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DEVELOPMENTAL DYNAMICS 233:390 – 406, 2005
RESEARCH ARTICLE
Genetic Locus half baked Is Necessary for
Morphogenesis of the Ectoderm
Karen N. McFarland,1 Rachel M. Warga,2 and Donald A. Kane2*
The zebrafish epiboly mutants partially block epiboly, the vegetalward movement of the blastoderm around
the giant yolk cell. Here, we show that the epiboly mutations are located near the centromere of Linkage
Group 7 in a single locus, termed the half baked locus. Nevertheless, except for the similar mutants lawine
and avalanche, we find the epiboly traits of each of the alleles to be distinguishable, forming an allelic
series. Using in situ analysis, we show that the specification and the formation of the germ layers is
unaffected. However, during early gastrulation, convergence movements are slowed in homozygous and
zygotic maternal dominant (ZMD) heterozygous mutants, especially in the epiblast layer of the blastoderm.
Using triple-mutant analysis with squint and cyclops, we show that ablating involution and hypoblast
formation in hab has no effect on the epiboly phenotype on the ventral and lateral sides of the embryo,
suggesting that the hypoblast has no role in epiboly. Moreover, the triple mutant enhances the depletion of
cells on the dorsal side of the embryo, consistent with the idea that convergence movements are defective.
Double-mutant analysis with one-eyed pinhead reveals that hab is necessary in the ectodermal portion of
the hatching gland. In ZMD heterozygotes, in addition to the slowing of epiboly, morphogenesis of the
neural tube is abnormal, with gaps forming in the midline during segmentation stages; later, ectopic rows
of neurons form in the widened spinal cord and hindbrain. Cell transplantation reveals that half baked acts
both autonomously and nonautonomously in interactions among cells of the forming neural tube. Together,
these results suggest that half baked is necessary within the epiblast for morphogenesis during both epiboly
and neurulation and suggest that the mechanisms that drive epiboly possess common elements with those
that underlie convergence and extension. Developmental Dynamics 233:390 – 406, 2005.
© 2005 Wiley-Liss, Inc.
Key words: epiboly; convergence; involution; half baked; epiblast; hypoblast; ectoderm; mesoderm; zebrafish
Received 30 August 2004; Revised 24 October 2004; Accepted 16 November 2004
INTRODUCTION
In teleosts, morphogenesis begins
with epiboly, the process in which the
blastoderm covers and engulfs the
large yolk cell. Here, we report our
analysis of the defects in the epiboly
mutants identified in the Tübingen
genetic screen for mutations that affect morphogenesis of the zebrafish
that was carried out at the Max
Planck Institut für Entwicklungsbiologie (Haffter et al., 1996). Epiboly is
blocked in the mutants half baked
(hab), lawine (law), avalanche (ava),
weg, and volcano (Kane et al., 1996;
Solnica-Krezel et al., 1996). In each of
these mutants, epiboly begins normally in the blastula and early gastrula stage, but by 70% to 80% epiboly, approximately 1.5 to 2 hr after
the onset of gastrulation, mutants begin to arrest their vegetalward
spreading and begin to dissociate,
usually with their blastoderm peeling
off the yolk.
The mutants hab, ava, and law also
display a zygotic maternal dominant
(ZMD) effect that is expressed when
both zygotic and maternal genomes
are heterozygous for the mutant locus.
The Supplementary material referred to in this article, can be found at http://www.interscience.wiley.com/jpages/1058-8388/suppmat
1
University of Virginia Health Systems, Department of Pathology, Charlottesville, Virginia
2
Department of Organismal Biology and Anatomy, University of Chicago, Chicago, Illinois
Grant sponsor: Pew Charitable Trust; Grant sponsor: National Institutes of Health; Grant number: R01-GM58513.
*Correspondence to: Don Kane, Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637.
E-mail: [email protected]
DOI 10.1002/dvdy.20325
Published online 14 March 2005 in Wiley InterScience (www.interscience.wiley.com).
© 2005 Wiley-Liss, Inc.
half baked ECTODERM 391
These embryos, termed ZMD mutants, display an intermediate rate of
epiboly between that of wild-type and
homozygous mutant siblings and complete epiboly approximately an hour
after wild-type siblings. Later, during
somitogenesis, cells dorsal to the developing neural tube round up and detach from the embryo (Kane et al.,
1996). In addition, hab mutants display a semidominant trait of an enlarged hatching gland. Although these
mutants fail to complement one another, the dominant and ZMD-dominant traits made it difficult to assess
whether they belong to one locus;
therefore, they were named separately.
The array of processes that drive
epiboly are not completely understood. Before epiboly begins, the zebrafish blastula is composed of three
domains: the epithelial-like enveloping layer (EVL) that covers the blastoderm, the deep cells of the blastoderm, and the yolk cell (Kane et al.,
1992). In Fundulus, after removing
both the EVL and deep cell layer from
the yolk cell, the syncytial layer of the
yolk completes epiboly autonomously
(Trinkaus, 1951). This movement is
dependent on a microtubule-based
motor. A highly organized microtubule array has been observed in the
zebrafish yolk cell (Strähle and Jesuthasan, 1993; Solnica-Krezel and
Driever, 1994), and treatments that
disrupt this microtubule network slow
or stop epiboly. At least part of the
blastoderm appears to be towed by
this yolk cell motor: the EVL is tightly
attached to the yolk cell (Betchaku
and Trinkaus, 1978), and when the
yolk syncytial layer begins to move,
the EVL follows in tandem. Curiously,
in the epiboly mutants, the EVL and
yolk syncytial layer are unaffected
and both complete epiboly normally.
The contribution— or lack thereof— of other morphogenetic movements to epiboly is not known. In the
mutants, only the movement of the
deep cells is arrested. To date, there is
no known role for the deep cells in
epiboly. Starting as an amorphous
mound at the animal pole, between
the EVL and the yolk cell, the deep
cell layers thin as they spread over the
yolk cell (Warga and Kimmel, 1990;
Wilson et al., 1995), and it is unclear if
the deep cells are active participants
in epiboly. Nevertheless, the epiboly
mutants demonstrate that at least
some aspects of the epiboly of the deep
cells are under a separate genetic control from that of the yolk cell and EVL.
Midway through epiboly, deep cells
at the margin of the blastoderm commence involution, separating the blastoderm into two layers: an outer epiblast layer, the future ectoderm, and
an inner hypoblast layer, the future
mesoderm and endoderm (Warga and
Kimmel, 1990; Warga and VolhardVolhard, 1999). Epiboly is normal in
squint/cyclops double mutants, where
the Nodal pathway is abolished, blocking mesoderm formation and involution, suggesting that these processes
have no effect on epiboly (Feldman et
al., 2000; Dougan et al., 2003). However, epiboly is arrested in lefty1/2 antisense knockdown embryos, where
the Nodal pathway is up-regulated
and the rate of involution is increased
(Branford and Yost, 2002; Chen and
Schier, 2002; Feldman et al., 2002).
Shortly after involution commences,
cells begin to converge and migrate
from lateral positions toward the dorsal side of the embryo (Warga and
Kimmel, 1990; Schmitz et al., 1993;
Kimmel et al., 1994), except for the
ventral-most region of the embryo
where convergence does not occur
(Myers et al., 2002). At the cellular
level, cohorts of cells extend anteroposteriorly and narrow mediolaterally
as they move dorsally. Whether in the
epiblast, where cells move as an epithelial sheet, or in the hypoblast,
where cells move as individuals (Jessen et al., 2002), these cells mediolaterally intercalate among themselves
as they enter the body axis (Warga
and Kimmel, 1990; Sepich et al.,
2000). Although convergence is occurring during epiboly, the mechanistic
relationships between the two processes are not understood. However,
these movements must be somewhat
interdependent, as epiboly is slowed
in frogs by treatments that block convergence (Hikasa et al., 2002).
Here, we document that the epiboly
mutations are all located on a small
region of Linkage Group 7, strongly
suggesting that the mutants map to a
single locus. Nevertheless, we find
that each allele displays specific differences in their effect on epiboly,
which correlates with differences in
aspects of their dominant phenotypes.
We then document that specification
of the three primary germ layers and
subsequent tissue differentiation is
normal in hab mutants and that involution of the mesendoderm occurs normally. However, we find that the axis
of homozygous mutants is wider, particularly in the epiblast layer, suggesting that convergence processes
may be defective during gastrulation.
In ZMD mutants, we also document
that, during segmentation stages, the
formation of the neural keel is wider
and contains ectopic neural structures. Using triple-mutant analysis of
hab with squint and cyclops, we confirm that involution and mesendoderm formation is unrelated to the
hab epiboly phenotype on the ventral
and lateral sides of the embryo but
find a depletion of cells on the dorsal
side of the triple mutants, further indicating that convergence movements
are defective. Examination of double
mutants of hab and one-eyed pinhead
reveals that hab is necessary in the
ectodermal portion of the hatching
gland. Furthermore, cell transplantation studies reveal that the hab gene
product acts autonomously as well as
nonautonomously in the neuroectoderm to produce aspects of the neural
tube defect. Altogether, our data suggest that hab is required in the epiblast of the embryo for morphogenetic
movements that drive epiboly and
neural morphogenesis.
RESULTS
Loci of the Epiboly Mutants
Map Closely to the Linkage
Group 7 Centromere
Because of the dominant effects of
many of the epiboly mutants, the results from the initial complementation matrix were uninterpretable.
Like the Minute loci of Drosophila
(Lindsley and Grell, 1968) and the
swirl-somitabun loci of zebrafish
(Mullins et al., 1996), it was quite possible that the epiboly mutants were
intra-allelic noncomplementing dominant mutations. To test if they represented different loci, we positioned the
mutant loci onto the zebrafish genetic
map. Initial mapping, using half-tetrad analysis (not shown), loosely
linked all of the mutants to the cen-
392 MCFARLAND ET AL.
tromere of Linkage Group 7. Unfortunately, this method was inadequate
for fine mapping of the epiboly mutants because of scoring artifacts. The
high hydrostatic pressure used for the
half-tetrad method affects the microtubule network of the embryo, slowing
epiboly and creating other unrelated
defects (Hatta and Kimmel, 1993).
This effect caused a fraction of the
ZMD heterozygous embryos to resemble the homozygous recessive phenotype and a fraction of the wild-type
embryos to resemble the ZMD phenotype. Moreover, many of the homozygous recessive mutants tended to prematurely dissociate and not be scored
at all. Hence, the number of heterozygous embryos was overrepresented
and the distance from the centromere
to the hab locus exaggerated. Genetic
distances also varied because of experimental variations of the treatment on
different clutches of embryos from
slightly different backgrounds.
Using haploid mapping panels to
carefully map the individual mutant
loci in relation to microsatellite markers, we first confirmed the locations of
available markers, creating the mitotic map shown in Figure 1A. Because the mitotic events in a haploid
panel occurred exclusively in females,
this map has longer intramarker distances than the sex-averaged mitotic
map from Boston (Shimoda et al.,
1999) and resolved many areas of
genetic compression. Furthermore,
markers that had been linked previously to the center of Linkage Group 7
using the radiation hybrid panel were
remapped, adding to the number of
useful markers. We also used half-tetrad mapping panels that were generated for the mapping of unrelated
(and unlinked) mutant loci and found
no recombinants in a mapping panel
of over 125 individuals between the
markers z7958 and z9869 or either
marker and the centromere; this finding established these markers as reliable markers of the centromere for
Linkage Group 7 (Fig. 1B) and closer
than markers that we and others have
suggested previously (Kane et al.,
1999; Mohideen et al., 2000).
As part of a related project, to identify the molecular nature of the hab
locus, linked candidate genes were
eliminated based on their recombination with the hab locus. These genes
became valuable markers in the process of mapping the hab locus. Typically, using the 3⬘ region of a cloned
fragment, we identified a size polymorphism that could be visualized on
an agarose gel and then checked if the
candidate was recombinant in individuals that were known to have recombination events close to the hab locus.
Such an example is shown in Figure
1E, where we identified recombination events between hab and the
closely linked gene FGF4. In some
cases, when no polymorphism to the
gene was present in the test cross, we
identified a clone containing the gene
of interest from a large-insert genomic
PAC library (Amemiya and Zon, 1999)
and then identified a polymorphism in
the ends of the clone or used a closely
linked microsatellite marker on the
same clone. The microsatellite marker
was then used to test linkage of the
candidate gene to the hab locus as is
shown in Figure 1D, where VN cadherin, which was linked to the marker
z3008 on PAC clone 30C24, was subsequently shown to be over 20 cM
from the hab locus. When no polymorphisms could be found using simple
size polymorphisms on agarose gels,
we used single-stranded conformation
polymorphisms (SSCP) methodology,
which successfully identified polymorphisms in approximately 90% of cases
attempted. Such an example is shown
in Figure 1F, where we identified recombination events between hab and
the very closely linked gene FGF3.
Finally, using the individual haploid
mapping panels, we established that
each of the mutant loci map between
marker z1239 and the centromeric
marker z7958, a distance of approximately 20 cM. Additional mapping
placed all the mutants at approximately the center of this interval, suggesting that they belong to the same
locus, which we term the half baked
locus (Fig. 1C). Because hab and ava
were tightly linked to marker z6852,
linkage to this marker subsequently
was used to genotype individual embryos for the hab locus.
Epiboly Mutants Show Allele
Specific Epiboly Phenotypes
The epiboly mutants display an array
of dominant phenotypes that are specific to each individual allele, and we
have suggested previously that the recessive epiboly phenotypes may display differences specific to each allele
as well (Kane et al., 1996). To measure
the fluid changes of epiboly, we staged
clutches at the eight-cell stage; as the
first embryos of the clutch reached the
100% epiboly stage, the phenotypes of
the remainder of the clutch were
quickly scored as to the degree of epiboly using the photographs in Figure
2A as guides. Afterward, we genotyped the embryos based on the segregation of closely linked markers that
flank the hab locus.
Analysis of three of the “wild-type”
strains maintained in the laboratory
showed that, as the first embryos
reach the end of epiboly, less than 10%
were delayed, and in the puma strain,
the usual outcross strain for the epiboly mutants, less than 2% are delayed (Fig. 2B). Analysis of the weg
allele showed that the mutant phenotype was completely recessive and
that no slowing of epiboly could be
seen in either heterozygous or homozygous wild-type embryos (Fig.
2C). Interestingly, the early dissociation phenotype of the weg⫺/⫺ mutant
is not evident in this work; this represents a shift in the phenotype of the
mutant, perhaps caused by moving
the allele to the puma background.
(The name “Weg” originates from a
German idiom for “gone” or “disappeared”.)
When homozygous, the epiboly alleles expressed a slowing of epiboly
followed by a retraction of the blastoderm (Fig. 2D,E). In these cases, the
mutants attained approximately 60 to
70% epiboly and then their blastoderms begin to retract. This phenotype was less severe in ava and law
compared with hab and only rarely
expressed in the weg phenotype. During this process, many embryos dissociated before completing epiboly, giving the “0” phenotype in Figure 2. This
dissociation phenotype was variable:
typically the blastoderm peeled off the
yolk cell, but sometimes large numbers of cells detached from the blastoderm and the yolk cell lysed. When
heterozygous, the ZMD mutants also
expressed a slowing of epiboly, reaching 70% to 90% epiboly when wildtype siblings were completing epiboly;
however, in these cases, the mutant
blastoderm did not retract, and the
half baked ECTODERM 393
Fig. 1. Mapping of the half-baked locus. A: Mitotic map for Linkage Group 7 constructed from haploid analysis using a panel of 2,916 individuals.
B: Mitotic map constructed from half-tetrad analysis using a panel of 125 individuals. Numbering begins at the centromere and proceeds along each
arm. C: Order of microsatellite markers and location of mutant loci. Numbers ⫾ standard deviation indicates recombination events between individual
markers or between markers and mutation. Black boxes indicate location of individual mutant loci and size of 95% confidence limits inferred from
numbers of recombinants. The haploid mapping panel sizes are as follows: hab, 222; ava, 90; law, 163; and weg, 114. Markers not shown were
monomorphic. D–F: Testing linkage of candidate genes to the hab locus by polymorphism in the haploid mapping panel. The first lane in each gel is
a molecular weight marker, except in F, and the phenotype of each haploid individual is indicated above its lane on the gel. Arrows indicate
recombination individuals for the hab locus and the candidate. D: Mapping of ventral neural cadherin (VNcad) to the hab locus using the size
polymorphism in microsatellite marker Z3008. PAC clone 30C24 contains VNcad (not shown) as well as microsatellite marker Z3008, whereas a control
PAC clone, 133F4, does not (lanes 2 and 3). E: Mapping of fibroblast growth factor4 (fgf4) to the hab locus using the size polymorphism in expressed
sequence tag AI957973 on the recombinants from the hab haploid panel. Cutting with AciI revealed a restriction fragment length polymorphism
between hab and wild-type embryos (lanes 3 and 4). F: Mapping of fibroblast growth factor3 (fgf3) to the hab locus using a single-stranded
conformation polymorphism. WT, wild-type.
mutants slowly completed epiboly.
ZMD mutants only rarely dissociated.
The ZMD epiboly phenotypes of hab
was the most severe of all the dominant alleles (compare D to E in Fig. 2);
indeed, the similarity between the
phenotype distributions of the ZMD
hab⫺/⫹ mutant to the simple recessive weg⫺/⫺ mutant is striking. Such
correlations were also seen among the
neural tube phenotypes of the mutants, e.g., the dominant phenotype of
hab is more severe than that of law
and ava. However, there was a curious
exception to this correlation of severity: whereas ava⫺/⫺, law⫺/⫺, and
weg⫺/⫺ mutants never survive past
the five-somite stage, some hab⫺/⫺
394 MCFARLAND ET AL.
Fig. 2. The epiboly phenotypes of the epiboly
mutant alleles. Comparison of epiboly phenotypes between the different epiboly mutants,
scoring the range in epiboly defects observed
within a single clutch at 100% epiboly stage,
grouping embryos by genotype. A: Photographs
of “guide embryos” used for scoring. Phenotype
“0” indicates that embryos dissociated before
100% epiboly, usually because the blastoderm
peeled off of the yolk cell. B: Variation in epiboly
of wild-type (WT) strains. C: Variation of the weg
allele. D: Variation of the hab allele. E: Variation
of the ava allele. Note that, in C, D, and E, the
results are compiled from multiple clutches, and
the embryos are selected before genotype or
phenotype is known. Note that the expected
ratio is 1:2:1 for wild-type: ZMD heterozygotes,
zygotic maternal dominant homozygotes.
Fig. 3.
half baked ECTODERM 395
embryos survived through segmentation stages and did not dissociate until
after 24 hr.
Note that, for all of the epiboly-dominant alleles, the wild-type progeny of
heterozygous females expressed a
slight slowing of epiboly (Fig. 2), a
dominant maternal effect. Regardless
of the genotype, progeny from heterozygous males and homozygous
wild-type females never expressed a
slowing of epiboly (data not shown).
half baked Mutants Have a
Widened Dorsal Axis in
Ectodermal Structures
To determine whether the epiboly arrest was the result of a misallocation
of cell fate, we examined patterns of
RNA expression in wild-type and mutant embryos during the epiboly and
segmentation stages, a period extending from approximately 4 to 20 hr of
development. Fate map experiments
using lineage tracing techniques were
not attempted, because the early lethality of mutants hindered documentation. Our studies can be roughly divided into an analysis of germ layer
specification during gastrulation and
analysis of tissue differentiation during somitogenesis. Note that, in all the
experiments, the wild-type embryos
are siblings of the mutant embryos
and were produced from heterozygous
females. However, we have not found
any differences in either such wildtype embryos or ZMD heterozygote
embryos before 75% epiboly, and we
believe that the expression patterns in
these embryos appear completely
wild-type.
To investigate axial tissues from all
three germ layers, we examined the
expression of goosecoid (SchulteMerker et al., 1994a; Thisse et al.,
1994) and axial/forkhead1/FoxA2
(Strähle et al., 1996; Odenthal and
Volhard-Volhard, 1998) gene products. At 40% epiboly, goosecoid expression in the dorsal axis was identical between hab⫺/⫺ and wild-type
embryos (Fig. 3A), indicating that the
axial layers of the mutant were normal in early epiboly. However, by late
epiboly, the expression pattern of
FoxA2 (Fig. 3B) was noticeably
shorter anteroposteriorly and slightly
wider mediolaterally compared with
wild-type siblings. In contrast, the
nonaxial endodermal expression domain of axial appeared normal.
We examined ectodermal fates in
hab⫺/⫺ mutants using the expression
of gata2 (Read et al., 1998), forkhead
3/mariposa/FoxB1 (Odenthal and Volhard-Volhard, 1998; Varga et al.,
1999), and sonic hedgehog (Krauss et
al., 1993) gene products. At 75% epiboly, gata2 is expressed in the ventral
non-neural ectoderm; this domain appeared smaller yet more-intensely
stained in hab⫺/⫺mutants (Fig. 3C).
sonic hedgehog, which is expressed in
the ventral neuroectoderm, revealed
related but more-severe defects. There
was a failure of the posterior neuroectoderm to reach the midline, causing
the axis to be bifurcated posteriorly,
with each posterior half wrapping
around the yolk cell at the approximate location of the germ ring (Fig.
3D). Also, expression of sonic hedgehog was absent anteriorly, detected in
what is normally the forebrain region
(compare Fig. 3D with 3F), indicating
perturbations in anterior forebrain
patterning. FoxB1 expression, which
is expressed in the dorsal neuroectoderm, reveals that neural fates were
mediolaterally wider in hab⫺/⫺mutants by late epiboly (Fig. 3E) and
markedly wider by tail bud (Fig. 3F).
Altogether, in the ectoderm, there is a
general impression of a failure of convergence to move fate domains toward
the dorsal side of the embryo. Also,
subtle defects in patterning at the animal pole may be present.
We examined mesodermal tissues
with the expression of no tail, the homologue of the Brachyury gene
(Schulte-Merker et al., 1994b), spadetail (Griffin et al., 1998), and its downstream target gene paraxial protocadherin (Yamamoto et al., 1998).
Expression of no tail mRNA illustrated that the notochord anlage
was shorter anteroposteriorly in
hab⫺/⫺mutants by 75% epiboly (Fig.
3G). By tail bud, these defects were
more marked (Fig. 3H) and the notochord anlage was slightly wider mediolaterally. Complementary defects
were seen in the paraxial mesoderm
with the expression of both spadetail
(Fig. 3I,J) and paraxial protocadherin
(Fig. 3K,L).
The expression of no tail (Fig. 3G,H)
also revealed a defect in a population
of cells, termed the dorsal forerunner
cells, that normally migrate as a cohesive group just vegetal of the deep cell
margin (Cooper and D’Amico, 1996;
Melby et al., 1996). It has been documented that at least a portion of these
forerunner cells appear to migrate
ahead of the blastoderm in volcano
mutants, a weg-like mutant isolated
in the Boston Screen (Solnica-Krezel
et al., 1996). We found that, rather
than forming a single group of cells,
forerunner cells in the hab mutants
coalesce into multiple clusters (asterisks in Fig. 3G,H⬘), which migrate
ahead of the blastoderm margin. Interestingly, the forerunner defect segregates as a maternal phenotype: all
embryos derived from habdtv43/⫹ females, regardless of genotype, have
Fig. 3. Specification in hab mutants before and during gastrulation. In the following experiments, all hab⫺/⫹ embryos are zygotic maternal dominant
(ZMD) embryos; during these stages, in situ analysis cannot distinguish between the wild-type and dominant phenotypes. Embryos were genotyped
after completion of photography as outlined in the Experimental Procedures section. All embryos are dorsal views with the animal pole to the top, and
arrows indicate the width of the axial expression pattern, except C, which is a side view with dorsal to the right and arrows indicate the anteroposterior
extent of expression. Arrowheads indicate the edge of the blastoderm margin in mutant embryos. A: Expression of goosecoid, 40% epiboly, in
wild-type and hab⫺/⫺, dorsal view. B: Expression of FoxA2/axial/forkhead 1, 75% epiboly in wild-type and hab⫺/⫺. C: Expression of gata2, 75% epiboly
in wild-type and hab⫺/⫺ (C). Arrows indicate the expression domain. D: sonic hedgehog, tail bud stage in wild-type and hab⫺/⫺. Note the absence of
expression in the anterior neural plate of the mutant. E,F: FoxB1/mariposa/forkhead 3, 75% epiboly and tail bud stage in ZMD hab⫺/⫹ and hab⫺/⫺. E⬘
and F⬘: Arrows indicate widening of the midline in the mutants. F⬘: Note gaps in the midline and absence of anterior forebrain expression. G,H: no tail,
75% epiboly and tail bud stage in wild-type and hab⫺/⫺. Ectopic forerunner cell clusters are indicated by asterisks. Wild-type embryos in G are from
hab⫺/⫹ mothers. I,J: spadetail, 75% epiboly and tail bud stage in wild-type, ZMD hab⫺/⫹ and hab⫺/⫺. K,L: paraxial protocadherin, 75% epiboly and
tail bud stage in wild-type, ZMD hab⫺/⫹and hab⫺/⫺. Note that, in the hab⫺/⫺ mutants, expression of both spadetail and paraxial protocadherin in the
prechordal plate is retained longer.
396 MCFARLAND ET AL.
multiple forerunner clusters. This
finding is shown in a homozygous
wild-type embryo in Figure 3G.
We examined the effects of the hab
mutation on later development using
the expression patterns of the gene
products hatching gland gene (Thisse et
al., 1994), Krox 20 (Oxtoby and Jowett,
1993), forkhead 6/FoxD3 (Odenthal and
Volhard-Volhard, 1998), alpha-collagen
2a (Yan et al., 1995), deltaA (Appel and
Eisen, 1998; Haddon et al., 1998), and
Zn-12 (Trevarrow et al., 1990), all of
which mark differentiated tissues. The
Fig. 5.
Fig. 4.
Fig. 6
half baked ECTODERM 397
expression of hatching gland gene in the
hatching gland cells of the prechordal
plate of hab⫺/⫺mutants at the fivesomite stage indicated that these cells
have begun to differentiate normally.
However, the area of expression was
larger and the borders uneven, and often, there were ectopic cells posterior to
the normal region of expression (Fig.
4A). Such changes in distribution may
be related to the semidominant enlarged hatching gland trait in heterozygous siblings at 24 hr. Krox 20 expression in rhombomeres 3 and 5 of the
mutant also revealed that segmentation of the hindbrain was occurring
properly. Similarly, at later stages, expression of forkhead 6/FoxD3, alphacollagen 2a, and deltaA (Fig. 4B–D)
revealed that somites, floor plate, notochord, hypochord, and many specific
neuroectodermal structures differentiate properly, albeit within the context of
the arrested epiboly phenotype. For example, somites and spinal cord, which
have lateral and ventral progenitors, always bifurcated and extended both
ways around the equator of the embryo.
However, the notochord, which originates from midline progenitors, extended randomly to either side of the
midline, along the equator. Moreover,
in surviving 1-day hab⫺/⫺ mutants,
some individual neurons formed axon
tracks extending around this germ ring,
revealed by the Zn12 antibody (Fig. 3D).
These neurons must form normal synapses on muscle tissue, for such survivors display normal rhythmic twitching
movements.
In summary, although epiboly is arrested during gastrulation, the effects
on specification of germ layers or differentiation of tissues in hab⫺/⫺ mutants are subtle. Indeed, the differentiation of posterior structures occurs
in situ at positions reminiscent of
their origin in the fate map. The slight
perturbations observed in spatial patterns, such as the shortened but widened axis, appear to be the result of
morphological changes of two sorts in
mutant embryos: the block in epiboly,
which stops the vegetalward extension of tissues around the yolk, and a
reduction in convergence movements,
which broadens the axis mediolaterally. Moreover, the defects in convergence seem to appear earlier and be
more severe in the epiblast than in the
hypoblast.
Ectoderm Is Perturbed in
the Enlarged Hatching
Gland
hab⫺/⫹ mutants exhibit an enlarged
and ragged hatching gland, a trait
that segregates as a semidominant
phenotype (Fig. 5A,B). In the zebrafish, the hatching gland is composed of two cell populations: meso-
dermally derived hatching gland cells,
which express the hatching gland
gene (hgg), and ectodermally derived
support cells (Kimmel et al., 1990;
Thisse et al., 1994). Because the number of hatching gland cells is unaffected in hab⫺/⫹ mutants (Kane et al.,
1996), it is possible that the defect is
due to loss of hab function in the ectodermal portion rather than the mesodermal portion of the hatching gland.
Therefore, we made double mutants of
hab to one-eyed pinhead (oep), a mutation that perturbs mesendoderm
formation, lacks the prechordal plate,
and lacks hatching gland cells (Hammerschmidt et al., 1996; Schier et al.,
1997a; Strähle et al., 1997; Warga and
Kane, 2003). At 24 hr of development,
we examined the embryos for the presence of the hatching gland defect in
the double mutants, individuals that
should be devoid of hatching gland
cells. From a cross of an oeptz257/⫹;
hab⫹/⫹ female to an oeptz257/⫹;
habdtv43/⫹ male, one quarter of the
progeny segregated with the oep phenotype, and half of these, the expected
number, displayed the enlarged
hatching gland-dominant phenotype
(Fig. 5A–D). All the embryos were assayed for hgg expression to verify the
lack of hatching gland progenitors.
Siblings from both of the oep classes
lacked hgg-positive cells (Fig. 5C⬘,D⬘),
including those that displayed the hab
Fig. 4. Differentiation in hab mutants after gastrulation. Embryos were genotyped after completion of photography as outlined in the Experimental
Procedures section. A: Expression of hatching gland gene and Krox20, five-somite stage in wild-type (A) and hab⫺/⫺(A⬘), dorsoanterior view. Note
ectopic hatching gland cell (arrow) below pollster in the mutant. B: FoxD3/forkhead 6, 10-somite stage in zygotic maternal dominant hab⫺/⫹ and
hab⫺/⫺. Side view except for B⬙, which is a dorsal view. Note weak expression in mutant tail bud. Dorsal view shows bilateral somites (indicated by
asterisks) forming on either side of the germ ring. C: alpha-collagen2a, 14-somite stage in wild-type (side view) and hab⫺/⫺ (dorsal view of kinked
notochord). C⬘: Higher magnification image of the boxed regions. D: delta A,18-somite stage in wild-type (dorsal view, D) and hab⫺/⫺ (dorsal view and
side view, D⬘). E: Zn12 antibody, 24 hr in wild-type (E) and rare hab⫺/⫺ escaper (E⬘), dorsal view. Note bifurcation of axis in mutant. cg, cranial ganglia;
fp, floor plate; hb, hindbrain; hcd, hypochord; llf, lateral longitudinal fascicle; mb, midbrain; ncd, notochord; ncr, neural crest; os, optic stalk; ov, otic
vesicles; pol, polster; r3, rhombomere3; r5, rhombomere5; rb, Rohan–Beard cells; sc, spinal cord; tbm, tail bud mesenchyme; tg, trigeminal. Arrows
indicate the width of the axis.
Fig. 5. half baked acts in the ectoderm of the hatching gland. A–D: Twenty-four hour progeny of oep/⫹ female ⫻ oep/⫹; hab/⫹ male: wild-type (A),
hab/⫹ (B), oep (C), and hab/⫹; oep (D). Note that hab/⫹ embryos are not zygotic maternal dominant mutants. A⬘–D⬘: Expression of hgg at 24 hr in
the hatching gland of similar embryos after in situ hybridization; ventral view. Arrows indicate semidominant enlarged hatching gland trait of the hab⫺/⫹
mutant. Note absence of expression in the oep mutants and the double mutants.
Fig. 6. half baked is not necessary for involution of the mesendoderm or epiboly of the epithelial-like enveloping layer (EVL). A,B: Cell movement during
involution, showing 30 min, approximately from germ ring to shield stage in hab and wild-type (WT) siblings. Face view recorded Z-stacks were
geometrically reoriented to appear in optical cross-section; EVL cell trajectories (thick yellow), yolk syncytial layer nuclei (thick blue– green), and deep
cells (thin multicolored; blue, 5 ␮m deep to red, 50 ␮m deep). Inset in B indicates location of recordings; arrows indicate general direction of movement
of like-colored tracings; ordinate (red numbers) are percentage epiboly. C: Animal pole view of a shield stage hab embryo showing thickness of the
germ ring. Slashes indicate location of measurements at dorsal (top), dorsolateral, and ventral positions on the blastoderm. D: Average ⫾ standard
error germ ring thickness in hab⫺/⫺ (clear), zygotic maternal dominant (ZMD) hab⫺/⫹ (red), and wild-type (green) embryos. E–H: Lateral views of
progeny from a sqt/⫹; cyc/⫹; hab/⫹ incross at tail bud stage: wild-type (E), sqt; cyc (F), hab (G), sqt; cyc; hab (H). Arrows indicate the extent of epiboly.
G⬘,H⬘: Dorsal views of G and H. I: Face view of EVL trajectories from four hab⫺/⫺ embryos. The interval is 30 minutes, and marks along the axes (red
numbers) indicate latitude (percentage epiboly) and longitude (degrees from dorsal). Inset in I indicates location of recording; arrow indicates direction
of movement. J: Average ⫾ standard error rate of EVL epiboly (y-vector) movement vs. time postfertilization in hab⫺/⫺ (open squares), ZMD hab⫺/⫹
(red triangles), and wild-type (green circles) embryos.
398 MCFARLAND ET AL.
enlarged “hatching gland.” This result
indicates that the hgg-positive mesodermal portion of the hatching gland
is not necessary for the enlarged
hatching gland phenotype, and
strongly suggests that hab is necessary in the support cells of the hatching gland, cells that are ectodermally
derived.
Involution of Marginal Deep
Cells and Epiboly of the
EVL Is Unaffected in hab
⫺/⫺
The block of epiboly in hab
mutants might result from an increase in
the number of cells that involute during epiboly as seen in the lefty1/2 antisense knockdown embryos (Branford
et al., 2000; Feldman et al., 2002). Although such a phenotype should cause
a marked reapportionment of cells between the ectoderm and mesendoderm—which is seen in the lefty1/2
knockdowns but not in hab⫺/⫺ mutants—we checked this possibility using four-dimensional (4D) Nomarski
time-lapse analysis. The involution of
marginal epiblast cells was recorded
just after the onset of involution. Because each time point contained a
stack of 10 to 15 focal planes, we could
compute the x, y, and z coordinates for
the nuclei of each individual cell, and
using these data, geometrically reorient the embryos to show a cross-section at the blastoderm margin. This
strategy is shown for a period of 30
min for the hab⫺/⫺ and wild-type embryos (Fig. 6A,B, and Supplementary
Figure S1, which can be viewed at
http://www.interscience.wiley.com/
jpages/1058-8388/suppmat). These experiments found no defects in the involution of marginal mesendodermal
cells (n ⫽ 3 hab and 3 wild-type embryos).
To quantify involution in a larger
number of embryos, we measured the
thickness of the germ ring at shield
stage (Fig. 6C), a method that was
used to demonstrate an increase in
the rate of involution in lefty1/2 antisense knockdown embryos (Feldman
et al., 2002). The thickness of the germ
ring was measured at the dorsal midline, as well as at 45 degrees and 180
degrees from dorsal. No significant
differences were found in the thickness of the germ ring between
hab⫺/⫺, ZMD hab⫺/⫹, and wild-type
embryos, regardless of location (Fig.
6D), indicating that involution and
mesendoderm formation are morphologically normal at this stage.
In squint; cyclops double mutants,
mesendodermal tissues fail to be specified normally and involution movements are absent. Also, the axes of
such double mutants are shortened
and do not extend to the animal pole.
Still, except for a small notch that
forms on the dorsal side, they complete epiboly normally (Feldman et
al., 1998, 2000). To test whether a reduction in involution movements affects the epiboly phenotype of hab⫺/⫺
mutants, we made triple mutants of
hab with squint and cyclops (Fig. 6E–
H). In the triple mutants, the absence
of involution had no effect on the hab
epiboly phenotype on the ventral and
lateral sides of the blastoderm (arrows
in Fig. 6G,H), suggesting that involution is not necessary in these regions
for expression of the hab phenotype.
However, on the dorsal side of the
triple mutants, the epiblast margin
arched toward the animal pole (Fig.
6H⬘) and the large gap was covered
only by the thin epithelium of the
EVL. This phenotype is a very strong
enhancement of the notching phenotype expressed in the sqt; cyc double
mutants at the same stage (not
shown). The widening of the notch on
the dorsal side may be related to the
perturbations in mesendodermal cell
fates in the triple mutants; however, it
also may be due to an impairment of
convergence. In any case, in the local
region of the dorsal side, squint; cyclops function is necessary for the normal hab phenotype and lack of squint;
cyclops enhances the hab⫺/⫺ epiboly
defect.
Using low-power magnification recordings of the shape of the embryo,
we previously noted that the yolk cell
continued epiboly normally in hab
mutants, and by inference, that the
EVL was normal as well (Kane et al.,
1996). To confirm directly that the
EVL was moving normally over the
epiblast, we analyzed the epiboly of
this layer in hab⫺/⫺, ZMD hab⫺/⫹,
and wild-type embryos using 4D Nomarski time-lapse analysis. After recording, analyzing, and geometrically
re-orienting the embryo with the animal pole to the top as described above,
we measured the rate of migration of
cells at approximately the equator of
the blastula. A face view is displayed
in Figure 6I, showing the combined
trajectories of EVL cells from four different hab⫺/⫺ mutants. We could discern no difference in the rate of EVL
epiboly between the 50% and 90% epiboly stages in hab⫺/⫺, ZMD hab⫺/⫹,
and wild-type embryos (Fig. 6J),
which agrees with our earlier observations (Kane et al., 1996).
Abnormal Neural Tube of
the ZMD Phenotype
ZMD hab⫺/⫹ mutants often exhibit
gaps along the midline of the neural
keel, with dorsal detached cells rounding up and accumulating along the
trunk and tail of the embryo in the
early segmentation stages (Kane et
al., 1996). These dorsal detached cells
are a very reliable indicator of the heterozygous genotype and, in certain
backgrounds, are useful for sorting
embryos to be raised for stocks. Although subtle, other aspects of the
Fig. 7. Convergence and extension of the neural axis is defective in zygotic maternal dominant (ZMD) heterozygotes. A,B: Wild-type (WT,
A) and severely affected (B) ZMD ava⫺/⫹ sibling
embryos at 10-somite stage; side view; white
arrowheads demarcate the head to tail distance
of the embryo. A⬙,B⬙: Dorsal view focused deep,
showing the neural and somitic tissue in optical
cross-section. A⬘⬙,B⬘⬙: Dorsal view focused
shallowly, showing the width of the neural (arrows) and somitic (arrowheads) tissue along the
mediolateral axis. A⬘,B⬘: Outline of the notochord (n), neural keel (nt), and somites (s), as
traced from view in orientation (A⬙,B⬙), and of
the eye (e), as traced from view in orientation A
and B. C: Expression of N-cadherin (cdh2), in
wild-type (C) and ZMD hab⫺/⫹ (C⬘) embryos,
10-somite stage, dorsal view. Arrows indicate
width of the neural tube and arrowheads width
of somitic mesoderm. White arrowhead points
out the gap in the midline of the neural tube.
D,E: Expression of deltaA in wild-type (D) and
ZMD hab⫺/⫹ (E) embryos, 10-somite stage, dorsal view. Arrow indicates an extra row of cells in
the midline of the neural tube, and the arrowhead points out the gap in the midline of the
neural tube. D⬘–E⬘⬙: Transverse cross-sections
as indicated for wild-type (D⬘,D⬙) and ZMD
hab⫺/⫹ (E⬘,E⬙,E⬘⬙). Outlines below the crosssections demarcate the boundaries of deltaA
staining in each neural cross-section. For reference, the dorsal detached cells and notochord,
which do not express deltaA, are also outlined.
F,G: Anti-acetylated tubulin staining (␣AT), in
wild-type (F) and ZMD hab⫺/⫹ (G) embryos, 24
hr, dorsal view. Arrows indicate extra rows of
cells in the midline of the neural tube.
half baked ECTODERM 399
ZMD neural tube defect could be identified in mid-segmentation stages. Before the tail bud begins to evert, when
the embryo is still wrapped around
the yolk cell, the head to tail distances
were longer in ZMD mutants and the
embryos correspondingly shorter (Fig.
7A,B). In ZMD siblings, rather than
the oval shape of the wild-type neural
tube (Fig. 7A⬙), the anterior neural
tube of the mutant appeared triangular in shape when viewed in deep optical cross-section and the dorsal portion of the neural tube was flatter
Fig. 7.
(Fig. 7B⬙) When viewed from the dorsal side of the embryo, the neural keel
appeared wider (Fig. 7B⬘⬙). Similar defects were also observed for the
somites, albeit to a lesser degree. Both
the widening of the neural tube and
the somites could also be visualized
with probes to N-cadherin mRNA
(Fig. 7C), which is expressed in the
neural tube and the somites at this
stage (Bitzur et al., 1994). This
marker further revealed the gaps that
form along the midline of the mutant
neural tube (Fig. 7C⬘, white arrow-
head), a phenotype not observed in the
convergence mutants trilobite, silberblick, or knypek. In ZMD law⫺/⫹ and
ZMD ava⫺/⫹ mutants, the deformities
in neural tube morphogenesis, as well
as the detached cell phenotype, occurred most strongly near the tail, approximately at the location of the yolk
plug, suggesting a connection between
the retardation of epiboly and the neural tube defects. However, in ZMD
hab⫺/⫹ mutants, these defects were
much more extensive and often occurred the entire length of the neural
Fig. 8. half baked acts autonomously and nonautonomously. A: Experimental approach for moving wild-type (WT) cells into zygotic maternal
dominant (ZMD) ava⫺/⫹ embryos. B–D: Three examples of wild-type cells
transplanted into ZMD ava⫺/⫹ embryos. B⬘–D⬘: Chimeras are shown at the
10-somite stage, and boxes indicate the detached cell area, shown at
higher magnification. E: Experimental approach for moving ZMD ava⫺/⫹
and ava⫺/⫺ cells into wild-type embryos. Note that, in contrast to the
wild-type hosts, the wild-type donors are produced from ava⫺/⫹ parents.
F,G: Two examples of ava⫺/⫺ mutant cells transplanted into wild-type
embryos. F⬘,G⬘: Chimeras are shown at the 10-somite stage, and boxes
indicate the detached cell area, shown at higher magnification. In A, the
hosts were genotyped based on phenotype. In E, the genotypes of the
donors were determined as described in the Experimental Procedures
section.
400 MCFARLAND ET AL.
tube. Correlating with the severity of
the neural phenotype, other ectodermal structures, such as the eye and
ear placodes, tended to be smaller and
misshapen in more severe ZMD mutants (Fig. 7A⬘,B⬘).
The neural tube phenotype expresses
considerable variability among ZMD
mutants, both between the alleles
themselves and between individual
clutches of embryos. The variability of
the phenotype could be seen in survival
rates: whereas survival to adulthood
could be as high as 50% of the ZMD
mutants, usually it was less than 5%,
especially with the hab allele. Weak
ZMD law⫺/⫹ and ZMD ava⫺/⫹ mutants slowly recovered, so that the neural tube defects were relatively mild by
24 hr. Most of these embryos survived
to adulthood; in a manner, this finding
is similar to the silberblick homozygote,
which suffers mild defects in dorsal convergence but later recovers (Heisenberg
et al., 2000). In aquaculture, we have
noticed no apparent changes in the behavior of the adult heterozygotes, e.g.,
in feeding or evasion to capture.
In ZMD hab⫺/⫹ individuals that
displayed stronger phenotypes, we examined the pattern of deltaA (Appel
and Eisner, 1998; Haddon et al.,
1998), which at 24 hr, labels most neurons weakly and nascent neurons
strongly. This experiment showed
that the neural tube of the strong
ZMD mutant was much broader than
normal (Fig. 7D,E), especially in the
trunk and tail regions of the axis.
Moreover, the width of the neural keel
was variable within individual embryos, sometimes being 50% wider in
some regions compared with others.
Closer examination of the cells that
strongly express deltaA revealed dramatic disorganization of the neural
tube, which can also be seen in hand
sections through various levels of the
axis. In particular, there were often
ectopic rows of cells in the midline of
the neural tube (Fig. 7E). Staining
with an antibody to acetylated tubulin, which visualizes differentiating
neurons, confirmed these observations
(Fig. 7F,G). Most of these strong ZMD
mutants do not survive; thus, it is unclear to what extent the neural tube
ultimately closes and, subsequently,
what morphological consequences
might be retained at the larval stage
of development.
Cell Autonomy of half baked
To determine the site and mode of hab
gene function, we moved cells of different genotypes by transplantation
either into ZMD mutants or wild-type
embryos. Previously, we had transplanted cells between hab⫺/⫺ and
wild-type embryos and then examined
the resulting chimerae during late
gastrula stages and at 24 hr. However, no differences were found between these experiments and their
controls (data not shown). In the following experiments, we focused our
analysis on the altered morphogenesis
of the neural tube in ZMD ava⫺/⫹ mutants, selecting heterozygous females
that produced embryos with extreme
dorsal detached cell phenotypes. We
used the ava allele for these experiments because of the local differences
where the neural tube phenotype was
expressed, i.e., in the tail and not in
the trunk, and because of the increased survival of the ZMD ava heterozygotes.
We first tested whether wild-type
cells transplanted into ZMD ava⫺/⫹
mutants could incorporate into the
dorsal detached cell cluster (Fig. 8A).
Of a total of 21 ZMD ava⫺/⫹ mutant
chimerae, 7 of these contained wildtype donor cells within the detached
cell clusters (Fig. 8B–D). In these
cases, donor wild-type cells were always intermingled among the mutant
cells (Fig. 8B⬘–D⬘). The mutant dorsal
detached cells must have originated,
at least in part, from the deep cell
domain, because EVL cells are not
moved or created in transplant operations (Ho and Kimmel, 1993). Moreover, in these experiments, wild-type
cells were capable of acquiring aspects
of the heterozygous phenotype when
transplanted into ZMD ava⫺/⫹ mutants, a nonautonomous result.
To test whether ava mutant cells
could integrate into tissues of a wildtype host, we moved cells from embryos derived from an incross of heterozygous parents into wild-type
embryos (Fig. 8E). Of 24 chimerae, 6
contained ava⫺/⫺ mutant cells, and 2
of these had small clusters of dorsal
detached cells (Fig. 8F,G). Whereas
the detached cells were composed primarily of mutant donor cells, in both
cases, they included wild-type cells
from the host. At 24 hr, the remaining
mutant donor cells (those that did not
detach) were found primarily in ectodermally derived tissues. These results suggest that the ava gene product can act autonomously, most
probably in the ectoderm, to produce
the detached cell phenotype. The mutation also acts nonautonomously to
recruit wild-type cells into the detached cell cluster. We also observed
that the mutant donor cells can integrate normally into the host embryo, a
result that has been noted previously
(Kane et al., 1996).
DISCUSSION
half baked locus
Our mapping of the epiboly mutants
places the genes near the centromere
of Linkage Group 7. This region is in a
neighborhood of many important
genes that act in early development,
including sonic you (sonic hedgehog),
engrailed 2a, cyclin E, cyclin D1,
FGF3, and FGF4, sox17, tc4, apolipoprotein Eb, achaete-scute complexlike 1b, and ubiquitin-conjugating enzyme E2I (Postlethwait and Talbot,
1997; Shimoda et al., 1999; Postlethwait et al., 2000; Woods et al., 2000).
We propose that the epiboly mutants
are at a single locus, termed the half
baked locus, named after the first mutant isolated. The mutations must be
in a single gene or in a complex of
closely linked, functionally similar
genes. If in a single gene, there are
three arrays of phenotypes that have
been separated previously on the basis of their dominant phenotypes: weg,
which has no dominant phenotype;
law and ava, which have ZMD phenotypes; and hab, which has an extreme
ZMD phenotype and partial dominant
phenotypes. In this report, we have
added to these characterizations,
showing that these three arrays of
phenotypes extend to expression of
the recessive epiboly phenotype.
Although not the normal case, Drosophila has many loci that have multiple phenotypes. For example, the
Notch locus has an intimidating array
of diversely named alleles, including
deletions that express dominant
haplo-insufficient phenotypes, and
nucleotide transversions some that
express dominant antimorphic phenotypes and some that express recessive
hypomorphic phenotypes, and re-
half baked ECTODERM 401
markably, some of these alleles even
complement each another. In fact,
even after the nucleotide transversions of alleles at the Notch locus are
identified, it takes a complex series of
genetic tests to sort out the hypomorphic and hypermorphic natures of
each allele (Go and Artavanis-Tsakonas, 1998).
If a single gene, how would the epiboly alleles be ordered? One possibility is that hab, lab ⫽ ava, and weg
represent a series of hypomorphic alleles, with hab being the strongest, an
explanation based on the hypothesis
that the hab locus would be haploinsufficient. In this case, the hab allele itself may not be null, for true null
alleles may be heterozygous lethals
and not represented in the mutant collection. Another possible hypothesis is
that weg is a hypomorph and law, ava,
and hab are hypermorphic gain-offunction alleles, which are normally
dominant. At the present time, we
cannot distinguish among these or
other possibilities.
The combination of recessive and
ZMD phenotypes forms an impressive
phenotypic series, and such a series is
a useful genetic tool in judging the
response of the hab locus to other mutants or treatments. In order of increasing severity, the recessive mutants would be ordered as weg, ava ⫽
law, and hab. The ZMD mutants
would be ordered as ZMD ava ⫽ ZMD
law, and ZMD hab. However, because
the ZMD hab appears similar to the
weg recessive, the entire series, from
weakest to strongest, would be ZMD
ava ⫽ ZMD law, ZMD hab ⫽ weg,
ava ⫽ law, and hab.
half baked Has Minor
Effects on Tissue
Specification and
Differentiation
Based on in situ hybridization with a
variety of markers encompassing all
three germ layers, specification appears largely unaffected in hab mutants. Hence, hab falls into a class of
mutants such as trilobite and knypek,
which are necessary for morphogenesis of the embryo but tend to have
subtle secondary effects on other developmental processes. For example,
changes such as the shortened and
widened axis are a likely consequence
of a slowing of convergence. Similar
changes in gene expression were seen
in volcano, another epiboly mutant
not yet shown to be allelic to hab
(Solnica-Krezel et al., 1996). Because
of the epiboly arrest in hab mutants,
lateral and posterior tissues differentiate in situ around the germ ring,
reminiscent of their origins in the
early gastrula stage fate map (Kimmel et al., 1990; Warga and VolhardVolhard, 1999). Moreover, the defects
in the morphogenesis of the neural
tube in the ZMD mutants indicate
that hab also acts in later types of
cell movement, because convergence
movements are necessary during neural tube formation (Keller et al., 1992;
Schmitz et al., 1993; Kimmel et al.,
1994; Papan and Campos-Ortega,
1994; Elul et al., 1997; Concha and
Adams, 1998; Goto and Keller, 2002).
Differentiation of tissues around
the germ ring brings to mind the curious idea of “concrescence,” proposed
for the teleost embryo by T.H. Morgan
(Morgan, 1895), who suggested that
the axis of the embryo formed independently as two separate fields, and
this “germ ring” then zippered together at the midline, beginning at
the shield, by the combined actions of
epiboly and convergence of lateral regions to the posterior dorsal midline.
In mutants such as hab, where there
is a cessation of deep cell epiboly, the
two posterior halves of the body axis
do indeed develop independently of
each other on separate halves of the
yolk cell, lending support to such a
hypothesis. Interestingly, these germ
ring tissues of the hab mutants lack
important interactions normally obtained at the midline; consequently,
they are not normal in terms of size or
shape.
Relationship Between
Epiboly and Other
Morphogenetic Movements
Besides the widened axial structures
shown in our in situ analysis, the results with the sqt; cyc; hab triple mutants lend further support to the idea
that convergence is slowed in hab mutants. This experiment, done to examine the effects of the ablation of involution on the hab phenotype as
discussed below, unexpectedly revealed that the phenotype of the triple
mutant expressed a large gap on the
dorsal side of the epiblast. Formally,
this gap is a local slowing of epiboly
and demonstrates that sqt; cyc function is necessary on the dorsal side of
the gastrula to compensate for the
lack of hab function. In sqt; cyc mutants, the Nodal pathway is down-regulated, causing the ablation of the majority of the mesendoderm. Moreover,
the residual morphogenetic movements on the dorsal side seem affected
more severely than elsewhere in the
double mutant, with dorsal cells moving away from the midline in a seemingly haphazard way (Warga and
Kane, 2003). Nevertheless, lateral
cells persist in their movement toward
the dorsal side, creating a large
mound of cells centered approximately in the position of the future
hindbrain. This finding suggests that,
in the double mutants convergence, a
lateral cell behavior is normal, but, in
the triple mutants, it is not. An absence of convergence on the lateral
side (from the hab phenotype) eliminates the “pile of cells” on the dorsal
side, and in conjunction with the abnormal behavior of cells on the dorsal
side (from the sqt; cyc phenotype), a
large gap forms at the midline. No
such gap forms in the hab mutant.
Therefore, Nodal-dependent movements must compensate for the lack of
hab function on the dorsal side of hab
mutants. Altogether, the analysis of
the triple mutants suggests two
points. First, an interconnection may
exist between the hab and Nodal pathways or the movements they control.
Second, some of the components of
morphogenesis that power epiboly on
the dorsal side may be different than
those on the lateral and ventral sides.
Disruption of convergence would be
an ideal candidate to explain an epiboly phenotype where the epiblast is
slowed but the EVL and yolk are not.
Convergence in the epiblast is thought
to be powered by mediolateral intercalation, a movement that would act autonomous to the tissue that it shapes.
In Xenopus, late convergence movements were suggested to be sufficient
to drive blastopore closure (Keller et
al., 1985), and indeed, experiments in
Xenopus suggest an interconnection
between convergence and extension
movements and epiboly. For example,
the overexpression of Xror2, a recep-
402 MCFARLAND ET AL.
tor tyrosine kinase that affects convergence movements through the noncanonical Wnt pathway, also causes an
epiboly arrest (Hikasa et al., 2002). In
contrast, fish mutants that specifically slow convergence—many of
which disturb the WNT signaling
pathway— do not slow epiboly. However, this comparison is not completely appropriate, as the convergence defects in these convergence
mutants tend to occur toward the end
of or after the epiboly period.
Convergence could help propel the
latter advance of epiboly through a
type of purse-string movement: the
forces acting in the epiblast, which
move cells dorsally, also could help to
contract the marginal region of the
blastoderm, pinching the blastoderm
over the ventral yolk cell, engulfing it
(Keller and Trinkaus, 1987; Fink and
Cooper, 1996). Indeed, a cortical belt
has been observed in the zebrafish
EVL, which could facilitate such a
model (Zalik et al., 1999). Although no
such belt has been found in the epiblast, the cells of the ventral side of
the embryo are aligned parallel with
the blastoderm margin, as if under
tension, and when they divide, their
divisions tend to align along those presumed lines of force (Concha and Adams, 1998). Hence, if chains of cells
wrapping around the ventral side of
the blastoderm are the string of the
purse, it is possible that such a mechanism might be operating in the epiblast of the blastoderm.
A model we have discarded is one
where the epiblast is somehow attached to the EVL–yolk junction and
pulled along by means of the yolk cell
motor of Trinkaus (1951). This process
would involve a connection between
the site where involution into the hypoblast is occurring in the blastoderm
and the site where the EVL attaches
to the yolk cell membrane. Because of
the complexity of this interaction,
comprising cells that are participating
in an epithelial to mesenchymal transition as they move from the epiblast
into the hypoblast, it is difficult to
speculate on the myriad of attachment
possibilities at this particular location. Nevertheless, this model was
considered based on the analysis of
Lefty1/2 morpholino knockdown embryos (Feldman et al., 2000), which
magnify involution and slow epiboly,
notwithstanding that, in this case, the
balance between epiblast and hypoblast is extremely upset, and the slowing effect on epiboly could actually result from the diminutive epiblast. We
find that involution and epiboly seem
independent, based on several experiments and observations. First, when
involution movements were examined
in the hab mutants, they are normal.
Second, the expression of mesoderm
and endodermal markers is normal in
hab mutants, suggesting that the involuted germ layers contain the normal amount of tissue. Third, mutants
that completely block involution such
as squint/cyclops double mutants
(Feldman et al., 2000) or MZoep mutants (Gritsman et al., 1999; Carmany-Rampey and Schier, 2001) have
no effect on epiboly, and we have
shown here, using double- and triplemutant combinations of these mutants with hab, that the mutant involution phenotype is independent of the
hab epiboly phenotype, at least on the
ventral side. Last, in ZMD heterozygotes, where the zone of involution
lags far behind the yolk–EVL junction, the blastoderm is slowed but it
continues to move vegetally, suggesting that it is not necessary that involution occur at the EVL–yolk junction,
as one might imagine for the involution zone to be towed.
Widened Neural Tube: The
Result of Lack of
Convergence?
Compared with the wild-type neural
tube, the ZMD mutant neural tube is
up to 50% wider. The hab homozygote
neural plate is wider still, at least in
the head regions, and posterior of the
hindbrain region, the neural plate bifurcates along the midline with both
halves extending to opposite sides of
the arrested germ ring. There are interesting consequences on cell specification in both of the mutants. In the
ZMD mutant, ectopic rows of neurons
form near the midline, suggesting
that the widened neural tube has perturbed the short-range morphogenetic
fields that pattern the neural tube. In
the case of the bifurcated neural tube
of the homozygous mutant, only one
side receives the proper signals from
the notochord, which only extends
along one side. In both cases, studies
of the changes in fate made in the
forming neural tube will be instructive as to pattern formation in the
CNS.
Lack of cell convergence in the epiblast is the most likely explanation for
the neural tube defects in both the hab
homozygote and the ZMD heterozygote. Normally, cells of the lateral regions of the neural plate converge to
the midline and then sink into the
forming neural keel. If either of these
processes do not occur, then the neural plate would remain mediolaterally
splayed out. Defects in such a movement would be autonomous to the
neural tube. It seems that the cells of
the neural tube must be defective in
cell adhesion, because based on our
transplantation experiments in the
ZMD mutants, the detached cells originate from the neural tube.
half baked Acts in the
Ectoderm, Autonomously
and Nonautonomously
One objective of these studies was to
pinpoint the location where hab is required. We now favor the hypothesis
that hab is acting in the epiblast of the
gastrula, the future ectoderm, an idea
supported by our epistasis experiments with one-eyed pinhead (oep)
and squint; cyclops, and our transplantation experiments. oep mutants
lack hatching gland cells (Hammerschmidt et al., 1996; Schier et al.,
1997b; Strähle et al., 1997). Here, we
show that, when the semidominant
hab⫺/⫹ is genetically combined with
oep, which lacks mesodermally derived hatching gland cells, the double
mutant retains the enlarged “hatching gland” phenotype in the absence of
the mesodermal component. Hence,
the normal function of hab must be
required in the ectodermally derived
support cells of the hatching gland
and not in the mesodermally derived
cells. In the case of the hab; squint;
cyclops triple, the mutant lacks almost all mesoderm. There is no hypoblast apparent and little involution, if
any, occurring at the margin of the
epiblast. Yet, the hab epiboly arrest
phenotype is unchanged, again suggesting that the formation and presence of the hypoblast is not required to
express the mutant phenotype.
Also, the cell transplantation exper-
half baked ECTODERM 403
iments indicate that hab is acting in
the epiblast. In this work, we report
on the behavior of cell transplantations into ZMD mutants, focusing on
the presence of wild-type donor cells
in the dorsal detached cell clusters.
These experiments illustrate that the
detached cells of the ZMD hab⫺/⫹ mutants are most probably of ectodermal
origin, because this is the predominant fate of the remaining transplanted cells in these particular experiments. This finding was the
logical expected result, as ectoderm is
the most superficial germ layer. The
experiments further demonstrate that
the dorsal detached cells are not derived exclusively from EVL cells, because, in our experience, EVL cells are
never transplanted in these types of
experiments.
Because the transplanted wild-type
deep cells integrate into detached cell
clusters, mutant host cells must
change the fate of neighboring wildtype cells, a nonautonomous action.
On the other hand, when hab homozygous mutant cells are transplanted
beneath wild-type EVL cells, they do
not change their fate, but assume the
detached cell phenotype seen in the
ZMD heterozygote, an autonomous action. Similar mixes of results are seen
in oep (Warga and Kane, 2003), trilobite (Jessen et al., 2002), and glass
onion (Malicki et al., 2003), evoking
the idea that, like all of these other
genes, hab acts at or near the cell surface.
Whereas our experiments demonstrate that hab acts in the ectoderm,
the gene product of hab may be autonomously required in tissues other
than the epiblast. For example, the
defects seen in forerunner and prechordal plate morphogenesis may reflect a requirement for hab in the mesendoderm. Of interest, in both of these
cases, the tissues are more epithelial
in nature compared with the loose organization typical of the early mesendoderm. Hence, perhaps hab is autonomously required for epithelial
tissues, which would of course include
the entire epiblast field. Alternatively,
the effects seen on nonectodermal
sites could be the failure of interactions between the mesoderm and the
ectoderm; this finding would be a nonautonomous effect.
In hab embryos, the defects in epi-
boly, convergence, and neural tube
formation provide the first genetic evidence of a common element connecting these forms of morphogenesis.
What is the hab gene product? If hab
acts at the cell surface, it would seem
that cadherins, nerve cell adhesion
molecules, integrins, and other cell
surface molecules would be likely candidates and would be expected to have
many effects on different morphogenetic processes throughout the embryo. Whereas we eliminated VN-cadherin, there are other cadherins that
are present. Of note is E-cadherin,
which has been shown recently to
have antisense knockdown phenotypes similar to hab (Babb et al., 2001;
Babb and Marrs, 2004). We are working currently to identify the hab gene
product. Understanding the function
of this gene will be essential for understanding the control of cell movement at the molecular level and how
this control choreographs cells to play
morphogenesis.
EXPERIMENTAL
PROCEDURES
Zebrafish Strains
The habdtv42, avatm94, lawts18, and
wegtx230 mutations were isolated in a
large-scale mutagenesis screen (Haffter et al., 1996) and initially outcrossed to the polymorphic WIK (L11)
strain of wild-type fish for mapping.
Subsequent generations were outcrossed to the puma wild-type strain
of fish, selecting individuals that were
prescreened for polymorphisms in
closely linked microsatellite markers
on Linkage Group 7. The ZMD hab;
oep double mutants were produced by
mating males doubly heterozygous for
habdtv43 and oeptz257 to females heterozygous for oeptz257. The hab;
squint; cyclops triple mutants were
produced by mating identified pairs of
fish that were triply heterozygous
for habdtv43, squintcz35, and cyclopsmm294.
Mapping Panels
The epiboly mutants were mapped to
the centromere of Linkage Group 7 by
half-tetrad analysis (Streisinger et al.,
1986; Johnson et al., 1995). The halftetrad mapping panel was generated
by subjecting a heterozygous female to
EP-parthenogenetic reproduction as
described in Westerfield (1995). Halftetrad mapping panels were also generated from unrelated and unlinked
mutations to provide additional information about the centromere on Linkage Group 7. Mapping resolution was
increased using a haploid mapping
panel for each of the mutations. Haploid embryos were generated as described by Westerfield (1995).
DNA Extraction
Half-tetrad and haploid embryos were
allowed to develop to 10 hr of development, sorted by phenotype, and then
harvested for DNA by placing in 50 ␮l
of lysis buffer (1.5 mM MgCl2, 10 mM
Tris-HCl pH 8.3, 50 mM KCl, 0.3%
Tween 20, 0.3% Triton X-100). The extraction protocol includes incubating
at 98°C for 20 min, followed by incubating at 55°C for 1.5–2 hr, while
treating with 1 mg/ml proteinase K
(Roche Biochemicals), and finally incubating at 98°C for 20 min. All DNA
extractions were performed in individual wells of a 96-well polymerase
chain reaction (PCR) plate (LPS Plastics), sealed with PCR tape (Nunc) using a Hot Bonnet thermal cycle machine (MJ Research). The resulting
extraction was diluted directly into
sterile distilled H2O at 1/200.
Linkage Analysis
In general, microsatellite markers
were mapped as simple sequence
length polymorphisms (SSLP). PCR
reactions were carried out in 30-␮l
volumes containing: 10 mM Tris-HCl
pH 8.3, 50 mM KCl, 1.5 mM MgCl2,
0.0001% gelatin, 100 ␮l/ml bovine serum albumin, 100␮M concentrations
of each dNTP, 1 ␮M concentrations of
each primer, 0.06 ␮l of Taq DNA polymerase, and 5 ␮l of the diluted DNA
(5–50 ng). PCR: initial denaturation,
94°C, 5 min; 45 cycles: denaturation,
94°C, 30 sec; annealing, 55°C, 30 sec;
extension, 72°C, 1 min; final extension, 72°C, 7 min. Polymorphisms
were scored by gel electrophoresis on
2.5 to 4% agarose gels.
Many of the zebrafish genes and expressed sequence tags (ESTs) were
mapped as restriction fragment length
polymorphisms. These were amplified
404 MCFARLAND ET AL.
and scored as above for SSLP, only before loading onto a gel, 12 ␮l of the PCR
reaction was digested for 3 hr in a final
volume of 25 ␮l, with the appropriate
restriction enzyme for revealing a polymorphism. Subsequently, the digest
was loaded onto a 2.5% agarose gel.
Some microsatellite markers, genes,
and ESTs were mapped as SSCP. These
were amplified and scored on a polyacrylamide gel using the protocols described for SSCP (Fornzler et al., 1998),
except that ⬃4 Ci/mmol of 32P dATP
was added to the dNTP mixture per
reaction instead of being used to label
one primer.
Primers for microsatellite markers
designed and used as described (Knapik
et al., 1996, 1998; Shimoda et al., 1999)
and can be found at http://zebrafish.
mgh.harvard.edu/mapping/ssr_map_
index.html. Primers for Danio rerio
genes and ESTs were designed from sequence deposited at NCBI and are
available upon request.
Immunohistochemistry and
RNA In Situ Hybridization
Antibody staining was performed as
described in Warga and Nüsslein Volhard (1998). RNA in situ hybridization was carried out as described in
Thisse et al. (1993). Embryos were
cleared in 70% glycerol and photographed on a Zeiss AxioPhot.
Genotyping of Individual
Embryos
Genomic DNA extraction and PCR reactions were carried out as described
above for SSLP, except that, after in
situ hybridization experiments, DNA
was not diluted. Embryos were genotyped using a closely linked z-marker,
Z6852 (F-CAT GTG GTA CAG TTG
AAG GGG, R-ATC ATT GGA AAC
CAT CCA TAC A), which is less than
0.5 cM from the hab locus. cyclops and
squint embryos were genotyped as described in Warga and Kane (2003).
Cell Transplantation
Cell transplantations were carried out
between sphere and 30% epiboly
stages as previously described in Ho
and Kane (1990); also, some transplantations were performed on a dissection scope, using ⫻50 power. Do-
nors were examined at tail bud stage
for phenotype and then later genotyped. Hosts were examined between
6- and 10-somite stages for the presence of dorsal detached cells. Embryos
were recorded using Nomarski and
fluorescence microscopy as described
in Warga and Kane (2003).
4D Nomarski Time-Lapse
Analysis
Embryos were mounted and timelapse recordings were performed as
described (Kane et al., 1996), except
that images were digitally stored. Embryos were mounted before germ ring
stage in 0.08% agarose between coverslips and sealed with Vaseline. Lowmagnification recordings were acquired initially to determine the
orientation of the embryos, and then
they were recorded using a Zeiss DIC
⫻40 or ⫻63 water immersion lens at
10 to 15 planes using a 6 to 8 ␮m
z-spacing for periods of 3 to 4 hr. In a
single experiment, groups of 10 to 15
individuals were recorded using a
computer controlled stage, and each
location was visited every 150 to 180
sec. Afterward, low magnification recordings were again acquired to determine the latitude and longitude of the
recording. Later, the embryos were
carefully removed from between the
coverslips and their genotypes were
determined by segregation of linked
markers or by phenotype. Using NIH
Image 1.63, movements of individual
cells were traced throughout the analysis period as has been previously described in Warga and Kane (2003).
EVL cells, yolk syncytial layer nuclei,
and deep cells were followed beginning at the start of involution movements for a total of 40 ⫾ 2 min. For a
“global” view, data from multiple embryos were combined, using the latitude and longitudes of the recordings
to place and orient the cells in the
field.
ACKNOWLEDGMENTS
We thank Lynne Angerer, Cheeptip
Benyajati, and Fred Hagen for comments on early versions of this manuscript, and Lyndsay Field and Esther
Liu for their contributions to Figure 7.
We also thank Yi-Lin Yan, Sharon
Amacher, Ashley Bruce, and Yun-Jin
Jiang, as well as many other members
of the zebrafish community for reagents. D.A.K. was funded by the Pew
Charitable Trust and the National Institutes of Health.
REFERENCES
Amemiya CT, Zon LI. 1999. Generation of
a zebrafish P1 artificial chromosome library. Genomics 58:211–213.
Appel B, Eisen JS. 1998. Regulation of neuronal specification in the zebrafish spinal
cord by Delta function. Development 125:
371–380.
Babb SG, Marrs JA. 2004. E-cadherin regulates cell movements and tissue formation in early zebrafish embryos. Dev Dyn
230:263–277.
Babb SG, Barnett J, Doedens AL, Cobb N,
Liu Q, Sorkin BC, Yelick PC, Raymond
PA, Marrs JA. 2001. Zebrafish E-cadherin: expression during early embryogenesis and regulation during brain development. Dev Dyn 221:231–237.
Betchaku T, Trinkaus JP. 1978. Contact
relations, surface activity, and cortical
microfilaments of marginal cells of the
enveloping layer and of the yolk syncytial and yolk cytoplasmic layers of Fundulus before and during epiboly. J Exp
Zool 206:381–426.
Bitzur S, Kam Z, Geiger B. 1994. Structure
and distribution of N-cadherin in developing zebrafish embryos: morphogenetic
effects of ectopic over-expression. Dev
Dyn 201:121–136.
Branford WW, Yost HJ. 2002. Lefty-dependent inhibition of Nodal- and Wnt-responsive organizer gene expression is essential for normal gastrulation. Curr
Biol 12:2136 –2141.
Branford WW, Essner JJ, Yost HJ. 2000.
Regulation of gut and heart left-right
asymmetry by context-dependent interactions between Xenopus lefty and
BMP4 signaling. Dev Biol 223:291–306.
Carmany-Rampey A, Schier AF. 2001. Single-cell internalization during zebrafish
gastrulation. Curr Biol 11:1261–1265.
Chen Y, Schier AF. 2002. Lefty proteins
are long-range inhibitors of squint-mediated nodal signaling. Curr Biol 12:2124 –
2128.
Concha ML, Adams RJ. 1998. Oriented cell
divisions and cellular morphogenesis in
the zebrafish gastrula and neurula: a
time-lapse analysis. Development 125:
983–994.
Cooper MS, D’Amico LA. 1996. A cluster of
noninvoluting endocytic cells at the margin of the zebrafish blastoderm marks
the site of embryonic shield formation.
Dev Biol 180:184 –198.
Dougan ST, Warga RM, Kane DA, Schier
AF, Talbot WS. 2003. The role of the
zebrafish nodal-related genes squint and
cyclops in patterning of mesendoderm.
Development 130:1837–1851.
Elul T, Koehl MAR, Keller R. 1997. Cellular mechanisms underlying neural con-
half baked ECTODERM 405
vergent extension in Xenopus laevis embryos. Dev Biol 191:243–258.
Feldman B, Gates MA, Egan ES, Dougan
ST, Rennebeck G, Sirotkin HI, Schier
AF, Talbot WS. 1998. Zebrafish organizer development and germ-layer formation require nodal-related signals.
Nature 395:181–185.
Feldman B, Dougan ST, Schier AF, Talbot
WS. 2000. Nodal-related signals establish mesendodermal fate and trunk neural identity in zebrafish. Curr Biol 10:
531–534.
Feldman B, Concha ML, Saude L, Parsons
MJ, Adams RJ, Wilson SW, Stemple DL.
2002. Lefty antagonism of Squint is essential for normal gastrulation. Curr
Biol 12:2129 –2135.
Fink RD, Cooper MS. 1996. Apical membrane turnover is accelerated near cell–
cell contacts in an embryonic epithelium.
Dev Biol 174:180 –189.
Fornzler D, Her H, Knapik EW, Clark M,
Lehrach H, Postlethwait JH, Zon LI,
Beier DR. 1998. Gene mapping in zebrafish using single-strand conformation
polymorphism analysis. Genomics 51:
216 –222.
Go MJ, Artavanis-Tsakonas S. 1998. A genetic screen for novel components of the
notch signaling pathway during Drosophilabristledevelopment.Genetics150:
211–220.
Goto T, Keller R. 2002. The planar cell
polarity gene strabismus regulates convergence and extension and neural fold
closure in Xenopus. Dev Biol 247:165–
181.
Griffin KJ, Amacher SL, Kimmel CB,
Kimelman D. 1998. Molecular identification of spadetail: regulation of zebrafish
trunk and tail mesoderm formation by
T-box genes. Dev Suppl 125:3379 –3388.
Gritsman K, Zhang J, Cheng S, Heckscher
E, Talbot WS, Schier AF. 1999. The
EGF-CFC protein one-eyed pinhead is
essential for nodal signaling. Cell 97:121–
132.
Haddon C, Smithers L, Schneider-Maunoury S, Coche T, Henrique D, Lewis J.
1998. Multiple delta genes and lateral
inhibition in zebrafish primary neurogenesis. Development 125:359 –370.
Haffter P, Granato M, Brand M, Mullins
MC, Hammerschmidt M, Kane DA,
Odenthal J, van Eeden FJ, Jiang YJ,
Heisenberg CP, Kelsh RN, FurutaniSeiki M, Vogelsang E, Beuchle D, Schach
U, Fabian C, Nüsslein-Volhard C. 1996.
The identification of genes with unique
and essential functions in the development of the zebrafish, Danio rerio. Development 123:1–36.
Hammerschmidt M, Pelegri F, Mullins
MC, Kane DA, Brand M, van Eeden FJ,
Furutani-Seiki M, Granato M, Haffter P,
Heisenberg CP, Jiang YJ, Kelsh RN,
Odenthal J, Warga RM, Volhard-Volhard C. 1996. Mutations affecting morphogenesis during gastrulation and tail
formation in the zebrafish, Danio rerio.
Development 123:143–151.
Hatta K, Kimmel CB. 1993. Midline structures and central nervous system coordi-
nates in zebrafish. Perspect Dev Neurobiol 1:257–268.
Heisenberg CP, Tada M, Rauch GJ, Saude
L, Concha ML, Geisler R, Stemple DL,
Smith JC, Wilson SW. 2000. Silberblick/
Wnt11 mediates convergent extension
movements during zebrafish gastrulation. Nature 405:76 –81.
Hikasa H, Shibata M, Hiratani I, Taira M.
2002. The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic
movements of the axial mesoderm and
neuroectoderm via Wnt signaling. Development 129:5227–5239.
Ho RK, Kane DA. 1990. Cell-autonomous
action of zebrafish spt-1 mutation in specific mesodermal precursors. Nature 348:
728 –730.
Ho RK, Kimmel CB. 1993. Commitment of
cell fate in the early zebrafish embryo.
Science 261:109 –111.
Jessen JR, Topczewski J, Bingham S,
Sepich DS, Marlow F, Chandrasekhar A,
Solnica-Krezel L. 2002. Zebrafish trilobite identifies new roles for Strabismus
in gastrulation and neuronal movements. Nat Cell Biol 4:610 –615.
Johnson SL, Africa D, Horne S, Postlethwait JH. 1995. Half-tetrad analysis in
zebrafish: mapping the ros mutation and
the centromere of linkage group I. Genetics 139:1727–1735.
Kane DA, Warga RM, Kimmel CB. 1992.
Mitotic domains in the early embryo of
the zebrafish. Nature 360:735–737.
Kane DA, Hammerschmidt M, Mullins
MC, Maischein HM, Brand M, van Eeden FJ, Furutani-Seiki M, Granato M,
Haffter P, Heisenberg CP, Jiang YJ,
Kelsh RN, Odenthal J, Warga RM, Volhard-Volhard C. 1996. The zebrafish epiboly mutants. Development 123:47–55.
Kane DA, Zon LI, Detrich HW III. 1999.
Centromeric markers in the zebrafish.
In: Detrich HW III, Westerfield M, Zon
L, editors. The zebrafish: genetics and
genomics. New York: Academic Press. p
361–363.
Keller RE, Trinkaus JP. 1987. Rearrangement of enveloping layer cells without
disruption of the epithelial permeability
barrier as a factor in Fundulus epiboly.
Dev Biol 120:12–24.
Keller RE, Danilchik M, Gimlich R, Shih J.
1985. The function and mechanism of
convergent extension during gastrulation of Xenopus laevis. J Embryol Exp
Morphol 89:185–209.
Keller R, Shih J, Sater A. 1992. The cellular basis of the convergence and extension of the Xenopus neural plate. Dev
Dyn 193:199 –217.
Kimmel CB, Warga RM, Schilling TF.
1990. Origin and organization of the zebrafish fate map. Development 108:581–
594.
Kimmel CB, Warga RM, Kane DA. 1994.
Cell cycles and clonal strings during formation of the zebrafish central nervous
system. Development 120:265–276.
Knapik EW, Goodman A, Atkinson OS,
Roberts CT, Shiozawa M, Sim CU, Weksler-Zangen S, Trolliet MR, Futrell C,
Innes BA, Koike G, McLaughlin MG,
Pierre L, Simon JS, Vilallonga E, Roy M,
Chiang PW, Fishman MC, Driever W,
Jacob HJ. 1996. A reference cross DNA
panel for zebrafish (Danio rerio) anchored with simple sequence length polymorphisms. Development 123:451–460.
Knapik EW, Goodman A, Ekker M, Chevrette M, Delgado J, Neuhauss S, Shimoda N, Driever W, Fishman MC, Jacob
HJ. 1998. A microsatellite genetic linkage map for zebrafish (Danio rerio). Nat
Genet 18:328 –343.
Krauss S, Concordet JP, Ingham PW.
1993. A functionally conserved homolog
of the Drosophila segment polarity gene
hh is expressed in tissues with polarizing
activity in zebrafish embryos. Cell 75:
1431–1444.
Lindsley DL, Grell EH. 1968. Genetic
variations of Drosophila melanogaster.
Washington, DC: Carnegie Institute
Washington.
Malicki J, Jo H, Pujic Z. 2003. Zebrafish
N-cadherin, encoded by the glass onion
locus, plays an essential role in retinal
patterning. Dev Biol 259:95–108.
Melby AE, Warga RM, Kimmel CB. 1996.
Specification of cell fates at the dorsal
margin of the zebrafish gastrula. Development 122:2225–2237.
Mohideen MA, Moore JL, Cheng KC. 2000.
Centromere-linked microsatellite markers for linkage groups 3, 4, 6, 7, 13, and
20 of zebrafish (Danio rerio). Genomics
67:102–106.
Morgan TH. 1895. The formation of the fish
embryo. J Morphol 10:419 –472.
Mullins MC, Hammerschmidt M, Kane
DA, Odenthal J, Brand M, van Eeden FJ,
Furutani-Seiki M, Granato M, Haffter P,
Heisenberg CP, Jiang YJ, Kelsh RN, Volhard-Volhard C. 1996. Genes establishing dorsoventral pattern formation in
the zebrafish embryo: the ventral specifying genes. Development 123:81–93.
Myers DC, Sepich DS, Solnica-Krezel L.
2002. Bmp activity gradient regulates
convergent extension during zebrafish
gastrulation. Dev Biol 243:81–98.
Odenthal J, Volhard-Volhard C. 1998. fork
head domain genes in zebrafish. Dev
Genes Evol 208:245–258.
Oxtoby E, Jowett T. 1993. Cloning of the
zebrafish krox-20 gene (krx-20) and its
expression during hindbrain development. Nucleic Acids Res 21:1087–1095.
Papan C, Campos-Ortega JA. 1994. On the
formation of the neural keel and neural
tube in the zebrafish Danio (Brachydanio) rerio. Rouxs Arch Dev Biol 203:
178 –186.
Postlethwait JH, Talbot WS. 1997. Zebrafish genomics: from mutants to genes.
Trends Genet 13:183–190.
Postlethwait JH, Woods IG, Ngo-Hazelett
P, Yan YL, Kelly PD, Chu F, Huang H,
Hill-Force A, Talbot WS. 2000. Zebrafish
comparative genomics and the origins of
vertebrate chromosomes. Genome Res 10:
1890 –1902.
Read EM, Rodaway AR, Neave B, Brandon
N, Holder N, Patient RK, Walmsley ME.
1998. Evidence for non-axial A/P patterning in the non-neural ectoderm of
406 MCFARLAND ET AL.
Xenopus and zebrafish pregastrula embryos. Int J Dev Biol 42:763–774.
Schier A, Neuhauss S, Helde K, Talbot W,
Driever W. 1997a. The one-eyed pinhead
gene functions in mesendoderm and
endoderm in zebrafish and interacts
with no tail. Development 124:327–342.
Schier AF, Neuhauss SC, Helde KA, Talbot
WS, Driever W. 1997b. The one-eyed pinhead gene functions in mesoderm and
endoderm formation in zebrafish and interacts with no tail. Development 124:
327–342.
Schmitz B, Papan C, Campos-Ortega JA.
1993. Neurulation in the anterior trunk
region of the zebrafish Brachydanio rerio. Rouxs Arch Dev Biol 202:250 –259.
Schulte-Merker S, Hammerschmidt M,
Beuchle D, Cho KW, De Robertis EM,
Volhard-Volhard C. 1994a. Expression of
zebrafish goosecoid and no tail gene
products in wild-type and mutant no tail
embryos. Development 120:843–852.
Schulte-Merker S, van Eeden FJ, Halpern
ME, Kimmel CB, Volhard-Volhard C.
1994b. no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene.
Development 120:1009 –1015.
Sepich DS, Myers DC, Short R, Topczewski
J, Marlow F, Solnica-Krezel L. 2000.
Role of the zebrafish trilobite locus in
gastrulation movements of convergence
and extension. Genesis 27:159 –173.
Shimoda N, Knapik EW, Ziniti J, Sim C,
Yamada E, Kaplan S, Jackson D, de Sauvage F, Jacob H, Fishman MC. 1999. Zebrafish genetic map with 2000 microsatellite markers. Genomics 58:219 –232.
Solnica-Krezel L, Driever W. 1994. Microtubule arrays of the zebrafish yolk cell:
organization and function during epiboly. Development 120:2443–2455.
Solnica-Krezel L, Stemple DL, Mountcastle-Shah E, Rangini Z, Neuhauss SC,
Malicki J, Schier AF, Stainier DY,
Zwartkruis F, Abdelilah S, Driever W.
1996. Mutations affecting cell fates and
cellular rearrangements during gastrulation in zebrafish. Development 123:67–
80.
Strähle U, Jesuthasan S. 1993. Ultraviolet
irradiation impairs epiboly in zebrafish
embryos: evidence for a microtubule-dependent mechanism of epiboly. Development 119:909 –919.
Strähle U, Blader P, Ingham PW. 1996.
Expression of axial and sonic hedgehog
in wildtype and midline defective zebrafish embryos. Int J Dev Biol 40:929 –
940.
Strähle U, Jesuthasan S, Blader P, GarciaVillalba P, Hatta K, Ingham PW. 1997.
one-eyed pinhead is required for development of the ventral midline of the zebrafish (Danio rerio) neural tube. Genes
Function 1:131–148.
Streisinger G, Singer F, Walker C, Dower
N. 1986. Segregation analysis and genecentromere distances in zebrafish. Genetics 112:311–319.
Thisse C, Thisse B, Schilling TF, Postlethwait JH. 1993. Structure of the zebrafish
snail1 gene and its expression in wildtype, spadetail and no tail mutant embryos. Development 119:1203–1215.
Thisse C, Thisse B, Halpern ME, Postlethwait JH. 1994. Goosecoid expression in
neurectoderm and mesendoderm is disrupted in zebrafish cyclops gastrulas.
Dev Biol 164:420 –429.
Trevarrow B, Marks DL, Kimmel CB.
1990. Organization of hindbrain segments in the zebrafish embryo. Neuron
4:669 –679.
Trinkaus JP. 1951. A study of the mechanism of epiboly in the egg of Fundulus
heteroclitus. J Exp Zool 118:269 –320.
Varga ZM, Wegner J, Westerfield M. 1999.
Anterior movement of ventral diencephalic precursors separates the primor-
dial eye field in the neural plate and
requires cyclops. Dev Suppl 126:5533–
5546.
Warga RM, Kane DA. 2003. One-eyed pinhead regulates cell motility independent
of Squint/Cyclops signaling. Dev Biol 261:
391–411.
Warga RM, Kimmel CB. 1990. Cell movements during epiboly and gastrulation in
zebrafish. Development 108:569 –580.
Warga RM, Volhard-Volhard C. 1999. Origin and development of the zebrafish
endoderm. Development 126:827–838.
Warga RM, Nüsslein Volhard C. 1998. spadetail-dependent cell compaction of the
dorsal zebrafish blastula. Dev Biol 203:
116 –121.
Westerfield M. 1995. The zebrafish book. A
guide for the laboratory use of zebrafish
(Danio rerio). Eugene: University of Oregon Press.
Wilson ET, Cretekos CJ, Helde KA. 1995.
Cell mixing during early epiboly in the
zebrafish embryo. Dev Genet 17:6 –15.
Woods IG, Kelly PD, Chu F, Ngo-Hazelett
P, Yan YL, Huang H, Postlethwait JH,
Talbot WS. 2000. A comparative map of
the zebrafish genome. Genome Res 10:
1903–1914.
Yamamoto A, Amacher SL, Kim SH, Geissert D, Kimmel CB, De Robertis EM.
1998. Zebrafish paraxial protocadherin
is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm. Dev Suppl 125:3389 –3397.
Yan YL, Hatta K, Riggleman B, Postlethwait JH. 1995. Expression of a type II
collagen gene in the zebrafish embryonic
axis. Dev Dyn 203:363–376.
Zalik SE, Lewandowski E, Kam Z, Geiger
B. 1999. Cell adhesion and the actin cytoskeleton of the enveloping layer in the
zebrafish embryo during epiboly. Biochem Cell Biol 77:527–542.