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First published online 3 May 2006
doi: 10.1242/dev.02386
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1 Department of Developmental and Molecular Biology, Albert Einstein College of
Medicine, Bronx, NY 10461, USA.
2 Division of Hematology/Oncology, Children's Hospital of Boston, Department of
Pediatrics, Howard Hughes Medical Institute, Harvard Medical School, Boston,
MA 02115, USA.
* Author for correspondence (e-mail: tevans{at}aecom.yu.edu)
Accepted 31 March 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hematopoiesis, Endothelial, Pronephros, Transgenic, Zebrafish, lmo2
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), in association with other major developmental
pathways regulated by WNT and FGF ligands
(Munoz-Sanjuan and Hemmati-Brivanlou,
2001). Ventral mesoderm is subsequently patterned during
post-gastrulation stages to distinguish lateral and intermediate derivatives.
Lateral mesoderm gives rise to hematopoietic and vascular lineages, while
intermediate mesoderm generates pronephric progenitors
(Drummond, 2000;
Thisse and Zon, 2002).
These secondary patterning events are less well defined, being
characterized mainly by the expression of genes that are hallmarks for the
differentiation of hematopoietic, vascular and pronephric progenitors from
lateral and intermediate mesoderm (Davidson
and Zon, 2000; Serluca and
Fishman, 2001). By early somitogenesis, distinct stripes of cells
can be distinguished in posterior mesoderm that express either the pronephric
marker pax2.1 (pax2a – Zebrafish Information Network)
or, immediately more medial, the hemato-vascular markers scl
(tal1 – Zebrafish Information Network) and gata2
(Drummond, 2003). The
developmental pathways that control these sub-divisions are also not well
characterized, although it seems reasonable to consider that lateral and
intermediate mesoderm continue to develop under the influence of the same
predominant signaling pathways. It is, however, a challenge to test this, as
disruption of the BMP, WNT or FGF pathways alters the initial specification
events and this precludes analysis of subsequent developmental transitions.
The fate of these cells has been considered to be not fully committed during
somitogenesis, as forced expression of Scl is sufficient to convert the entire
region to Gata1-expressing hematopoietic cells
(Gering et al., 1998).
However, this interpretation is complicated because it is not known at what
stage of development Scl exerts this effect.
BMPs are members of the TGFß super-family of secreted ligands and they
bind type I and type II receptor complexes to initiate a signaling cascade
that activates transcription of downstream `ventral' target genes. This is a
highly conserved pathway and loss of BMP signaling in fish
(Mintzer et al., 2001;
Schmid et al., 2000), frog
(Graff et al., 1994) or mouse
(Winnier et al., 1995) leads
to expansion of dorsal mesoderm (for example trunk muscle) at the expense of
ventral mesoderm (blood, vasculature and pronephros). The early requirement
for BMP signaling was confirmed by the identification in zebrafish of numerous
`dorsalized' mutants that are defective for genes in the BMP pathway (for a
review, see Hammerschmidt and Mullins,
2002), including lost-a-fin [alk8
(Bauer et al., 2001)],
swirl [bmp2b (Kishimoto
et al., 1997; Nguyen et al.,
1998)], snailhouse [bmp7
(Dick et al., 2000)], mini
fin [tolloid (Connors et al.,
1999)] and somitabun [smad5
(Hild et al., 1999)].
Hyper-activation of the pathway leads to an opposite phenotype; Bmp4
overexpression expands the expression domain of hematopoietic-, vascular- and
pronephric-specific markers, including scl, flk1 (kdr
– Zebrafish Information Network) and pax2.1.
In contrast to the generation of early ventral mesoderm, it is not known if
BMP signaling is directly involved in the further development of lateral
mesoderm derivatives. For example, the cloche and bloodless
mutants are characterized by defective differentiation of hematopoietic cells
from lateral mesoderm, despite apparently normal patterning of ventral
mesoderm. These mutants are rescued by forced expression of Scl but not Bmp4
(Liao et al., 1998). However,
zygotic loss of the Smad5 gene product, which is a direct mediator of the BMP
signaling pathway, causes defective differentiation in the blood and
vasculature (Chang et al.,
1999; Yang et al.,
1999). This is consistent with a patterning role for BMP signaling
post-gastrulation, e.g. to maintain, promote or restrict expression of the
transcriptional regulatory programs that control the survival and
differentiation of hematopoietic, vascular and pronephric progenitors from
lateral mesoderm.
The receptors and the signaling components for the BMP pathway are expressed in the embryo post-gastrulation. However, manipulation of the pathway by overexpression of constitutively active or dominant-negative components, or loss of function through genetic and morpholino experiments cannot accurately interrogate later functions without disturbing early embryogenesis. To address function at subsequent stages, we developed an approach for conditional modulation of BMP signaling in lateral mesoderm of developing zebrafish embryos. We show that the BMP signaling pathway continues to function during somitogenesis to regulate the development of hematopoietic, vascular and pronephric lineages from lateral mesoderm. The data supports a model in which BMP signaling at this stage promotes pronephric fate while restricting the generation of hemato-vascular derivatives.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Most
experiments used embryos derived from parents of a hybrid strain, itself
derived from crossing wild-type AB and TUB strains, obtained from the
Zebrafish International Research Center (Eugene, OR). The gata1:gfp
reporter strain (Long et al.,
1997) was provided by Dr Shou Lin (UCLA, Los Angeles, CA). The
fli:gfp line (Lawson and
Weinstein, 2002) was obtained from the Zebrafish International
Research Center.
Plasmids
The parental I-SceI plasmid vector was assembled in two steps.
First, oligonucleotides with the sequence containing the I-SceI sites
were annealed and used to replace the pBluescript SKII+ backbone previously
digested with MluI and BssHII restriction enzymes. Next, the
entire polylinker of pBluescript SK+ was cloned back into the first vector
(BssHII/BssHII). Each of the subsequent expression plasmids
were then generated by subcloning into the parental I-SceI plasmid,
using inserts derived from plasmids for lmo2:gfp
(Zhu et al., 2005),
gata1:gfp (Long et al.,
1997), CA-XBMPR (Candia et al.,
1997) and pTurbo-Cre (Hug et
al., 1996) inserts. The lmo2:
BR and
gata1:BR vectors were generated by PCR amplifying the
BR insert described previously
(Zhang and Evans, 1996) in
place of GFP. Likewise, the lmo2:caBR and lmo2:cre vectors
were generated by subcloning the respective cDNAs in place of GFP.
The sequence of the I-SceI oligomers are: FP, 5'-CGCGTTAGGGATAACAGGGTAATGCGCGCGAGCTCGAATTCGGTACCGCGCGCTAGGGATAACAGGGTAATA-3'; RP, 5'-CGCGTATTACCCTGTTATCCCTAGCGCGCGGTACCGAATTCGAGCTCGCGCGCATTACCCTGTTATCCCTAA-3';
Primers used to amplify the BR sequence are: FP,
5'-ATAGAATTCACCATGAGAGAACGACTTTTC-3'; RP,
5'-ATAGGATCCCCTTTGTAAATCCATATGATAAG-3'.
Microinjection
The I-SceI injection mixture was assembled on ice until just
before injection. Plasmids were mixed with or without I-SceI
meganuclease (1 µg/µl; New England Biolabs) in 0.5x
I-SceI meganuclease restriction buffer. Each vector (40-80 pg) was
injected in a volume of 2.3 pl at the one-cell stage. RNA encoding the
dominant-negative BMP receptor (BR) was generated in vitro from
expression vector pSP64T using mMessage mMachine (Ambion) and 60 pg was
injected into one- to two-cell stage embryos.
Whole mount in situ hybridization
Whole mount in situ hybridization was preformed essentially as described
(Alexander et al., 1998).
Digoxigenin-labeled RNA antisense probes were described previously for
gata1 (Detrich et al.,
1995); scl (Liao et
al., 1998); pax2.1
(Krauss et al., 1991);
lmo2 (Zhu et al.,
2005); flk1 (Thompson
et al., 1998); L-plastin (lcp1 – Zebrafish
Information Network) (Herbomel et al.,
1999); mpo (Bennett et
al., 2001); and no tail
(Schulte-Merker et al., 1992).
The br digoxigenin-labeled antisense RNA probe was generated
from the Xenopus BR cDNA clone
(Zhang and Evans, 1996).
Two-color whole-mount in situ hybridization was preformed essentially as
described previously (Westerfield,
1995), although instead of Fast Red, INT Red (Sigma) was used to
develop the red color. Fluorescein-12-UTP-labeled gata1 probe and
digoxigenin-labeled pax2.1 probe were used sequentially.
Flow cytometry
Fluorescence activated cell sorting (FACS) was performed as described
(Long et al., 1997). For each
experiment, a clutch of embryos from the gata1:gfp transgenic line
was divided into two sets, with one batch being injected and other uninjected
batch serving as a control. Both groups were then dissociated at the 16- to
17-somite stage and cells analyzed by FACS under standard FITC conditions.
Cell transplantation and imaging
Donor embryos were microinjected at the one- to two-cell stage with a
solution of 5% lysine fixable tetramethylrhodamine dextran (Molecular Probes)
and 0.2 M KCl, either alone or including 100 pg RNA encoding Bmp4. Cell
transplantation was preformed as described
(Ho and Kane, 1990). Donor
cells (20-50) from high-sphere staged embryos were transplanted into the
margin of similarly staged hosts. Host embryos were derived from the
gata1:gfp line. Chimeric embryos were cultured in embryo media + 0.2%
penicillin-streptomycin until the 10- to 12-somite stage, fixed overnight in
4% paraformaldehyde and screened for localization of fluorescently labeled
(red) donor to lateral mesoderm. Embryos with similar numbers of red cells
were then stained with Hoechst, de-yolked, flat-mounted and analyzed on a
Zeiss Axiovert (200M) microscope. Criteria used to score the host embryo is as
follows: an event is scored as positive when a green (GFP+) cell is in direct
proximity with a red (donor) cell and by the absence of a GFP-negative but
blue (Hoechst) cell between the two. Raw z-series of images were
processed in Image-J to clearly analyze cell boundaries.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
pattern marks the hematopoietic, vascular and pronephric progenitors, of both
rostral and caudal origin. A 2.5 kb promoter fragment derived from sequences
upstream of the lmo2 transcriptional start site is sufficient to direct
expression of GFP to recapitulate the endogenous expression pattern of
lmo2 in lateral mesoderm (Zhu et
al., 2005). This promoter therefore provides an appropriate tool
for targeting gene expression to lateral mesoderm by transgenesis.
Transient transgenic expression in zebrafish is characterized typically by
substantial mosaicism, which complicates interpreting any phenotypes caused by
transgenic expression. To address this problem, we flanked transgenes with
restriction sites for the I-SceI meganuclease. Co-injection of
transgenes flanked with these restriction sites along with the I-SceI
enzyme was shown previously in Medaka to decrease mosaicism and increase
germline transmission (Thermes et al.,
2002). We confirmed the utility of this approach in zebrafish by
injecting a transgenic construct with the lmo2 promoter directing
expression of GFP. This reporter transgene flanked by I-SceI sites
[designated by I(transgene)I] was co-injected either alone or with
the meganuclease, and the resulting expression pattern of GFP was observed. In
30% of the co-injected embryos an essentially uniform pattern of GFP
expression is seen in lateral mesoderm, which effectively recapitulates the
normal expression pattern for the lmo2 gene
(Fig. 1). This uniform pattern
is never seen in embryos that are injected with the reporter in the absence of
the meganuclease. The remaining co-injected embryos either express no GFP, or
most often can still be characterized as displaying mosaic expression, similar
to those that did not receive the meganuclease.
To determine if generation of this uniform expression pattern was unique to
the lmo2 promoter, we tested another construct using, instead, the
promoter for the gata1 gene. Gata1 is also expressed in lateral
mesoderm, but labels at a later stage (five somites) more specifically the
progenitors for the caudal embryonic hematopoietic program and subsequently
the differentiated erythroid cells. When this reporter gene was co-injected
with the I-SceI meganuclease the results were similar, in that again
30% of the embryos showed uniform expression of GFP in the gata1
expression domain, similar to stably transgenic gata1:gfp lines
(Fig. 1D). Compared with the
GFP pattern observed using the lmo2 promoter, the gata1
promoter directs expression of GFP at a slightly later stage (observed first
around the five-somite stage, compared with one somite for lmo2:gfp),
which again is consistent with the normal expression patterns for these genes.
In both cases, GFP expression is not observed outside of the normal domains
for the endogenous genes. In summary, although the majority of the transient
transgenic embryos still show mosaic expression, using the I-SceI
approach, these promoters reliably target expression of transgenes throughout
the normal expression domains in lateral mesoderm for about 30% of the
injected embryos.
Inhibition of BMP signaling in lateral mesoderm alters hematopoietic and vascular development
An established approach to reduce specifically BMP signaling in developing
embryos is by overexpression of a truncated BMP receptor. A BMP type I
receptor that is deleted of the C-terminal kinase domain acts in a
dominant-negative fashion by associating with BMP type II receptors and
thereby blocking subsequent cellular signaling to the regulatory SMADs
(Graff et al., 1994;
Maeno et al., 1994). In
previous studies, we used a truncated Xenopus BMP receptor to inhibit
BMP signaling in developing Xenopus embryos, and showed that BMP
signaling is required during gastrulation for establishment of the embryonic
hematopoietic program (Zhang and Evans,
1996). This mutant receptor, called BR, is truncated of
C-terminal sequences just after the transmembrane domain and therefore lacks
the kinase domain entirely. In order to confirm that this mutant receptor is
functional in zebrafish embryos, we injected purified RNA encoding BR
into fertilized zebrafish eggs. Embryos expressing BR develop with
substantial axial defects (not shown), including the lack of normal tail
structures, and in this manner resembles the phenotype of swirl
embryos, which are mutant for the bmp2 gene
(Schmid et al., 2000).
However, the phenotype, as also described previously
(Hammerschmidt et al., 1996),
is more severe than the bmp2 mutant, and is more similar to embryos depleted
of both Smad1 and Smad5 (L. McReynolds and T.E., unpublished). Therefore,
forced expression of the BR mutant receptor can be used to block BMP
signaling during zebrafish development.
Thus, to investigate if there is a continued or later function for active
BMP signaling in lateral mesoderm subsequent to ventral specification, our
strategy was to use the lmo2 promoter to direct expression of
BR in developing embryos using transgenic constructs flanked with
I-SceI restriction sites. To ensure the fidelity of the approach, we
first injected the I(lmo2:BR)I transgene into
wild-type embryos and analyzed the expression of the BR cDNA by in situ
hybridization. Because the transgene is derived from the Xenopus
gene, the antisense probe is specific for the expressed transgene. As shown in
Fig. 2A, there is no expression
of the transgene detected at the tailbud stage, consistent with the expected
activation of the transgene at around the one- to two-somite stage. Indeed by
the three-somite stage, transgene expression is detected specifically in the
emerging two stripes of lateral mesoderm
(Fig. 2B). This pattern is
extended by the eight-somite stage to recapitulate entirely the normal pattern
of expression for lmo2 (Fig.
2C). The pattern appears essentially uniform in 25% of the
embryos, although even in these embryos there often appears some mosaicism
(`patchiness') compared with what would be expected from a stable transgenic
line. However, the approach is sufficient to delay expression of the mutant
receptor until somitogenesis and to target it specifically to much of the
lateral mesoderm.
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BR throughout early
embryogenesis is sufficient to block completely the expression of scl
(Zhang and Evans, 1996).
However, activation of scl expression should not be altered if the
lmo2 transgene delays expression of the mutant receptor. Indeed, as
shown in Fig. 3 (summarized
also in Table 1), embryos
transgenic for I(lmo2:BR)I activate normally the
initial pattern of scl expression. The transcripts are detected as
early as the one- to two-somite stage in short parallel stripes of lateral
mesoderm in both transgenic and control embryos. Therefore, we are confident
that the transgene is not affecting initial stages of mesoderm patterning or
the initial specification of hematopoietic progenitors.
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BR)I
transgene into embryos that are transgenic for the gata1:gfp
reporter. This transgenic line expresses GFP in primitive erythroid
progenitors from the five-somite stage, and can be used to visualize the
emergence of the committed embryonic hematopoietic program. If BMP signaling
is required in Lmo2+ cells for erythroid development, we expected at least 30%
of the injected embryos to show substantial defects in GFP expression. Instead
the exact opposite result was observed. In multiple independent experiments,
30% of the embryos derived from fertilized eggs injected with
I(lmo2:BR)I show markedly enhanced levels of GFP in
the intermediate cell mass (ICM), the normal location of primitive
hematopoiesis. Representative embryos are shown in
Fig. 4, and the data are
summarized in Table 1. In order
to quantify the phenotype and to determine if the enhanced levels of GFP are
due to increased transcript or protein levels or instead represent an increase
in numbers of Gata1+ cells, batches of injected embryos were
dissociated and GFP+ cells scored by FACS analysis. Embryos derived from
fertilized eggs injected with I(lmo2:BR)I show two-
to threefold more GFP+ cells compared with equal numbers of control uninjected
gata1:GFP embryos. This represents a conservative estimate of the
expansion of the erythroid population originating from lateral mesoderm when
BMP signaling is inhibited, as in this case GFP+ cells from the entire batch
of injected embryos was scored, rather than selecting the 30% expected to
generate the most significant phenotype. This result indicates that BMP
signaling normally restricts the number of Gata1+ cells that develop
from Lmo2+ lateral mesoderm progenitors.
|
). We extended our initial observations by characterizing
gene expression patterns by in situ hybridization for various blood and
vascular markers in both posterior and anterior regions. As expected from the
analysis of the gata1:gfp transgenic fish, the expression pattern of
the erythroid marker gata1 is expanded in the ICM of embryos injected
with I(lmo2:BR)I compared with controls
(Fig. 5A-C). The expression
pattern at the 12-somite stage is expanded laterally in the characteristic
bilateral stripes, but also towards the most caudal region of the embryos,
forming a complete loop. This loop pattern is never observed in the uninjected
or control injected embryos. However, changes in gene expression were not
limited to the posterior domain or to erythroid cells. In addition, the
expression patterns for the macrophage marker l-plastin
(Fig. 5D,E) and the granulocyte
marker mpo (Fig. 5F,G)
were each strikingly enhanced and expanded in the transient transgenic embryos
with an observed frequency between 20-30% (summarized in
Table 1). Other sets of control
embryos that were injected with a I(lmo2:cre)I transgene displayed
normal patterns of gene expression, showing that the phenotypes are dependent
on expression of the mutant BMP receptor (as shown, for example, in
Fig. 5C).
The vascular endothelial cell marker flk1 is expressed in both
anterior and posterior lateral mesoderm and blocking BMP signaling expands the
pattern in both domains, although more markedly in tail and trunk regions
(Fig. 5H,I). The effect on
vascular endothelium was further investigated by injecting the
I(lmo2:BR)I transgene into embryos transgenic for the
fli1:gfp reporter. Fli1 is a member of the ets family of
transcription factors that is expressed throughout lateral mesoderm during
early development, and exclusively in vascular endothelium as lateral mesoderm
differentiates (Isogai et al.,
2003). We find that inhibiting BMP signaling in Lmo2+ lateral
mesoderm results in dilation of major vessels by 1.5 days post-fertilization
(Fig. 5J,K). We conclude that
blocking BMP signaling specifically in lateral mesoderm results in the
expansion of markers representing both hematopoietic and vascular
lineages.
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|
). When analyzed by in situ hybridization at the 10- to
12-somite stage, the expression patterns for lmo2 and scl
are both expanded in embryos injected with the
I(lmo2:BR)I transgene
(Fig. 6A-D). This is in
contrast to the initial pattern of scl earlier at the one-somite
stage (Fig. 3). Expansion of a
hemangioblast population is not expected to occur using a block that is more
specific for the hematopoietic lineage. To test this, we analyzed embryos that
had been injected instead with the I(gata1:BR)I
transgene. In this case the mutant receptor is expressed in the committed
hematopoietic progenitors localized in lateral mesoderm at the five-somite
stage, compared with when the lmo2 promoter is used to express the
mutant receptor (throughout lateral mesoderm) starting around the one-somite
stage. In this case we found that gata1 expression was still enhanced
indicating that even in the Gata1+ cells the BMP signaling pathway is
functioning to restrict hematopoiesis (Fig.
6E,F). However, in this case, the expansion of the endothelial
marker flk1 does not occur, as we see no consistent difference in the
flk1 expression pattern comparing the transgenic and control embryos
(Fig. 6G,H), Therefore, the
results are consistent with a function for the BMP signaling pathway to
restrict the expansion of both blood and vascular lineages in lateral mesoderm
from the earliest stages, including potentially through regulation of the
common progenitor, the hemangioblast. However, the effect could just as well
occur independently in both committed vascular and hematopoietic progenitors,
as suggested by the fact that responsiveness to BMP signaling is still
maintained in a subset of progenitor cells from which erythroid cells
differentiate in lateral mesoderm.
|
). For
this analysis we analyzed fully the numbers of associated GFP+ cells from six
chimeras each, choosing those that had incorporated approximately equal
numbers of donor cells in the lateral mesoderm. Representative results for one
test and control embryo are shown in Fig.
7, and the data from the full analysis is tabulated in
Table 2. The data show that
cells within lateral mesoderm that express exogenous Bmp4 are surrounded by a
significantly fewer number of GFP+ cells (on average 65%), compared with
control transplanted cells that do not express exogenous Bmp4
(P<0.002). The data are fully consistent with the interpretation
based on the effect of expressing the mutant receptor, and indicate that BMP
signaling restricts the number of hematopoietic cells that develop from
progenitors in the lateral mesoderm.
|
|
).
Therefore we used the I-SceI-mediated approach to generate transgenic
embryos that express caBR in Lmo2+ lateral mesoderm. As shown in
Fig. 8, embryos injected with
the I(lmo2:caBR)I transgene develop a phenotype that is precisely the
opposite of embryos injected with the I(lmo2:BR)I
transgene. In this case, there is a relative block of expression of
hematopoietic markers, including gata1 and scl (data also
summarized in Table 1).
Therefore, the balance of BMP signaling in lateral mesoderm is necessary for
normal hematopoietic development, such that repressed or hyperactive signaling
leads to subsequent enhancement or restriction, respectively, in subsequent
development of hematopoietic progenitors.
|
BR)I transgene for expression of the
pronephric marker pax2.1. The pax2.1 expression domain
overlaps with that for lmo2 and is immediately more ventral to the
expression domain for scl in lateral mesoderm (note that in many species this
is usually called `intermediate mesoderm' and lies medial to the lateral
hemato-vascular mesoderm). Transient transgenic embryos were analyzed at the
12 somite stage for pax2.1 transcripts by in situ hybridization. In
contrast to the results obtained for hemato-vascular markers, pax2.1
expression levels are reduced by forced expression in lateral mesoderm of the
mutant receptor, again observed most clearly with the expected frequency of
30% of the embryos (Fig.
9A,B; Table 1). To
confirm that BMP signaling actively promotes pronephric development, we tested
the effect of expressing in lateral mesoderm the transgene that encodes a
constitutively active BMP receptor (caBR). Approximately 30% of the embryos
derived from eggs injected with the I(lmo2:caBR)I transgene display
an expansion of the pax2.1 expression domain
(Fig. 9C).
|
BR)I
transgene. As shown in representative examples
(Fig. 9D,E), transgenic embryos
that show an expansion of the signal for gata1, show a corresponding
decrease in signal for the pronephric marker pax2.1. Therefore, the
level of active BMP signaling in lateral mesoderm regulates the relative
numbers of progenitors that commit to either hemato-vascular or pronephric
fate. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Previous studies have addressed the temporal requirement for active BMP
signaling, particularly in the Xenopus model system
(Kikkawa et al., 2001;
Kumano et al., 1999;
Miyanaga et al., 1998;
Xu et al., 1999;
Zhang and Evans, 1996), and
have clearly shown that the pathway functions late relative to the initial
steps of mesoderm induction. By using a conditionally active form of Smad6, a
negative regulator of the pathway, we were able to confirm that even
post-gastrulation stages of mesoderm development require active signaling in
order for the subsequent development of the primitive erythroid blood island
(Schmerer and Evans, 2003).
However, each of the approaches used previously was limited to blocking BMP
signaling during the pre-commitment phase of hemato-vascular development, and
thus could not interrogate accurately any subsequent requirement after
progenitors are specified.
In order to probe functions for the pathway in the specified progenitors
for these specific lineages, it was necessary to generate a conditional
approach to block BMP signaling during somitogenesis within lateral mesoderm.
Our results demonstrate that BMP signaling functions in lateral mesoderm to
affect the decision of progenitors to commit either to a hemato-vascular fate,
or to a pronephric fate. Transgenesis facilitated by the I-SceI
meganuclease provides an effective approach that is limited only by the
availability of appropriate promoters. The lmo2 promoter was ideal
for our purpose as it is not expressed until around the one-somite stage, and
therefore genes expressed using this promoter will not interfere with
development prior to this point. However, the lmo2 gene is
subsequently activated throughout lateral mesoderm and so can be used to
regulate simultaneously gene expression in the progenitors for hematopoietic,
vascular and pronephric lineages. Our results indicate that BMP signaling does
continue to regulate development of lateral mesoderm within Lmo2+
cells and acts at this stage to restrict hemato-vascular development and
promote pronephric mesoderm. Although the increased numbers of hematopoietic
cells could be caused by changes in cell proliferation, immunohistochemistry
using anti-phospho-histone H3 antibodies failed to show any significant
increase in mitotic cell numbers in embryos expressing the mutant BMP receptor
(data not shown). Instead, the data support a model in which decreased levels
of BMP signaling within lateral mesoderm results in changes of lineage fate,
with increased numbers of hemato-vascular progenitors occurring at the expense
of pronephric progenitors. This interpretation relies on the assumption that
BMP ligands are normally expressed and active at these later stages of
development. Although the expression patterns for a number of potential
ligands are not fully described, there are clear candidates for the functional
signal, including Bmp2b [in ventral mesoderm
(Kishimoto et al., 1997)],
Bmp4 [in posterior epidermis, tailbud and lateral mesoderm
(Dick et al., 1999)] and,
perhaps most strikingly, Bmp6, which is expressed in the two stripes of
ventral mesendoderm during early stages leading into somitogenesis
(Thisse and Thisse, 2005).
The effects caused by manipulating BMP signaling could be mediated by the
direct regulation of key transcription factors such as scl. Much like
the mutant BMP receptor, the forced expression of scl expands both
blood and vascular lineages and at the same time is capable of suppressing the
development of pronephric mesoderm (Gering
et al., 2003). Moreover, expansion of the hematopoietic program
caused by co-injection of RNA encoding scl and lmo2 is
restricted to pronephric mesoderm, consistent with an inherent plasticity of
lateral mesoderm (Gering et al.,
2003). Enhanced expression of scl could also explain the
corresponding expansion in markers specific to differentiated blood and
vascular cells, including gata1 and flk1. In addition,
myelopoiesis that occurs in the anterior part of the lateral mesoderm is also
dependent on scl, as there is a strong reduction in
l-plastin expression in the scl morphant
(Dooley et al., 2005).
Depletion of BMP signaling in our experiments also occurs in anterior lateral
mesoderm and results in enhanced expression of both l-plastin and
myeloperoxidase, markers for macrophage and granulocytes,
respectively. The development of myeloid progenitors in the rostral domain has
previously been shown to be resistant to alterations in BMP signaling prior to
gastrulation, compared with the caudal erythroid domain
(Lieschke et al., 2002). By
contrast, the enhanced levels of the anterior hematopoietic markers caused by
inhibiting BMP signaling during somitogenesis is particularly robust. This
might be consistent with a lower level of endogenous signaling in the anterior
region, corresponding with a gradient of BMP signaling from high posterior to
low anterior, as proposed previously
(Lieschke et al., 2002), so
that the dominant-negative receptor most effectively decreases the levels
below a particular threshold in the rostral domain. As scl expression
is initially activated in ventral mesoderm by BMP signaling
(Maeno et al., 1996;
Zhang and Evans, 1996), if it
remains a relevant target gene during somitogenesis, our data suggest that in
defined progenitors the BMP pathway switches, instead, to downregulate or
restrict scl levels.
According to our results, cells in the lateral mesoderm respond to
different threshold levels of BMP signaling to determine cell fate. This is
consistent with results in the chick embryo obtained by exposing presomitic
regions with different levels of Bmp4
(Tonegawa et al., 1997).
Somites exposed to the highest levels of signal are transformed entirely to
lateral plate, whereas those cells exposed to a lower level express a distinct
lateral somatic program. Similarly, in ectoderm the expression of a
constitutively active BMP receptor is sufficient to convert in a
cell-autonomous matter progenitors from a neural to an epidermal fate
(Nikaido et al., 1999). With
respect to lateral mesoderm, the results are consistent with a threshold that
influences specified but uncommitted lateral mesoderm to distinguish
intermediate pronephric mesoderm, which below this threshold would otherwise
commit to a hemato-vascular fate. This is consistent with a gradient of BMP
signaling that is highest in the most lateral regions and lower at more medial
positions, being lowest in presomitic mesoderm owing to midline-derived
inhibitors. We note that the pax2.1 signal is never lost completely
in the transgenic embryos expressing the dominant-negative BMP receptor in
Lmo2+ cells, but this probably reflects the lack of a complete overlap in
lmo2 and pax2.1 expression, so that the most lateral
pronephric progenitors avoid the transgene effect.
Although the level of BMP signaling may act to specify lineage at the level
of the hemangioblast, for several reasons we suggest that it functions at a
later stage to restrict the development of committed endothelial and
hematopoietic cells. If the expression of lmo2 defines the
short-lived hemangioblast, then our experiments using the lmo2
promoter are by definition manipulating expression at subsequent stages. Using
the gata1 promoter we show that the pathway continues to restrict the
development of committed hematopoietic progenitors. It is perhaps relevant
that the analysis of the mouse Smad5 knockout
(Yang et al., 1999) showed a
twofold increase in the numbers of myeloid progenitors on the yolk sac
(although in this case erythroid progenitors were unchanged). The direct
application of Bmp2 or Bmp7 to a highly enriched population of stem cells
inhibits the proliferation of hematopoietic progenitors
(Bhatia et al., 1999). Thus,
although there is much evidence that BMPs support the development of
hematopoietic mesoderm (e.g. Li et al.,
2001), the pathway may then help to maintain a stem or early
progenitor state and restrict the emergence of more differentiated
hematopoietic cells. Although we did not test if the pathway is also
restrictive to the development of committed endothelial progenitors, there is
a large literature documenting both stimulatory and inhibitory effects of the
TGFß pathway on angiogenesis (Goumans
et al., 2003). Notably, violet beuregarde embryos
(vgb), carrying a mutation of the ALK1 (acvrl1)
gene, are characterized by vessel dilations caused by increased numbers of
endothelial cells (Roman et al.,
2002). Although ALK1 is a TGFß/activin receptor, it signals
via Smad1 and Smad5 (Goumans et al.,
2002), and therefore this pathway could be influenced by the
expression in angioblasts of the dominant-negative BMP receptor.
Recently, a distinct but related approach was developed to manipulate
conditionally BMP signaling in zebrafish, using a heat shock-inducible
promoter to express a dominant-negative receptor
(Pyati et al., 2005). This
approach was also successful at distinguishing specific roles for BMP
signaling subsequent to early patterning and into early stages somitogenesis.
Expression of the mutant receptor by heat-shock caused defects in ventral tail
fin development and tail organizer development. However, in this study,
specific alterations in hemato-vascular development were not noted, in
contrast to the results we present. The generation of phenotypes we describe
may be facilitated by the targeted expression of the mutant receptor to
lateral mesoderm, and perhaps more importantly by the sustained expression of
the mutant receptor using the lmo2 promoter, compared with the
relatively transient expression generated by a heat-shock. Combinations of
these two independent approaches for conditional gene expression should help
in dissecting the functions of important developmental signaling pathways in
specific lineages and at defined embryonic transition states.
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ACKNOWLEDGMENTS |
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