Probing gene activity in Drosophila embryos

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

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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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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

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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

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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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