Department of Biology, University of Rochester, Rochester, NY 14627, USA
Author for correspondence (e-mail:
ddke{at}mail.rochester.edu)
Accepted 29 December 2004
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SUMMARY |
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Key words: Epiboly, Radial intercalation, E-cadherin, Epiblast, Teleost, Morphogenesis, Genetics, Antisense, Zebrafish
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Introduction |
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Additionally, three of the five epiboly mutants display dominant
phenotypes. The mutants habdtv43, avalanchetm94
and lawinets18 display a zygotic-maternal dominant (ZMD)
effect that is expressed when both zygotic and maternal genomes are
heterozygous for the mutant locus. This phenotype is displayed as an
intermediate rate of epiboly between that of wild-type and homozygous mutant
siblings. These embryos, termed ZMD mutants, complete epiboly about an hour
after wild-type siblings, and later, during somitogenesis, cells dorsal to the
developing neural tube round up and detach from the embryo
(Kane et al., 1996). Also,
habdtv43 mutants display a semi-dominant trait of an
enlarged hatching gland.
Although the epiboly mutants failed to complement one another, they were
named separately because complementation testing was complicated by the
dominant effects. In recent work, submitted elsewhere, we have shown that all
the mutants (with the exception of vol) map to a single locus near
the centromere of Linkage Group 7, and the locus is provisionally named the
hab locus. Here we establish that all of the mutations at the
hab locus are in a single gene. Hence, all the alleles have been
renamed hab, following the precedent for the nomenclature of
somitabun/captain hook (Kramer et
al., 2002), which also includes dominant and recessive
alleles.
In the first portion of this work, we demonstrate that the alleles of
hab are mutants in a single gene that encodes a zebrafish homolog of
E-cadherin, an important membrane protein necessary for homotypic cell
adhesion (Takeichi, 1987). In
the mouse, E-cadherin mRNA is present maternally and E-cadherin function is
necessary for the process of cell compaction in the 8-cell stage embryo
(Ao and Erickson, 1992
). Later,
as zygotic expression of E-cadherin begins, it is necessary for the expansion
of the trophectoderm (Larue et al.,
1994
). In the zebrafish, E-cadherin mRNA is present maternally
(Babb et al., 2001
), and
experiments based on antisense oligonucleotides have shown that E-cadherin is
necessary for blastomere adhesion during the cleavage stage, and later for
aspects of morphogenesis during gastrulation and epiboly
(Babb and Marrs, 2004
). In
frogs, based on dominant negative analysis, E-cadherin seems more important
for tissue integrity during early development
(Heasman et al., 1994
;
Levine et al., 1994
), whereas
the closely related C-cadherin is necessary for morphogenesis
(Lee and Gumbiner, 1995
).
However, the two cadherins overlap in their expression patterns and possibly
functionally as well. In all creatures, during and after gastrulation,
E-cadherin is expressed in the epidermal ectoderm, in a pattern complementary
to the expression of N-cadherin in the neural ectoderm, and in the endoderm
(Rutishauser et al., 1988
;
Takeichi, 1995
). Later,
reflecting its diverse role in numerous developmental and epithelial tissue
functions, E-cadherin is also expressed in many epithelial tissues, including
some that are mesodermally derived.
At the structural level, the protein is characterized by five extracellular
cadherin (EC) repeats, a single pass transmembrane domain and a cytoplasmic
domain. The EC repeats are extremely conserved among their homologs in other
species, and are necessary for the specific adhesion properties of E-cadherin
(Blaschuk et al., 1990). The
cytoplasmic domain of the protein binds
- and ß-catenin, and
indirectly actin, simultaneously anchoring the protein to the cytoskeleton of
the cell and connecting the molecule to the WNT signaling pathway
(Gumbiner and McCrea, 1993
;
Herrenknecht et al., 1991
;
Kintner, 1992
;
McCrea et al., 1991
;
Sanson et al., 1996
). The
protein is thought to function as a dimer, and the EC repeats are necessary
for this dimerization (Brieher et al.,
1996
; Nagar et al.,
1996
). This fact has been the basis for the creation of transgenes
that abate gene function: deleting one or two of the exterior EC domains
allows dimerization between mutant and wild-type E-cadherins, blocking
E-cadherin function and causing a very effective dominant negative effect.
In the second portion of this work, we examine the role of hab in
the morphogenesis of the epiblast during zebrafish epiboly, focusing on the
differences in cell movement between hab mutants and wild-type
embryos. The epiblast forms from all the deep cells of the blastoderm, and,
somewhat similarly to the amniote epiblast, contributes cells to the hypoblast
throughout gastrulation. Afterward, the epiblast forms the anlage for the
ectodermal derivatives, whereas the hypoblast forms the anlage for mesodermal
and endodermal derivatives. The epiblast thins as it spreads over the yolk
cell, from about five cells thick at doming stage to about two cells thick at
100% epiboly, and finally to one cell thick in all but the axial region in the
early segmentation stages. We hypothesized that this thinning may be due to
the morphogenetic process of radial intercalation, the means where two or more
layers of cells thin into a single layer, thus causing an expansion in surface
area of the resulting layer. Using scanning electron micrographs of
freeze-fractured blastulae, radial intercalation was classically described in
the amphibian blastocoel roof (Keller,
1980), and this process is thought to be a driving force for the
expansion of the amphibian animal cap. Radial intercalation is also known to
occur in the zebrafish blastula as the blastoderm thins during the doming
stage, and is renowned for its annoying property of mixing blastula cell
lineages, thus causing the indeterminate early fate map of zebrafish
(Helde et al., 1994
;
Kimmel and Warga, 1987
;
Warga and Kimmel, 1990
;
Wilson et al., 1995
). It is
notable that in all the above examples, frog and fish, it is not clear if
radial intercalation by itself is a force generating movement or if the
movement is a passive response of a tissue to outside forces, e.g. being
stretched or being compressed between two opposing tissues, as has been
suggested by Wilson et al. (Wilson et al.,
1995
).
Here we describe the cell behaviors that drive late epiboly in the zebrafish embryo. We find two previously unrecognized layers in the zebrafish epiblast, the exterior layer of the epiblast and the interior layer of the epiblast, and we show that each of these layers have unique functions in this zebrafish version of radial interaction. In hab mutant embryos, these layers do not form completely and other cellular behaviors involved with radial intercalation do not occur normally. Finally, using cell transplantation, we show that the hab defects occur cell-autonomously, demonstrating that defects in radial intercalation are the most likely cause for the hab epiboly defect.
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Materials and methods |
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Positional cloning of the hab locus
hab was initially mapped to the centromere of Linkage Group 7 by
half tetrad analysis (Johnson et al.,
1995). For fine-resolution mapping we used a panel of 2916 haploid
embryos and identified two closely linked microsatellite markers on either
side of the hab locus (Knapik et
al., 1998
). These markers were used to isolate clones from a
large-insert genomic library (Amemiya et
al., 1999
) and construct a contig, as has been described
(Wei and Malicki, 2002
).
Clones spanning the critical region of hab were sequenced as part of
the ongoing Zebrafish Genome Project (GenBank Accession numbers: PAC180O5:
AL929461; PAC109L11: AL929295). To determine the site of the hab
mutations we isolated total RNA from wild-type and hab embryo pools
and generated E-cadherin cDNA by reverse transcription and PCR amplification
using five overlapping primer pairs designed from the published cDNA sequence
and compared sequence. We confirmed each mutation by isolating total RNA or
genomic DNA from individual mutant and wild-type embryos to generate sequence
for the site of lesion. Sequences of PCR primers are available from the
corresponding author upon request.
hab morpholino oligonucleotides
hab/E-cadherin splice site-targeting morpholino oligonucleotides
were designed from trace sequences deposited by Sanger before the draft
sequence was available. Exons were identified by comparison of the mRNA
sequence to the genomic sequence and numbered according to mouse terminology
(Ringwald et al., 1991).
Morpholinos were purchased from Gene Tools, the sequence of MO1 is:
5'-GTAACACACAGTAACCTTTACAGTGG-3'; and the sequence of MO2 is:
5'-AAGCATTTCTCACCTCTCTGTCCAG-3'. We monitored splicing events by
RT-PCR using pools of five embryos for each developmental timepoint. Sequences
of PCR primers used for monitoring are available from the corresponding author
upon request.
Embryo manipulations and genotypic characterization
For targeted gene-knockdown, we injected wild-type embryos with morpholino
oligonucleotides (Gene-Tools) at the 1-cell stage. For transplants, we labeled
donor embryos derived from crosses of habtm94
heterozygotes with a mixture of 3% rhodamine-dextran and 3% biotin-dextran
(Molecular Probes) at the 1- to 8-cell stage and transplanted donor cells into
wild-type embryos at the onset of epiboly using standard procedures
(Ho and Kane, 1990). Donor
embryos were harvested and genotyped using the closely linked microsatellite
marker Z20715 (F-5'-CATCTGTAAGTGCCCAGCAA-3',
R-5'-GTGTCCGGTTAGGCTACAAT-3'). Host embryos were fixed at 80%
epiboly and processed as below for the co-injected biotin-dextran. For in vivo
cell labeling, we injected a single superficial blastomere with 3%
rhodamine-dextran at the 2k- to 4k-cell stage using standard procedures
(Warga and Nüsslein-Volhard,
1999
) in embryos derived from crosses of
habdtv43 heterozygotes. For these experiments we selected
parental strains that gave less severe epiboly phenotypes so that survival of
homozygous offspring persisted through the epiboly period. Embryos were
genotyped by phenotype at the 100% epiboly stage based on the epiboly arrest
trait of homozygous embryos and the detached cell trait of heterozygous
embryos. Heterozygous embryos were reconfirmed at 24 hours by the enlarged
hatching gland trait.
Immunohistochemistry and RNA in situ hybridization
Embryos injected with biotinylated-dextran were processed as described in
the Zebrafish Book (Westerfield,
1993), cleared, mounted in PermountTM (Fischer Scientific)
and photographed.
Antibody staining was carried out as described
(Warga and Nüsslein-Volhard,
1999); briefly, embryos derived from either wild-type parents or
habdtv43 heterozygous parents were stained with
anti-ß-catenin (Sigma), embedded in 17% gelatin: 50% glycerol and
bisected along the 90° meridian by hand using a razor blade. Afterward,
these hand-sections were cleared, mounted in Permount and photographed.
RNA in situ hybridization was carried out as described
(Thisse et al., 1993) using
embryos derived from wild-type parents. Afterward, hand-sections were cut as
described above, cleared in 70% glycerol and photographed.
Time-lapse and data analysis
For in vivo observations, embryos derived from habdtv43
heterozygotes were mounted and recorded in multi-plane as previously described
(Warga and Kane, 2003).
Afterward, the recordings were analyzed using Cytos Software, looping
half-hour segments from the time-lapse recording. In the case of
Fig. 7, black and white images
from one plane of the time-lapse video recording were imported into Adobe
Photoshop and pseudo-colored to aid in presentation, matching the color of
cells shown in the model in Fig.
9.
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Results |
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A radial gradient of hab expression in the non axial blastoderm during epiboly
To analyze the distribution of the hab gene product, we performed
whole-mount in situ hybridizations, focusing our investigation on the
embryonic epiblast before and during late epiboly, the time when the epiboly
arrest trait of the mutant is most strongly expressed. At this stage, the
epiblast is sandwiched between the very thin epithelium of the EVL, and, on
the inside of the embryo, the yolk cell or, if present, the hypoblast.
Using a probe made to the full-length hab cDNA sequence, we found
that cleavage stage embryos expressed hab ubiquitously, showing that
the mRNA was present maternally (Fig.
4A), as shown previously (Babb
et al., 2001). At 30% epiboly, hab expression began to
fade, revealing a very subtle radial gradient of expression
(Fig. 4B) from the deepest
layers of the blastoderm (lowest expression) to the superficial cells of the
blastoderm (highest expression). By shield stage, the epiblast began to clear
on the dorsal side of the embryo and the gradient became steeper elsewhere
(Fig. 4C). At the same time,
hab was strongly expressed in the entire EVL layer
(Fig. 4C,D), forming a ring of
expression around the nuclei of these thin epithelial cells. Expression was
especially high in a subpopulation of EVL cells that become the forerunner
cells. At 70% epiboly, hab expression was maintained on the lateral
and ventral sides of the epiblast, and, within those regions, formed a steep
radial gradient that was highest in the cells just beneath the EVL
(Fig. 4E,F), and was absent in
the yolk cell. Expression of hab was maintained in the EVL and
cleared in the axial portion of the epiblast
(Fig. 4F',G); interior to
this zone of clearing, expression was seen in the anterior edge of the
hypoblast (Fig. 4F',G), as shown previously (Babb et al.,
2001
), and is presented as an ad hoc control to demonstrate the
penetration of the whole-mount in situ probe.
|
Identification of the exterior layer and interior layer of the epiblast
During the examination of the above in situ experiments, there seemed to be
slight differences in cell morphology that correlated with the concentration
of hab in the superficial and deep cell layers of the epiblast. After
fixing homozygous mutant and wild-type embryos at 80 to 90% epiboly, staining
to visualize the cell membranes, and sectioning the embryos, two distinct
layers of the epiblast could be distinguished on the ventral and lateral sides
of the embryo (Fig. 5). The
exterior layer of the epiblast was epithelial-like, containing thin cuboidal
cells that spread under, and perhaps against, the EVL. In between the exterior
layer and the hypoblast, there was an interior layer of the epiblast, which
consisted of disorganized cells that tended to be oriented with their long
axis perpendicular to the surface of the epiblast. In general, the border
between the two layers was indistinct, and often individual cells were
spanning the border. In homozygous hab mutants, cells in both layers
were rounder than their wild-type counterparts: the cells of the exterior
layer of the epiblast were less spread out, and the cells of the interior
layer were less elliptical. Indeed, the mutant layers were so similar that it
was difficult to say which layer contained the extra cells. Also, when
compared with wild-type embryos, the exterior epiblast formed a less
continuous epithelium in hab mutants (which can be seen nicely in
comparisons among homozygous mutants and wild-type embryos in
Fig. 8).
|
|
To test the cell-autonomy of hab, we transplanted cells from homozygous mutant and wild-type siblings into the epiblast of wild-type hosts, and fixed the embryos at 80 to 90% epiboly. Examination of the morphology of the donor cells in the fixed material showed that cells from the mutant donors had strikingly identical sizes and aspect ratios to the measurements in the live mutants (Fig. 6C,D), demonstrating that the mutation acts cell-autonomously. We also noted the relative contributions from the homozygous mutant and wild-type donors to the exterior and interior layers of the host epiblast. Wild-type cells tended to contribute to the exterior layer of the epiblast (286 exterior:104 interior), whereas homozygous mutant cells did not (111 exterior:196 interior). Hence, although mutant cells are able to contribute to the exterior layer of the epiblast, they tend not to, and this bias is cell-autonomous.
Abnormal radial intercalation in hab mutants
We hypothesized that a combination of radial intercalation and cell shape
changes drive the spreading of the blastoderm during epiboly. To directly
observe these processes, we recorded homozygous mutant, ZMD mutant and
wild-type siblings during late epiboly and documented occurrences of cells
intercalating into - and out of - the exterior layer of the epiblast
(Fig. 7). In ZMD mutants and
wild-type embryos, as newly intercalated cells entered the exterior layer,
they flattened to the dimensions typical of cells in that layer. This is shown
for a ZMD habdtv43/+ mutant in
Fig. 7A. The process of
entering the exterior layer and flattening typically took about 15 minutes and
is shown in the supplementary material (Movies 1-3).
In homozygous mutants, cells from the interior layer also intercalated into the exterior layer (Fig. 7B), although the cells never completely flattened out. This process occurred somewhat quicker than in wild-type embryos, taking about 8 to 10 minutes. Surprisingly, in the homozygous mutants, exterior layer cells often returned to the interior layer (Fig. 7C). Examining time-lapse records over a 15 minute interval, we measured intercalation and de-intercalation events in homozygous mutants, ZMD mutants and wild-type embryos (Table 1). De-intercalation events were never seen in wild-type embryos. However, in homozygous mutants, the rate of de-intercalation was almost equal to the rate of intercalation, suggesting that loss of the cells in the exterior layer of the epiblast is the physical basis for the arrest of epiboly. In ZMD mutants, there was approximately one de-intercalation event for every three or four intercalation events, which roughly correlates with the slowing of epiboly observed in the ZMD mutants.
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Discussion |
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The recessive alleles of hab act as nulls for zygotically expressed E-cadherin
The cell membrane protein E-cadherin requires the function of five EC
domains, which facilitate the homophilic binding among cells, and a
cytoplasmic portion necessary for interactions with the cytoskeleton. The
recessive alleles of hab and the MO1 antisense oligonucleotide
knockdown product all result in truncated proteins due to premature stop
codons in the EC3 or EC4 domains. These mutations should produce proteins that
contain the signal sequence and the pro domain and the first two or three EC
domains, but lack the inner EC domains, the transmembrane domain and the
cytoplasmic portion of the protein. Hence, we imagine that the hab
recessive alleles are functional nulls. Interestingly, both of the
hab recessive alleles were truncated in EC4, similar to two of the
three recessive alleles of parachute (pac), which encodes the
zebrafish homolog of N-cadherin (Lele et
al., 2002); these pac alleles are thought to be recessive
nulls. Nevertheless, in the cases of either hab or pac, it
is possible that the truncated protein products are secreted and have some
unknown function.
Even if null, probably none of the hab phenotypes display the
lack-of-function phenotype of the gene, for hab is maternally
supplied. The lack-of-function phenotype may resemble the knockdown phenotype
of using 5' antisense oligonucleotides seen by Babb and Marrs
(Babb and Marrs, 2004), which
should knock down maternal mRNA expression, or the phenotype could be more
severe.
The ZMD alleles of hab act as dominant negatives
Our interpretation of the dominant phenotypes is that they are caused by
mutations acting as antimorphic alleles. It is thought that cadherins act as
dimers, and the EC repeats are necessary for dimerization
(Brieher et al., 1996;
Nagar et al., 1996
;
Takeda et al., 1999
). Based on
sequence analysis, all the dominant alleles and the MO2 antisense knockdown
product should produce proteins that are properly inserted into the cell
membrane of the cells that normally express wild-type hab, but
possess one or more nonfunctional EC domains. These mutant proteins should
dimerize with wild-type proteins, and thus interfere with gene function in a
dominant negative fashion.
The dominant negative effects of the ZMD alleles are consistent with the
strengths and behaviors of their phenotypes. First, in the case of
heterozygous embryos derived from a wild-type mother and a father heterozygous
for a ZMD allele, there is no dominance. In this case, we imagine that the
maternally encoded protein is capable of competing the effect of the one
zygotic copy of the dominant allele. By this argument, hab is the
strongest of the dominant alleles, as it alone has a dominant effect in the
absence of a heterozygous mother. Second, when homozygous, the dominant
alleles express more severe epiboly defects than the recessive alleles of
hab (Kane et al.,
1996). Here we imagine that the zygotic products of the dominant
allele partially inactivate the maternally supplied products, and the
phenotype is shifting toward what we would imagine is the maternal
phenotype.
In the case of the ZMD alleles, the location of the mutation within the
protein correlates with specific aspects of the dominant phenotypes,
suggesting that domains of the protein may possess different functions in
different regions of the embryo. For example, both habts18
and habtm94 possess mutations in the EC1 domain, and both
their ZMD neural tube phenotypes look very similar. Both phenotypes express
defects focused in the tail, and there, the defects are severe. However, the
habdtv43 allele possesses a mutation in the EC5 domain.
Its ZMD neural tube phenotype is expressed the entire length of the neural
axis, but, in general, the effect is not locally severe. Moreover, the
habdtv43 allele has the dominant hatching gland defect;
habtm94 and habts18 do not.
Interestingly, the one pac allele that is reported to possess some
dominant effects is a missense mutant in EC1 of N-cadherin
(Malicki et al., 2003). This
particular mutant also has allele-specific phenotypes, expressing a more
severe phenotype in the eye than the recessive pac alleles.
In humans, gastric cancers that are particularly invasive have been traced
to familial inherited mutations in E-cadherin
(Hajra and Fearon, 2002), and
some of these mutations are transversions that result in amino acid
substitutions in the EC1 and EC5 domains. Many of our fish lines that carry
the hab dominant alleles show reduced long-term survival, and it will
be of interest to examine the fish that survive only to early adulthood.
A model for expansion of the epiblast in the zebrafish embryo
Our results suggest that a combination of radial intercalation with other
cell behaviors drive the spreading of the non-axial epiblast during late
epiboly. We have shown the existence of two cell layers in the epiblast, which
we term the `exterior layer' and `interior layer'
(Fig. 9). Closely opposed to
the well-organized epithelium of the EVL, cells of the exterior layer formed a
continuous layer that roughly resembled a cuboidal epithelium. Underlying the
exterior layer was the interior layer, a somewhat disorganized multilayer
consisting of cells that tended to be aligned in a radial direction. Here we
have documented that the cells of the interior layer of the epiblast moved
into the exterior layer via radial intercalation. Once there, cells of the
exterior layer tended to remain in the exterior layer, and became restricted
to that layer.
We propose that the expansion of the epiblast has three components: first, cells of the interior layer radially intercalate into the exterior layer; second, the cells flatten as they join the epithelium of the exterior layer; third, these cells become restricted to the exterior layer.
Biomechanically, the model spreads the epiblast by two means. First, the process of radial intercalation increases the number of cells in the exterior layer of the epiblast, and thus expands the surface area of the epithelium, similar to the effect of radial intercalation in amphibians. Note that to secure this effect the cells should not leave the exterior layer. Second, the flattening of the cells further increases the surface area of the exterior epiblast. It is of interest that the exterior layer of the epiblast appeared to spread out against the EVL, as a substrate of sorts. Whereas this attachment was quite weak, for the epiblast freely slid under the EVL, the subtle interactions between the two layers might have mechanical consequences that are underappreciated.
The expansion of the zebrafish epiblast seemed to show a remarkable blend
of Xenopus and mouse morphogenesis. The cell shape changes reported
here for the exterior layer of the epiblast during teleost epiboly appeared
very similar to those thought to occur during the expansion of the
trophectoderm epithelium (Barcroft et al.,
1998; Fleming et al.,
2001
; Reima et al.,
1993
). Based on morphogenetic arguments, the formation of the
mammalian trophectoderm and the epiboly of the teleost blastoderm over the
yolk have been suggested to be similar events
(Kane and Adams, 2002
).
Interestingly, both these structures fail to form normally after zygotic
E-cadherin is abrogated by mutation in either species
(Kane et al., 1996
;
Larue et al., 1994
),
demonstrating that they are among the first structures that require the
zygotic expression of E-cadherin. Observations such as these open questions as
to the homology of these two structures.
Compared with radial intercalation in other model systems, teleost radial
intercalation displays a number of unique differences. First, teleost epiboly
may involve substrates, such as the EVL, that could organize radial
intercalation. In urodeles, with the underlining of the animal cap by the void
of the blastocoel, all the cell layers of the animal cap appear to participate
equally in radial intercalation (Keller
and Shook, 1994), resulting in a single layer at the completion of
the process, and demonstrating that radial intercalation can be a
substrate-free process. In this regard, zebrafish may have some similarities
to Xenopus, which also possesses an outer epithelial layer that does
not participate in radial intercalation
(Keller, 1980
). Second,
teleost epiboly is markedly asymmetric. In amphibians, and especially in
urodeles, there is an impression of two or three somewhat similar layers
merging into a single epithelium. In teleosts, the exterior and the interior
layers of the epiblast appear very different from one another. On the one
hand, the exterior layer acts as a sink for the intercalation of cells and
participates in cell shape changes, both processes that act to drive the
epiblast over the yolk cell. On the other hand, the interior layer acts only
as a reservoir, contributing cells as needed to the exterior layer. Such a
mechanism might be more appropriate for teleost epiboly, where a relatively
small but solid blastoderm must cover the huge yolk cell.
hab is necessary for restrictions and cell shape changes in the epiblast
Almost all the cell behaviors described by the above model exhibit defects
in hab mutants. First, distinguishable exterior and interior layers
of the epiblast did not form in the mutant. The cells of what would be the
exterior layer fail to flatten and never form a continuous epithelium, and
albeit more subtly, the cells of the interior layer failed to attain a radial
aspect ratio. Second, radial intercalation in the mutant was negated by the
inability of the cells to remain in the exterior layer. This aberrant cell
movement was probably the direct cause for the arrest of epiboly.
The transplantation experiments demonstrate that hab acts in a cell-autonomous fashion. Hence, hab function is necessary within the cells themselves for their participation in the cell behaviors of the epiblast. More importantly, when mutant cells are placed in a wild-type embryo, both the cell shape phenotype of hab and the inability of hab cells to remain restricted to the exterior layer are virtually identical to the behavior of cells in mutant embryos. This result strongly indicates that the hab defects in cell behavior are not the result of other morphogenetic processes in the epiblast. We note that an important control experiment has not yet been done: that is, placing wild-type cells in mutant embryos. This experiment would test whether wild-type cells can undergo normal cell behaviors in an embryo devoid of any global clues or forces that may be necessary for such cell behaviors, a non-autonomous effect. We had poor success with this experiment because of the general lethality of hab host embryos, which seems to be exacerbated by the process of transplantation itself. However, in other experiments, submitted elsewhere, we have seen that when wild-type cells are transplanted into neural tubes of ZMD mutants, the wild-type cells often assume mutant phenotype, suggesting that in certain situations, hab can act in a non-autonomous fashion. We are now breeding lines of the mutant habtx230, which has the least severe epiboly phenotype, in order to produce a recessive mutant that reliably survives into early segmentation stages.
Our observations regarding the radial concentration gradient of
hab mRNA are in line with the defects observed in hab
mutants. hab mRNA is expressed in the exterior layer of the epiblast,
and that layer is missing in hab mutants. Possibly, cells of the
exterior layer require additional hab gene product because they are
forming an epithelium, and if they are unable to produce additional gene
product, they do not integrate into the exterior layer and slip back into the
interior layer. The radial gradient could also control the ordering and
layering of cells during epiboly. Cells sort into layers dependent on adhesive
activity (Duguay et al., 2003;
Steinberg and Takeichi, 1994
).
One could imagine that as the blastoderm thins, cells with the highest
concentration of hab gene product would move to the outside, against
the EVL, which continuously expresses high levels of hab mRNA; cells
with low concentrations would move to the yolk cell, which has downregulated
hab mRNA. Indeed, in the MO2 antisense knockdown experiment we
observed unevenness in the thicknesses of the blastoderm. Antisense injections
are known to mix unevenly in the early cleavage embryo, and irregularities in
oligonucleotide concentration would cause local differences in hab
function. Hence, because cells would tend to adhere differently in regions of
differing Hab concentrations, some regions would tend to be thicker than
others.
In hab mutants, cells move, but many tissues do not. For example, radial intercalation occurs at the same rate in hab mutants as in wild-type embryos, but the mutant cells de-intercalate, unable to remain in the exterior epiblast, and the epiblast ceases movement. In work to be presented elsewhere, we have found that many aspects of global convergence are affected in hab mutants, but the cells themselves continue to move at the same rate as cells in wild-type embryos. Hab seems specifically required for the normal integration of cells into embryonic epithelia and continues to be required for the normal behavior of the cells within the epithelia. Hence, in hab mutants, such epithelia form poorly or not at all; when they do form, the epithelia behave abnormally, causing the slowing or arrest of large-scale movements. Unraveling the roles that Hab and other molecules play in these early cell interactions that bind together the cells of the gastrula will be instructive, as we untangle these subtle forces that act to shape the vertebrate embryo.
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: University of Virginia Health Systems, Department of
Pathlogy, 415 Lane Road, Charlottesville, VA 22908, USA
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REFERENCES |
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