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First published online 14 May 2008
doi: 10.1242/dev.022673
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Research Report |
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
* Author for correspondence (e-mail: scott.holley{at}yale.edu)
Accepted 25 April 2008
SUMMARY
Cell division, differentiation and morphogenesis are coordinated during embryonic development, and frequently are in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but that undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and use the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is driven substantially by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression, and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1-/-. The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize, as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for the normal segmental arrangement of epithelial borders and internal mesenchymal cells.
Key words: Somitogenesis, Cell cycle, Zebrafish, emi1, Somite morphogenesis
INTRODUCTION
Somites are the segmented precursors to the axial skeleton and musculature
created as the trunk and tail elongate. The periodic formation of somites is
governed by the segmentation clock, which creates oscillations in gene
expression in the presomitic mesoderm (PSM)
(Pourquié, 2003
). In
zebrafish, the segmentation clock requires Notch signaling, while the amniote
clocks also incorporate Wnt and Fgf signaling
(Holley, 2007). It is debated
whether the Notch, Wnt or Fgf pathways constitute core components of the
clock, or whether they are a readout of a global clock that governs all of
embryonic development (Aulehla et al.,
2003; Dequeant et al.,
2006; Niwa et al.,
2007; Wahl et al.,
2007). For instance, some models link the segmentation clock to
the cell cycle oscillator (Collier et al.,
2000; McInerney et al.,
2004; Primmett et al.,
1989; Primmett et al.,
1988).
Somite morphogenesis occurs as the segment boundary cells undergo a
mesenchymal to epithelial transition (MET), forming a ball of cells with an
epithelial surface and a core of mesenchyme
(Holley, 2007). Zebrafish
somite morphogenesis requires the transcription factor fused somites
(fss; also known as tbx24), Eph/Ephrin signaling and
integrin 
5/fibronectin function
(Barrios et al., 2003;
Durbin et al., 1998;
Durbin et al., 2000;
Jülich et al., 2005a;
Koshida et al., 2005;
Nikaido et al., 2002;
van Eeden et al., 1996).
fss links the segmentation clock and somite morphogenesis
(Holley et al., 2000).
fss mutants fail to maintain the segmentation clock in the anterior
PSM, lack segment polarity and ephA4 (epha4a - Zebrafish
Information Network) expression, and exhibit a complete loss of MET in the
paraxial (somitic) mesoderm (Durbin et al.,
2000; Holley et al.,
2000; Oates et al.,
2005b; van Eeden et al.,
1998). However, exogenous expression of ephA4 in genetic
mosaics can induce boundaries in fss-/- embryos
(Barrios et al., 2003).
Similarly in mouse genetic mosaics, EphA4 expression correlates with boundary
formation (Nakajima et al.,
2006). Integrin 5-GFP clusters along the basal side of
nascent somite boundary cells, and integrin 5 and its
ligand fibronectin are required for the maintenance and full
maturation of the boundary in zebrafish, mice and Xenopus
(Georges-Labouesse et al.,
1996; Goh et al.,
1997; Jülich et al.,
2005a; Koshida et al.,
2005; Kragtorp and Miller,
2007; Yang et al.,
1993). Double mutants between integrin 5
and the Notch pathway lead to a complete loss of MET in the paraxial mesoderm
(Jülich et al., 2005a).
Simultaneous loss of ephrin B2a, a ligand for ephA4, and
integrin 5 leads to a synergistic defect in somite
boundary morphogenesis (Koshida et al.,
2005). Ena/Vasp and Fak, which function in Integrin signaling, are
necessary for somite formation in Xenopus
(Kragtorp and Miller, 2006).
Chick somite morphogenesis is regulated by Snail2 and Cdc42, which promote
mesenchymal cell morphology, and Rac1, which fosters epithelial cell
morphology (Dale et al., 2006;
Nakaya et al., 2004).
Emi1 is a negative regulator of the Anaphase Promoting Complex (APC) and is
required for entry into mitosis in Xenopus embryos
(Reimann et al., 2001). APC,
an E3 ubiquitin ligase, also functions in post-mitotic cells. In
Drosophila and C. elegans neurons, APC localizes to the
synapse and regulates the turnover of glutamate receptors
(Juo and Kaplan, 2004;
van Roessel et al., 2004). In
vertebrate neurons, inhibition of APC by RNA interference or overexpression of
Emi1 increases axonal growth and overcomes much of the growth-inhibitory
effects of myelin. In contrast to the synapse studies, virtually all of the
APC is located in the nuclei of these neurons, and the axon growth phenotype
appears to be due to stabilization of Id2 and SnoN
(Lasorella et al., 2006;
Stegmuller et al., 2006).
Here, we identify a zebrafish mutant for emi1 that ceases mitosis at the beginning of gastrulation. Using this mutant, we find that normal cell cycle progression is not required for segmentation clock function, but rather that mitosis is a modest source of noise for the clock. Finally, we show that the cell cycle defect leads to hyper-epithelialization of the somites after the initiation of morphological segmentation.
MATERIALS AND METHODS
Zebrafish breeding, mapping and cloning
Breeding and meiotic mapping followed standard protocols
(Geisler, 2002;
Nüsslein-Volhard and Dahm,
2002). The coding sequence of emi1 (GenBank NM_001003869)
was isolated via RT-PCR and cloned into pCS2+. This clone was used to generate
sense mRNA using the Ambion SP6 mMessage Machine kit, and an antisense
riboprobe using the Roche digoxigenin-labeling mix. For allele sequencing, we
used an emi1 template from two independently derived tiy121
RT-PCRs. Wild-type embryos were injected with 0.5 mM emi1 morpholino
targeting the splice donor of the second intron
(5'-TGATTGTCGTTTCACCTCATCATCT-3').
Immunohistochemistry, in situ hybridization
Fibronectin, phalloidin, S58 staining
(Jülich et al., 2005a),
and fluorescent in situ hybridization with β-catenin immunohistochemistry
(Jülich et al., 2005b),
were performed as previously described. All in situ hybridizations were
performed with digoxigenin-labeled riboprobes. her1 and
deltaC antisense probes were made from plasmid clones, as previously
described (Holley et al.,
2000; Holley et al.,
2002). The tbx18, mesogenin, mespb and ripply1
coding sequences were isolated via RT-PCR and subjected to an additional round
of PCR in which a T7 promoter was added in the antisense orientation.
Antisense riboprobes were then created using T7 RNA polymerase (NEB). Integrin
5-GFP (Jülich et al.,
2005a) and YFP-Emi1 were visualized with rabbit anti-GFP (1:1000,
Invitrogen) and anti-rabbit Alexa 488 (1:200, Invitrogen). Goat anti-EphrinB2
(1:500, R&D Systems) was paired with anti-goat Alexa 647 (1:200,
Invitrogen). Rabbit anti-Phospho-Histone H3 (PHH3) antibody (1:1000, Sigma)
was used with goat anti-rabbit-HRP (1:400, Invitrogen) and Fluorescein TSA
(Perkin Elmer).
We analyzed her1 expression in emi1 mutant and sibling
embryos injected with translation-blocking morpholinos against either
deltaC or deltaD (Holley
et al., 2002). Three independent trials were performed with
embryos derived from different parents and injected on different days.
deltaC morpholino-injected and deltaD morpholino-injected
embryos were fixed in 4% paraformaldehyde (PFA) at the 
2-somite and
5-somite stages, respectively. Embryos were co-stained for her1
expression with NBT/BCIP and for PHH3 by immunofluorescence. Absence of PHH3
staining was used to sort emi1-/- from sibling
embryos.
Drug treatment and BrdU labeling
Embryos were incubated in 150 µM aphidicolin and 20 mM hydroxyurea
(Sigma) in 4% DMSO, from the germ ring/early shield stage until fixation
(Harris and Hartenstein, 1991;
Lyons et al., 2005). Drug
treatment at this stage blocked mitosis by the late shield stage, mimicking
the onset of the emi1-/- phenotype. To assay for DNA
synthesis, 10 mM BrdU was injected into the yolk just after the shield stage,
at the 1-somite stage or at the 8-somite stage. Embryos were fixed in 4% PFA
at the 14- to 15-somite stage. BrdU incorporation was visualized using a mouse
anti-BrdU antibody (1:200, Sigma) and an Alexa 647-labeled goat anti-mouse
antibody (1:200, Invitrogen). Embryos injected at each stage showed BrdU
incorporation, indicating that endoreplication occurs continuously during late
gastrulation and trunk segmentation in emi1 mutants.
RESULTS AND DISCUSSION
We identified a zebrafish mutant, tiy121, which exhibits a mitotic
block (Fig. 1A-D). By the
shield stage, mutant embryos cease all mitosis, as visualized by
immunostaining for phosphorylated Histone H3. Despite the mitotic arrest,
mutant embryos undergo gastrulation and axis elongation
(Fig. 1E,F). Measurement of the
distance from the otic vesicle to the tip of the tail indicates that
tiy121 embryos (n=15) are on average 22% (s.d. ±3.2%)
shorter than their wild-type siblings (n=17). After the mitotic
block, mutant embryos continue endoreplication, as indicated by BrdU labeling
(Fig. 1G,H). tiy121
embryos ultimately develop a pericardial edema and extensive necrosis in the
head, and die 2-3 days post-fertilization. The relatively normal progression
of early development in tiy121 embryos parallels the finding that
early Xenopus development is unperturbed by the chemical inhibition
of mitosis (Cooke, 1973;
Harris and Hartenstein, 1991;
Rollins and Andrews,
1991).
|
). Together, these data indicate that the tiy121
phenotype is due to perturbation of emi1.
|
Although tiy121 embryos are short, the mutant phenotype indicates
that cell proliferation is not absolutely required for trunk and tail
extension. However, the mutants display irregularly sized and partially fused
somites and myotomes (Fig.
3A-D; see also Fig. S1 in the supplementary material). The
segmentation clock creates oscillations in transcription that manifest as
stripes of expression sweeping through the cells of the PSM in a wave-like
fashion. We examined the expression of three oscillating genes, her1,
her7 and deltaC, at the 3-, 8- and 15-somite stage and found no
appreciable defect in their expression in emi1-/-
(Fig. 3E-J; data not shown).
Note that, at the 8- and 15-somite stage, the tailbud of
emi1-/- embryos is smaller than normal (compare Fig.
3F and G to
3I and J, respectively). This
decrease is reflected in the reduction of the domain of mesogenin
expression (see Fig. S1 in the supplementary material). Although emi1
mutants undergo mitosis at the beginning of gastrulation when oscillations are
first seen (Riedel-Kruse et al.,
2007), our data indicate that continued oscillation of the
segmentation clock is not dependent upon the cell cycle.
In contrast to models that link the cell cycle to the segmentation clock,
it has been postulated that mitosis is actually a source of noise for the
clock (Horikawa et al., 2006).
To test this hypothesis, we examined the effect of inhibiting mitosis in
embryos lacking either of the Notch ligands deltaC or
deltaD. The deltaC and deltaD mutants form the
first 3-5 and 7-9 somites, respectively, as the oscillating pattern of gene
expression gradually breaks down, leading to the segmentation defect
(Fig. 3K-N; see also Fig. S1 in
the supplementary material) (Holley et
al., 2000; Jiang et al.,
2000; Jülich et al.,
2005b; Oates et al.,
2005a; van Eeden et al.,
1996; van Eeden et al.,
1998). This breakdown may be accelerated because of noise. Thus,
if mitosis is a source of noise in the segmentation program, one would predict
that the breakdown would decelerate in the absence of cell division. We
assayed the expression of her1 mRNA in deltaD or
deltaC morpholino-injected embryos that were either wild type or
mutant for emi1 (Fig.
3N, Fig. S1 in the supplementary material). The difference between
the mutants and siblings was not immediately apparent. However, upon careful
categorization of the expression patterns, we found a subtle improvement in
the integrity of the her1 stripes in embryos lacking emi1
compared with sibling embryos. For each trial, the more organized stripe
patterns are biased towards the injected emi1 mutants, and the two
more disorganized expression categories are biased towards the injected
siblings. In summary, these results are consistent with mitosis being a modest
source of noise in the segmentation clock.
Further examination of the segmentation defect in
emi1-/- embryos revealed profound abnormalities in somite
morphology. Although emi1-/- somites initially contain
internal mesenchymal cells, these cells leave the core of the somite and at
least some integrate into the epithelial somite boundary
(Fig. 4A,B,D,E). We have
observed other cells migrating to the lateral surface of the paraxial
mesoderm. The somite boundary cells then appear to elongate and meet in the
middle of each segment, creating somites solely consisting of two rows of
boundary cells (Fig. 4B,E,G,I).
These hyper-epithelialized somites, having no internal mesenchyme and
abnormally elongated epithelial border cells, often fuse to create irregularly
sized segments. The nuclei of the boundary cells show a basal localization, as
does Integrin 5-GFP clustering (Fig.
4A,B). Fibronectin matrix is also assembled along the somite
boundaries (Fig. 4F,G). This
maintenance of border cell polarity distinguishes the
emi1-/- phenotype from that of integrin 5
and fibronectin1a mutants
(Jülich et al., 2005a;
Koshida et al., 2005). Ephrin
B2 is localized to the cortex of the somite cells, with slightly higher levels
in the posterior somite cells, and this pattern appears largely intact in
emi1-/- (Fig.
4H,I; see also Fig. S1 in the supplementary material). Expression
of mespb, ripply1 and tbx18, myod and deltaC is
clearly segmental, although there is some aberrant expression of
deltaC in the mutant embryos (Fig.
4J-M; Fig. S1 in the supplementary material). The segment polarity
alterations observed in emi1 mutants are slight in comparison to
those defects seen in fss and the Notch pathway mutants, and seem
unlikely to be the cause of the morphological phenotype.
|
;
Konishi et al., 2004;
Lasorella et al., 2006;
Stegmuller et al., 2006;
van Roessel et al., 2004). To
test this hypothesis, we blocked mitosis using a combination of hydroxyurea
and aphidicolin (Harris and Hartenstein,
1991; Lyons et al.,
2005). Addition of the compounds at the germ ring/early shield
stage blocked all mitosis by the late shield stage and resulted in embryos
lacking internal mesenchymal cells in their somites, strongly phenocopying
emi1-/- (Fig.
4C; see also Fig. S1 in the supplementary material). These data
suggest that the segmentation defect in emi1-/- mutant
embryos is primarily due to the lack of normal cell cycle progression and not
to a cell cycle-independent function of emi1 or APC. Note that in
both mutant and drug-treated embryos, the cells and nuclei are larger than in
wild type (Fig. 4). The
increase in cell size, along with the decrease in cell number, might also be
causally linked to the somite morphogenesis defect.
The mitotic defect in emi1-/- embryos arises after the
midblastula transition (MBT). MBT initiates during the tenth cell cycle [3
hours post-fertilization (hpf)], when divisions become asynchronous and
zygotic transcription commences (Kane,
1999; Kane and Kimmel,
1993). During cycles 11 and 12, the blastula forms three domains,
the extra-embryonic yolk syncytial layer and enveloping layer, and the deep
cells that give rise to the embryo proper
(Kane, 1999). At 5.5 hpf,
gastrulation starts, as most of the deep cells are in cell cycle 14
(Kane, 1999;
Kane et al., 1992).
emi1-/- embryos cease cell division around this time. In
wild-type embryos, the cell cycle lengthens during this period, with the
thirteenth, fourteenth, fifteenth and sixteenth cycles averaging 54, 78, 151
and 240 minutes, respectively. During segmentation, most cells are in either
cell cycle 16 or 17 (Kane,
1999). The mild elongation defect in emi1-/-
is likely to be due to the fact that mitosis is normally not a great
contributor to axial growth during the segmentation period. This conclusion
was also reached by examining the elongation of clonal strings of cells in the
CNS: the exponential lengthening of the string suggested that it was largely
due to cell intercalation and not cell division
(Kimmel et al., 1994). The
relatively normal differentiation in emi1-/- embryos can
be explained by the fact that many cells undergo a terminal differentiation
during cell cycle 15, 8-10 hpf, and a major wave of differentiation occurs
during cycle 16 (Kane, 1999;
Kimmel et al., 1994;
Kimmel and Warga, 1987). Thus,
for many cell lineages, the mitotic defect in emi1 embryos does not
reduce dramatically the number of cell cycles that these cells would normally
undergo.
|
). Experiments in ascidians have suggested that the timing of
myogenesis may depend upon the number of cycles of DNA synthesis that a
myogenic progenitor experiences (Satoh,
1987). However, previous cell labeling experiments indicate that
zebrafish myofiber differentiation is not regulated by such a cell
cycle-counting mechanism (Kimmel and
Warga, 1987). Similarly, the expression of differentiation markers
in the C. elegans gut occurs independently of cell cycle counting
(Edgar and McGhee, 1988). The
segmentation clock has been suggested to be linked to the cell cycle
oscillator. Reiterated segmentation defects are seen in chick embryos after
treatment with cell-cycle inhibitors, and the periodicity of this defect is
equal to the cell cycle length at that stage of development
(Primmett et al., 1989).
Similar periodic defects were seen after a single heat shock
(Primmett et al., 1988). More
recently, this cell cycle model has been formalized mathematically
(Collier et al., 2000;
McInerney et al., 2004). In
the zebrafish, a single heat shock can produce reiterated segmentation
defects, but the periodicity of the defect does not correlate with the length
of the cell cycle during segmentation (Roy
et al., 1999). Additionally, there is no organized pattern of cell
proliferation in the zebrafish tailbud
(Kanki and Ho, 1997). Our
analysis of the emi1 mutant indicates that cell cycle progression is
not required for zebrafish segmentation clock function. Conversely, our data
are consistent with the hypothesis that mitosis is a modest source of noise
for the segmentation clock (Horikawa et
al., 2006).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/12/2065/DC1
ACKNOWLEDGMENTS
We thank Hiroyuki Takeda and members of the lab for comments on the manuscript. The tiy121 mutant was isolated in the Tübingen 2000 Screen, and we acknowledge all participants of the consortium: F. Van Bebber, E. Busch-Nentwich, R. Dahm, O. Frank, H.-G. Fronhöfer, H. Geiger, D. Gilmour, S. Holley, J. Hooge, D. Jülich, H. Knaut, F. Maderspacher, H.-M. Maischein, C. Neumann, C. Nüsslein-Volhard, H. Roehl, U. Schönberger, C. Seiler, S. Sidi, M. Sonawane, A. Wehner, P. Erker, H. Habeck, U. Hagner, C. E. Hennen Kaps, A. Kirchner, T. Koblizek, U. Langheinrich, C. Loeschke, C. Metzger, R. Nordin, J. Odenthal, M. Pezzuti, K. Schlombs, J. de Santana-Stamm, T. Trowe, G. Vacun, B. Walderich, A. Walker and C. Weiler. We thank Nancy Hopkins for the hi2648 allele. We thank Joseph Wolenski for assistance with the confocal microscopy. This work was supported by the March of Dimes Foundation, research grant number 1-FY07-424, and the NICHD, R01 HD045738.
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