globin expression in embryos lineage traced with lacZ
following in situ hybridization with an aT1 globin probe. b-gal
staining gives a blue precipitate while a-globin staining is purple.
(A) Embryos with a b-gal staining pattern typical of dorsally injected
pigmented controls. Arrowhead indicates a-globin expression.
(B) Embryos with b-gal staining typically seen in ventrally injected
pigmented controls. Arrowhead indicates a-globin expression.
(C) Ventral view of an embryo treated as in A. Note the significant
overlap of the b-gal staining and the a-globin staining. (D) Ventral
view of embryo shown in B.
Fig. 7. Benzidine staining of larvae (stage 45-50) injected in the
VMZ, arrows point to the heart. (A) Control injected larva. (B) Larva
injected with full-length Xaml mRNA. (C) Larva injected with Xrd
mRNA. (D) Northern blot of RNA harvested from stage 38 embryos
probed with a-globin and histone H4 (globin is reduced 26-fold in
the bloodless embryos). Note that this is an earlier stage than is
shown in A, B or C.
Fig. 8. Embryonic a-globin staining in
embryos injected with XRD+lacZ mRNA (BH)
or lacZ alone(A). Blue staining indicates
cells that received the lacZ message, purple
staining indicates cells expressing a-globin.
(A) embryo injected ventrally with lacZ
alone, note the significant overlap of blue and
purple staining. (B) Embryo injected with
XRD+lacZ and processed simultaneously
with the embryos in C-H; note the normal a-
globin expression in the VBI and the lack of
blue staining in the VBI. (C,D) Embryos
injected with lacZ staining overlapping the
posterior VBI (i.e.. ventrally injected)
showing significant inhibition of posterior a-
globin. (E) Detail of embryo shown in D; note the small patch of a-globin-positive cells in the posterior of the VBI demonstrating that this
embryo is at a similar developmental stage as that in A. (F,G) Embryos injected with XRD+lacZ with anterior targeting of the VBI typically
seen with dorsally injected pigmented controls. Note the absence of a-globin in the anterior VBI (n=19). (H) Detail of embryo shown in G.
Note the complimentary pattern of blue and purple staining. The blue cells are in close proximity to purple cells suggesting that XRD acts cell
autonomously. In this experiment, we injected 60 embryos with either lacZ alone or with XRD+lacZ. Of these embryos, 54 injected with lacZ
alone survived the entire procedure while 51 injected with XRD+lacZ survived. All 54 embryos injected with lacZ alone showed normal a-
globin staining. Of the 51 embryos injected with Xrd+lacZ, 16 had normal a-globin expression and did not show XRD targetting to the VBI. 28
embryos (54%) showed abnormal a-globin staining and all of these showed b-gal staining in the VBI. In these experiments, there were an
additional seven embryos injected with Xrd+lacZ that demonstrated normal a-globin staining and also contained b-gal in the VBI. This
incomplete penetrance could be due to variations in the effective levels of XRD in these embryos. Consistent with this, we did not observe
overlaps with b-gal and a-globin when higher levels of XRD mRNA (2 ng/blastomere) were injected.