/. Embryol. exp. Morph. 97 Supplement, 157-168 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
157
Probing gene activity in Drosophila embryos
HERBERT JACKLE, EVELINE SEIFERT, ANETTE PREISS AND
URSB. ROSENBERG
Max-Planck-Institut fur Entwicklungsbiologie, Abteilung Biochemie, Spemannstrafie
35/11, 7400 Tubingen, Federal Republic of Germany
INTRODUCTION
The segmentation pattern of the Drosophila wild-type embryo is characterized
by a number of easily identifiable cuticular structures. They include skeletal
elements of the involuted head and ventral denticle belts that define by size,
pattern and orientation the anterior part of the three thoracic and eight abdominal
segments. Further landmarks such as sensory organs and the posterior tracheal
endings ('Filzkorper'), in combination with the denticle belts, allow one to unequivocally determine the polarity and quality of each segment in preparations of
the larval cuticle (see Fig. ID).
The segmentation pattern of Drosophila is established at about blastoderm stage
and it requires both maternally and zygotically active genes. Genetic analysis has
identified a number of genes with zygotic activity that regulate key steps during
pattern formation. Mutations in these genes cause specific defects in the segmental
pattern of the embryo that allow the definition of classes of segmentation genes
required for the subdivision of the embryo into segmental units (Nusslein-Volhard
&Wieschaus, 1980).
Kriippel (Kr) is a member of the gap class of segmentation genes that are
characterized by a deletion of adjacent body segments in the mutant embryo.
Embryos homozygous for Kr mutations die before hatching and show a unique
phenotype. A total of twenty-eight alleles can be ordered into a phenotypic series.
In amorphic alleles, all three thoracic and five out of eight anterior abdominal
segments are deleted. Deleted segments are partially replaced by a mirror-image
duplication of parts of the normal posterior abdomen (compare Fig. 1A and D)
often including the dorsally located Filzkorper. Some intermediate alleles have all
thoracic and four abdominal segments deleted but no duplication except that
ectopic Filzkorper develop frequently close to the head region (Fig. IB). In
weaker alleles, progressively fewer segments are deleted and the prothorax is
always developed (Fig. 1C-G). The weakest detectable phenotype is observed in
heterozygous Kr embryos showing small defects in the denticle bands of thoracic
Key words: Drosophila, gene activity, segmentation, Kriippel (Kr), phenotypic rescue, antisense RNA.
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H. JACKLE, E. SEIFERT, A. PREISS AND U. B. ROSENBERG
Fig. 1. Cuticular pattern of Kr mutant embryos aligned into a phenotype series. (A)
Amorphic Kr allele; not the lack of the three thoracic and five anterior abdominal
segments being replaced by a mirror-image duplication of the normal sixth abdominal
segment. (B,E) Intermediate Kr phenotype; note the presence of a normal fifth
abdominal segment. (D) Wild-type cuticular pattern of a Drosophila larva showing
skeletal structures of the involuted head, three thoracic (T1-T3) and eight abdominal
(A1-A8) segments that can be distinguished by denticle bands marking the anterior
boundary of each segment, and a pair of posterior tracheal endings, the Filzkorper.
(E-G) Weak Kr phenotype; note the presence of Tl and the increasing number of
anterior abdominal segments. Dark-field photographs; the wild-type embryo is about
1 mm long.
Probing gene activity in Drosophila embryos
159
and anterior abdominal segments. Such embryos may hatch and survive to become
adults. The common motif of all alleles so far analysed is the defect in the thorax
region and as the alleles get stronger, a deletion of progressively larger regions in
the segment pattern up to eight segments in the strongest amorphic alleles. The
interpretation of this phenotypic series is a lack of Kr function in strong alleles,
increasing residual Kr+ activity in intermediate and weak alleles and half the
normal Kr+ activity in heterozygous Kr embryos, which are almost normal. Aside
from the fact that the Kr gene, its requirement and possible interaction with other
genes for normal segmentation is interesting in its own right, it appeared sensible
to use the Kr mutant embryos as a biological assay system for Kr+ activity provided
by injected material, and to use changes along the phenotypic Kr series as an
indicator for inhibition of Kr+ activity in wild-type embryos being injected with
gene-specific probes. This experimental design is especially promising in viewing
the accessibility of Drosophila eggs and embryos for injection studies (see
Anderson & Niisslein-Volhard, 1984 for details).
PHENOTYPIC RESCUE AFTER INJECTION OF WILD-TYPE CYTOPLASM INTO KT
MUTANT EMBRYOS
Injection of wild-type cytoplasm provides phenotypic rescue in mutant Kr
embryos. Embryos from a Kr SMI mating (SMI is a balancer chromosome to
maintain Kr stocks) were injected. To distinguish homozygous Kr embryos from
their siblings, the mutant Kr1 chromosome was marked in all experiments with a
dopadecarboxylase mutation (Ddc) as it renders the cuticle and mouth parts of
homozygous Kr1 larvae unpigmented. Such embryos express the strong Kr phenotype (Fig. IB) which is always associated with a duplication of the sixth abdominal
segment in reversed polarity (Fig. 2A). Injection of cytoplasm taken from whole
wild-type embryos up to the late blastoderm stage into stages younger than late
blastoderm stage had no effect on this phenotype, independent of where it was
injected into the Kr mutant embryos. By contrast, when cytoplasm was taken from
a middle region of blastoderm-stage wild-type embryos and transferred into a
middle region of Kr embryos, up to 40 % of these developed segments with normal
polarity anterior to the sixth abdominal segment ('phenotypic rescue', Fig. 2B).
This indicates weak but significant Kr+ activity in Kr mutant embryos provided by
the transferred cytoplasm. The weak phenotypic rescue encouraged us to analyse,
under standardized conditions, the developmental profile of Kr+ activity in wildtype cytoplasm, its spatial distribution and the region responding to rescue in Kr
mutant embryos.
STAGE-DEPENDENCE OF KT+ ACTIVITY IN WILD-TYPE EMBRYOS
Cytoplasm from the 45-55 % egg region (0 % is the posterior pole) was taken
from wild-type embryos at stages between egg deposition and late blastoderm, and
transferred into the same region of Kr embryos at pole cell to migration stages. As
160
H. JACKLE, E. SEIFERT, A. PREISS AND U. B. ROSENBERG
shown in Fig. 3A, phenotypic rescue was obtained with cytoplasm from blastoderm stage donors, but not with cytoplasm from younger embryos. Furthermore,
the rescue response was increased by use of older cytoplasm indicating the Kr+
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Fig. 2. Enlarged abdominal region of an amorphic Kr allele showing (A) reversed
polarity (arrow) in the duplicated sixth abdominal segment. A6 marks the normal sixth
abdominal segment. Orientation of denticles can be taken to establish the polarity of a
given denticle row. (B) Same region of an amorphic Kr allele injected with wild-type
cytoplasm as described in the text. Note the normal polarity (arrow) of an additional
segment anterior to A6 which is taken as a criterion for 'phenotypic rescue'.
Probing gene activity in Drosophila embryos
161
activity accumulates during blastoderm stage. However, when the highly active
cytoplasm was injected into Kr embryos at late blastoderm stage, no rescue was
observed. This indicates that Kr+ activity was injected after the phenocritical
period and/or that the molecules ultimately providing Kr+ activity require some
time to accumulate the minimum level of activity that is necessary for phenotypic
rescue. In the light of (i) a correlation of Kr+ activity in the cytoplasm and Kr+
mRNA accumulation at the respective stages (see Knipple et al. 1985 for details),
(ii) first indirect evidence for the Kr product being a DNA-binding protein which
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Fig. 3. Phenotypic rescue of Kr mutant embryos after injection of wild-type
cytoplasm. (A) Cytoplasm was taken from the middle of wild-type embryos at different
stages of development (abscissa) and injected into the middle region of early cleavage
stage embryos from Ddc Krl/SM1 parents. Note that first rescue effects were seen with
cytoplasm from embryos at blastoderm stage. (B) Cytoplasm taken from late
blastoderm-stage wild-type embryos was effective when injected in Kr embryos at pole
cell to cleavage stage but not at late blastoderm stage. Ordinate: % of rescued Kr /Kr1
embryos that can be distinguished from their injected siblings by the Ddc phenotype
(skeletal parts and denticles unpigmented). For details of injection and analysis see
Knipple etal. (1985). n, number of injected Kr^/Kr1 embryos; r, number of rescued
embryos.
162
H. JACKLE, E. SEIFERT, A. PREISS AND U. B.
ROSENBERG
should be present in nuclei (see Rosenberg etal. 1986 for details) and (iii) the
protein product is excluded from the injection experiments, we favour the view
that Kr mRNA is transferred with cytoplasm into the Kr embryo and it requires
there amplification of the Kr gene products by translation before it provides Kr
rescuing activity.
KT+ ACTIVITY IS BOTH ACCUMULATED AND REQUIRED IN THE MIDDLE OF THE
EMBRYO
Kr mutant embryos were injected, at pole cell stage, with cytoplasm taken from
different regions of wild-type embryos. Cytoplasm from any region of embryos
younger than syncytial blastoderm stage was ineffective (Fig. 4A), while cytoplasm taken from the middle region but not from 0-30 % or 75-100 % of egg
length of blastoderm-stage embryos, showed phenotypic rescue. This indicates
that Kr+ activity is at least enriched, if not exclusively present, in the middle of the
wild-type embryos (Fig. 4B), and possibly available at about blastoderm stage.
Cytoplasm was taken from the middle region of blastoderm-stage wild-type
embryos and then transferred into different regions of Kr embryos. Phenotypic
rescue response was only seen between 30-70 % of the egg length (Fig. 4C) which
is the region where Kr+ activity accumulates in wild-type embryos. This demonstrates that both Kr+ activity in wild-type embryos and the rescue responsive
region in Kr embryos coincide within the limits of resolution.
The position of both Kr+ activity accumulation and Kr+ requirement in the
middle region of the embryo correlates with the region affected in weak Kr mutant
embryos (Fig. 1), the blastoderm fate map position of thoracic and anterior
abdominal anlagen (see Fig. 4D), and the region where Kr+ transcripts accumulate during blastoderm stage (see Knipple etal. 1985).
PROBING CLONED DNA IN Kr MUTANT EMBRYOS
The finding that Kr+ activity can be transferred into Kr mutant embryos (its
effect being to weaken the strong Kr phenotype) clearly demonstrates that low
Kr+ activity provokes biological resonance. Considering that only about 2 % of
the total egg volume was transferred and the Kr gene product is required in more
than 20 % of the wild-type eggs, the effective dilution of Kr+ activity is by more
than one order of magnitude. This means that the weak phenotypic rescue
observed is within the expected range of biological response, provided that 50 %
gene activity in Kr heterozygous embryos already express a weak Kr phenotype.
Based on this, we felt encouraged to use Kr mutant embryos as a diagnostic tool to
identify the Kr coding gene sequences on cloned genomic DNA which should
cover the Kr region.
Genetics and deletion mapping placed the Kr locus in polytene chromosome
band 60 F3 at the tip of the right arm of chromosome two. Clones obtained from
microdissected DNA of the corresponding band facilitated the isolation of some 50
163
Probing gene activity in Drosophila embryos
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Fig. 4. Localization of Kr activity in wild-type and the rescue responsive regions in Kr
mutant embryos. Ordinate for A-C: % of rescued Kr embryos; see legend to Fig. 3.
Abscissa for A-E: region (in % of egg length) where cytoplasm was taken from (A)
early cleavage or (B) blastoderm-stage wild-type embryos to be injected into the
middle of early cleavage stage Kr embryos or (C) the region where cytoplasm from the
middle of blastoderm-stage wild-type embryos was injected in Kr embryos, n, r: see
legend Fig. 3. Note that rescue was only obtained when cytoplasm from the middle of
blastoderm-stage wild-type embryos (see A,B) was injected into the middle region (C)
of Kr embryos. (D) Correlation of the blastoderm fate map (left half) with the Kr
responsive region (from C) and localization of Kr+ activity (from B). Note that both
regions are almost identical and smaller than the gap seen in the segment pattern of
amorphic Kr embryos.
164
H. JACKLE, E. SEIFERT, A. PREISS AND U. B. ROSENBERG
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Fig. 5. Molecular map of Kr region. The coordinates (in kb) are based on an EcoRI
site at the start point of the chromosomal walk. (A) Restriction map of DNA from the
// chromosome used for mutagenesis. The same map was found for the Bl If
chromosomes used to make chromosomal rearrangements. The four restriction
enzymes used for mapping were: R, EcoRI; B, BamHI; S, Sal I; H, Hindlll.
(B) Individual phage clones covering the Kr region. The cloned DNA originated from
homozygous Ifflies(designation AI, each insert ends with a true EcoRI site), Canton S
embryos (designation cc), or Oregon R embryos (designation ER; each insert ends
with a Sau3A site due to partial digestion of genomic embryo DNA and ligation into
the BamHi site of vector DNA). Note polymorphic restriction sites in Oregon R DNA,
that is, two additional EcoRI sites and the absence of one Sail and one Hindlll site also
absent in CyO DNA. Fragments smaller than 0-5 kb and other clones overlapping the
indicated clones are not shown. Vectors used for library construction were EMBL 4
(ER), Charon 4 (cc) and Charon 4A (AI). Note that only clone ER3 DNA provides
Kr+ rescuing activity as described in the text. Methods: DNAs from phages were
mapped with four restriction enzymes after separation on 0-8 % agarose gel. End
fragments of DNA inserts were labelled by nick translation to screen the three libraries
for homologous sequences flanking either side of the original clone. For each walking
step, the presence of repetitive sequences was tested by reverse Southern analysis using
32
P-labelled genomic DNA as probe. As an additional control, the size of each
restriction fragment was checked using Southern blots of genomic DNA.
kilobases (kb) of genomic DNA in a series of overlapping clones (Fig. 5; Preiss
etal. 1985 for details). The use of cytologically mapped deletions enabled us to
identify DNA sequences within a 4 kb interval as those required for Kr+ gene
function. Both molecular analysis and subsequent transcript mapping were consistent with the localization of the Kr locus in or close to the region 0 to +10 shown
in Fig. 5. To identify the Kr+ gene function, we injected cloned DNA into mutant
embryos and scored them for possible phenotypic rescue effects resulting from
Kr+ activity provided by the injected DNA.
DNAs from various clones for most of the cloned region were injected into
various regions of pole cell stage embryos. The segment anterior to the sixth
abdominal segment showed normal polarity in about 40 % of the homozygous Kr
embryos only after injection of clone ER3 (which contains an 18 kb segment of
genomic Drosophila DNA) into the middle of the embryo. These embryos more
closely resembled the intermediate rather than the strong Kr phenotype of
uninjected Kr embryos. A small fraction of injected Kr embryos (about 5 %)
Probing gene activity in Drosophila embryos
165
developed more than two additional segments with normal polarity (see also
Fig. 2), indicative of a substantial weakening of the amorphic phenotype.
In our most successful experiments, we injected about 300 pi of DNA
(130//g ml" 1 ) which corresponds to about 106 molecules. This number is about 100
times the number of Kr+ gene copies of normal blastoderm-stage embryos, or 500
times the number of Kr+ gene copies being expressed (see Preiss etal 1985).
Earlier experiments demonstrated that about 80 % of the injected DNA is rapidly
degraded and that transient transcription from the remaining DNA is more than
one order below the efficiency of endogenous gene transcription.
The degree of rescue response, as in the case of cytoplasm transfer (see above)
was in the expected range. These experiments therefore suggest that ER3 clone
DNA contains Kr+ sequences and that our transient expression assay, based on
phenotypic rescue, identifies the function of cloned DNA.
PROBING Kr FUNCTION BY INJECTION OF Kr ANTI-SENSE RNA
The above experiments used Kr mutant embryos as an indicator for Kr+ activity
contained in the injected material which weakened the strong Kr phenotype.
However, this way of identifying genes and their activity requires the prerequisite
of genetical and cytological analysis in combination with a refined set of molecular
techniques. This approach is, therefore, limited to a small number of biological
systems where the combination of genetics, cytology and transfer of macromolecules and/or recombinant DNA transformation is accessible. To overcome
this limitation and to establish a general tool for probing gene function in less
fortunate systems, we designed experiments to inhibit a specific gene function.
This assay involves the production of RNA containing the complementary
sequences to the natural mRNA ('anti-sense RNA'). Upon injection, both RNAs
should form duplexes by hybridization in vivo and thus prevent the mRNA from
being normally translated.
Several features make the Kr gene useful for assessing this sequence-specific
inhibition of gene function. First, Kr gene function is defined by the number of
phenotypes that can be aligned in an allelic series (see above). Second, the Kr
transcripts accumulate in a defined region of the embryo. During the period of first
expression, at syncytial blastoderm stage, nuclei and their surrounding cytoplasmic islands may be accessible to injected anti-sense RNA while individual cells
that form during blastoderm stage may not. Third, the Kr+ gene is only transcribed
between the syncytial blastoderm stage and the beginning of germband extension,
producing a rare 2-5-kb poly(A) + RNA transcript. Most of this transcript has been
recovered in a 2-3kb cDNA clone (Rosenberg etal. 1985). This cDNA was
subcloned, in both orientations, into plasmid DNA containing the SP6 promoter
which allows Kr sense and anti-sense RNA to be transcribed in vitro using SP6
RNA polymerase which specifically starts transcription at the SP6 promoter site
(see Rosenberg et al. 1985, for a detailed description).
166
H. JACKLE, E. SEIFERT, A. PREISS AND U. B. ROSENBERG
Wild-type embryos were injected with either sense or anti-sense RNA at the
syncytial blastoderm stage. Sense RNA, which contained only part of the mRNA
sequence, had no specific effect on the embryonic phenotype. By contrast,
injected Kr anti-sense RNA had a dramatic effect on genetically wild-type
embryos, i.e. they developed lethal Kr phenocopies (see Fig. 6) with up to 30 %
frequency. Some of the phenocopies developed ectopic Filzkorper close to the
head region, as found in Kr mutants developing the intermediate phenotype. This
new potential of the thoracic region to develop a structure from a dorsal-posterior
Fig. 6. Kruppel phenocopy produced by injection of anti-sense RNA to the Kr mRNA.
Note that this embryo which is genotypically wild type closely resembles an
intermediate Kr phenotype (see Fig. 1C) showing an ectopic pair of Filzkorper close to
the head region (arrow).
Probing gene activity in Drosophila embryos
167
position on the blastoderm fate map demonstrates unequivocally the production of
Kr phenocopies in wild-type embryos. Extreme Kr phenocopies resembling the
amorphic Kr phenotype (Fig. 1A), however, were not observed, indicating that
Kr+ activity was not abolished completely (for details see Rosenberg etal. 1985).
CONCLUSIONS
The experiments with Kr embryos demonstrate the use of Drosophila mutant
embryos for injection studies on the activity of the wild-type gene, localization of
and local requirement for the gene product in vivo. Moreover, cloned DNA can be
identified as functional sequences thus facilitating the delimitation of sequences
being required for at least the coding region of a given gene. In this respect, a
simple assay system allowed us to confirm predictions (Wieschaus, NiissleinVolhard & Kluding, 1984) made from genetic analysis: the middle region of the
embryo accumulates Kr+ activity. This region coincides with the anlagen of thorax
and anterior abdomen on the blastoderm fate map and appears to be most
sensitive to the absence of the gene product, as reflected in the lack of thoracic and
anterior abdominal segments in weak Kr alleles. The results of the injection
experiments were the first indication of localized Kr+ gene product requirement
during blastoderm stage which are possibly reflected in the localized expression of
the Kr gene revealed by in situ hybridization of the molecular Kr probe to sections
of embryos (see Knipple et al. 1985).
The reverse experiment, trying to inhibit a specific gene function by injection of
anti-sense RNA offers a great potential not limited to Drosophila embryos. While
in principle it is possible to physically isolate and clone almost any gene, it is often
difficult or even impossible to ascribe a discrete function to a cloned DNA
sequence, except for organisms where classical genetics has identified a gene
function, and transformation of cells with foreign DNA is well established.
Although far from being optimized, the potential of anti-sense RNA inhibition,
especially in combination with transformation and amplification of 'flipped gene
constructs' transcribing from strong promoters (for an example see Kim & Wold,
1985) is clear and needs no further explanation.
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