Ectopic segmentation gene expression and metameric regulation in Drosophila (1988)

Embryonic pattern in Drosophila is organized by a hierarchy of maternal and zygotic segmentation genes that interact to define increasingly precise spatial domains (reviewed by Scott & O’Farrell, 1986; Akam, 1987). A metameric body plan is already established by the onset of gastrulation (3 h postfertilization) such that each segment expresses the segment-polarity genes engrailed (en) and wingless (wg) in single-cell-wide stripes (Kornberg, Sidon, O’Farrell & Simon, 1985; Fjose, McGinnis & Gehring, 1985; DiNardo, Kuner, Theis & O’Farrell, 1985; Baker, 1987). The current challenge is to define specific segmentation gene interactions, to determine how they achieve this spatial refinement and how their spatial relationships give rise to the structural complexities of the mature larva.The pattern of en expression defines two metameric registers: the en domain lies at the posterior of each segment and at the anterior of each parasegment (Martinez-Arias & Lawrence, 1985; Fig. 1C). It has been suggested that the parasegment is the primary metamere of the early embryo and that establishing en domains is crucial in defining metameric units (see Howard & Ingham, 1986; Ingham & Martinez-Arias, 1986; Lawrence, this volume). Moreover, initial selector gene expression appears parasegmental (Akam & Martinez-Arias, 1985; Carroll et al. 1988). However, a second feature of establishing metamerism is ensuring the stability of en domains. Various mutant conditions cause the establishment of unstable en patterns that underlie subsequent loss of pattern elements (DiNardo & O’Farrell, 1987; Ish-Horowicz & Pinchin, 1987; Martinez-Arias, Baker & Ingham, 1988; DiNardo et al. 1988; Ish-Horowicz, Gyurkovics, Pinchin & Ingham, 1988).The hierarchy of segmentation genes can be ranked according to their interactions. Gap genes act before the pair-rule genes; pair-rule genes act to establish the patterns of segment-polarity genes (Howard & Ingham, 1986; Caroll & Scott, 1986; Ingham, Ish-Horowicz & Howard, 1986; Ingham & Martinez-Arias, 1986; Harding et al. 1986; DiNardo & O’Farrell, 1987; Martinez-Arias & White, 1988; Ingham, Baker & Martinez-Arias, 1988). The hierarchy is further subdivided by distinctions between the pair-rule genes, hairy (h), even-skipped and runt are required for the patterning of fushi tarazu (ftz) expression, indicating that they act earlier in establishing pair-rule spatial domains (Howard & Ingham, 1986; Carroll & Scott, 1986; Ingham & Gergen, this volume).Genetic analysis of epistatic relationships favour simple hierarchical models. The large number of segmentation genes and the rapidity with which they organize themselves make it clear that pattern cannot be the result of strictly sequential gene action. As gene interactions become more complicated, in number and temporal complexity, it becomes increasingly difficult to distinguish between direct interactions and indirect consequences.An alternative approach to conventional genetic analysis is the deliberate mis-regulation of segmentation gene expression. Struhl (1985) has constructed a hybrid gene (HSF) in which the coding region of the pair-rule gene ftz is under the control of the (hsp70) heat-shock promoter. The latter is inducible in essentially all tissues, overcoming the spatial specificity of the endogenous ftz promoter. Indeed, heat shock of HSF blastoderm embryos causes specific pair-rule pattern defects, indicating that the striped pattern of ftz is critical in establishing proper metameric pattern. We have shown that similar ectopic expression of the pair-rule gene (h) also causes pair-rule defects (Ish-Horowicz & Pinchin, 1987).Struhl (1985) has interpreted the HSF phenotype in terms of a combinatorial role for ftz, suggesting that it is required for the development of the ftz-expressing domains and prohibited in the alternate non-expressing metameres (see also Gergen & Wieschaus, 1986; Gergen, Coulter & Wieschaus, 1986). However, Duncan (1986) has proposed an alternative interpretation of the HSF pair-rule defects whereby ectopic ftz interferes with the establishment of parasegmental metamerism. Such a model demands that the metameres affected in pair-rule HSF embryos be parasegmental, not segmental as described by Struhl. A further expectation is that the loss of pattern elements would be due to size regulation within abnormal metameres and that the apparent deletion need not derive exclusively from cells expressing ectopic ftz.We have therefore re-examined the cuticular phenotypes of heat-shocked HSF embryos in order to define more precisely the affected structures. In this paper, we present evidence that the pair-rule HSF phenotype is indeed reciprocal to that of ftz mutant embryos. We also show that the odd-numbered parasegments are still represented within such HSF embryos. Thus, the ultimate developmental fates of many cells appears to depend on patterning within a metamere, rather than their precise blastoderm identities.Struhl (1985) showed that heat shock of blastoderm HSF embryos causes specific pair-rule pattern defects, describing the HSF cuticular phenotype as deletions of alternate segments. He suggested that such embryos retain segments T2, Al, A3 etc. and that they are not reciprocal to the (roughly) parasegmental deletions in ftz mutant embryos (Fig. 1C).This would also argue against the parasegment as the primary embryonic metamere. For example, retaining a complete Al segment would require the fusion of two adjacent parasegments, posterior PS6 and anterior PS7.It is difficult to distinguish whether the metameres deleted in pair-rule HSF embryos are segmental or parasegmental. Both phasings predict similar ventral cuticular phenotypes as the losses of pT2 (posterior second thoracic segment - lost if the deletions are parasegmental) and pT3 (lost if the deletions are segmental) are indistinguishable (Fig. 1C).However, we have been able to show that the HSF pair-rule phenotype affects parasegmental metameres by analysing the dorsal cuticular patterns. In wild-type embryos, each segment includes a wide anterior band of fine posterior-orientated hairs (for a detailed description of the dorsal pattern, see Lohs-Schardin, Cremer & Nüsslein-Volhard, 1983). The bands are separated by a naked region, except for pA2 to pA7 which develop a row of broader anterior-pointing triangular hairs (Figs 2A and 3). Such a row distinguishes pA2 (PS8) from pAl (PS7) which occasionally shows individual hairs but never a row.The dorsal pattern of pair-rule HSF embryos demonstrates that the apparent segments display mixed segmental character. The ‘Al-like’ segment includes the posterior row of hairs characteristic of pA2 (Fig. 2B). Analysis of heat-shocked HSF;Ubx embryos, in which PS6 is transformed to PS5, confirm that the affected metameres are parasegmental. In such embryos, aAl (PS6) becomes thoracic (PS5) and includes an extra pair of partial Keilin’s organs at the parasegmental boundary (see Struhl, 1984) (Fig. 2C). As expected, the pA2 posterior row is unaffected (Fig. 2D). Both experiments indicate that pair-rule HSF embryos retain PS6 and PS8 while lacking PS7.The patterns of dorsal thoracic hairs in pair-rule HSF embryos are also consistent with apparent loss of the odd-numbered parasegments. In the ‘T2-like’ segment, the anterior band of hairs extends past the dorsal sense organs as is usually found in pT3 (PS6), but not pT2 (PS5) (Fig. 2A,E).In some heat-shocked HSF embryos, the pattern deletions extend over slightly more than a metamere. In such embryos, the posterior row is often lacking, not only in pA2 and but also in more posterior segments. We do not understand the basis for these more extensive deletions, but such embryos may explain the discrepancy between Struhl’s and our interpretations of the HSF phenotype.ftz mutant embryos display apparent deletion of the even-numbered parasegments (Nüsslein-Volhard & Wieschaus, 1980; Wakimoto, Turner & Kaufman, 1984; Ingham & Martinez-Arias, 1986). Thus, the pair-rule HSF cuticular phenotype represents their exact reciprocal. However, we have analysed the patterns of segmentation gene expression in heat-shocked HSF embryos and shown that the mechanisms leading to the two phenotypes are very different. The pair-rule ftz phenotype can be attributed to the absence of the even-numbered en stripes, i.e. misestablishment of the initial embryonic metameres (Ingham & Martinez-Arias, 1986). In heat-shocked HSF embryos, all en stripes are initiated but alternate domains become unstable (Ish-Horowicz et al. 1988). This appears to be due to the repression of alternate stripes of wg, leading to decay of the even-numbered en stripes at the end of germ-band extension. The same bands are lost in ftz mutant embryos; thus the pair-rule metameric organizations of HSF and ftz embryos are the same, not reciprocal. The different cuticular phenotypes are due to differential selector gene expression (Ish-Horowicz et al. submitted). The final HSF cuticular phenotype is the result of metameric instability and homoeotic transformations.Clearly, the integrity and stability of parasegmental domains are critical to the establishment of metamerism. Loss of alternate en stripes leads to loss of alternate metameres. Nevertheless, the double-metameric primordia must regulate to give approximately normal-sized cuticular segments. It has not yet proved possible to follow the fates of individual cells during this process. If size regulation is due to an intrinsic response of cells to mutant patterns of gene expression, their fates will depend on their individual blastoderm identities. In pair-rule HSF embryos, the deleted structures would derive from the non ftz- expressing, odd-numbered parasegment, as envisaged in the combinatorial model. Alternatively, if the cells are responding to external cues consequent on inappropriate metameric size, regulation will involve all regions of the metamere. In this latter case, both parasegments will be represented in the pair-rule embryos.We used the ftz-lacZ fusion gene of Hiromi, Kuroiwa & Gehring (1985) to mark the even-numbered parasegments in heat-shocked HSF embryos. This fusion gene directs the synthesis of E. coli β -galactosidase (lacZ) under the control of the ftz promoter. lacZ persists through germ-band shortening and retraction, so that it is still present when segmental organization is overt (9h).In wild-type embryos that have completed germband shortening, the lacZ staining boundary extends to the parasegmental boundary, anterior to the segmental groove (Lawrence, Johnston, MacDonald & Struhl, 1987; Fig. 3B). The stained domain appears narrower than a metamere due to weaker staining in the posterior of the parasegment.In pair-rule HSF embryos, there is a single stained domain in each segment by the end of germ-band retraction, reflecting the loss of alternate metameres. The centre of each segment is stained, but not the anterior or posterior edges (Fig. 4A). The staining is neither segmental nor parasegmental, and both parasegments contribute to the fused metamere.Thus, the parasegmental deletions in heat-shocked HSF embryos are misleading. They do not reflect the loss of a parasegmental metamere, but rather its inappropriate development. That the final cuticular defects appear to be deletions of precise metameres illustrates the importance of the parasegment as a regulative unit in early development.Although we have not followed the detailed time course of lacZ staining, size regulation in pair-rule HSF embryos must occur during germ-band shortening. The pattern is roughly normal at 5-6 h (Fig. 3C; see below) and we have been unable to visualize any cell death in 5 h embryos (not shown). The pattern of lacZ staining suggests that the two parasegments do not contribute equally to the final metamere. The lacZ domain occupies considerably more than half the final metamere (Fig. 3C), indicating that the majority of each segment derives from the even-numbered parasegments.We have found that the heat-induced ftz expression acts at blastoderm to advance the anterior margin of ftz expression by one cell (Ish-Horowicz et al. 1988). This leads to the broader lacZ staining in HSF embryos than in wild-type (Fig. 3C, D). The anterior margins of the even-numbered en stripes advances coincident with the change in ftz expression, causing narrowing and widening of the odd- and even-numbered parasegmental anlagen respectively (Fig. 5). Despite recruitment of extra cells into the even-numbered parasegments, they do not contribute exclusively to the final pair-rule HSF pattern.The major advances in studying embryonic pattern formation have resulted from realization of the importance of the early analysis of developmental perturbations. Lewis (1978) and Nüsslein-Volhard & Wieschaus (1980) pioneered the combination of genetic analysis and an emphasis on the study of larval cuticular phenotypes. The current use of segmentation gene probes as molecular markers is a natural extension of this philosophy and reflects the need to overcome the inherent ambiguities of cuticle pattern phenotypes as markers for very early events.The ftz and HSF cuticular phenotypes are reciprocal; the same is true for the ectopic h (HSH) and h mutant phenotypes (Ish-Horowicz & Pinchin, 1987). However, the underlying causes of the complementary pattern defects differ completely. Ectopic h acts to repress the pair-rule gene ftz, thereby affecting the establishment of en pattern. Ectopic ftz represses wg, a segment polarity gene, giving rise to instability of en expression (Ish-Horowicz et al. 1988). Cuticular phenotypes alone provided no distinction between these different mechanisms. Only the analysis of initial stages of embryogenesis, when pattern is being established, allows us to define the direct interactions that lead to the establishment of spatial domains.We should like to thank Dr Gary Struhl for the HSF flies, which he generously made available for our analysis. We should also like to thank Phil Ingham for discussions in the course of this work and our colleagues in the laboratory for criticisms of the manuscript.