The maternal determinant Bicoid (Bcd) represents the paradigm of a morphogen that provides positional information for pattern formation. However, since bicoid seems to be a recently acquired gene in flies, the question has been raised as to how embryonic patterning is achieved in organisms with more ancestral modes of development. Because the phylogenetically conserved Hunchback (Hb) protein acts as a morphogen in abdominal patterning, it was 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, it has been shown that Hb is able to form thoracic segments in the absence of Bcd (Wimmer, 2000).

A functional hb transgene has been generated that is missing all P2 promoter sequences and relies solely on the P1 promoter (hbP1only). hbP1only constructs do not respond to bcd and do not mediate gene expression in the anterior cap domain. Therefore, hbP1only uncouples the direct link between the Bcd and Hb morphogen systems. Zygotic hb mutants derived from heterozygous parents do not develop labial or thoracic structures, and they also show a fusion of abdominal segments A7 and A8. When one copy of the hbP1only transgene is provided zygotically (by the father) to a hb mutant embryo, it rescues the posterior phenotype, and A7 and A8 developed normally. The labial/thoracic phenotype is not rescued. However, when hbP1only is provided as one copy by the mother to a hb mutant embryo, the posterior and part of the anterior phenotype are rescued. These embryos exhibit normal labial and prothoracic (T1) segments, and only lack meso- and meta-thoracic segments (T2 and T3). The anterior rescue is due to the maternal contribution of hbP1only because sibling embryos that do not inherit the hbP1only construct zygotically also exhibit the partial anterior (but not the posterior) rescue. 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. Therefore, the lack of zygotic hb leads only to the loss of T2 and T3 and to the fusion of A7 and A8, whereas the previously reported zygotic hb phenotype represents a combination of a haploinsufficient maternal plus a zygotic phenotype (Wimmer, 2000).

The loss of zygotic hb activity affects regions of the embryo that correspond to the two late stripes of zygotic hb expression: The A7-A8 fusion corresponds to the posterior stripe, whereas the loss of T2 and T3 corresponds to the PS4 stripe, which starts as a fairly wide domain covering the anlagen of T2 and T3. This correlation between the zygotic hb phenotype and the late stripe expression pattern led to a reconsideration of 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, the hb PS4 stripe is activated without bcd-dependent hb expression. This stripe is repressed by the knirps abdominal gap-gene product and is activated by high levels of Hb itself, either directly or indirectly (through repression of kni). Embryos that lack the bcd-dependent hb cap domain have been generated that contain an increased maternal hb contribution (to four copies) and kni reduced to one copy. These embryos display a range of partially rescued hb phenotypes, including some embryos with a full set of head and thoracic segments. 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 maternal hb contribution in species where zygotic hb is not under the control of bcd or where a bcd homolog might not exist (Wimmer, 2000).

The rescue of T2 and T3 structures by bcd-independent hb expression raises 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. Although previous work has shown that, in the absence of bcd, high levels of maternal hb can rescue a normal abdomen and some thoracic structures, no complete thoracic segments can be induced. A bcd-independent source for high levels of zygotic hb expression was introduced into a bcd mutant background. By establishing this artificial zygotic Hb gradient, two notable results were obtained, with variable expressivity: (1) about 20% of the embryos exhibit rescued T2 and T3 segments. The maintenance of high Hb levels that lead to the rescue of thoracic segments is likely due to the activation of the hb stripe element because the hbP1AB reporter is activated as a stripe where T2 and T3 form. (2) Most of the ectopic tail structures that are anteriorly duplicated in bcd mutants are 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: Bcd acts by repressing cad mRNA translation, whereas Hb does not 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 and represents another variation as to how cad activity is suppressed at the anterior of the embryo (Wimmer, 2000).

Different species use various strategies for repression of Cad function: In Drosophila, translational repression of CAD mRNA involves the Bcd homeoprotein, whereas in Caenorhabditis elegans repression involves the KH-domain protein MEX-3. In vertebrates, a mutually antagonistic relation between otd-like and cad-like genes has been proposed to reflect an ancestral system to pattern the anteroposterior axis of the embryo. 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, 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, because it allowed Bcd to function both as an RNA binding protein 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 these 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) and supports anterior head development by synergizing with Bcd. It will be interesting to see whether such a synergism can also take place between Hb and other more ancestral head determinants (Wimmer, 2000).

These 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. In Drosophila, the abundance of bcd-dependent hb expression eventually renders superfluous the maternal hb contribution, which is widespread within arthropods. Consistent with the idea that the bcd-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. 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. By tinkering with the rather plastic mechanisms of early development, the ontogeny of Drosophila could be changed 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; this presents the most likely variation on which evolutionary processes are based (Wimmer, 2000).

Although gastrulation is regarded as the stage during Drosophila development when the AP patterning system first influences morphological processes, transcription is already regulated in complex patterns by cycle 10. How soon this transcriptional complexity produces spatial differences in morphology has been unclear. Two new processes are described that establish visible morphological inhomogeneities before the onset of gastrulation. The first of these is the regulation of syncytial nuclear densities in the anterior end of the egg and represents the first zygotically driven AP asymmetry in the embryo. The second process is the generation of a fine-scale pattern in the actin/myosin array during cellularization. Three domains of different yolk stalk diameters as well as depths of cellularization along the AP axis have been found. These domains are established under the control of the AP patterning system and require bicoid activity. The anterior-most domain is a region of large yolk stalk diameters and corresponds to the region of decreased nuclear densities observed during syncytial stages. The middle domain shows smaller yolk stalk diameters and more rapid cellularization. Its establishment requires wild-type paired activity and thus indirectly requires bicoid. It occurs in a region of the embryo that ultimately gives rise to the cephalic furrow and may account for the effect of paired on that structure during gastrulation. These results therefore suggest a link between cytoskeletal organization during cellularization and subsequent morphogenetic processes of gastrulation (Blankenship, 2001).

To assay the degree of uniformity along the AP axis during cellularization, the furrow canals of wild-type embryos were visualized by staining for Myosin. By mid-cellularization, three domains of differing diameters of yolk stalks could be observed. The first domain is centered around the anterior pole of the embryo, and possesses the largest diameters. The second domain, immediately posterior to the first anterior domain, has the smallest diameters. This domain is centered around the location where the cephalic furrow will eventually form. The third domain lies posterior to this pre-CF domain and has furrow canals of intermediate diameter (Blankenship, 2001).

These three domains also differ in the depth of the cellularization front. The anterior domain, which has large yolk stalk diameters, is the shallowest part of the embryo in its depth of cellularization. By the end of cellularization, the most anterior part of this domain can possess a depth of cellularization (15-20 µm) half that of the rest of the embryo (~35 µm on average). The region posterior to the pre-CF domain has an intermediate depth, whereas the pre-CF domain, which has the smallest yolk stalk diameters, has the greatest depth of cellularization. The regions of differing yolk stalk diameters correspond to the regions of varying depth during cellularization (Blankenship, 2001).

To test whether these cellularization phenomena are due simply to the position or geometry of the embryo (e.g. the curvature of the anterior part of the egg), or if these phenomena are being specified and positioned by the AP patterning system, the three domains were sought in embryos that lacked all positional information along this axis. In embryos derived from females triply mutant for bicoid nanos torso-like, yolk stalk diameters are uniform, even though other aspects of cellularization occur normally. The shallow cellularization front of the anterior domain, as well as the greater depth of the pre-CF, is also lost. Thus, these phenomena require the activity of the AP patterning system for their morphogenesis (Blankenship, 2001).

These cellularization phenomena respond to specific levels of the bicoid gradient. Embryos that carry six copies of bicoid produce more Bicoid protein, shifting any given concentration of Bicoid to a position posterior to where it would be in a wild-type embryo. In embryos carrying six copies of bicoid, the pre-CF domain of small yolk stalk diameters and greater depth is shifted posteriorly, while the anterior domain of large yolk stalk diameters expands to cover close to half of the embryo. bicoid appears to be the important factor specifying these phenomena, since embryos derived from females mutant for bicoid lack the anterior and pre-CF domains (Blankenship, 2001).

Since the pre-CF domain is a relatively narrow domain, it seemed unlikely that the broad Bicoid gradient would be directly specifying the pre-CF domain. Several pair-rule genes are expressed in approximately the right time and place to be mediating the formation of the pre-CF domain. While Even-skipped (Eve) is expressed in the posterior half of the pre-CF domain, Prd expression is directly centered on the pre-CF domain. Prd expression also has an unusual feature. When Prd expression is first detected during cycle 13, it is observed in a single, gap gene-like domain. Thus, Prd is expressed before the pre-CF domain is formed, and in the right location in the embryo. Moreover, the early expression of Prd in a single stripe, rather than in the stereotypical seven-stripe pattern, suggests that it could specify the pre-CF domain independent of other factors (Blankenship, 2001).

Various patterning mutants were examined for a disruption of the pre-CF domain. prd homozygotes show a normal anterior domain of larger yolks, but there is very little difference in the yolk stalk diameters between the pre-CF domain and the posterior domain. The relatively subtle appearance of the pre-CF domain in different genetic backgrounds required the scoring of embryos in a blind test. On a classification scale of 0-5, with 0 indicating a complete absence of the pre-CF domain, prd homozygotes scored a 1.2, while their non-homozygous siblings scored a 3.3. These classifications are significant (P <0.001, n=61). Thus, prd is already active in the regulation of morphogenesis during the process of cellularization (Blankenship, 2001).

The pre-CF domain is first visible when the cellularization front has reached about 25% of its depth. However, the larger yolk stalks of the anterior domain are visible throughout cellularization. The density of nuclei in the anterior is reduced by about 30% from the nuclear density that is found in the rest of the embryo. This anterior domain of lower nuclear density is centered around the anterior pole and extends to approximately two to three nuclei in front of the first stripe of Eve, or ~70% EL. Judging from the staining patterns of Eve and Prd, this is the same area of the embryo in which the anterior domain of large yolk stalk diameters meets the pre-CF domain. During cellularization the anterior nuclear domain is consistently observed in OreR embryos, with an average decrease in nuclear density of 27.6%. In this sample, the values for individual embryos ranged from 34% to 20%. This domain of lower nuclear densities is maintained and stays constant throughout the process of cellularization, and can still be observed in gastrulating embryos. This greater spacing of nuclei in the anterior would necessarily lead to a cellularization network with larger diameters. Additionally, by mid-cellularization, when a pre-CF domain is already visible, a slight clustering of nuclei in this region occurs (Blankenship, 2001).

Since the anterior domain of lower nuclear densities is present at the start of cycle 14, it was asked when this AP asymmetry arises. While both the sphere of nuclei that migrate to the surface of the embryo during cycle 9, and the cortical nuclei of cycle 10 are uniform along the AP axis, the first sign of increased nuclear spacing in the anterior can be observed in cycle 11 embryos. By cycle 12, a pronounced AP asymmetry is observed, which is maintained through the subsequent mitosis to the start of cellularization. The anterior domain of lower nuclear densities is also observable in living embryos. Nuclear counts on GFP-histone embryos demonstrate a similar ~30% reduction in nuclear density in vivo. These observations on living embryos also reveal that, except for when the nuclei are undergoing mitosis and the associated yolk contractions, the anterior nuclear domain is present throughout the rest of the cell cycle (Blankenship, 2001).

Before cycle 14, cortical nuclei are associated with large actin caps. In embryos without actin caps, the regular spacing of nuclei is disrupted. One possible mechanism for the formation of the anterior domain of lower nuclear densities would use the regulation of the size of these actin caps. To see if asymmetries in actin cap size occur along the AP axis, wild-type embryos were stained with fluorescently labeled phalloidin to visualize F-actin. Measurements show that anterior actin caps are larger than caps in the rest of the embryo in cycle 11 and cycle 12, but not during cycle 10. This difference, although small, is highly reproducible from embryo to embryo (Blankenship, 2001).

The exceptionally early appearance of the AP asymmetries in nuclear distributions led to an examination of whether the anterior domain of lower nuclear densities is specified by the AP patterning system. In embryos derived from bicoid nanos torso-like mutant females, nuclear spacing is uniform along the AP axis. In addition, the anterior nuclear domain can be expanded posteriorly in embryos carrying six copies of bicoid. Additionally, embryos from bicoid females are uniform in their nuclear densities, arguing that the anterior domain of lower nuclear densities is set up by the Bicoid gradient. Finally, the larger diameters of the actin caps in the anterior do not occur in embryos from bicoid nanos torso-like females (Blankenship, 2001).

Since zygotic transcription only starts around cycle 10, the early appearance of the anterior domain by cycle 11 raised the question of whether zygotic transcription is required for this domain's formation. It is possible that bicoid might be regulating nuclear spacing through a post-transcriptional mechanism. To assay the effect of transcription on nuclear spacing, embryos were injected with alpha-amanitin to block RNA polymerase II. Consistent with earlier studies, such embryos develop to cycle 14 with normal gross morphology. However, when these embryos were examined with nuclear stains, they showed a total absence of asymmetric nuclear distributions. Embryos injected with alpha-amanitin fail to form the anterior domain of lower nuclear densities. It is concluded that bicoid regulates the zygotic transcription to bring about the formation of the anterior domain of lower nuclear densities (Blankenship, 2001).

Two genes necessary for cephalic furrow formation have been identified. Embryos mutant for either eve or buttonhead (btd) lack cephalic furrows. Both eve and btd have a defect in the earliest phase of CF formation. At no point during development are the stereotypical cell shape changes of shortening along the apical-basal axis and widening of the cell observed in these mutants. Mutations in prd also cause a disruption in CF formation. At the onset of gastrulation, the cephalic furrow does not form, nor do initiator cells undergo their characteristic cell shape change. Because there is an absolute correlation between initiator cell behavior and the middle nucleus of the first stripe of Eve in wild-type embryos, the location where initiator cells should form in prd embryos can still be identified. In prd embryos these Eve-marked cells are indistinguishable from their neighbors at stages during gastrulation when the ventral furrow has formed and the CF would normally be visible in wild-type embryos. However, by mid-germband extension (GBE), prd embryos have formed a regular, CF-like invagination. This late fold is thought to arises through the same mechanisms that govern normal CF formation, but these processes are delayed relative to the development of wild-type embryos (in the stage 7 prd embryo there are no cell shape changes in the region where the CF would form, while the stage 6 wild-type embryo has an obvious CF). Initiator cells form in wild-type embryos at stage 6, at the onset of gastrulation. Imaging of prd embryos reveals initiator cell activities beginning at stage 7. At this stage, wild-type embryos have already begun to deform the yolk sack with a basal bulge of the epithelium. At the beginning of stage 8, or germband extension, wild-type embryos have a furrow that is many cells deep, while prd embryos have just begun to deform the yolk sack. It is only by mid-GBE that most, but not all, prd embryos have a regular CF stretching around the entire circumference of the embryo (Blankenship, 2001).

The abnormalities in CF formation observed at the beginning of gastrulation in prd embryos are superficially similar to those observed in embryos mutant for eve or btd. In the absence of the activity of eve, btd or prd, the cell shape changes that occur in the row of cells that initiate CF formation at the beginning of gastrulation do not occur. In contrast to eve or btd, however, early activities of prd during cellularization have been identified that may account for the later differences observed in CF formation. Consistent with this view the analysis indicates that prd embryos often recover and form a fairly regular CF by mid-germband extension, unlike the severe disruption of the CF in eve and btd embryos. While the start of gastrulation occurs normally in prd embryos, CF formation and initiator cell behavior is delayed. It is proposed that this delay is due not to a function of prd during gastrulation in the specification of initiator cells, as has been proposed proposed for eve and btd function. These latter mutants completely block initiator cell and CF formation. Instead, prd function is necessary for the formation of the pre-CF domain, and it is suggested that prd functions in CF formation only through this disruption of the pre-CF domain. The recovery of the CF observed in prd embryos may be a reflection that cellularization throughout the embryo has finally reached the point that the pre-CF domain reaches at the very beginning of gastrulation, and so CF formation, although delayed, may be correctly initiated. The advanced rate of cellularization in the pre-CF domain may reflect a required premature closing of the base of the initiator cells so that cell volumes may be maintained during the severe cell shape changes of gastrulation, or that cytoskeletal components involved in cellularization must be freed for initiator cell movements. Because little deformation, or loss of volume, occurs in the presumptive initiator cells of early prd embryos, the latter of these two possibilities is favored. Thus, the subtle regulation of one stage of development can have profound effects upon a later, seemingly discrete, process of development (Blankenship, 2001).

The cellularization front that arises during early cycle 14 is rich in actin and myosin, and is thought to provide a contractile force that orients and drives the process of cellularization. This network, as well as cellularization itself, proceeds through a two-phase process. The first phase is a basal movement of the cellularization front towards the interior of the embryo. During this phase, the furrow canals stay constant in their small size, and the actin/myosin array has a hexagonal shape. The second phase is a lateral movement of the cellularization front, which creates a pinching off at the bases of cells in the newly forming cell sheet. The morphology of the cellularization front is not uniform along the AP axis. The pre-CF domain is distinguished from other regions of the embryo by its small yolk stalk diameters and greater depth of cellularization (Blankenship, 2001).

These results suggest the following model for the generation of AP asymmetries during cellularization. In the pre-CF domain, the Bicoid gradient directs the correct localization of the early gap gene-like expression domain of prd, which, in turn, directs a greater local contraction of the cellularization network. When cellularization is in its first phase of inward directed movement, the greater contraction of the pre-CF domain leads to a greater advance inwards of the cellularization front, thus creating the greater depth of the pre-CF domain. Then, when cellularization shifts to the second phase of a lateral, pinching-off movement, the greater contractility of the pre-CF domain leads to a greater widening of the furrow canals, which creates the smaller yolk stalk diameters observed in the pre-CF domain. The creation of this domain of advanced cellularization may be necessary for the initiator cell shape change required for cephalic furrow formation (Blankenship, 2001).

In certain respects, the anterior domain of large yolk stalk diameters and shallow cellularization appears to be the opposite of the pre-CF domain, and thus might be produced by a downregulation of the same contractile mechanisms that are proposed to operate in the pre-CF domain. However, the formation of the larger yolk stalk diameters in the anterior domain clearly involves a different mechanism. The anterior cellularization domain is pre-figured by an anterior domain of lower nuclear densities, while the pre-CF domain initiates in a region where nuclear densities are uniform. The greater spacing of nuclei in the anterior necessarily causes the formation of actin/myosin arrays of greater diameter during cellularization. A model for the formation of the anterior domain is favored in which the bicoid gradient, by cycle 11, has regulated the transcription of a set of zygotic genes, which in turn regulate the size of the actin caps overlaying the nuclei. This regulation of the actin caps results in larger caps in the anterior that necessitates a greater spacing of nuclei. By cycle 14, when cellularization is initiated, the greater spacing of nuclei dictates the generation of a cellularization network in the anterior in which the hexagonal components are larger in diameter. The initiation of large hexagons would thus lead to larger yolk stalks. It may be that the larger hexagons of the cellularization network contract less efficiently, thus generating the shallower depth of cellularization that is observed for the anterior domain (Blankenship, 2001).

The concept of a mid-blastula transition (MBT) has generally referred to a time when the genome of an embryo begins to exert an influence on development, presumably through zygotic transcription. The best-defined MBT is for Xenopus, where the MBT was characterized by a cessation of synchronous mitotic cycles, the start of zygotic transcription, and a change in the morphology of blastula cells (i.e., the acquisition of cell motility). Various aspects of this characterization have since been called into question. There is low level zygotic transcription before the MBT, and it appears as though cell motility may not be a function of zygotic transcription, but of slower mitosis. Although these discrepancies call into question the usefulness of the MBT as a concept, the idea of an MBT remains an attractive model for explaining the changing morphology of the Drosophila embryo (Blankenship, 2001).

In flies, the MBT has traditionally been discussed in terms of cycle 14 development. It is at this point that the fly embryo ceases its synchronous syncytial divisions and several morphogenetic processes require zygotic transcription for their genesis. At a superficial level, the first observable defects in embryos deficient for zygotic transcription is at cycle 14, when alpha-amanitin-injected embryos show defects in the processes of lipid droplet clearing and cellularization. However, as in Xenopus, defining a precise MBT presents some difficulties. Although cycle 14 marks a major increase in transcriptional efficiency, the start of zygotic transcription occurs in different nuclear cycles for different genes. In general, for the early acting genes involved in patterning, transcription begins around cycle 10, although some genes are transcribed as early as cycle 8, and mutations in specific segmentation genes show subtle disruption as early as cycle 10. What is striking in the results reported for Bicoid and Prd is the correspondence between spatial patterns of expression and regional alterations in morphology. These results show that the genome of the embryo is active in the spatial regulation of morphogenesis at a much earlier time than previously described. The genome directs reproducible asymmetries in morphology by cycle 11. The generation of an anterior domain of lower nuclear densities argues against a single discrete MBT at cycle 14. These results further suggest a more refined view of the Drosophila MBT in which there is a gradual shifting in the guidance of development from maternal to zygotic gene products, one that stretches from as early as cycle 8 until the cessation of mitosis and formation of a cellularized embryo at cycle 14 (Blankenship, 2001).

The origin of evolutionary novelty is believed to involve both positive selection and relaxed developmental constraint. In flies, the redesign of anterior patterning during embryogenesis is a major developmental innovation and the rapidly evolving Hox gene bicoid plays a critical role. This study reports evidence for relaxation of selective constraint acting on bicoid as a result of its maternal pattern of gene expression. Evolutionary theory predicts 2-fold greater sequence diversity for maternal effect genes than for zygotically expressed genes, because natural selection is only half as effective acting on autosomal genes expressed in one sex as it is on genes expressed in both sexes. Single individuals have been sampled from ten populations of Drosophila melanogaster and nine populations of D. simulans for polymorphism in the tandem gene duplicates bcd, which is maternally expressed, and zerknüllt, which is zygotically expressed. In both species, the ratio of bcd to zen nucleotide diversity was found to be two or more in the coding regions but one in the noncoding regions, providing the first quantitative support for the theoretical prediction of relaxed selective constraint on maternal-effect genes resulting from sex-limited expression. These results suggest that the accelerated rate of evolution observed for bcd is owing, at least partly, to variation generated by relaxed selective constraint (Barker, 2005).

Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is a particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. A subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. Evidence is provided that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation (Werz, 2005).

Although this study largely focus on the maternal effect mutants bicoid and oskar, it is likely that the principles uncovered are of broader significance. Segmentation mutants acting downstream of bicoid and oskar, including mutants of gap genes (Krüppel, knirps), pair-rule genes (odd, fushi-tarazu) and segment polarity genes (wg, hedgehog, engrailed) induce expression of hid. These mutants are characterized by loss of larval tissue. As in the case of bicoid and oskar, hid expression is upregulated during stage 9 of embryogenesis in the regions of the mutant embryos that are later deleted in the larvae. In addition, hid mutants rescue the cuticle phenotype of armadillo mutants. Finally, hid expression accompanied by TUNEL-positive cell death was found in dorsal and Toll^10b mutants, which cause dorsalizing and ventralizing phenotypes, respectively, along the dorsoventral axis of Drosophila embryos. Thus, these data support the notion that upregulation of hid appears to be a common trigger for a caspase-dependent cell death program in mis-specified cells of patterning mutants (Werz, 2005).

Furthermore, mutations affecting imaginal disc development result in loss of the adult appendage due to inappropriate cell death. It is currently being determined whether these mutants also require hid expression to develop the final phenotypes. Moreover, many gene disruptions in mice result in inappropriate cell death in the tissue that requires the function of the disrupted gene, suggesting that similar mechanisms might exist in mammalian development. Finally, cell death may be an important contributing factor to human congenital birth defects. Thus, an understanding of the underlying mechanisms is of general interest (Werz, 2005).

Interestingly, not all segment polarity mutants analyzed induce hid expression and cell death. Embryos mutant for patched, which encodes the hedgehog receptor, were not found to express hid and do not exhibit increased cell death, although hedgehog mutants both upregulate hid and contain increased amounts of cell death. The reasons for these differences are not known, but partial redundancy might account for lack of hid expression in patched mutants. The Drosophila genome encodes another patched homolog, patched-related, which might provide the survival requirement for mis-specified cells in patched mutants (Werz, 2005).

Mis-specified cells in bicoid and oskar mutants induce expression of hid. No increased reaper or grim expression was observed in these mutants. However, expression of reaper has been reported in crumbs mutants, which affect epithelial integrity. X-ray-treated embryos also preferentially respond by upregulation of reaper, rather than hid. Although crumbs mutants were not analyzed for hid expression, it appears that cells contain several developmental checkpoints, which activate different cell death-inducing regulators depending on the type of abnormal cellular development (Werz, 2005 and references therein).

Mis-specified cells can survive if an alternative survival pathway is provided. The example presented here is the acron into telson transformation in bicoid mutants, which is mediated by the torso signaling pathway. Although the cells giving rise to telson structures at the anterior tip are mis-specified based on Abd-B-labeling experiments, they survive because they receive a survival signal from the torso signaling system. In this case, transformation rather than cell death is favored. It has been shown that activation of the Ras/Mapk pathway protects cells from hid-induced apoptosis, both by transcriptional repression of hid and by phosphorylation of Hid protein by Mapk. Because Torso, which encodes a receptor tyrosine kinase (RTK), is known to activate Ras and Mapk, tests were performed to see whether manipulation of active Mapk levels using a gain-of-function allele, Mapk^Sem, can suppress hid expression and cell death in bicoid mutants. However, this was found not to be the case. Thus, torso appears to protect mis-specified cells independently of Mapk activation (Werz, 2005).

The hid bicoid double mutant analysis reveals that the transformation of anterior into posterior identity expands beyond the telson, and that this expansion undergoes hid-induced cell death in bicoid single mutants. The rescued cells secrete larval cuticle elements, suggesting that mis-specified cells have the developmental capacity to terminally differentiate. However, in hid⁺ background, they instead die, presumably because equivalent survival signals are lacking. It is proposed that mis-specified cells undergo cell death if no alternative survival pathway is provided to protect them (Werz, 2005).

An alternative survival mechanism might also operate in other developmental mutants where transformation rather than cell death occurs. Mutations in the sev RTK and its ligand boss result in transformation of the R7 photoreceptor cell into a non-neuronal cone cell. Survival of this cell could be mediated by the Drosophila Egf receptor (Egfr), another RTK, which is required to maintain cell survival in the developing eye disc. Accordingly, activation of the Ras/Mapk pathway by Egfr would inhibit hid expression and support survival of the presumptive R7 photoreceptor cell. This interpretation is also consistent with observations that egfr^- clones are small and undergo cell death, and that this death can be suppressed in hid mutants. Thus, transformation of the R7 photoreceptor to a cone cell rather than R7 cell death in sev and boss mutants could occur because of survival signaling by the Egfr (Werz, 2005).

The hid bicoid double mutant analysis suggests that mis-specified cells can continue to develop and differentiate. Yet, they die. Presumably, this cell death protects the organism from potentially dangerous cells. For example, it is conceivable that in mammals, surviving mis-specified cells might lie dormant in the host organism for years. During this time, they might acquire additional genetic alterations that could drive the progressive transformation of these cells into malignant cancer. In wild-type embryos, mis-specification probably occurs in cells in isolation, and elimination of these cells does not interfere with development and survival of the organism. Only in extreme situations, such as the patterning mutants analyzed in this study, is the mis-specification caused by aberrant development so severe that the affected organism dies (Werz, 2005).

The cause of mis-specification in each segmentation mutant is different. Usually, the expression of other segmentation genes is shifted and expanded, resulting in flattened gradients. Yet, irrespective of the cause of mis-specification, most of these mutants have in common that they induce hid expression. It is currently unknown how the mis-specified fate of cells is recognized, and how hid expression is induced. One possibility might be that the protein gradients established by bicoid⁺ and oskar⁺, as well as other segmentation genes are used as readout for proper cellular specification. The steepness of protein gradients as a means to determine life or death decisions has recently been proposed. Such a model would imply that cells are able to determine their position in a graded field and compare this readout with their neighbors. Because in bicoid and oskar mutants these gradients do not form, the concentration difference between neighboring cells would be zero. If the concentration difference between two neighboring cells is below a crucial threshold, they induce the expression of hid and undergo cell death. This model could also explain embryonic pattern repair, which was described in embryos that express six copies of the bicoid gene. In these embryos, the head and thorax primordia are expanded because of the presence of six copies of bicoid. However, this expansion is corrected for by induction of cell death, and relatively normal larvae develop. In this case, the Bicoid protein gradient does form, but would be flatter compared with wild type. Thus, the concentration difference between neighboring cells would be below a critical threshold, sufficient to induce hid-dependent cell death. However, it is largely unknown how cells compare their position in a graded field with those of their neighbors. It has been proposed that short-range cell interactions mediated via the cell-surface proteins Capricious and Tartan provide cues that support cell survival during wing development. Cells unable to participate in these interactions are eliminated by cell death. It is unclear, however, whether short-range interactions are sufficient to explain the cell death phenotype in bicoid and oskar mutants (Werz, 2005).

Irrespective of the underlying mechanism for sensing mis-specification, the current results highlight the role of an active gene-directed process that removes mis-specified cells during development. However, if a survival mechanism is provided, mis-specified cells can survive and adopt a different fate. In wild-type embryos, mis-specification probably occurs in cells in isolation, and hence is difficult to study. However, in bicoid and oskar mutants, large regions of neighboring cells are mis-specified and undergo cell death simultaneously, providing a unique opportunity to clarify the signals that initiate cell death in situations where cells are developmentally mis-specified (Werz, 2005).