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doi: 10.1242/10.1242/dev.00548
Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York NY 10029, USA
* Author for correspondence (e-mail: manfred.frasch{at}mssm.edu)
Accepted 17 April 2003
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: T-box, Amnioserosa, Dorsal ectoderm, Dpp, wingless, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ray et al., 1991;
Ferguson and Anderson, 1992a;
Staehling-Hampton et al.,
1994; Frasch,
1995). In addition to promoting dorsal epidermal and PNS fates in
the dorsolateral ectoderm, Dpp acts to suppress the formation of neurons of
the central nervous system in the same area.
For a better understanding of these activities, we need to consider that
Dpp exercises some of its functions sequentially at different stages of
development, during which dpp changes its own pattern of expression
(St Johnston and Gelbart,
1987). In particular, during blastoderm and gastrulation stages,
Dpp acts in a dose-dependent fashion to establish positional information in
dorsal and lateral areas of the embryo and to specify amnioserosa tissue
(Ferguson and Anderson, 1992a;
Ashe et al., 2000). Although
dpp is expressed uniformly around 
40% of the dorsal
circumference of the embryos during this stage, the activity of Dpp is
modulated along the dorsoventral axis by diffusion of the secreted gene
product as well as by positive and negative regulators of the signaling
pathway. Negative regulators include Short gastrulation (Sog) and Brinker
(Brk), both of which are expressed ventrolaterally
(Ferguson and Anderson, 1992b;
Francois et al., 1994;
Jazwinska et al., 1999).
Whereas Sog and its vertebrate homolog Chordin are secreted molecules that
inhibit BMP signaling via binding to the ligand (reviewed by
Garcia Abreu et al., 2002),
Brk appears to be a nuclear factor that interferes with the signaling output
via binding to regulatory sequences of Dpp target genes
(Sivasankaran et al., 2000;
Kirkpatrick et al., 2001;
Rushlow et al., 2001;
Zhang et al., 2001). By
contrast, specification of amnioserosa fates in the dorsal 10% of embryonic
cells requires maximal signaling activities that involve Sog as a positive
regulator of Dpp in conjunction with Twisted gastrulation (Tsg) as well as a
second, uniformly-distributed BMP ligand, Screw (Scw)
(Arora et al., 1994;
Mason et al., 1994;
Ashe and Levine, 1999;
Decotto and Ferguson, 2001).
Dpp, Sog and Tsg are thought to be present in a diffusible trimolecular
complex that serves to carry and release active Dpp prefentially into
dorsalmost areas where tsg is expressed
(Decotto and Ferguson,
2001).
After gastrulation, dpp expression ceases in the developing
amnioserosa and becomes restricted to a broad stripe of cells in the
dorsolateral ectoderm along the elongated germ band
(St Johnston and Gelbart,
1987). During this period, the dorsally migrating cells of the
mesoderm reach the dpp-expressing area of the ectoderm, thus allowing
Dpp to induce dorsal mesodermal cell fates across germ layers
(Staehling-Hampton et al.,
1994; Frasch,
1995). In addition, Dpp is thought to act in the continuing
patterning processes within the dorsolateral ectoderm during this stage, which
lead to the specification of tracheal as well as particular epidermal and
sensory organ progenitors. Both in the dorsal mesoderm and dorsolateral
ectoderm, Dpp must act in combination with additional patterning molecules
that provoke differential responses of cells to the Dpp signal. For example,
in the dorsal mesoderm, the presence or absence of Wingless (Wg) activity
determines whether cells will respond to Dpp by forming heart and dorsal
somatic muscle progenitors versus visceral muscle progenitors
(Wu et al., 1995;
Azpiazu et al., 1996;
Carmena et al., 1998).
In order to obtain more insight into the mechanisms of how Dpp signals
pattern the embryo and how they are integrated with other patterning
processes, it is crucial to study the regulation of Dpp target genes. To date,
detailed molecular studies have been described for three targets that are
induced during early embryogenesis, namely the homeobox genes
zerknüllt (zen), tinman (tin) and
even-skipped (eve). zen is required for the
specification of the amnioserosa downstream of Dpp. Accordingly, the
expression of zen in a dorsal on/ventral off pattern, although
initially Dpp-independent, requires low levels of Dpp activity for its
maintenance and high Dpp activities for its subsequent refinement to areas of
the prospective amnioserosa (Doyle et al.,
1986; Rushlow and Levine,
1990). Likewise, tin is required for the specification of
all dorsal mesodermal tissues and eve for the normal differentiation
of specific pericardial cells and dorsal somatic muscles in a Dpp-dependent
manner. (Bodmer, 1993;
Azpiazu and Frasch, 1993;
Su et al., 1999). All three
genes have in common the presence of multiple binding sites for intracellular
Dpp effectors, the Smad proteins Mad and Medea, in their regulatory regions,
which are essential for mediating the inductive activity of Dpp. However, in
addition to these Smad-binding sites, each of these genes has a characteristic
set of additional regulatory sequences that, at least in part, explain its
particular spatial and tissue-specific response to Dpp signals. For example,
zen contains binding sites for Brk in addition to the Smad sites
(Rushlow et al., 2001). It
appears that the antagonistic activities of the Brk and Smad sites and the
differential ratios of Brk versus active Smad proteins along the dorsoventral
embryo axis determine the ventral border of Dpp-dependent zen domain
during cellularization stages. The Smad sites but not the Brk sites are also
required for zen induction in the prospective amnioserosa during the
cellularized blastoderm stage (Rushlow et
al., 2001). The mesodermal Dpp targets tin and
eve require Smad-binding sites and, in addition, binding sites for
Tin, which serve to target the Dpp response to the mesoderm
(Xu et al., 1998;
Halfon et al., 2000;
Knirr and Frasch, 2001).
Further, the Dpp-responsive enhancer of eve contains functionally
important binding sites for regulators that restrict its activity to segmental
subsets of dorsal mesodermal cells, including the Wg effector Pangolin (Pan)
(Halfon et al., 2000;
Knirr and Frasch, 2001).
In the present study, we introduce three novel genes that respond to Dpp
signals in the prospective amnioserosa, dorsal ectoderm and dorsal mesoderm,
and are good candidates for being direct targets of the Dpp signaling cascade.
The three genes, Dorsocross1 (Doc1), Dorsocross2
(Doc2) and Dorsocross3 (Doc3), which are present in
a gene cluster, are closely related members of the T-box family of genes and
presumably arose by relatively recent duplications from a common ancestor. The
Dorsocross (Doc) genes are expressed in essentially identical patterns within
several areas that receive high levels of Dpp signals, including the
prospective amnioserosa during the cellularized blastoderm stage, the
dorsolateral ectoderm and dorsal mesoderm during germ band elongated stages
and areas that span the compartment border in wing discs. We show that Doc
expression in the prospective amnioserosa depends on dpp and
zen, whereas the metameric expression in the dorsolateral ectoderm
and dorsal mesoderm depends on a combination of dpp and wg.
Our genetic analysis demonstrates that the three Doc genes have largely
redundant functions during amnioserosa development, as well as during
dorsolateral ectoderm and dorsal mesoderm patterning. We focus on the role of
the Doc genes in the amnioserosa and dorsolateral ectoderm. We show that they
are essential for full differentiation and maintenance of the amnioserosa,
including the arrest of cell proliferation in this tissue. Owing to the
requirement of a functional amnioserosa for normal germ band retraction, loss
of Doc activity produces embryos with a permanently extended germ band. Hence,
Doc genes are new members of the u-shaped family of genes. All genes of this
family, which also includes hindsight (hnt; peb
– FlyBase), serpent (srp), tail-up
(tup), u-shaped (ush), epidermal growth factor
receptor (Egfr) and insulin-like receptor
(InR), are components of a regulatory network that controls normal
development and functioning of the amnioserosa
(Frank and Rushlow, 1996;
Goldman-Levi et al., 1996;
Yip et al., 1997;
Lamka and Lipshitz, 1999). In
addition to the amnioserosa, the Doc genes are required for the normal
patterning of the dorsolateral ectoderm, which includes the repression of
wg and ladybird (lb) expression within this area.
These findings provide valuable insight into the mechanisms of how Dpp signals
are executed during the development of the amnioserosa and the patterning of
dorsolateral areas of the embryonic germ band.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was
screened with the respective radioactively labeled fragments and the plasmid
DNAs of isolated clones were sequenced (GeneWiz, New York). The cDNAs shown in
Fig. 1A correspond, from top to
bottom, to clones Doc1-a1.1, Doc2-c6.2, Doc2-c1.1, Doc2-c12.2 and
Doc3-b4.1.
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Generation of UAS-Dorsocross transgenes
Transformation plasmids were constructed by subcloning the cDNAs Doc1-a1.1,
Doc2-c6.2 and Doc3-b4.1 into pP{UAST}
(Brand and Perrimon, 1993)
using EcoRI or blunted EcoRI and NotI cloning
sites. Doc1-a1.1 was cloned as EcoRI-NotI fragment,
Doc2-c6.2 as SmaI-NotI fragment and Doc3-b4.1 as
HindIII (blunted)-NotI fragment. The HindIII and
NotI sites are pNB40 vector-derived and the EcoRI and
SmaI sites are located in the 5' UTR of Doc1 and
Doc2, respectively. Several independent lines were established for
each construct using standard transformation methods
(Rubin and Spradling, 1982).
GAL4 inducible expression of Dorsocross proteins was confirmed by
immunostaining with the antibodies described above.
Mutagenesis by male recombination
In order to create deletions uncovering all three Dorsocross genes a
mutagenesis screen was performed using the male recombination method
(Preston et al., 1996). The
closest available P-insertion, EP(3)3556, was used to
trigger male recombination in the presence of active transposase.
EP(3)3556 is a homozygous viable insertion in the 5' region of
smg (Dahanukar et al.,
1999), which is located about 8 kb upstream of Doc3 (see
Fig. 6A). F0 males subject to
recombination/mutagenesis carried both a X-chromosomal transposase source and
the targeted P-insertion flanked by genetic markers on the third
chromosome (y w H{P
2-3} HoP8/Y; ru EP(3)3556 th st cu sr
es ca/+). F0 males were crossed to ru h th st cu sr
es Pr ca/TM6B, Bri Tb females. The F1 generation was
screened for non-balancer males carrying a recombinant third chromosome. The
ru marker is expected to be retained along with the
P-insertion if recombination causes a deletion extending towards the
Dorsocross gene cluster (Preston et al.,
1996). Recombinants were crossed individually to
Df(3L)Scf-R11/TM3, Sb eve-lacZ females for producing
TM3, eve-lacZ balanced stocks and for complementation analysis with
Df(3L)Scf-R11.
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80 kb downstream of Doc1, yielded
Df(3L)EP584MR2 (Fig.
5A).
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) using Pwht1 and Plac1 primers. The removal of genes by
various deficiencies (Fig. 5A)
was confirmed by PCR from homozygous mutant embryos as described in Duan et
al. (Duan et al., 2001). PCR
from equally treated heterozygous embryos and PCR amplification of sequences
not affected by the deficiencies served as positive controls for primers and
template, respectively. PCR amplification and sequencing of DNA to the left of
EP(3)3556 demonstrated no change in sequences flanking the 3'
P-end.
TUNEL staining and BrdU labeling of embryos
Apoptotic cells were labeled by terminal deoxynucleotidyl transferase
(TdT)-mediated dUTP nick end-labeling (TUNEL) using components of the
ApopTag® Peroxidase kit S7101 (Intergen Company/Serologicals Corporation).
Rehydrated embryos (about 30 µl) were treated with 10 µg/ml Proteinase K
for 1 minute, rinsed quickly three times and washed another five times for 3
minutes with PBT. Embryos were postfixed in 3.7% formaldehyde in PBT for 20
minutes, washed five times with PBT and twice for 20 minutes with 30 µl
equlibration buffer. TdT reaction was performed over night at 37°C using
50 µl buffer/TdT mixed in a ratio 7:3 and supplemented with 0.3% Triton
X-100. The reaction was stopped by a 20 minute wash in 1/34 diluted stop
buffer. Detection of incorporated Digoxigenin-nucleotides using sheep-anti-Dig
and biotinylated anti-sheep antibodies, the VectaStain ABC elite kit (Vector
Laboratories) and Tyramide Signal Amplification (TSA) reagents (NEN/Perkin
Elmer Life Sciences) was essentially as for in situ hybridization using
digoxigenin-labeled probes (Knirr et al.,
1999).
BrdU labeling and detection was as described by Shermoen (Shermoen, 2000). Embryos were labeled by 30 minutes incubation in 1 mg/ml BrdU in PBS. For detection mouse anti-BrdU antibody (Becton-Dickinson, 1:200) and Cy3-anti-mouse antibody (Jackson ImmunoResearch Laboratories, 1:200) were used. For double staining, standard antibody staining using the VectaStain ABC elite kit and TSA fluorescence substrates were performed prior to the TUNEL reaction or BrdU detection.
Drosophila strains and crosses
y w or Oregon R were used as wild-type controls and stocks were
obtained from the Bloomington stock collection unless noted otherwise. The
following UAS/GAL4 driver lines were used: P{GAL4-nanos.NGT} 40
(Tracey et al., 2000),
P{en2.4-GAL4} e22c, P{ZKr-GAL4}#8
(Frasch, 1995),
P{dpp.blk1-GAL4} 40C.6, P{w+mW.hs=GawB}c381
(Manseau et al., 1997) (which
drives amnioserosa expression from stage 9; I.R. and M.F., unpublished) and
UAS-dpp#5 (Frasch,
1995). The following previously described mutant alleles were also
used: dppH46, hntE8,
pnr1, CyO
slp34B,
srpP{PZ}01549, tup1,
ush2, wgCX4 and
zen7. For male recombination and mapping experiments, we
used the lines y w H{P2-3} HoP8, `ru cu ca'
and `ru Pr ca', ru Df(3L)Scf-R11, Scf-R6 th st cu sr e
ca (Kopp and Duncan,
1997), smg1
(Dahanukar et al., 1999),
Df(3L)29A6 kniri-1 pp, EP(3)3556, and
EP(3)584 (Exelixis). For ectopic expression experiments, embryos were
collected at 28°C from crosses of GAL4-carrying females with UAS
construct-carrying males. All other crosses were performed at room temperature
(22-25°C).
RNA interference experiments
Sense and antisense RNA was transcribed from non-conserved 3'
fragments of Doc1, Doc2 and Doc3 in pCRII-TOPO (see above)
and hybridized in injection buffer (5 mM KCl, 10 mM sodium phosphate, pH 7.8)
to generate dsRNA (Kennerdell and Carthew,
1998). A mix of Doc1, Doc2 and Doc3 dsRNA
(100-300 pl of 4 mg/ml each) was injected ventrally into pre-blastoderm
embryos that had been dechorionated and mounted onto double-sided sticky tape
under Halocarbon 700 oil. An Eppendorf FemtoJet automatic injector and
Eppendorf Femtotip injection needles were used for injections. Embryos were
allowed to develop at 18°C until the desired stage was reached. For
immunostaining, embryos were transferred to standard heptane/formaldehyde
fixation solution in a small drop of oil. The heptane phase and total fixation
solution were exchanged twice to remove traces of oil. After 20 minutes
incubation, the formaldehyde solution was replaced by PBS. For the manual
removal of the vitelline membranes, embryos were spread on agar plates,
transferred to double-sided sticky tape and covered with PBS. After
devitellinization 0.05% Tween was added and embryos were transferred into
reaction tubes for standard staining procedures. Cuticle preparations were
made 2-3 days after injection. Embryos were passed through acetic
acid/glycerol (4:1) overlaid with heptane, transferred to a mesh, rinsed with
heptane and PBT and mounted in standard Hoyer's medium.
Staining of embryos and imaginal discs
Antibody staining of embryos using DAB, double fluorescent staining and in
situ hybridization in combination with fluorescent antibody staining were
carried out as described previously (Knirr
et al., 1999). Dorsocross in situ hybridization probes were made
by in vitro transcription from 3' fragments cloned into pCRII-TOPO (see
above). The race in situ hybridization probe and zen cDNA
were a gift from C. Rushlow (Frank and
Rushlow, 1996). Antibody staining of imaginal discs was
essentially the same as for embryos, except that imaginal discs attached to
inverted larval heads were fixed in 3.7% formaldehyde in PBS, dehydrated in
methanol, and rehydrated using 70%, 50% and 30% methanol in PBT before
blocking and staining.
The following antibodies were used: rabbit anti-Doc2 (1:2000), guinea pig
anti-Doc2+3 (1:400 to 1:600), guinea pig anti-Doc3+2 (1:600), rat anti-Doc1
(1:200), rabbit anti-Bap (1: 500) (Zaffran
et al., 2001), rabbit anti-ß-galactosidase (Promega; 1:1500),
rabbit anti-Phospho-Smad1/PMad (1:2000; gift from C.-H. Heldin), rat anti-Cf1a
(1:3500; gift from W. A. Johnson, University of Iowa), guinea pig anti-Kr
(1:400) (Kosman et al., 1998),
guinea pig anti-Slp (1:20) (Kosman et al.,
1998), affinity-purified rabbit anti-C15 (1:25 to 1:50,
TSA-indirect, NEN; S. Grimm and M.F., unpublished), mouse monoclonal anti-Lbe
(1:10) (Jagla et al., 1997),
and rabbit anti-phospho-Histone H3 (1:600; Upstate Biotechnology, NY).
Monoclonal mouse antibodies obtained from the Developmental Studies Hybridoma
Bank, University of Iowa: anti-
-tubulin 12G10 (1:10), anti-Futsch 22C10
(1:250), anti-Wg 4D4 (1:40, in imaginal discs 1:400), anti-En/Inv 4D9 (1:4),
anti-Hnt 27B8 1G9 (1:30) and anti-ß-gal 40-1a (1:40).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) revealed
that three novel, closely related T-box encoding genes are clustered within
40 kb of genomic sequences at 66F1 to 66F2 on chromosome arm 3L. In
reference to their peculiar patterns of expression in blastoderm embryos (see
below), these genes have been named, from proximal to distal,
Dorsocross1 (Doc1; previously Tb66F2)
(Lo and Frasch, 2001),
Dorsocross2 (Doc2) and Dorsocross3 (Doc3)
(Fig. 1A). The gene cluster
also includes an unrelated predicted gene, CG5194, which maps between
Doc2 and Doc3. cDNAs for the three Doc genes, which were isolated from an early embryonic cDNA library (see Materials and Methods), encode proteins of 391 amino acids (Doc1), 469 amino acids (Doc2) and 424 amino acids (Doc3), respectively. Comparisons between cDNA and genomic sequences indicate that Doc2 encodes at least three different mRNA products, which appear to be generated from alternative transcription start sites. Among these, Doc2 variants A and B encode identical polypeptides, whereas variant C does not encode any long open reading frame (Fig. 1A). The data from northern analysis indicate that the longest cDNAs obtained for each gene are close to full length if the polyA tails are taken into account (1.7 kb transcripts versus 1500 bp cDNA for Doc1, 2.0 kb transcripts versus 1759 bp cDNA for Doc2A, and 1.8 kb transcripts versus 1681 bp cDNA for Doc3) (data not shown). For Doc2, these data indicate that variant A (1.75 kb, presumably corresponding to the 2.0 kb transcripts) is expressed much more strongly than the other two variants. In addition, we note that splicing occurs at identical positions within the open reading frames of Doc1, Doc2 and Doc3, although most introns in Doc3 are much smaller as compared with those in the other two genes (Fig. 1A).
Sequence comparisons show that the three Doc proteins share high degrees of
similarity within their T-box domain sequences (>95% amino acid identities)
as well as within short sequence stretches extending N- and C-terminally from
these domains (Fig. 1B). The
N-terminal regions of the polypeptides up to the T-box domains are moderately
conserved (>40% amino acid identities), whereas the C-terminal regions
contain only few short stretches of additional sequence similarity (data not
shown). Additional sequence comparisons with T-box domains from vertebrates
and phylogenetic analysis show that the Doc T-box domains are most closely
related to those from members of the Tbx6 subfamily of T-box proteins
(Fig. 1B,C)
(Papaioannou, 2001) (see
Discussion).
Dorsocross expression is prominent in dorsal tissues during
embryogenesis
Northern analysis with gene-specific probes showed that all three Doc genes
display similar expression profiles during development with maximal levels
occurring between 2 and 12 hours of embryonic development and lower levels
during late embryonic, larval and pupal stages. The only significant
difference among the three genes in this assay was the expression of
Doc1 mRNA in adult males, which was not observed for Doc2
and Doc3 (data not shown).
The spatial expression patterns of Doc products in embryos were examined by whole-mount in situ hybridization with gene-specific probes and whole-mount immunocytochemistry using antibodies raised against the unique C-terminal regions of the Doc proteins (see Materials and Methods). As all three genes were found to have essentially identical expression patterns (with some minor differences regarding the relative levels of expression in different tissues; data not shown), we will henceforth collectively refer to them as `Doc genes'. As expected, Doc proteins are exclusively nuclear during interphase.
The initial expression of Doc genes is observed at the cellular blastoderm
stage in a transverse stripe encompassing the dorsal 40% of the embryonic
circumference within the prospective head region. Shortly later, a narrow
longitudinal stripe of expression appears, which ultimately extends all along
the dorsal midline of the embryo, and the joint domains form a cross-shaped
pattern of Doc expression in dorsal areas of the early embryo
(Fig. 2A). The domain of the
transverse stripe is located anteriorly to the cephalic furrow forming during
gastrulation (Fig. 2B;
Fig. 3G) and largely
corresponds to procephalic neuroectoderm. The cells of this domain continue
Doc expression until stage 11, when the segregation of procephalic neuroblasts
is completed (Fig. 2B-D)
(Campos-Ortega and Hartenstein,
1997). By contrast, the cells from the dorsal longitudinal domain
within the trunk region give rise to amnioserosa, which maintains strong Doc
expression until stage 15 (Fig.
2C-F). In addition, the cells from the anterior and posterior
termini within this longitudinal stripe contribute to regions of the anterior
and posterior digestive tract and maintain expression until stage 11.
|
|
). As
shown in Fig. 2E, Doc and Cf1a
are expressed in mutually exclusive domains, which implies that Doc expression
encompasses prospective tissues of the lateral epidermis and dorsolateral
sensory organs. After stage 11, the segmental expression in the epidermis is
modified to form segmental stripes that are interrupted in dorsolateral
regions. Within these stripes, Doc expression is largely found in posterior
areas of the anterior compartments of each segment and there is a graded
distribution of Doc expression with increasing levels towards the posterior of
each stripe (Fig. 2F,G). The
dorsal epidermal expression domains now extend to the amnioserosa, and after
dorsal closure the bilateral domains merge at the dorsal midline
(Fig. 2F-H).
Additional sites of Doc expression during late embryogenesis include the
dorsal pouch in the embryonic head (Fig.
2H), the anterior pair of Malpighian tubules
(Fig. 2I) and the
pentascolopidial chordotonal sensory organs
(Fig. 2J). The mesodermal
expression of the Doc genes, which will be presented elsewhere in more detail
along with functional data, is observed in areas between the expression
domains of the homeobox gene bagpipe (bap) at stage 10
(Fig. 2K). This location
defines them as dorsal areas of the mesodermal A (or slp) domains
(Azpiazu et al., 1996;
Riechmann et al., 1997), which
include the dorsal somatic and cardiogenic mesoderm. During early stage 11,
additional Doc expression initiates in the caudal visceral mesoderm, which
contains the founder cells of the longitudinal muscles of the midgut
(Fig. 2L)
(San Martin et al., 2001;
Klapper et al., 2002). As
reported previously for Doc1, two out of six bilateral cardioblasts
in each segment of the dorsal vessel, which are tin negative and
svp positive, also express the Doc genes
(Fig. 2H)
(Lo and Frasch, 2001).
Doc expression along the dorsal midline depends on dpp and
zen
As peak levels of Dpp activity are known to be required for cell fate
determination at the dorsal midline, we tested whether there is a correlation
between Dpp activity and dorsal longitudinal Doc expression during blastoderm
stages. As shown in Fig. 3A,
double-staining for Doc mRNA and phosphorylated Mad (PMad) indicates a close
correlation between cells containing high levels of PMad and Doc products
within the dorsal-longitudinal stripe. In addition, faint Doc signals that are
modulated in a pair-rule pattern extend into areas that receive lower Dpp
inputs and lack detectable PMad (Fig.
3A). As predicted, both PMad and Doc expression in the
dorsal-longitudinal stripe, but not the dorsal-transverse head stripe of Doc
expression, are absent in dpp-null mutant embryos
(Fig. 3B). Conversely, in
blastoderm embryos with ubiquitous Dpp expression (UAS-dpp activated
by maternally provided nanos-GAL4), we observe a significant widening
of the dorsal-longitudinal stripes of PMad and Doc expression, during which
the correlation between high PMad and Doc mRNA levels is still maintained
(Fig. 3C).
The expansion of PMad upon uniform ectopic expression of dpp includes the prospective mesoderm, although not ventrolateral areas of the blastoderm embryo (Fig. 3D). However, high PMad in the prospective mesoderm does not trigger ectopic Doc expression, suggesting either the presence of a ventral repressor or the requirement for a co-activator in dorsal areas. A candidate for a co-activator is the homeobox gene zerknüllt (zen). Double in situ hybridization shows that the appearance of dorsal Doc mRNAs coincides with the time when zen mRNA levels increase in the areas of the presumptive amnioserosa as a result of high Dpp inputs (Fig. 3E). When the refinement of zen expression is completed, there is an exact correspondence in the widths of the Doc and zen expression domains, although Doc expression extends more posteriorly (Fig. 3F). As shown in Fig. 3H (compare with Fig. 3G), the activity of zen is necessary for normal levels of Doc expression in the dorsal-longitudinal stripe, because in zen mutant embryos there are only low residual levels of Doc products present in this domain. These observations suggest that Doc expression along the dorsal midline of blastoderm embryos requires the combined activities of dpp and zen.
Metameric Doc expression in dorsal ectoderm and mesoderm requires Dpp
+ Wg
The known distribution of dpp mRNA during its second phase of
expression in the dorsolateral ectoderm of stage 9-11 embryos
(St Johnston and Gelbart,
1987) suggests that Doc expression in the dorsolateral ectoderm
and mesoderm during these stages is also dependent on Dpp activity. As
expected from the known fate map shifts in dpp mutants, these domains
of Doc expression are missing in dpp-null mutant embryos (data not
shown). Notably, the exact coincidence between the ventral borders of the
domains of dorsolateral Doc expression and high nuclear PMad
(Fig. 4A,B) suggests that Doc
expression is directly controlled by Dpp-activated Smad proteins in the
ectoderm and mesoderm during this stage. Additional evidence for this
hypothesis comes from experiments with ectopic expression of dpp in
the ventral ectoderm of the Krüppel domain (by virtue of a modified
Kr-GAL4 driver) (Frasch,
1995), which results in the concomitant expansion of PMad and the
Doc expression stripes towards the ventral midline
(Fig. 4C).
|
), results in a reduction, but not a complete loss of
metameric DOC expression (data not shown). Hence, slp probably
affects Doc indirectly through its effect on ectodermal wg
expression. Altogether, our data suggest that metameric Doc expression in the
ectoderm and mesoderm is triggered by the intersecting activities of Wg and
Dpp.
The Doc genes are required for full differentiation and maintenance
of amnioserosa cells
The similarities in sequence and expression of the three Doc genes
suggested functional redundancy among these genes. Because our molecular
analysis of available deficiencies at 66E-F showed that none of them uncovered
all three genes (Fig. 5A) we
used the flanking P-insertions EP(3)3556 and EP(3)584 in
attempts to delete the entire Doc gene cluster via male recombination-induced
mutagenesis (see Materials and Methods). Molecular mapping of the obtained
deletions demonstrated that two of them, Df(3L)DocA and
Df(3L)DocB, which were generated with the distally located insertion
EP(3)3556 and cause embryonic lethality, deleted all three Doc genes
(Fig. 5A). As
Df(3L)DocA deletes the smallest number of additional genes
(CG5087, CG5194, CG5144, Argk and CG4911), we describe the
phenotypic analysis in the present study using this deficiency, although the
salient phenotypes are very similar between Df(3L)DocA and
Df(3L)DocB.
Additional genetic analysis showed that it is possible to obtain a small number of viable adult escapers with the genotype Df(3L)Scf-R11/Df(3L)DocA, which indicates that CG5087 is not absolutely required for viability, and that Doc1 and Doc2 can functionally substitute for the loss of Doc3. Similarly, the full viability of flies with the genotype Df(3L)DocA/Df(3L)EP584MR2 (Fig. 5A) shows that CG4911 and the 5' exons of Argk (preceding the large intron) are also not essential. Furthermore, we determined that embryos with the genotypes Df(3L)Scf-R11/Df(3L)DocA and Df(3L)DocA/Df(3L)29A6 (which causes pupal lethality) do not display any of the phenotypes described below for Df(3L)DocA homozygous embryos. In summary, our genetic analysis shows that the loss of either Doc3 or Doc1 can be compensated for by the remaining two Doc genes in embryos and that the phenotypes described herein are a consequence of the loss of all three of the Doc genes. However, we can not rule out a contribution of CG5194, which encodes a 128 amino acid predicted ORF with no known homology, to the observed phenotypes.
Because of the prominent Doc expression in the primordia and developing tissue of the amnioserosa we used the amnioserosa marker Krüppel (Kr) to examine whether the Doc genes are required for the development of this extra-embryonic tissue. These experiments demonstrated that homozygous Df(3L)DocA mutant embryos (henceforth called DocA mutants) fail to express Kr in the amnioserosa at any stage, whereas CNS expression of Kr is not affected (Fig. 5B). To confirm that this observed phenotype is due to the loss of Doc gene function we diminished Doc gene functions by using RNA interference (RNAi) as an independent assay and performed rescue experiments with DocA mutants (see below). As shown in the example of Fig. 5C, injection of a mixture of equimolar amounts of dsRNAs for all three Doc genes (see Materials and Methods) frequently results in a complete absence of Kr expression in the amnioserosa. The remaining embryos display strongly reduced numbers of Kr-containing nuclei in this tissue (data not shown). These phenotypes correlate with the observed absence or severe reduction of Doc protein levels in Doc RNAi embryos (data not shown). By contrast, mock-injected embryos display normal expression of Kr in the amnioserosa (Fig. 5D). Hence, the strongest phenotype obtained by RNAi mimics the observed DocA mutant phenotype, confirming that the lack of Kr expression in DocA mutant embryos is specifically due to the loss of the activity of all three Doc genes.
Besides the effects on Kr expression, DocA mutant and
RNAi-treated embryos share several morphological defects. The extending germ
band is unable to displace the amnioserosa fully towards the anterior and the
posterior germ band is therefore forced to bend underneath the amnioserosa. Of
note, germ band retraction is strongly disrupted, which can be clearly seen in
stage 14 embryos (Fig. 5E) and
in cuticle preparations of unhatched first instar larvae
(Fig. 5H,I; compare with J). This phenotype is shared with previously described genes of the u-shaped (ush)
group, which affect the maintenance of the amnioserosa
(Frank and Rushlow, 1996). Kr
expression and the germ band retraction defects in DocA mutant
embryos can be partially rescued by expressing any of the three Doc genes with
an early amnioserosa-specific driver (Fig.
5F,G). Rescue with Doc2
(Fig. 5F) is consistently more
efficient when compared with Doc1 (data not shown) and Doc3
(Fig. 5G), although it is not
known whether this difference is due to a higher intrinsic activity or a more
efficient expression of Doc2 protein in this assay.
An additional phenotype consists of reductions in the size of the embryonic
head in DocA mutants and RNAi-treated embryos, which is apparent from
stage 12 onwards and results in reduced head structures and a frequent failure
of head involution at later stages (Fig.
5B,C,H,I, and data not shown). This phenotype is probably due to
excessive cell death as a consequence of the absence of Doc activity in the
procephalic neuroectoderm and other dorsal areas of the embryonic head
(Fig. 2B,C and data not shown).
The observed head phenotypes, as well as the aberrant shape of the
filzkörper (Fig. 5I), are
also reminiscent of similar phenotypes of embryos mutant for genes of the ush
group (Frank and Rushlow,
1996).
To obtain more information about the particular role of the Doc genes in
the specification and/or differentiation of the amnioserosa we analyzed the
distribution of additional amnioserosa markers in DocA mutant
embryos. For the ush group gene hnt
(Yip et al., 1997) we find a
strong reduction of expression, with significant levels of Hnt protein only
being detected in nuclei along the posterior margin of the amnioserosa
(Fig. 6A,
Fig. 7A, compare with
Fig. 6B and
Fig. 7B, respectively). By
contrast, the expression of the amnioserosa marker race
(Ance – FlyBase) (Tatei et
al., 1995) is initiated normally in the primordium of the
amnioserosa of DocA mutant embryos, suggesting that the expression of
the race upstream activator zerknüllt (zen) is
also not disrupted (data not shown). However, after embryonic stage 9,
race expression is gradually lost in the amnioserosa of DocA
mutant embryos and its residual mRNA distribution closely follows that of Hnt
(Fig. 7A, compare with 7B).
|
). In addition, from early stage 10 onwards there is a narrow
domain of expression at the leading edge of the dorsal germ band, which later
becomes segmental (Fig. 6F,H).
In DocA mutant embryos, the level of C15 expression in early
amnioserosa cells is unaltered, which allows us to use C15 protein as a marker
for the development of this tissue in the absence of Doc activity.
Until stage 9, the large majority of amnioserosa nuclei in DocA
mutant embryos appear large and flattened as in wild-type embryos
(Fig. 6C, compare with 6D). Together with data from -tubulin staining (not shown), this observation
indicates that the amnioserosa cells begin to acquire the normal features of a
squamous epithelium (data not shown). However, the amnioserosa does not
display a properly folded morphology during stages 8-10, and the posterior
germ band is forced to bend towards the inside in DocA mutant embryos
(Fig. 6C, compare with 6D, and
data not shown). In addition, some small nuclei become detectable within the
amnioserosa during this stage (Fig.
6C, arrow). Altogether, these observations indicate that the
amnioserosa initiates its differentiation process in the absence of Doc gene
activity but fails to complete it, thus leading to morphological and
functional abnormalities of this tissue towards the end of germ band
elongation. Much stronger alterations can be observed during subsequent
stages, when there are an increasing number of C15-stained amnioserosa nuclei
with much smaller diameters than regular amnioserosa nuclei. At late stage 12,
almost all amnioserosa cells feature small nuclei that are difficult to
distinguish from dorsal epidermal cells
(Fig. 6E,
Fig. 7C, compare with
Fig. 6F,
Fig. 7D, respectively).
Co-staining for race indicates that it is predominantly the cells
with the small nuclei that lose race expression, while most
normally-sized nuclei are still surrounded by race signals
(Fig. 7C, compare with D). From
this stage onwards, non-stained `holes' appear in the amnioserosa and the
number of C15-stained amnioserosa nuclei decreases prematurely. Hence, unlike
wild-type embryos, stage 14 DocA mutant embryos are not covered
dorsally by C15-stained amnioserosa cells
(Fig. 6G, compare with 6H). In
addition to the observed alterations in the amnioserosa, the C15 expression
domain at the leading edge of the epidermis appears significantly broadened
(Fig. 6E).
We tested whether the increasing number of smaller nuclei in the amnioserosa of DocA mutant embryos is connected with abnormal cell divisions. As shown in Fig. 7E, the M-phase marker phospho-Histone H3 can be detected in numerous amnioserosa nuclei of DocA mutant embryos after stage 10, which is not seen in wild-type embryos (Fig. 7F). In addition, there is significant incorporation of BrdU in amnioserosa nuclei of DocA mutant embryos (particularly in the small nuclei; Fig. 7G), whereas no incorporation is observed in wild-type embryos (Fig. 7H). Mitotic spindles are also present in the amnioserosa of DocA mutants (Fig. 7I, compare with J). These observations indicate that the normal G2 arrest of amnioserosa cells has been released and the cells re-enter the cell cycle. We also examined whether the subsequent disappearance of small C15-stained amnioserosa nuclei in DocA mutant embryos is a result of premature apoptosis of cells in this tissue. This possibility was confirmed by the results of TUNEL labeling experiments, which produced signals in many amnioserosa nuclei from 12 onwards. Most of the TUNEL-labeled nuclei have reduced or are lacking C15 expression (Fig. 7K, compare with 7L, which shows that wild-type amnioserosa nuclei at late stage 12 are not apoptotic). Altogether, these observations suggest that loss of Doc activity prevents the normal differentiation of the amnioserosa to a fully functional tissue, suspends the cell cycle block of amnioserosa cells, and causes premature apoptotic cell death in this tissue.
Doc patterns the lateral ectoderm via repressing wg and
ladybird
The segmental stripes of wg expression in the embryonic trunk
segments initially span the entire dorsoventral extent of the ectoderm, but at
stage 11 they become interrupted in dorsolateral areas
(Baker, 1988). A comparison of
Wg and Doc expression at this stage shows that the positions of the metameric
ectodermal domains of Doc expression correspond to the areas in which the Wg
stripes become interrupted (Fig.
8A). Temporally, there is a brief overlap of ectodermal Wg and Doc
expression during stage 10 until Wg expression is downregulated within the Doc
domains (see Fig. 4E). In
contrast to the wild-type situation (Fig.
8A,C), the Wg stripes remain continuous in DocA mutant
embryos (Fig. 8B). Similar
observations were made with the homeobox gene product Ladybird (Lb=Lbe + Lbl)
(Jagla et al., 1997) as a
marker. In wild-type embryos after stage 11, Lb is also expressed in striped
domains that are interrupted at the positions of the ectodermal Doc domains
(Fig. 8D), whereas in
DocA mutant embryos there is ectopic expression in a pattern of
continuous stripes (Fig. 8E, compare with
F). These data show that Doc activity is required for patterning
events in the dorsolateral ectoderm, which include the repression of
wg and lb expression in these areas.
|
|
), its
repression by ectopic Doc results in the loss of distal structures of wings,
legs and antenna of adult animals (Fig.
9G-I). Analogous ectopic expression experiments with Doc1
and Doc3 in embryos and discs produced qualitatively similar,
although weaker, effects to Doc2. |
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A prominent feature of the Doc genes is their expression in areas that receive inputs from Dpp, including the dorsalmost cells in blastoderm embryos, the dorsolateral ectoderm and mesoderm in the elongated germband, and distinct domains spanning the compartment border of the wing disc. Indeed, our genetic data, together with the co-localization of Doc transcripts with active Mad in dorsal embryonic tissues, favor the possibility that the Doc genes are direct targets of the Dpp signaling cascade. However, the Dpp signals are required to act in combination with additional regulators during each of these inductive events.
Regulation and function of the Doc genes during amnioserosa
development
Our observations suggest that robust and stable induction of Doc expression
in a dorsal stripe requires the activity of the homeodomain protein Zen as a
co-activator of Dpp signals. The zen gene features an early, broad
expression domain along the dorsal embryonic circumference, which is initially
Dpp independent but subsequently requires Dpp for it to be maintained
(Rushlow et al., 1987;
Ray et al., 1991). Thereafter,
its expression refines into a narrow dorsal domain in a process that requires
peak levels of Dpp (Rushlow and Levine,
1990; Ray et al.,
1991; Rushlow et al.,
2001). The activation of Doc expression occurs at the same time as
the refinement of zen expression and within the same narrow domain,
which also coincides with high phospho-Mad levels
(Rushlow et al., 2001).
Although the maintenance and refinement of zen by Dpp is zen
independent (Ray et al.,
1991), we propose that Zen synergizes with peak signals of Dpp to
trigger Doc gene expression in a dorsal stripe. The requirement for this
proposed interaction between zen and dpp would explain the
failure of zen to activate Doc genes in an early, broad domain as
well as the observed low levels of residual Doc expression in zen
mutant embryos, which may be due to inputs from Dpp alone. Formally, this
proposed mechanism would be analogous to previously described inductive events
in the early dorsal mesoderm, where the synergistic activities of the
homeodomain protein Tinman and activated Smads induce the expression of
downstream targets such as even-skipped
(Halfon et al., 2000;
Knirr and Frasch, 2001). The
identification of functional binding sites for Zen and Smads in Doc enhancer
element(s) will be necessary for demonstrating that an analogous mechanism is
active during induction of Doc gene expression in a dorsal stripe. In the
absence of such data, we can not completely rule out that dorsal Doc
expression is controlled indirectly by Dpp, possibly via the
combinatorial activities of zen and another high-level target gene of
Dpp. As mutations in several other genes that are expressed in the early
amnioserosa, including pannier (pnr), hnt, srp, tup
and ush, do not affect Doc expression until at least stage 12 (I.R.
and M.F., unpublished), these genes can be excluded as candidates for early
upstream regulators of Doc.
Unlike zen, which is expressed only transiently, Doc expression is maintained throughout amnioserosa development. Hence, the Doc genes provide a functional link between the early patterning and specification events in dorsa
