Probing gene activity in Drosophila embryos

/. Embryol. exp. Morph. 97 Supplement, 157-168 (1986) 157Printed in Great Britain © The Company of Biologists Limited 1986

Probing gene activity in Drosophila embryos

HERBERT JACKLE, EVELINE SEIFERT, ANETTE PREISS ANDURSB. ROSENBERG

Max-Planck-Institut fur Entwicklungsbiologie, Abteilung Biochemie, Spemannstrafie35/11, 7400 Tubingen, Federal Republic of Germany

INTRODUCTION

The segmentation pattern of the Drosophila wild-type embryo is characterizedby a number of easily identifiable cuticular structures. They include skeletalelements of the involuted head and ventral denticle belts that define by size,pattern and orientation the anterior part of the three thoracic and eight abdominalsegments. Further landmarks such as sensory organs and the posterior trachealendings ('Filzkorper'), in combination with the denticle belts, allow one to un-equivocally determine the polarity and quality of each segment in preparations ofthe larval cuticle (see Fig. ID).

The segmentation pattern of Drosophila is established at about blastoderm stageand it requires both maternally and zygotically active genes. Genetic analysis hasidentified a number of genes with zygotic activity that regulate key steps duringpattern formation. Mutations in these genes cause specific defects in the segmentalpattern of the embryo that allow the definition of classes of segmentation genesrequired 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 arecharacterized by a deletion of adjacent body segments in the mutant embryo.Embryos homozygous for Kr mutations die before hatching and show a uniquephenotype. 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 abdominalsegments are deleted. Deleted segments are partially replaced by a mirror-imageduplication of parts of the normal posterior abdomen (compare Fig. 1A and D)often including the dorsally located Filzkorper. Some intermediate alleles have allthoracic and four abdominal segments deleted but no duplication except thatectopic Filzkorper develop frequently close to the head region (Fig. IB). Inweaker alleles, progressively fewer segments are deleted and the prothorax isalways developed (Fig. 1C-G). The weakest detectable phenotype is observed inheterozygous Kr embryos showing small defects in the denticle bands of thoracic

Key words: Drosophila, gene activity, segmentation, Kriippel (Kr), phenotypic rescue, anti-sense RNA.

158 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 abdominalsegments being replaced by a mirror-image duplication of the normal sixth abdominalsegment. (B,E) Intermediate Kr phenotype; note the presence of a normal fifthabdominal segment. (D) Wild-type cuticular pattern of a Drosophila larva showingskeletal structures of the involuted head, three thoracic (T1-T3) and eight abdominal(A1-A8) segments that can be distinguished by denticle bands marking the anteriorboundary 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 ofanterior abdominal segments. Dark-field photographs; the wild-type embryo is about1 mm long.

Probing gene activity in Drosophila embryos 159

and anterior abdominal segments. Such embryos may hatch and survive to becomeadults. The common motif of all alleles so far analysed is the defect in the thoraxregion and as the alleles get stronger, a deletion of progressively larger regions inthe segment pattern up to eight segments in the strongest amorphic alleles. Theinterpretation of this phenotypic series is a lack of Kr function in strong alleles,increasing residual Kr+ activity in intermediate and weak alleles and half thenormal Kr+ activity in heterozygous Kr embryos, which are almost normal. Asidefrom the fact that the Kr gene, its requirement and possible interaction with othergenes for normal segmentation is interesting in its own right, it appeared sensibleto use the Kr mutant embryos as a biological assay system for Kr+ activity providedby injected material, and to use changes along the phenotypic Kr series as anindicator for inhibition of Kr+ activity in wild-type embryos being injected withgene-specific probes. This experimental design is especially promising in viewingthe accessibility of Drosophila eggs and embryos for injection studies (seeAnderson & Niisslein-Volhard, 1984 for details).

PHENOTYPIC RESCUE AFTER INJECTION OF WILD-TYPE CYTOPLASM INTO KTMUTANT EMBRYOS

Injection of wild-type cytoplasm provides phenotypic rescue in mutant Krembryos. Embryos from a Kr SMI mating (SMI is a balancer chromosome tomaintain Kr stocks) were injected. To distinguish homozygous Kr embryos fromtheir siblings, the mutant Kr1 chromosome was marked in all experiments with adopadecarboxylase mutation (Ddc) as it renders the cuticle and mouth parts ofhomozygous Kr1 larvae unpigmented. Such embryos express the strong Kr pheno-type (Fig. IB) which is always associated with a duplication of the sixth abdominalsegment in reversed polarity (Fig. 2A). Injection of cytoplasm taken from wholewild-type embryos up to the late blastoderm stage into stages younger than lateblastoderm stage had no effect on this phenotype, independent of where it wasinjected into the Kr mutant embryos. By contrast, when cytoplasm was taken froma middle region of blastoderm-stage wild-type embryos and transferred into amiddle region of Kr embryos, up to 40 % of these developed segments with normalpolarity anterior to the sixth abdominal segment ('phenotypic rescue', Fig. 2B).This indicates weak but significant Kr+ activity in Kr mutant embryos provided bythe transferred cytoplasm. The weak phenotypic rescue encouraged us to analyse,under standardized conditions, the developmental profile of Kr+ activity in wild-type cytoplasm, its spatial distribution and the region responding to rescue in Krmutant embryos.

STAGE-DEPENDENCE OF KT+ ACTIVITY IN WILD-TYPE EMBRYOS

Cytoplasm from the 45-55 % egg region (0 % is the posterior pole) was takenfrom wild-type embryos at stages between egg deposition and late blastoderm, andtransferred into the same region of Kr embryos at pole cell to migration stages. As


shown in Fig. 3A, phenotypic rescue was obtained with cytoplasm from blasto-derm 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) reversedpolarity (arrow) in the duplicated sixth abdominal segment. A6 marks the normal sixthabdominal segment. Orientation of denticles can be taken to establish the polarity of agiven denticle row. (B) Same region of an amorphic Kr allele injected with wild-typecytoplasm as described in the text. Note the normal polarity (arrow) of an additionalsegment anterior to A6 which is taken as a criterion for 'phenotypic rescue'.


activity accumulates during blastoderm stage. However, when the highly activecytoplasm was injected into Kr embryos at late blastoderm stage, no rescue wasobserved. This indicates that Kr+ activity was injected after the phenocriticalperiod and/or that the molecules ultimately providing Kr+ activity require sometime to accumulate the minimum level of activity that is necessary for phenotypicrescue. 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-typecytoplasm. (A) Cytoplasm was taken from the middle of wild-type embryos at differentstages of development (abscissa) and injected into the middle region of early cleavagestage embryos from Ddc Krl/SM1 parents. Note that first rescue effects were seen withcytoplasm from embryos at blastoderm stage. (B) Cytoplasm taken from lateblastoderm-stage wild-type embryos was effective when injected in Kr embryos at polecell 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 seeKnipple etal. (1985). n, number of injected Kr^/Kr1 embryos; r, number of rescuedembryos.


should be present in nuclei (see Rosenberg etal. 1986 for details) and (iii) theprotein product is excluded from the injection experiments, we favour the viewthat Kr mRNA is transferred with cytoplasm into the Kr embryo and it requiresthere amplification of the Kr gene products by translation before it provides Krrescuing 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 fromdifferent regions of wild-type embryos. Cytoplasm from any region of embryosyounger than syncytial blastoderm stage was ineffective (Fig. 4A), while cyto-plasm taken from the middle region but not from 0-30 % or 75-100 % of egglength of blastoderm-stage embryos, showed phenotypic rescue. This indicatesthat Kr+ activity is at least enriched, if not exclusively present, in the middle of thewild-type embryos (Fig. 4B), and possibly available at about blastoderm stage.

Cytoplasm was taken from the middle region of blastoderm-stage wild-typeembryos and then transferred into different regions of Kr embryos. Phenotypicrescue response was only seen between 30-70 % of the egg length (Fig. 4C) whichis the region where Kr+ activity accumulates in wild-type embryos. This demon-strates that both Kr+ activity in wild-type embryos and the rescue responsiveregion in Kr embryos coincide within the limits of resolution.

The position of both Kr+ activity accumulation and Kr+ requirement in themiddle region of the embryo correlates with the region affected in weak Kr mutantembryos (Fig. 1), the blastoderm fate map position of thoracic and anteriorabdominal anlagen (see Fig. 4D), and the region where Kr+ transcripts accumu-late 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 (itseffect being to weaken the strong Kr phenotype) clearly demonstrates that lowKr+ activity provokes biological resonance. Considering that only about 2 % ofthe total egg volume was transferred and the Kr gene product is required in morethan 20 % of the wild-type eggs, the effective dilution of Kr+ activity is by morethan one order of magnitude. This means that the weak phenotypic rescueobserved 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 toidentify the Kr coding gene sequences on cloned genomic DNA which shouldcover the Kr region.

Genetics and deletion mapping placed the Kr locus in polytene chromosomeband 60 F3 at the tip of the right arm of chromosome two. Clones obtained frommicrodissected DNA of the corresponding band facilitated the isolation of some 50


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Fig. 4. Localization of Kr+ activity in wild-type and the rescue responsive regions in Krmutant 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 themiddle of early cleavage stage Kr embryos or (C) the region where cytoplasm from themiddle of blastoderm-stage wild-type embryos was injected in Kr embryos, n, r: seelegend Fig. 3. Note that rescue was only obtained when cytoplasm from the middle ofblastoderm-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 Krresponsive region (from C) and localization of Kr+ activity (from B). Note that bothregions are almost identical and smaller than the gap seen in the segment pattern ofamorphic Kr embryos.


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Fig. 5. Molecular map of Kr region. The coordinates (in kb) are based on an EcoRIsite 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 Ifchromosomes used to make chromosomal rearrangements. The four restrictionenzymes 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 fromhomozygous If flies (designation AI, each insert ends with a true EcoRI site), Canton Sembryos (designation cc), or Oregon R embryos (designation ER; each insert endswith a Sau3A site due to partial digestion of genomic embryo DNA and ligation intothe 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 alsoabsent in CyO DNA. Fragments smaller than 0-5 kb and other clones overlapping theindicated 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 providesKr+ rescuing activity as described in the text. Methods: DNAs from phages weremapped with four restriction enzymes after separation on 0-8 % agarose gel. Endfragments of DNA inserts were labelled by nick translation to screen the three librariesfor homologous sequences flanking either side of the original clone. For each walkingstep, the presence of repetitive sequences was tested by reverse Southern analysis using32P-labelled genomic DNA as probe. As an additional control, the size of eachrestriction 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 con-

sistent 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 %)


developed more than two additional segments with normal polarity (see alsoFig. 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 100times the number of Kr+ gene copies of normal blastoderm-stage embryos, or 500times the number of Kr+ gene copies being expressed (see Preiss etal 1985).Earlier experiments demonstrated that about 80 % of the injected DNA is rapidlydegraded and that transient transcription from the remaining DNA is more thanone 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 cloneDNA contains Kr+ sequences and that our transient expression assay, based onphenotypic 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+ activitycontained in the injected material which weakened the strong Kr phenotype.However, this way of identifying genes and their activity requires the prerequisiteof genetical and cytological analysis in combination with a refined set of moleculartechniques. This approach is, therefore, limited to a small number of biologicalsystems where the combination of genetics, cytology and transfer of macro-molecules and/or recombinant DNA transformation is accessible. To overcomethis limitation and to establish a general tool for probing gene function in lessfortunate systems, we designed experiments to inhibit a specific gene function.This assay involves the production of RNA containing the complementarysequences to the natural mRNA ('anti-sense RNA'). Upon injection, both RNAsshould form duplexes by hybridization in vivo and thus prevent the mRNA frombeing normally translated.

Several features make the Kr gene useful for assessing this sequence-specificinhibition of gene function. First, Kr gene function is defined by the number ofphenotypes that can be aligned in an allelic series (see above). Second, the Krtranscripts accumulate in a defined region of the embryo. During the period of firstexpression, at syncytial blastoderm stage, nuclei and their surrounding cyto-plasmic islands may be accessible to injected anti-sense RNA while individual cellsthat form during blastoderm stage may not. Third, the Kr+ gene is only transcribedbetween 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 beenrecovered in a 2-3kb cDNA clone (Rosenberg etal. 1985). This cDNA wassubcloned, in both orientations, into plasmid DNA containing the SP6 promoterwhich allows Kr sense and anti-sense RNA to be transcribed in vitro using SP6RNA polymerase which specifically starts transcription at the SP6 promoter site(see Rosenberg et al. 1985, for a detailed description).


Wild-type embryos were injected with either sense or anti-sense RNA at thesyncytial blastoderm stage. Sense RNA, which contained only part of the mRNAsequence, had no specific effect on the embryonic phenotype. By contrast,injected Kr anti-sense RNA had a dramatic effect on genetically wild-typeembryos, i.e. they developed lethal Kr phenocopies (see Fig. 6) with up to 30 %frequency. Some of the phenocopies developed ectopic Filzkorper close to thehead region, as found in Kr mutants developing the intermediate phenotype. Thisnew 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 anintermediate Kr phenotype (see Fig. 1C) showing an ectopic pair of Filzkorper close tothe head region (arrow).


position on the blastoderm fate map demonstrates unequivocally the production ofKr phenocopies in wild-type embryos. Extreme Kr phenocopies resembling theamorphic Kr phenotype (Fig. 1A), however, were not observed, indicating thatKr+ activity was not abolished completely (for details see Rosenberg etal. 1985).

CONCLUSIONS

The experiments with Kr embryos demonstrate the use of Drosophila mutantembryos for injection studies on the activity of the wild-type gene, localization ofand local requirement for the gene product in vivo. Moreover, cloned DNA can beidentified as functional sequences thus facilitating the delimitation of sequencesbeing required for at least the coding region of a given gene. In this respect, asimple assay system allowed us to confirm predictions (Wieschaus, Niisslein-Volhard & Kluding, 1984) made from genetic analysis: the middle region of theembryo accumulates Kr+ activity. This region coincides with the anlagen of thoraxand anterior abdomen on the blastoderm fate map and appears to be mostsensitive to the absence of the gene product, as reflected in the lack of thoracic andanterior abdominal segments in weak Kr alleles. The results of the injectionexperiments were the first indication of localized Kr+ gene product requirementduring blastoderm stage which are possibly reflected in the localized expression ofthe Kr gene revealed by in situ hybridization of the molecular Kr probe to sectionsof embryos (see Knipple et al. 1985).

The reverse experiment, trying to inhibit a specific gene function by injection ofanti-sense RNA offers a great potential not limited to Drosophila embryos. Whilein principle it is possible to physically isolate and clone almost any gene, it is oftendifficult or even impossible to ascribe a discrete function to a cloned DNAsequence, except for organisms where classical genetics has identified a genefunction, 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 geneconstructs' transcribing from strong promoters (for an example see Kim & Wold,1985) is clear and needs no further explanation.

REFERENCESANDERSON, K. V. &NUSSLEIN-VOLHARD, C. (1984). Information for the dorsal-ventral pattern of

the Drosophila embryo is stored as maternal mRNA. Nature, Lond. 311, 223-227.KIM, S. K. & WOLD, B. J. (1985). Stable reduction of thymidine kinase activity in cells expressing

high levels of anti-sense RNA. Cell 42, 129-138.KNIPPLE, D. C , SEIFERT, E., ROSENBERG, U. B., PREISS, A. & JACKLE, H. (1985). Spatial and

temporal patterns of Kriippel gene expression in early Drosophila embryos. Nature, Lond. 317,40-44.

NUSSLEIN-VOLHARD, C. & WIESCHAUS, E. (1980). Mutations affecting the segment number andpolarity in Drosophila. Nature, Lond. 287, 795-801.

PREISS, A., ROSENBERG, U. B., KIENLIN, A., SEIFERT, E. & JACKLE, H. (1985). Moleculargenetics of Kriippel, a gene required for segmentation of the Drosophila embryo. Nature,Lond. 313, 27-33.

168 H. J A C K L E , E. SEIFERT, A. P R E I S S AND U. B . ROSENBERG

ROSENBERG, U. B., PREISS, A., SEIFERT, E., JACKLE, H. & KNIPPLE, D. C. (1985). Production ofphenocopies by Kriippel antisense RNA injection into Drosophila embryos. Nature, Lond.313, 703-706.

ROSENBERG, U. B., SCHRODER, C , PREISS, A., KIENLIN, A., COTE, S., RIEDE, I. & JACKLE, H.(1986). Structural homology of the product of the Drosophila Kriippel gene with Xenopustranscription factor III A. Nature, Lond. 319, 336-339.

WIESCHAUS, E., NUSSLEIN-VOLHARD, C. & KLUDING, H. (1984). Kriippel, a gene whose activity isrequired early in the zygotic genome for normal embryonic segmentation. Devi Biol. 104,172-186.

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Probing gene activity in Drosophila embryos