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First published online 7 January 2004
doi: 10.1242/dev.00948
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1 Dipartimento di Biologia, Universita' di Padova, Via U. Bassi 58/B, 35131
Padova, Italy
2 Department of Molecular Biology and Functional Genomics, Università
Vita Salute San Raffaele and San Raffaele Scientific Institute, Via Olgettina,
58, 20132 Milano, Italy
3 Department of Developmental Biology, University of Freiburg, Haupstrasse 1,
D-79104 Freiburg, Germany
* Author for correspondence (e-mail: francesco.argenton{at}unipd.it)
Accepted 27 October 2003
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Vertebrate, Zebrafish, Hox, Pbx, Meis, Prep, Rhombomere, Segmentation, Neural Crest, Pharingeal arch, Morpholino
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Moens and Prince, 2002).
Spatial-specific and temporal-specific gene expression is regulated by
mechanisms generated by combinatorial interactions of individual transcription
factors. The segmentation of the vertebrate hindbrain, for example, relies on
the rhombomere-specific expression of a subset of Hox genes.
Hox gene products, in turn, gain high specificity for DNA target
sequences by interacting with Pbc (Pbx in vertebrates) members of the TALE
family of homeodomain proteins (Burglin,
1997; Kamps et al.,
1990; Nourse et al.,
1990). Four Pbx genes have been identified in mammals and
five in zebrafish (Mann and Chan,
1996; Moens and Prince,
2002; Waskiewicz et al.,
2002).
A further subfamily of homeodomain transcription factors, which are also
members of the TALE family, is involved in the Hox regulation machinery. These
are the Meinox proteins (Burglin,
1997). In vertebrates, the Meinox subfamily include Meis and Prep
proteins, whereas a single member occurs in Drosophila (Hth) and
Caenorhabditis (UNC-62) (Van
Auken et al., 2002). The interaction between Meinox and Pbc
proteins leads to the nuclear import of the former, which lack a nuclear
localization signal, and prevents the nuclear export of the latter
(Abu-Shaar et al., 1999;
Berthelsen et al., 1999).
Because Pbc uses different surfaces to interact with Hox and Meinox proteins,
Pbc-Hox and Meinox-Pbc molecular interactions direct the formation of
Meinox-Pbc-Hox trimers. Such trimeric complexes recognize a split, 16-bp
sequence on the regulatory regions of target genes
(Ferretti et al., 2000;
Jacobs et al., 1999;
Ryoo et al., 1999). Moreover,
Meinox proteins are important in stabilizing Pbc members. For example, Hth is
required to maintain the level of the Pbc protein Exd in D.
melanogaster (Kurant et al.,
2001) and overexpression of dominant negative Meis derivatives
reduce the level of Pbx proteins in higher vertebrates and zebrafish
(Capdevila and Belmonte, 1999;
Choe et al., 2002;
Mercader et al., 1999;
Waskiewicz et al., 2001),
whereas overexpression of Prep1 in mammalian cells increases the level of Pbx
proteins (Longobardi and Blasi,
2003).
A total of 14 hox paralogs (11 in the mouse), 2 pbx, 3
meis and 2 prep genes are expressed in zebrafish hindbrain
(Moens and Prince, 2002). The
inactivation of pbx4/lazarus (lzr) in zebrafish leads to a phenotype
that is characterized by embryonic death at 6-7 days post-fertilization (dpf)
with major developmental defects, in particular in hindbrain segmentation and
cranial neural crest determination
(Pöpperl et al., 2000).
This phenotype closely resembles that of the mouse Pbx1-null mutant
(Selleri et al., 2001)
because, in both cases, major developmental defects are observed in the
cartilages arising from the second branchial arch. In zebrafish, elimination
of hindbrain-expressed Pbx proteins (Pbx2 and Pbx4) uncovers a hindbrain
ground state in which rhombomeres 2 to 6 (r2-r6) acquire an r1 identity
(Waskiewicz et al., 2002). In
Xenopus, zebrafish and chicken, the role of Meis proteins has been
investigated by overexpression and dominant-negative mutant approaches
(Dibner et al., 2001;
Mercader et al., 1999;
Salzberg et al., 1999;
Vlachakis et al., 2001;
Waskiewicz et al., 2001).
However, the specific role of individual Meis or Prep proteins is unknown.
Prep1 was identified as a protein that copurifies with several Pbx proteins
in human cells, and Prep2 was identified from a search of the human genome
sequence and cloned subsequently
(Berthelsen et al., 1998a;
Berthelsen et al., 1998b;
Fognani et al., 2002;
Haller et al., 2002).
Together, they form a subgroup of Meinox proteins that share 
80% overall
amino acid sequence identity. By contrast, the Meis and Prep proteins share
high amino acid sequence conservation only in specific domains
(Fognani et al., 2002). An
additional difference between Prep and Meis might lie in Hox proteins binding
activity. Vertebrate Meis associates in vitro with Hox9-Hox13, which increases
the DNA-binding specificity of the Meis-Hox complex
(Shen et al., 1997); no such
properties appear to be present in Prep1
(Thorsteinsdottir et al.,
2001). Whereas prep1.1 and prep1.2 are expressed
almost ubiquitously in zebrafish, at least in early developmental stages up to
24 hpf (Choe et al., 2002),
the expression of meis genes is more restricted
(Biemar et al., 2001;
Waskiewicz et al., 2001).
However, both Meis and Prep bind to Pbx proteins, need Pbx for nuclear
localization and prevent export of Pbx from the nucleus.
In the present investigation we have studied the functional role of the prep1.1 gene during early zebrafish development using antisense morpholino technology. We demonstrate that prep1.1 is necessary for histogenesis of the pharyngeal skeleton and, interacting with Pbc, is crucial for hindbrain patterning. In addition, our results support the idea that segmentation of the hindbrain and pharyngeal arches are independent processes.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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RNA constructs and microinjections
To create the prep1.1-deletion constructs
(Fig. 2A), the full-length
prep1.1 cDNA (RZPD clone MPMGp609C2025Q8) was dissected by PCR
amplification. The primers used to prepare prep1.1
HR1-2 cDNA, in which HR1-2 (Meis family Homology Regions 1 and
2) are deleted, were:
The primers used to amplify prep1.1 HD cDNA, in
which the homedomain (HD) is deleted, were:
The primer used to prepare full coding prep1.l cDNA, which lack 5' and 3' non-coding ends, were:
In each case, BamHI and BglII sites are underlined. Constructs were subcloned in BamHI sites of pCS2+ and pCS2+GFP plasmid vectors and sequenced. For microinjection of mRNAs, plasmids containing coding sequences were linearized, and sense-strand capped mRNAs synthesized using SP6-dependent mMessage mMachine kit (Ambion). Subsequently, mRNAs were purified, tested by agarose-gel electrophoresis, diluted in PBS and microinjected into fertilized embryos at the one-cell stage. The amount of mRNA injected was determined by measuring the diameter of the drop injected. To confirm that mRNA cause no nonspecific effects during embryogenesis, control embryos were also injected with an mRNA encoding green fluorescent protein (GFP).
Morpholino antisense oligonucleotides were obtained from Gene Tools. The sequences of morpholinos used were as follows:
The pbx2-MO has been described
(Waskiewicz et al., 2002). The
stock solution was diluted to working concentrations of 0.5-3.0 mg
ml–1 in Danieau solution [58 mM NaCl, 0.7 mM KCl, 0.4 mM
MgSO4, 0.6 mM Ca(NO3)2, 5 mM HEPES pH 7.6],
before injection into the yolk of embryos at the one-cell stage. For the
rescue experiment, prep1.1-MOb was co-injected with synthetic
prep1.1 mRNA. To test the capacity of prep1.1-MOa to block
translation, zebrafish eggs were injected with 25 pg of prep1.1-GFP
mRNA and 2 ng of prep1.1-MOa (directed against the start codon and
fluorescein tagged at the 3' end). The embryos were then fixed at 80%
epiboly and GFP expression tested using anti-GFP antiserum (Biocat). Lack of
staining in embryos co-injected with prep1.1-GFP and
prep1.1-MOa, confirmed the specific targeting.
RT-PCR
Total RNA was extracted from 100 frozen embryos at each developmental
stage and from dissected ovaries using TRIzol (Gibco), purified with DNaseI
and quantified by agarose-gel electrophoresis. mRNA was then retrotranscribed
and amplified with the Access RT-PCR System kit (Promega) using oligos
specific for either prep1.1 (5'-CTCTTTTCCCTCTCCTGGCT-3'
and 5'-ATGAATCCTCAGCAGCTGGA-3'), which gave a 580-bp cDNA product,
or ß-actin (5'-TGTTTTCCCCTCCATTGTTGG-3' and
5'-TTCTCCTTGATGTCACGGAC-3'), which resulted in a 560-bp cDNA
product.
Cell extracts and immunoblotting
After dissection, 100 zebrafish embryos were resuspended in 60 µl lysis
buffer (10 mM HEPES pH 7.9, 30 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.5
mM PMSF, 1 mM Na2S2O5), kept in ice for 10
minutes and lysed with TritonX-100 to a final concentration of 0.1%. The
nuclei were washed and the cell debris collected by centrifugation. The
supernatant was removed into a new tube, added to 0.11 vol of 0.3 mM HEPES pH
7.9, 1.5 mM MgCl2 and centrifuged. The resulting supernatant
corresponds to the cytoplasmic extract (CE). Nuclear extract (NE) was prepared
by resuspending the nuclear pellet in 30 µl of 20 mM HEPES pH 7.9, 25%
glycerol (v/v), 0.42 M NaCl, 1.5 mM MgCl2, 1 mM DTT, 0.5 mM PMSF, 1
mM Na2S2O5, and incubating at 4°C with
shaking for 30 minutes. The extracts were cleared by centrifugation. Extracts
(30 µg of NE and 60 µg of CE) were separated by 10% SDS-PAGE and blotted
to PVDF membrane (Millipore).
Immunoblotting analysis was performed with the anti pan-Pbx antibodies
(1:5000) (Pöpperl et al.,
2000), kindly provided by H. Pöpperl. The final detection
utilized the Dura chemoluminescent kit (Pierce).
Immunohistochemistry and histology
Cartilage staining
Larvae were fixed overnight in 4% buffered p-formaldehyde, rinsed
in distilled water and stained overnight in a 0.1% Alcian blue solution.
Larvae were then cleared by washing sequentially in 3% hydrogen peroxide and
70% glycerol, and whole mounted.
Whole-mount in situ hybridization
Embryos were fixed in 4% buffered p-formaldehyde. RNA in situ
hybridizations were performed essentially as reported in
(Thisse et al., 1993).
Digoxigenin and fluorescein-labelled antisense probes were synthesized from
cDNAs of prep1.1 (RZPD clone MPMGp609C2025Q8), krox20
(Oxtoby and Jowett, 1993),
pax6.1 (Puschel et al.,
1992), pax2.1 (Krauss
et al., 1991), islet1
(Korzh et al., 1993),
snail1 (Thisse et al.,
1993), foxb1.2/mariposa
(Moens et al., 1996),
hoxb1b and hoxb1a
(McClintock et al., 2001),
hoxa2 and hoxb2 (Prince
et al., 1998), dlx2
(Akimenko et al., 1994),
col2a1 (Sachdev et al.,
2001) and myoD
(Weinberg et al., 1996).
Results of the hybridizations were analyzed statistically using Chi-square
analysis.
Immunohistochemistry
Antibody staining of whole-mounted embryos with acetylated tubulin, RMO-44
and Zn5, was performed essentially as described by
(Abdelilah et al., 1996;
Piotrowski and Nüsslein-Volhard,
2000; Waskiewicz et al.,
2001), respectively.
Detection of apoptotic cell death
For a preliminary analysis of cell death, embryos were stained with the
vital dye acridine orange (acridinium chloride hemi-zinc chloride; Sigma)
(Abrams, 1999). Embryos were
incubated for 10-15 minutes in 5 µg ml–1 acridine orange,
washed two times for 5 minutes in Fish Water (60 mg l–1;
Instant Ocean) and observed under a microscope (Leica MZFLIII) using a green
filter set to reveal labelled cells undergoing cell death. For further
analysis, apoptosis was detected by terminal transferase dUTP nick-end
labelling (TUNEL), as previously described
(Williams et al., 2000).
Retrograde labelling
RSNs were revealed by retrograde labelling from the spinal cord of 3- and
5-day-old larvae, as described (Alexandre
et al., 1996). Labelled brains were dissected free of surrounding
larval tissues, mounted in 50% glycerol in PBS and visualized by
epifluorescence with a compound microscope (Leica Diaplan) using a Texas-red
filter.
Image acquisition and elaboration
Subcellular dynamics of Prep1.1-GFP constructs were visualized, acquired
and elaborated with the Bio-Rad Radiance 2000 confocal system. All other
pictures were acquired from microscope phototubes using a Leica DC500
photocamera and processed with Adobe Photoshop software.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Waskiewicz et al., 2001). Our
RT-PCR analysis shows that prep1.1 cDNA could be amplified from total
RNA of mature ovaries and embryos at all stages studied, from 2 cells to
larval day 4 (Fig. 1B), which
confirms the occurrence of maternally expressed prep1.1 mRNA in
zebrafish embryos. Transcripts of prep1.1, revealed with an
anti-sense probe, were distributed ubiquitously in the embryo up to 24 hpf
(Fig. 1C-G), whereas no
labelling was detected with a sense probe (not shown). However staining in the
trunk and tail, was already weaker at 24 hpf, markedly reduced at 48 hpf
(Fig. 1H) and had completely
disappeared by 72 hpf (Fig.
1I). At 72 hpf, labelling was limited to the head, predominantly
in the brain and otic vesicles, the latter representing the posteriormost
boundary of prep1.1 expression. Hence, prep1.1 transcripts
were initially spread throughout the embryo, but became restricted to the head
in later developmental stages.
Prep1.1 may be translocated to the nucleus from gastrulation onwards in the whole embryo
Prep1.1 is a cofactor in transcriptional regulation, so its presence in the
nucleus is a prerequisite for its activity. To determine the timing and
regulation of the nuclear localization of Prep1.1, we injected fertilized eggs
with 10 pg of prep1.1-GFP mRNA, a dose that did not alter the
phenotype (Table 1) but allowed
the subcellular localization of the fluorescent protein to be tracked directly
in live embryos. We found that Prep1.1-GFP localized in the cytoplasm at early
developmental stages and that its nuclear import began during the blastula
period. In particular, the fluorescent protein was exclusively cytoplasmic
during the high stage (not shown) but soon after, during the sphere stage, was
traced both to the cytoplasm and nucleus
(Fig. 2B). Then, from 30% to
50% epiboly, Prep1.1-GFP became predominantly nuclear
(Fig. 2C) and, at the beginning
of gastrulation, was almost exclusively localized to the nucleus
(Fig. 2D). An identical pattern
was observed after injection of prep1.1HD-GFP mRNA
(Fig. 2I), which encodes a
GFP-linked derivative of Prep1.1 that lacks the homeodomain
(Fig. 2A). Conversely,
injection of prep1.1HR-GFP mRNA, which encodes a
GFP-linked Prep1.1 derivative that lacks HR1 and HR2 Pbx-binding regions,
resulted in the restriction of the fluorescent product to the cytoplasm
(Fig. 2J). Incidentally, this
also excluded the possibility that the nuclear localization of Prep1.1-GFP was
influenced by the GFP moiety. On the basis of these results we conclude that
(1) the mechanisms required for the nuclear translocation of Prep1.1 are fully
effective at the onset of gastrulation in the whole embryo, and (2) as for
other Meinox proteins, the Pbx-binding region, but not the homeodomain, is
required for its nuclear import.
|
;
Berthelsen et al., 1999).
Pbx4/Lzr is the Pbx member with higher levels of expression during zebrafish
embryogenesis (see Fig. 2K)
(Waskiewicz et al., 2002),
indicating that it might be the most important partner of Prep1.1 in early
development. To determine whether the two proteins interacted during
embryogenesis, we examined the subcellular localization of Prep1.1-GFP in live
embryos in which pbx4 expression was repressed using a pbx4
morpholino. The fact that the phenotype of nearly all embryos injected with 2
ng of pbx4-MO at the one-cell stage was altered, proved the
effectiveness of the morpholino treatment
(Table 1). The specificity of
the effects produced by the pbx4-MO was demonstrated by the
observation that pbx4 morphants displayed morphological anomalies
(Fig. 3I) and impaired
expression patterns of molecular markers (see below), identical to those
observed in the lzr mutant
(Pöpperl et al., 2000).
We found that in embryos that received 2-6 ng of pbx4-MO and 10 pg of
prep1.1-GFP mRNA, the fluorescent protein was translocated to the
nucleus with the same temporal pattern in both pbx4-MO-treated
embryos and untreated siblings (Fig.
2E-G). But, importantly, in all pbx4 morphants a
significant amount of Prep1.1-GFP was traced to the cytoplasm in stages when
it was restricted mostly to the nucleus in the controls
(Fig. 2D,G). This provided
evidence that Pbx4 is involved in the nuclear transport of Prep1.1. Moreover,
the nuclear signal was not abolished in pbx2/pbx4 double morphants
(not shown), suggesting that maternal Pbx4, which is still present at 10 hpf
in pbx4/lzr homozygous mutants
(Waskiewicz et al., 2002), was
responsible of the residual nuclear import of Prep1.1-GFP.
|
;
Berthelsen et al., 1999) and
the stability of their Exd/Pbx partners
(Kurant et al., 2001;
Waskiewicz et al., 2001;
Longobardi and Blasi, 2003).
Because this indicates that Prep1.1 interacts with Pbx4 and, possibly, other
Pbx proteins, we asked whether Prep1.1 might affect the levels and
localization of Pbx proteins. To this purpose, we performed an immunoblot
analysis, using a pan-Pbx antibody
(Pöpperl et al., 2000),
to test cytoplasmic and nuclear extracts of wild-type and
prep1.1-MO-treated embryos at 24 hpf. As a control, we employed human
Pbx1a and Pbx1b cotranslated in vitro with human Prep1. The pan-Pbx antibodies
identified the 47x103 and 39x103
Mr bands expected for the two Pbx proteins
(Fig. 2K). Moreover, the same
two bands were also identified in extracts from human HEK cells, whereas a
species of 38x103 Mr was revealed in
an extract from mouse testis (Wagner et
al., 2001). When tested with the zebrafish extracts, the pan-Pbx
antiserum revealed two major bands with mobilities that matched the
electrophoretic migration of in vitro translated mouse Pbx1b
(38.5x103 Mr) and Pbx1a
(46.5x103 Mr). This pattern is identical
to that obtained by Waskiewicz et al.
(Waskiewicz et al., 2002) in
identifying Pbx2 and Pbx4. Both species were more abundant in NEs than CEs
and, importantly, all the bands were much more intense in extracts from
wild-type embryos than prep1.1 morphants. As the assay was
standardized by loading an equal amount of protein and by Ponceau staining of
the gel (not shown), it appears that suppression of prep1.1
expression caused a significant decrease of the concentration of Pbx2 and Pbx4
in both nucleus and cytoplasm. This is consistent with the possibility that
Prep1.1 stabilizes and increases the nuclear localization of different Pbx
proteins.
Effects of prep1.1 mRNA inactivation and overexpression
It has been shown that appropriate amounts of antisense morpholinos
injected into zebrafish embryos at the one-cell stage might repress the
expression of both maternal and zygotic genes
(Nasevicius and Ekker, 2000).
Hence, as the first step in investigating the functional role of
prep1.1 during early embryonic development we adopted the morpholino
approach to examine the phenotypic effects induced by prep1.1
inactivation. We used two different morpholinos, complementary to either the
initial 25 bp of the translated region (MOa) or a 25-bp sequence from the
5'-untranslated region (MOb) of prep1.1 mRNA
(Fig. 2A). Embryos that
received either prep1.1-MOs developed normally, both morphologically
and temporally, up to early somitogenesis. However, at the 15-somite stage
(16.5 hpf), cell death was apparent in the form of an opacity in the
neuroectoderm. The effects of the two morpholinos were indistinguishable,
dose-dependent and synergistic: 2-3 ng of either morpholino injected
separately or 0.5 ng of each injected in combination, modified the phenotype
of virtually all embryos treated (Table
1). One-day old morphants were characterized by a prominent area
of degeneration, which was clearly visible bilaterally both inside and outside
the CNS at the level of the hindbrain (Fig.
3B,C,E,F). Morphant embryos were impaired in motor coordination,
so that most were unable to exit from the shell. At 5 dpf, the morphant
embryos had small heads and eyes, and atrophic pectoral fins; they lacked jaws
and a recognizable swim bladder and displayed an abnormal distribution of
melanocytes and pericardial edema (Fig.
3H). The morphant embryos did not survive after 6-7 dpf.
We next investigated the effects of Prep1.1 overexpression by injecting
one-cell stage embryos with either 50 pg or 100 pg of prep1.1 mRNA.
Embryos injected with either dose developed normally beyond gastrulation, but
the phenotype of >50% of the treated embryos was markedly affected at 24
hpf (Table 1). Injection of 50
pg prep1.1 mRNA induced two main phenotypes in 50% of embryos,
both characterized by a shortening of the head, as evidenced by a decreased
distance between the eye and the otic vesicle
(Fig. 3R). Most of these
embryos had a phenotype in which the eyes were closer one to another but still
distinct, whereas a few exhibited a more severe phenotype that was
characterized by a single cyclopic eye. In situ hybridization with a
krox20 antisense probe showed that the hindbrain was unaffected
(Fig. 3T) but a pax6.1
probe revealed a strong reduction of the forebrain
(Fig. 3V). Similar effects were
observed in embryos treated with 50 pg of
prep1.1HD-GFP mRNA, encoding a GFP-linked Prep1.1
derivative lacking the homeodomain (Table
1), but in this case most embryos were cyclopic. This indicates
that deletion of the homeodomain does not impair but rather increases the
activity of Prep1.1, reminiscent of the finding that deletion of the PREP1
homeodomain might enhance the transcriptional activity of mammalian
PBX1-HOXB1-PREP1 complexes (Berthelsen et
al., 1998b). Moreover, embryos that received 100 pg of
prep1.1 mRNA, displayed a even more extreme phenotype, characterized
by the absence of eyes and a marked reduction of all head structures.
Conversely, overexpression of prep1.1HR-GFP mRNA,
which encodes a GFP-linked Prep1.1 derivative that lacks the HR1 and HR2
regions, had no phenotypic consequences
(Table 1). This indicates that,
as with other members of the Meinox family
(Choe et al., 2002), the
Pbx-interacting domain is crucial to Prep1.1 activity. In summary, the results
of the overexpression of prep1.1 in zebrafish closely resemble those
obtained in Xenopus in which the overexpression of meis3
causes caudalization of anterior neural tissue
(Salzberg et al., 1999;
Dibner et al., 2001).
To verify the specificity of the morpholinos, we checked whether the mutant phenotype was rescued by coinjecting MOb with a prep1.1-expressing mRNA. To this purpose, we used an mRNA (prep1.1-GFP mRNA) that encodes wild-type Prep1.1 linked to GFP, so that expression of the chimeric protein could be ascertained visually in the embryos. This experiment could be devised for two reasons: first, when overexpressed in zebrafish embryos, Prep1.1-GFP and Prep1.1 produced qualitatively and quantitatively identical effects (Table 1), indicating that the activity of the chimeric protein was not significantly affected by the GFP moiety; second, because the prep1.1-MOb was complementary to a sequence of the 5' UTR, it blocked the endogenous prep1.1 mRNA but not the microinjected synthetic prep1.1-GFP mRNA (Fig. 2A). As shown (Table 1), The number of embryos displaying the wild-type phenotype increased from <2% of those injected with 2 ng of prep1.1-MOb alone to >54% of those that received in addition 50 pg of prep1.1-GFP mRNA, thereby demonstrating that Prep1.1-GFP was effective in rescuing the mutant phenotype. Thus, it appears that the prep1.1-MOs used in the present investigation inhibited specifically the expression of prep1.1.
Neural degeneration in prep1.1 morphants is caused by apoptosis
Visual inspection of prep1.1 morphants revealed a pronounced
process of degeneration that started during early somitogenesis and peaked at
about 24-36 hpf. Staining with the vital dye acridine orange showed a major
increase in cell death in prep1.1 morphants. Although this occurred
principally in the CNS, in particular the hindbrain and spinal cord, it was
also significant in other tissues (Fig.
3K). By contrast, acridine orange staining was inconsistent in
wild-type embryos and pbx4 morphants
(Fig. 3J,L). To establish
whether cell death induced by prep1.1 inactivation was due to
apoptosis, the embryos were analyzed by in situ TUNEL assay to detect DNA
fragmentation. As shown in Fig.
3 (Fig. 3N,P) TUNEL
labelling was evident throughout the brain of prep1.1-MOb-treated
embryos, especially in the hindbrain, but was limited in the brains of
controls (Fig. 3M,O).
Therefore, we, conclude that the pronounced cell death induced by
prep1.1 inactivation was due primarily to apoptosis.
prep1.1 knockdown disrupts hindbrain segmentation
In zebrafish, Pbx proteins are essential for hindbrain development
(Waskiewicz et al., 2002), but
overexpression of wild-type and dominant-negative Meis proteins also support
the idea of an important role of Meinox proteins in this process
(Choe et al., 2002;
Vlachakis et al., 2001). As
both the morphological inspection and TUNEL analysis showed that the hindbrain
is significantly affected in prep1.1 morphants, we addressed the role
of prep1.1 in hindbrain development by analysing the effects of its
knockdown on the expression of several hindbrain markers. The results were
compared to those observed in mutants that lack Pbx proteins
(Pöpperl et al., 2000;
Waskiewicz et al., 2002) and
embryos treated with a pbx4 morpholino.
In zebrafish, hoxb1b is indispensable for normal segmentation of
the hindbrain (McClintock et al.,
2001), and the onset of its expression represents the initial step
of hindbrain patterning (Waskiewicz et
al., 2002). Hence, we first checked whether the expression of
hoxb1b was altered in prep1.1 morphants. We observed that
expression of this gene was indistinguishable in wild-type and
prep1.1-MOb-injected embryos (Fig.
4A,B,). Because expression of hoxb1b is also unaffected
by eliminating both pbx2 and pbx4
(Waskiewicz et al., 2002), it
appears that neither Prep1.1 nor Pbx proteins are involved in the regulation
of this gene.
|
;
Choe et al., 2002). As shown
in Fig. 4
(Fig. 4C,D,G,H),
prep1.1 knockdown caused the loss of the six clear-cut boundaries
(from r1-r2 to r6-r7), evidenced by the expression of these two genes in
wild-type embryos. By contrast, in pbx4 morphants
(Fig. 4I) pax6.1
expression revealed that hindbrain segmentation was indistinct rostrally but
remained clearly identifiable in the three caudal boundaries (from r4-r5 to
r6-r7). This agrees with the expression pattern of pax6.1 observed in
embryos that express ectopically a dominant negative derivative of
pbx4 (Choe et al.,
2002), and correlates with the fact that in mutants such as
lzr (Pöpperl et al.,
2000) and MZlzr (which lacks both maternal and zygotic
pbx4) (Waskiewicz et al.,
2002), mariposa expression is eliminated rostrally but
maintained in the three caudal boundaries. However, expression of
mariposa in the hindbrain was suppressed completely by injecting
MZlzr embryos with a pbx2 morpholino
(Waskiewicz et al., 2002).
Because suppression of mariposa expression can be caused by the
simultaneous depletion of Pbx2 and Pbx4, we asked whether, in our morphants,
this phenotype could be caused by prep1.1 knockdown-mediated
reduction of Pbx proteins. However, this was not the case, because injection
of pbx4 mRNA did not rescue mariposa expression in
prep1.1 morphants (26 morphants observed, 21 deficient, data not
shown).
Further evidence of impaired hindbrain segmentation in prep1.1
morphants was provided by the expression of pax2.1. In wild-type
embryos, pax2.1 is expressed in segmental clusters of commissural
interneurons, arranged in two longitudinal rows extending from the hindbrain
to the whole spinal cord (Jiang et al.,
1996; Mikkola et al.,
1992; Schier et al.,
1996). Injection of prep1.1-MOb caused the selective
disappearance of pax2.1-expressing cells in the region from r2 to r6
(Fig. 4E,F), whereas injection
of pbx4-MO did not affect the wild-type phenotype (data not
shown).
We next investigated the effects of prep1.1 knockdown on the
expression of a series of markers that specify rhombomere identity. In
wild-type embryos at 24 hpf, hoxb1a is expressed in r4,
krox20 is expressed in r3 and r5, hoxa2 is expressed highly
in r2 and r3 and at lower levels in r4 and r5, and hoxb2 is expressed
highly in r3 and r4 and at a lower level in r5
(Prince et al., 1998).
Injection of prep1.1-MOb suppressed the expression of hoxb1a
in r4 (Fig. 4J,K). Because
expression of hoxb1a, like that of mariposa, is suppressed
by depletion of both Pbx2 and Pbx4
(Waskiewicz et al., 2002), we
checked the effects of Pbx4 overexpression on hoxb1a pattern in
prep1.1 morphants. Although hoxb1a expression was rescued in
a significant number of embryos, it always remained at minimal levels
(Fig. 4T). Hence, it appears
that the hoxb1a phenotype of the morphants was caused by the combined
effects of prep1.1 knockdown and reduced Pbx protein concentration.
Nonetheless, the fact that hoxb1a expression was only minimally
rescued by pbx4 overexpression demonstrates that prep1.1 is
crucial for the regulation of hoxb1a.
In prep1.1 morphants, krox20 expression was abolished in
r3, but not in r5 (Fig. 4Q).
This effect was rescued by coinjection of prep1.1 mRNA plus
prep1.1-MOb (Fig. 4R),
which confirms the morpholino specificity. Disappearance of krox20
expression in r3 was also observed in lzr and MZlzr mutants
(Pöpperl et al., 2000;
Waskiewicz et al., 2002) and
in embryos injected with pbx4-MO
(Fig. 4S). However,
krox20 expression in r3 was not rescued by pbx4 mRNA
overexpression in prep1.1 morphants
(Fig. 4U), indicating that
prep1.1 and pbx4 are both crucial for expression of
krox20 in r3. By contrast, depletion of both pbx4 and
pbx2 suppressed krox20 expression in r3 and r5
(Waskiewicz et al., 2002). In
prep1.1-injected embryos, expression of both hoxa2 and
hoxb2 was reduced markedly along the whole expression domain
(Fig. 4L-O), which resembles
the phenotype of lzr mutants
(Pöpperl et al., 2000).
In MZlzr mutants, hoxa2 expression is similarly reduced, and
eliminated entirely following injection with a pbx2 morpholino
(Waskiewicz et al., 2002).
Thus, the expression of krox20 and hoxa2 appears to be more
susceptible to the elimination of the two pbx genes than to
prep1.1 knockdown.
prep1.1 knockdown affects the migration of facial nerve motor neurons and causes disappearance of all reticulo-spinal neurons except Mauthner cells
In vertebrates, the position of the motor nuclei of cranial nerves mirrors
the rhombomeric organization of the hindbrain
(Lumsden and Keynes, 1989).
Because hindbrain segmentation and expression of rhombomere-specific genes
were severely perturbed in prep1.1 morphants, we asked whether the
patterning and localization of these nuclei was also affected. We examined the
espression of islet1 (isl1), which is widely expressed in
early postmitotic neurons (Korzh et al.,
1993) and whose expression pattern in the motor nuclei of cranial
nerves has been described in detail in zebrafish embryos
(Chandrasekhar et al., 1997;
Higashijima et al., 2000). In
wild-type embryos at 48 hpf, isl1 was detected in all motor nuclei of
the hindbrain and ganglia of cranial nerves
(Fig. 5A,C). In
prep1.1 morphants (Fig.
5B,D), the number of neurons that expressed isl1 neurons
in cranial nerve nuclei was not changed significantly, but their distribution
was altered. In particular, the motor neurons of the facial nerve (nVII) were
not located in the region that corresponded to r6 and r7 as in controls
(Fig. 5A), but were spread in
an elongated nucleus extending from r4-r6
(Fig. 5A-D). In zebrafish, the
nVII motor neurons originate in r4 and migrate caudally to r6 and r7
(Higashijima et al., 2000).
Therefore, the presence of this aggregate indicates that their migration is
impaired in prep1.1 morphants. The altered migration of nVII motor
neurons to caudal regions of the hindbrain correlated well with the
downregulation of hoxb1a in r4. In fact, knockdown of hoxb1a
inhibits migration of nVII motor neurons
(McClintock et al., 2002;
Cooper et al., 2003). Further
evidence of abnormal neuronal organization in the hindbrain of
prep1.1-MOb-injected embryos was revealed with antiserum to
acetylated tubulin. As shown in Fig.
5K,L, neuropils and commissural tracts of the rhombomeric segments
were clearly distinguishable in the controls but barely detected in
prep1.1 morphants. By contrast, the ganglion and sensory root of the
trigeminal nerve (nV), were apparently unaffected by prep1.1
inactivation, confirming the results of isl1 expression.
|
). A striking feature of prep1.1 morphants at
both 3 and 5 dpf, is the disappearance of most retrogradely labelled RSNs
apart from a pair of large, bilateral neurons that project controlaterally.
These are identifiable as Mauthner cells that normally develop in r4. Given
that general defects of the CNS might lead to the impossibility of backfilling
RSNs, we also examined the presence and morphology of RSNs at 48 hpf using the
RMO-44 antibody. The results confirmed the retrograde labelling analysis and
showed that the projection of the RMO-labelled cells in prep1.1
morphants was identical to that of Mauthner neurons in wild-type embryos
(Fig. 5I,J). The results of
this experiment demonstrate that Prep1.1 is necessary for the development of
all RNSs except Mauthner cells.
Head cartilage defects in prep1.1 morphants
As described above, prep1.1 morphants lacked the jaw, indicating a
defective development of neural-crest derivatives. To analyze the cranial
skeletal defects induced by prep1.1 inactivation, we examined the
skull morphology using Alcian Blue staining. Five-day-old morphants lacked all
neural crest-derived cartilages of the pharyngeal arches
(Fig. 6C,D). The skull
consisted of the neurocranium only in which the ethmoid plate and trabeculae
cranii, which are thought to derive from the neural crest, were misshaped and
reduced significantly in size, whereas the mesodermally-derived elements were
affected much less. The phenotype induced by the prep1.1-MOb could be
substantially rescued by co-injection of prep1.1
(Fig. 6E,F) but not
pbx4 mRNA (data not shown). The occurrence of the cartilaginous
neurocranium indicates that the lack of pharyngeal cartilages was not the
consequence of a generalized block in the chondrogenic process, but the result
of either patterning or specification defects. In pbx4 morphants, the
pharyngeal skeleton displayed the same defects as in lzr mutants
(Pöpperl et al., 2000).
In such embryos (Fig. 6G,H),
all the branchial cartilages were missing, and the skeletal elements of the
mandibular and hyoid arches were present but improperly shaped and abnormally
fused.
|
). In wild-type embryos, dlx2 is expressed in three
distinct streams of cranial neural crest cells that migrate to the mandibular
(m), hyoid (h) and five branchial (b) arches
(Fig. 7A). dlx2 is
also expressed in post-migratory neural crest cells within the arches
(Fig. 7C). In embryos injected
with prep1.1-MOb, dlx2-positive cranial neural crest cells
behaved like those of wild-type embryos, gathering in three migrating streams
that eventually populated the pharyngeal arches
(Fig. 7B,D). The correct
pattern of neural crest cell migration in prep1.1 morphants, was
further confirmed with the snail1 marker
(Fig. 7E,F), which is expressed
in the head mesenchyme, in neural crest cells that give rise to the pharyngeal
skeleton and in paraxial mesoderm cells that originate muscle cells
(Thisse et al., 1993). The
fact that dlx2-positive and snail1-positive postmigratory
cells were distributed similarly in separate clusters in the pharyngeal region
in both prep1.1 morphants and wild-type embryos indicated that
pharyngeal segmentation occurred normally in the morphants
(Fig. 7C-F). Indeed, staining
with the Zn5 antibody, confirmed that formation of the endodermal pouches and
segmentation of the pharyngeal region took place correctly in prep1.1
morphants (Fig. 7G,H).
|
;
Yan et al., 2002). Therefore,
we examined whether the expression of col2a1 was altered in
postmigratory neural crest cells of prep1.1 morphants. We found that
col2a1 was expressed in the mesenchyme that will give origin to the
neurocranium in prep1.1 morphants, though to a lesser degree than in
wild-type embryos. However, it was not expressed in the pharyngeal arches
(Fig. 7I-L).
Neural crest cells are known to pattern the muscles of the pharyngeal
region (Noden, 1983). In
zebrafish, pharyngeal chondroblasts and myoblasts differentiate synchronously,
which indicates interdependence of their patterning
(Schilling and Kimmel, 1994).
In both chinless (chn)
(Schilling et al., 1996b) and
jellyfish (jef) (Yan et
al., 2002) mutants, neural crest cells migrate normally to the
pharyngeal region but fail to differentiate into chondrocytes. However, the
pharyngeal musculature is absent in chn mutants but develops normally
in jef mutants, which indicates that chondrogenesis is not a
prerequisite for the differentiation of the pharyngeal musculature. To
determine whether prep1.1 knockdown affected the pharyngeal
musculature we examined the expression of myoD, the early marker of
myogenesis, in prep1.1 morphants. At a stage in which all head
muscles of wild-type larvae express myoD, expression of myoD
in the head of the morphants was detected in the eye muscles and in few
lateral elements, seemingly opercular muscles, but not in the pharyngeal
region (Fig. 7M,N).
In zebrafish, there are two independent phases of expression of the
homeobox gene goosecoid (gsc), a gene that is required for
cranio-facial development in mammals
(Clouthier et al., 2000;
Zhu et al., 1997): an early
phase in cells anterior to the presumptive notochord; and a late phase at 2-3
dpf in the brain and in neural crest derivatives of the mandibular and hyoid
arches (Schulte-Merker et al.,
1994). In zebrafish, expression of gsc in mandibular and
hyoid arches is dependant on Hoxa2 function
(Hunter and Prince, 2002). To
assess whether gsc expression was affected by prep1.1
knockdown we used transgenic zebrafish that contain a GFP construct driven by
a gsc promoter, which express GFP in the embryonal brain and
mandibular cartilage (Doitsidou et al.,
2002). Knockdown of prep1.1 in transgenic larvae
suppressed GFP expression in the first pharyngeal arch but not in the anterior
telencephalic area (Fig. 7O,P).
This result shows that Prep1.1 activity is required for the expression of
gsc in the mandibular arch, which indicates that prep1.1 is
necessary for both arch patterning and pharyngeal chondrogenesis.
Hence, in prep1.1 morphants, cranial neural crest cells were present and migrated correctly to the pharyngeal region, which was properly segmented by the endodermal pouches but failed to undergo chondrogenesis. In addition, the muscles connected to the pharyngeal cartilages did not develop.
|
Discussion |
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|
TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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The maternal expression of prep1.1 and its ubiquitous distribution
in zebrafish embryos up to 24 hpf indicated its involvement in early
embryogenetic processes. This assumption was confirmed by our finding that
inactivation of prep1.1 induced prominent apoptosis, which becomes
clearly visible during somitogenesis. Accordingly, the time course of the
nuclear translocation of Prep1.1-GFP, demonstrated that the mechanisms
required for the nuclear import of Prep1.1, which is a prerequisite for its
activity, are fully functional by the end of gastrulation. Like other Meinox
proteins, Prep1.1 lacks a nuclear-localization signal and relies on Pbc
partners for translocation to the nucleus
(Berthelsen et al., 1999).
Because Pbx4 and Pbx2 are the main Pbc members that are expressed early in
zebrafish embryogenesis (Pöpperl et
al., 2000; Waskiewicz et al.,
2002), they appear to be the major Prep1.1 partners in early
zebrafish development. Indeed, this is supported by the observation that
Prep1.1-GFP remains mostly cytoplasmic in both pbx4 and
pbx2/pbx4 double morphants. Hoever, in such morphants, the finding
that some Prep1.1-GFP was associated with the nucleus, is consistent with the
occurrence of maternal Pbx4 in early developmental stages
(Waskiewicz et al., 2002).
As evidenced by immunoblotting analysis, Prep1.1 affects the levels of Pbx2
and Pbx4 and, possibly, of other Pbx members. The effect is probably
post-transcriptional and might be caused by a longer half-life of Pbx proteins
when they form dimers with either Prep and Meis. In fact, in
Drosophila, Exd is destabilized and degraded in the absence of Hth
(Kurant et al., 2001), and
lack of Prep1 in mice coincides with a strong, widespread reduction of the
levels of all Pbx proteins (E.F. and F.B., unpublished). Moreover,
overexpression of Prep1 in mouse teratocarcinoma cells increases the half-life
and, therefore, the level of Pbx proteins
(Longobardi and Blasi, 2003).
As simultaneous depletion of Pbx2 and Pbx4 also causes a series of molecular
and morphological effects that are similar to the phenotypes observed in
prep1.1 morphants (Waskiewicz et
al., 2002), such a phenotype might be due to the reverberating
effects of Prep1.1 suppression on the levels of Pbx proteins. However, this
was apparently not the case. In fact, the inability of pbx4/lzr mRNA
to rescue apoptosis, expression of mariposa in rhombomere boundaries,
expression of krox20 in r3 and branchial cartilage formation in
prep1.1 morphants, reveals a direct role of prep1.1 in
embryo development other than the mere stabilization of Pbx proteins.
Suppression of prep1.1 induces apoptosis
Meinox proteins control differentiation through a wide array of
interactions with different homeodomain transcription factors. Thus, the
apoptotic process observed in prep1.1 morphants might be caused by
the programmed cell death of cells that fail to differentiate
(Ishizaki et al., 1995). It is
noteworthy that the morphological phenotype of prep1.1 morphants
shares similarities with the spacehead class (group II) of zebrafish
mutants described by Abdelilah et al.
(Abdelilah et al., 1996). In
particular, the onset of apoptosis coincides temporally and spatially in
prep1.1 morphants and spacehead mutants. Interestingly, it
was proposed that the genes affected in spacehead mutants are
involved in either the differentiation or maintenance of neural cell types,
suggesting that cells that are unable to conclude their differentiation
process are fated to death by apoptosis
(Abdelilah et al., 1996). Links
between Meinox partners such as mouse Hoxa1 and Hoxb1 (which are the
orthologous of zebrafish Hoxb1b and Hoxb1a, respectively) and apoptosis have
been established recently (Barrow et al.,
2000; Lohmann et al.,
2002). Thus, the prominent apoptotic process observed in
prep1.1 morphants might be related to an impaired activity of Hox
proteins due to prep1.1 inactivation.
Suppression of prep1.1 affects the expression of genes crucial for hindbrain development
Our results show that prep1.1 morphants exhibit major changes of
gene expression patterns in the hindbrain. First, although expression of
hoxb1b, the orthologue of mouse Hoxa1, is not affected,
hoxb1a, the orthologue of mouse Hoxb1, which is normally
expressed in r4, is absent. Moreover, we show that the expression of
hoxa2 and hoxb2 is strongly reduced. Meinox proteins form
functionally active heterotrimeric complexes with Pbx and Hox and these
complexes are functionally important in vivo in the expression of at least
Hoxb2 in r4 and r6-r8 (Jacobs et
al., 1999; Ryoo et al.,
1999; Ferretti et al.,
2000). However, the Meinox protein involved has not been
identified. In zebrafish, hoxb1a expression in r4 requires
pbx2/4 and hoxb1b
(Cooper et al., 2003;
Waskiewicz et al., 2001;
Waskiewicz et al., 2002).
Because prep1.1 is required for full expression of hoxb1a in
r4, the expression of this gene might also require the formation of a
heterotrimeric complex between prep1.1, pbx2/4 and hoxb1b
gene products. Although the role of heterotrimeric complexes was not apparent
in initial investigations of Hoxb1 expression
(Jacobs et al., 1999;
Ferretti et al., 2000), we
have observed that, in mice, the r4 Hoxb1 enhancer extends slightly
more 3' than previously defined, and heterotrimeric Meinox-Pbc-Hox
complexes might be required for Hoxb1 expression (E. Ferretti, R.
Krumlauf and F. Blasi, in preparation).
In the mouse, expression of Hoxb2 depends on krox20 in r3
and r5, and on the heterotrimeric Meinox-Pbx-Hox complex in r4
(Jacobs et al., 1999;
Ryoo et al., 1999; Ferretti et
al., 2001). Here, we show that hoxb2 expression requires
prep1.1 in r2, r4 and r3. The effect observed in r3 is probably due
to the absence of krox20, whereas in r4 it might depend directly on
the lack of prep1.1 as well as the induced decrease of pbx4.
In conclusion, because the absence of prep1.1 results in the absence
of hoxb1a, prep1.1 inactivation causes the decrease/absence of all
factors that are required for activity of the hoxb2 enhancer Krox20
in r3 and the heterotrimeric complex in r4. The lack of expression of
krox20 in prep1.1 morphants is also reflected by the absence
of hoxa2 expression in r3. Conversely, in prep1.1 morphants
krox20 is expressed normally in r5, in which hoxa2 and
hoxb2 are absent. These results show unique properties of the Prep1.1
protein in the specification of r2-r5.
prep1.1 is crucial for hindbrain patterning
The vertebrate hindbrain exerts a key function in patterning the developing
head through its segmental rhombomeric structure and its ability to generate
neural crest cells. Rhombomeres direct the proper organization of cranial
ganglia, branchiomotor nerves and the migration of neural crest cells
(Trainor and Krumlauf, 2000).
Our results show that Prep1.1 is crucial for hindbrain segmentation. This is
illustrated in prep1.1 morphants by the loss of the segmental
expression patterns of mariposa and pax6.1 throughout the
hindbrain, and the absence of pax2.1-positive commissural
interneurons in the r2-r6 region. Unlike Prep1.1, Pbx4 is necessary for
hindbrain segmentation only anteriorly to the r4-r5 boundary. In fact, in our
pbx4 morphants, and in embryos expressing a dominant negative
derivative of pbx4 (Choe et al.,
2002), pax6.1 segmentation is lost anteriorly but not
posteriorly to the r4-r5 boundary. This is in accord with the loss of
rhombomere segmentation anterior to the r4-r5 boundary in lzr mutants
revealed by mariposa expression
(Pöpperl et al.,
2000).
On the basis of the effects of prep1.1, pbx4 and pbx2
inactivation on hindbrain patterning (Fig.
4) (Waskiewicz et al.,
2002), and of the deficiencies in hox gene expression in
the hindbrain (Fig. 4), it can
be deduced that Prep1.1 regulates the process of rhombomeric segmentation and
specification by acting with Pbx4 rostral to the r4-r5 boundary, and with Pbx4
and Pbx2 caudal to that boundary.
Another striking effect of prep1.1 inactivation on hindbrain
development is the lack of RSNs except Mauthner cells. This feature indicates
that Prep1.1 is necessary for early differentiation and/or survival of most
RSNs. By contrast, pbx4 would be required subsequently for the
acquisition of the identity of RSNs, as shown in lzr mutants in which
all RSNs located posteriorly to r2 display r2 identity
(Pöpperl et al., 2000).
Although r4 identity is disrupted in prep1.1 morphants, as evidenced
by the absence of hoxb1a expression, the occurrence of r4-specific
Mauthner cells is not incongruous. In fact, Mauthner cells appear normally at
7.5 hpf, so their differentiation is independent of hoxb1a, whose
expression starts 2 hours later
(Prince et al., 1998
