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bicoid-Independent Formation of Thoracic Segments in Drosophila

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Science  31 Mar 2000:
Vol. 287, Issue 5462, pp. 2476-2479
DOI: 10.1126/science.287.5462.2476

Abstract

The maternal determinant Bicoid (Bcd) represents the paradigm of a morphogen that provides positional information for pattern formation. However, as bicoid seems to be a recently acquired gene in flies, the question was raised as to how embryonic patterning is achieved in organisms with more ancestral modes of development. Because the phylogenetically conserved Hunchback (Hb) protein had previously been shown to act as a morphogen in abdominal patterning, we asked which functions of Bcd could be performed by Hb. By reestablishing a proposed ancient regulatory circuitry in which maternal Hb controls zygotic hunchback expression, we show that Hb is able to form thoracic segments in the absence of Bcd.

In Drosophila, a key component of the anterior maternal system is Bcd (1). Embryos from mothers mutant for bicoid (bcd) lack head, thorax, and some abdominal segments. The maternal bcdmRNA is localized to the anterior pole of the egg and early embryo, and its translation generates an anteroposterior gradient of the Bcd homeoprotein (2). In the syncytial environment of the early fly embryo, the Bcd protein appears to act as a morphogen in dictating distinct developmental fates by providing a series of concentration thresholds: At low levels, Bcd acts through high-affinity binding sites to activate target genes like hunchback (hb) for the formation of the thorax, whereas high levels of Bcd activate head gap genes, such as orthodenticle (otd), which contain low-affinity Bcd binding sites (3). However,bcd seems to be the result of a recent gene duplication event in the Hox cluster of flies, which would explain why nobcd homologs have been identified outside higher Dipterans (4). Thus, different insect species may use other morphogens to pattern their embryos. One candidate morphogen that has been functionally conserved during evolution is the zinc-finger protein Hb (5–7).

In Drosophila, a maternal Hb gradient is established by the Nanos protein that blocks translation of the ubiquitously distributed hb mRNA in the posterior region of the embryo (8). This Hb gradient can by itself provide long-range polarity to the embryo and compensates for the absence of Bcd in the formation of abdominal segments (6). In addition, Hb synergizes with Bcd to pattern the anterior region of the embryo (9). However, the zygotic, bcd-dependent expression of hb (10) causes an intrinsic problem for the analysis of the specific roles of the two morphogens: Whenever Bcd activity is altered, Hb activity is changed (3). Thus, many of the effects attributed to Bcd might indirectly be caused by Hb. The hb gene is expressed from two independent promoters, P1 and P2 (Fig. 1A) (11): Maternal and late blastoderm expression in the central (parasegment 4, PS4) and posterior stripes are initiated at P1, whereas P2 mediates the zygotic,bcd-dependent expression at the early blastoderm stage (Fig. 1, B to D) (12).

Figure 1

hb genomic organization and expression pattern. (A) hb transcription is initiated at two different promoters, P1 and P2. Early embryonic expression is controlled by three separable enhancer elements: An oogenesis element (yellow box) (11) leads to the maternal contribution (B), thebcd-dependent element (red) (3) directs anterior cap expression (C), and a stripe element (green) (11) controls the central hb-dependent PS4 and the posterior stripes (D). In (B) to (D), wild-typehb expression was detected by in situ hybridization (12). (E to G) hbP1AB was constructed by replacing the hb coding region ofhbP1only with a lacZ gene (13); embryos were immunostained using antibody to β-galactosidase (12). When provided maternally, this construct leads to strong expression throughout the embryo (E). When provided only zygotically, no anterior cap domain is observed, but a wide stripe forms (F, borders indicated by two lines) that later refines to the PS4 stripe. In the late blastoderm, both the PS4 and the posterior stripes are formed (G).

We have generated a functional hb transgene that is missing all P2 promoter sequences and relies solely on the P1 promoter (13) (hbP1only, Fig. 1A). hbP1onlyconstructs (14) do not respond to bcd and do not mediate gene expression in the anterior cap domain (Fig. 1, E to G). Therefore, hbP1only uncouples the direct link between the Bcd and Hb morphogen systems. Zygotic hb mutants derived from heterozygous parents (14) do not develop labial or thoracic structures, and they also show a fusion of abdominal segments A7 and A8 (5) (Fig. 2, A and B). When one copy of the hbP1only transgene was provided zygotically to a hb mutant embryo (by the father), it rescued the posterior phenotype, and A7 and A8 developed normally (Fig. 2C). The labial/thoracic phenotype was not rescued. However, whenhbP1only was provided as one copy by the mother to ahb mutant embryo, the posterior and part of the anterior phenotype were rescued. These embryos exhibited normal labial and prothoracic (T1) segments, and only lacked meso- and metathoracic segments (T2 and T3) (Fig. 3A). The anterior rescue is due to the maternal contribution of hbP1onlybecause sibling embryos that did not inherit the hbP1onlyconstruct zygotically also exhibited the partial anterior (but not the posterior) rescue (Fig. 2D). This indicates that restoring high levels of maternal hb expression (i.e., two copies: one wild type plus one copy of hbP1only) is sufficient to rescue the labial and prothoracic segments in the zygotic hb mutant progeny (Fig. 2D) (6). Therefore, the lack of zygotichb (Fig. 2D) leads only to the loss of T2 and T3 and to the fusion of A7 and A8, whereas the previously reported zygotichb phenotype (Fig. 2B) (5) represents a combination of a haploinsufficient maternal plus a zygotic phenotype.

Figure 2

Rescue of zygotic hb mutants byhbP1only (14). (A) Wild-type cuticle (hs, head skeleton; Lb, labial; T1, prothoracic; T2, mesothoracic; T3, metathoracic; A1 to A8, abdominal segments; sp, spiracles). (B) hb zygotic mutant embryo derived from heterozygous hb mother. (C) hbzygotic mutant with A7 and A8 rescued (arrowhead) by thehbP1only transgene provided paternally. (D)hb zygotic mutant with labial and T1 segments (arrow) rescued by maternal expression of hbP1only in addition to one wild-type maternal copy of hb.

Figure 3

Rescue of thorax withoutbcd-dependent hb expression (12, 14). (A) In 30% of embryos, only the maternal rescue of the labial and T1 segments is seen (compare with Fig. 2D). Most embryos, however, show additional rescue of thoracic segments, varying from an additional set of thoracic sensory organs to an additional thoracic denticle belt, or both (B). These structures are most likely derived from the T3 segment, which is less sensitive than the T2 segment to lack of hb function (5). (C) In 5 to 10% of the embryos, a complete thorax (consisting of T1, T2, and T3 segments) is seen.

The loss of zygotic hb activity affects regions of the embryo that correspond to the two late stripes of zygotic hbexpression: The A7-A8 fusion corresponds to the posterior stripe, whereas the loss of T2 and T3 corresponds to the PS4 stripe (Fig. 1D), which starts as a fairly wide domain covering the anlagen of T2 and T3 (11) (Fig. 1F). This correlation between the zygotichb phenotype and the late stripe expression pattern led us to reconsider the importance of the early bcd-dependent anterior cap domain. Under some conditions, hbP1only(maternal hb contribution plus stripe expression) might suffice for normal segmentation of head and thorax, making superfluous the bcd-dependent anterior cap domain. Hence, we activated the hb PS4 stripe without bcd-dependenthb expression. This stripe is repressed by the knirps abdominal gap-gene product (15) and is activated by high levels of Hb itself, either directly or indirectly (through repression of kni) (16). We generated embryos that lacked the bcd-dependenthb cap domain but contained an increased maternalhb contribution (to four copies) and kni reduced to one copy (14). These embryos displayed a range of partially rescued hb phenotypes, including embryos (5 to 10%) with a full set of head and thoracic segments (Fig. 3). Thus,bcd-dependent hb expression can in principle be dispensable for embryonic segmentation, and the only critical anterior domain of zygotic hb expression appears to be the PS4 stripe, with the bcd-dependent cap domain serving to activate this stripe. This role is likely achieved by the maternalhb contribution in species where zygotic hb is not under the control of bcd or where a bcdhomolog might not exist (17, 18).

The rescue of T2 and T3 structures by bcd-independenthb expression raised the question of whether these structures could develop in a completely bcd-independent manner. Embryos derived from bcd mutant mothers develop ectopic tail structures that replace head and thorax and exhibit a disruption of some abdominal segments (Fig. 4, A and B). Although previous work (6) has shown that, in the absence ofbcd, high levels of maternal hb can rescue a normal abdomen and some thoracic structures, no complete thoracic segments could be induced. We therefore introduced into abcd mutant background a bcd-independent source for high levels of zygotic hb expression (Fig. 4) (14). By establishing this artificial zygotic Hb gradient, we obtained, with variable expressivity, two notable results: First, about 20% of the embryos exhibited rescued T2 and T3 segments (Fig. 4C). The maintenance of high Hb levels that lead to the rescue of thoracic segments is likely due to the activation of the hbstripe element (Fig. 1A) because the hbP1AB reporter (13) is activated as a stripe where T2 and T3 form (Fig. 4D). Second, most of the ectopic tail structures that are anteriorly duplicated in bcd mutants were suppressed, suggesting further redundancy between Hb and Bcd. However, Hb and Bcd must act at different levels in suppressing these tail structures, which depend on the activity of the caudal (cad) gene (19): Bcd acts by repressing cad mRNA translation (20), whereas Hb does not (19) but might instead interfere with Cad protein function. This bcd-independent suppression of cad function might be important in organisms where the Cad gradient only forms late (21) and represents another variation as to how cad activity is suppressed at the anterior of the embryo.

Figure 4

Rescue of T2 and T3 segments in the absence of bcd activity (13, 14). (A) Wild-type cuticle preparation (t, tuft; other abbreviations as in Fig. 2). (B) Embryo derived from abcd mutant mother. At the anterior end of the embryo, the head and thorax are replaced by tail structures (sp and t). The embryo also lacks abdominal segments A2 and A4. (C andD) Embryos derived from a cross between bcdmutant mothers carrying four copies of thenos-GAL4GCN4-3′bcd driver and fathers carryingUAS-HB transgenes homozygous on both the second and third chromosomes. In (C), most of the anterior duplicated tail structures, except for the most terminal structure (t), are suppressed by the induced zygotic hb expression. Moreover, thoracic structures are rescued to a variable extent, up to a complete rescue of the T2 and T3 segments. In (D), anterior expression of zygotically provided hbP1AB, detected by lacZin situ hybridization (11) in a blastoderm-stage embryo, indicates that zygotic hb expression is mediated in this thoracic rescue situation by regulatory elements upstream of the P1 promoter.

Different species use various strategies for repression of Cad function: In Drosophila, translational repression ofcad mRNA involves the Bcd homeoprotein (19), whereas in Caenorhabditis elegans repression involves the KH-domain protein MEX-3 (22). In vertebrates, a mutually antagonistic relation between otd-like andcad-like genes has been proposed to reflect an ancestral system to pattern the anteroposterior axis of the embryo (23). In arthropods, ancestral head determinants are probably encoded by otd-like genes as well. Thus, in the beetle Tribolium, where no bcd homologs but Bcd-like activities have been found (18), these activities are probably also covered by Otd or KH-domain proteins. This is consistent with the Otd-like DNA binding specificity of Bcd, which is atypical for a factor encoded by a gene duplication in the Hox cluster. This change in specificity was probably crucial for Bcd to acquire its key role in anterior patterning, as it allowed Bcd to function both as an RNA binding protein (20) and as an Otd-like transcription factor. In this respect, it is not surprising that the zinc-finger protein Hb cannot completely replace Bcd in the head region. Even the highest levels of Hb obtained in our experiments were not able to induce head formation in the absence of Bcd. However, Hb is required for the posterior head region (maxillary and labial segment) (5) and supports anterior head development by synergizing with Bcd (9). It will be interesting to see whether such a synergism can also take place between Hb and other more ancestral head determinants.

Our results indicate that the two morphogenetic systems Bcd and Hb do not need to be directly linked. Hence, the direct regulation of hb by Bcd might represent a recent evolutionary addition to the insect body plan (17, 18). InDrosophila, the abundance of bcd-dependenthb expression eventually rendered superfluous the maternalhb contribution (8), which is widespread within arthropods (24). Consistent with the idea that thebcd-dependent hb expression represents a recent evolutionary acquisition, the P2 promoter contains only activator sites that allow the direct response to a specific threshold level of a morphogen (3, 16). This might be a unique situation, given that most other developmentally regulated promoters contain, in addition to activator sites, repressor elements for setting the exact borders of gene expression (25). By tinkering with the rather plastic mechanisms of early development, we could change the ontogeny of Drosophila toward an inferred ancestral state where maternal Hb controls zygotic hb. This change could be brought about by altering patterns and levels of gene expression, which presents the most likely variation on which evolutionary processes are based (26).

  • * To whom correspondence should be addressed. E-mail: claude.desplan{at}nyu.edu

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