Dorsoventral Axis Formation in the Drosophila Embryo—Shaping and Transducing a Morphogen Gradient

Current Biology, Vol. 15, R887–R899, November 8, 2005, ©2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2005.10.026

Dorsoventral Axis Formation in the Drosophila Embryo — Shaping and Transducing a Morphogen Gradient Bernard Moussian1 and Siegfried Roth2

The graded nuclear location of the transcription factor Dorsal along the dorsoventral axis of the early Drosophila embryo provides positional information for the determination of different cell fates. Nuclear uptake of Dorsal depends on a complex signalling pathway comprising two parts: an extracellular proteolytic cascade transmits the dorsoventral polarity of the egg chamber to the early embryo and generates a gradient of active Spätzle protein, the ligand of the receptor Toll; an intracellular cascade downstream of Toll relays this graded signal to embryonic nuclei. The slope of the Dorsal gradient is not determined by diffusion of extracellular or intracellular components from a local source, but results from self-organised patterning, in which positive and negative feedback is essential to create and maintain the ratio of key factors at different levels, thereby establishing and stabilising the graded spatial information for Dorsal nuclear uptake.

In the past 10 years, compelling evidence has highlighted the importance of morphogens for pattern formation during development [1,2]. A morphogen is defined by two crucial properties. First, it exhibits a graded distribution across a developmental field, so that the value of morphogen concentration at any given point of the gradient corresponds to a particular position within that field. Second, distinct ranges of morphogen concentrations elicit different cell fates. The combination of these two properties provides an elegant and powerful mechanism for specifying cell fate as a function of space. Recent work has focused on the cellular mechanisms of morphogen spreading and long-range gradient formation. A surprising diversity of cellular processes and mechanisms of considerable kinetic complexity have been found to be required for morphogen transport and for the shaping of morphogen gradients [3–7]. Despite the highly complex picture that has emerged from these studies, all cases considered so far have one feature in common: gradient formation depends either on spreading of the morphogen from a local source or on the spreading of a localised inhibitor, which forms a gradient opposing that of the morphogen. As morphogen (or inhibitor) production is localised while its degradation occurs within the entire developmental 1Department

of Genetics, Max-Planck Institute for Developmental Biology, Spemannstr. 35, 72076 Tübingen, Germany. 2Institute for Developmental Biology, University of Köln, Gyrhofstr. 17, 50923 Köln, Germany. E-mail: [email protected]

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field, these cases can be classified as ‘local-sourcedispersed-sink’ (LSDS) mechanisms [8–10]. Here, we summarise recent findings on the establishment of the morphogen gradient that patterns the dorsoventral axis of the early Drosophila embryo, the formation of which can apparently not be explained by the LSDS model. This gradient forms in the fluid-filled perivitelline space surrounding the embryo in which the key components required for gradient formation are evenly distributed. Positive and negative feedback loops among these compounds, together with their differential spreading within the perivitelline fluid, appear to constitute a reaction-diffusion system with self-organising properties. The Ventralising Cascade of Drosophila — an Overview Establishment of dorsoventral asymmetry in the early Drosophila embryo is largely under maternal control (Figure 1) [11]. The initial cue for axis formation arises at the ventral side of the embryo and is stably transduced into the embryo via the Toll pathway (Figures 2 and 3). This pathway is evolutionarily conserved and also acts during insect and mammalian immune responses, where it is activated upon microbial challenge and induces the expression of antimicrobial peptides or inflammatory cytokines [12–14]. During Drosophila embryogenesis, the Toll pathway establishes at least three different regions along the dorsoventral axis, resulting in the separation of the mesoderm and the neuroectoderm from the non-neurogenic (dorsal) ectoderm (Figure 4) [15,16]. The dorsal ectoderm is patterned by the zygotically regulated Decapentaplegic (Dpp) pathway [17]. Generation of the ventralising information involves 17 maternally provided factors, which were identified in various genetic screens (Table 1) [18–23]. The spatial cues for the embryonic dorsoventral axis originate during oogenesis in the follicle cell layer, a somatic, monolayered epithelium, which surrounds the oocyte–nurse cell complex (Figure 1) [24–27]. Factors derived from the follicle cells comprise the intracellular proteins Pipe (Pip), Slalom (Sll) and Windbeutel (Wbl) and the secreted protease Nudel (Ndl). Together they are required to activate a cascade of proteases which are secreted as inactive precursors by the oocyte or the early embryo. This cascade is composed of Gastrulation defective (GD), Snake (Snk), and Easter (Ea) and has similarity to the blood clotting and complement activating cascades of vertebrates [28]. Three-dimensional modelling of the proteasesubstrate complexes supports the sequence of action of the proteases (GD–Snk–Ea), which was derived by genetic epistasis analysis [29] and suggests that all components of the cascade have been identified. The protease cascade is modulated by several intrinsic

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A Restriction of Pipe expression Nurse cells

Dorsal

Nucleus

Oocyte Oocyte

Follicle epithelium

B ECM modification Perivitelline space

Perivitelline space

Embryo

Embryo

C Ventralising signal and nuclear Dorsal gradient Perivitelline space

Perivitelline space

Nuclei

Embryo

Lateral view

Embryo

Ventral view

Cross section Current Biology

Figure 1. Major events during dorsoventral axis formation in Drosophila. α protein emanating from a dorsal source in the oocyte, represses pipe (orange) in the follicular epithelium, (A) Gurken (green), a TGFα restricting its expression to a ventral stripe. (B) Pipe leads to the modification of an unknown ECM component (red) which is secreted by the follicle cells and maintained within the extracellular space surrounding the late oocyte and early embryo. (C) Subsequently, an extracellular signal is generated within the region defined by the modified ECM component. This signal induces the nuclear transport of the transcription factor Dorsal. The signal has peak levels within a narrow stripe straddling the ventral midline (dark blue). In the area between the dark blue stripe and the border of the red stripe in (B) a gradient is established, which accounts for the different ventrolateral cell fates of the embryo (compare to Figure 6).

positive and negative feedback loops, as well as by the Easter inhibitor Serpin27A. The activation of the cascade culminates in the generation of the morphogen Spätzle (Spz), which binds to the transmembrane receptor Toll (Tl). The signal is transduced into the embryo by the adaptor proteins Krapfen/dMyd88 (Kra/dMyd88) and Tube (Tub) and the kinase Pelle κB homo(Pll). This leads to the degradation of the Iκ κB transcriplogue Cactus (Cact), which allows the NFκ tion factor Dorsal (Dl) to enter the nucleus and activate or inhibit zygotic genes required for dorsoventral cell fate specification. At several positions of the pathway, the means of signal transduction is not yet understood. In particular, the substrate of Pipe specifying the ventral domain remains to be identified, and the role of Nudel is controversial as neither its activating factors nor its substrates are known. Furthermore, it is not clear how Pelle transmits the signal to Cactus. Finally, two new maternal factors, Weckle (Wek) and Seele (Sle) have been found which have loss-of-function phenotypes similar to those of other pathway components. Their role within the pathway remains to be established [19].

In order to generate a pattern of distinct cell types along the dorsoventral axis, the intensity of the signal regulating the entry of Dorsal into the nucleus is high in the ventral most region and drops at the lateral positions, whereas in the dorsal region Dorsal continues to be sequestered by Cactus and remains in the cytoplasm [30–32] (Figure 4 and 5B). High nuclear concentrations of Dorsal activate the transcription of twist (twi) and snail (sna) in the ventralmost 18–20 cells (20%), which invaginate into the embryo to form the mesoderm [16] (Figure 5C). Abutting this zone, the lateral cells (16–18 cells on each side) at intermediate and low Dorsal concentration express single minded (sim), rhomboid (rho) and short gastrulation (sog) and a number of other genes required for the establishment and patterning of the neuroectoderm [33–35]. The neuroectoderm gives rise to the ventral nerve cord and the ventral epidermis; finally, the remaining dorsal portion of the embryo (40%) lacks Dorsal activity and expresses genes required for the establishment of the amnioserosa and the dorsal epidermis, like zerknüllt (zen) and dpp. These genes are repressed by Dorsal in the ventrolateral and ventral cells. A combination of microarray experiments and

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Table 1. Genes involved in dorsoventral axis formation in Drosophila. Gene

Protein

Molecular function

Biological function

pipe

Heparan-sulfate 2-sulfotransferase ECM modification

Specification of the ventral domain

slalom

Translocator

Import of PAPS1 into Golgi

Specification of the ventral domain

windbeutel

ER protein

Localisation of Pipe to the Golgi

Specification of the ventral domain

nudel

Serine protease

?

Specification of the ventral domain

gastrulation defective

Serine protease

Activation of Snake

Generation of the ventralising signal

snake

Serine protease

Activation of Easter

Generation of the ventralising signal

easter

Serine protease

Interaction with Gd Processing of Spätzle

Generation and refinement of the ventralising signal

serpin27A

Serine protease inhibitor

Inhibition of Easter

Modulation of Easter activity

spätzle

NGF-like

Ligand of Toll

Ventralising signal, morphogen

Toll

Receptor

Receptor for Spätzle

Transmission into the embryo

tube

Adaptor

Recruitment of Pelle

Signal transduction

dMyd88/krapfen

Adaptor

Binding of Toll and Tube

Localisation of Tube

pelle

Serine/threonine kinase

Phosphorylation leading to Cactus degradation Inactivation of Toll and Tube

Stable copy of gradient within the embryo

cactus

κB homologue Iκ

Inhibition of Dorsal

Suppression of the ventralising signal

dorsal

κB homologue NFκ

Regulation of zygotic genes

Morphogen

weckle

Zinc-finger transcription factor

Regulation of transcription

?

seele

?

?

?

bioinformatics has recently been used to identify a large number of new Dorsal target genes and their cis-regulatory elements, which mediate concentration dependent regulation by Dorsal [33,34,36,37]. Thus, Dorsal and its target genes constitute one of the best-understood gene regulatory networks [38]. Origin of the Ventralising Information The ventralising signal is derived from the dorsoventral asymmetry of the egg chamber. Four factors have been identified so far, which are required to transmit this asymmetry to the embryo by specifying a domain at the ventral side of the egg where the Toll ligand is generated (Figure 2) [21,39–42]. The dorsoventral asymmetry arises from the repression of pipe transcription in the dorsal half of the follicular epithelium by Gurken–EGF receptor signalling during oogenesis [39,43], which is a direct readout of a gradient of EGF receptor activity and does not require secondary signalling processes (Figure 1) [44–46]. Pipe expression at the ventral side of the developing oocyte is sufficient to induce axis formation in the embryo, as ectopic expression of pipe in the follicular epithelium activates the Toll pathway [43,45]. However, it is crucial to note that expression of pipe is insufficient to explain the shape of the Dorsal gradient. Pipe is expressed evenly in a domain encompassing ~40% of the circumference of the egg chamber with sharp lateral borders (Figure 1 and Figure 5A,B). During egg maturation, this domain maintains its size relative to the egg circumference [43,45]. The entire bell-shaped Dorsal gradient spans 40% of the egg circumference and thus lies within the domain of uniform

pipe expression (Figure 1 and Figure 5A,B). More importantly, in mutants of grk or egfr, pipe repression is compromised and the pipe domain expands. The Dorsal gradient is not just expanding, however, but rather splits and acquires two peaks, one at each side of the ventral midline [39,43] (Figure 5C,D and Figure 6). These observations demonstrate that pipe does not just localise a source, from which a gradient forms by diffusion. Pipe rather defines a ventral region of the egg, in which a complex patterning system leads to gradient formation by the dynamic interactions of its components. Therefore, a LSDS model cannot be used to explain the formation of the Dorsal gradient. The pipe locus encodes ten related proteins, which show similarity to heparansulfate 2-O-sulfotransferases of vertebrates [39,47]. Only one of the protein isoforms is expressed in the ventral follicle cells and responsible for dorsoventral axis formation. Other isoforms appear to be required for the late morphogenesis of the salivary glands. As mentioned above, the substrate modified by Pipe is not known. A functional analysis of enzymes essential for glycosaminoglycan synthesis showed that heparan sulfate and other glycosaminoglycans are not required for dorsoventral patterning within follicle cells and thus are unlikely to be substrates for Pipe [39,47]. However, several observations suggest that Pipe indeed acts as a sulfotransferase. The universal sulfate donor in sulfotransferase reactions is 3´-phosphoadenosine 5´-phosphosulfate (PAPS). Both the PAPS synthetase [47] and Slalom, which imports PAPS from the cytoplasm into the Golgi apparatus [21], are required for dorsoventral pattern formation.

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Figure 2. A model for the activation of the Toll pathway. Slalom The ventral domain (light grey) of the embryo is specified by Pipe in the follicPipe ular epithelium. Windbeutel is required to allow the export of Pipe from ER to Serpin27A ? Nudel Golgi. Slalom is required to transport the Easter sulfate donor PAPS into the Golgi where ? Perivitelline fluid it functions as substrate for the sulfotransferase reaction carried out by Pipe. Spätzle The autocatalytically processed Nudel Gd central protease domain either pro? ? ECM cesses the ECM component modified by Toll Snake ? Pipe or directly triggers the protease Plasma membrane cascade through the activation of GD. Embryo GD activates Snake, which in turn activates Easter to process Spätzle, the Current Biology ligand for the Toll receptor. Easter activity is spatially and temporally regulated by Serpin27A. Moreover, Easter might control its own activation through inactivation of GD. Finally, the amino-terminal fragment of Spätzle interferes with Toll activation through an unknown mechanism, possibly by activating Serpin27A. Ventral domain

Follicular epithelium

Heparan

Windbeutel

In addition, the molecular analysis of different pipe alleles and feeding experiments with an inhibitor of PAPS synthetase support the assumption that Pipe transfers sulfate groups to the carbohydrate moieties of a yet unknown protein or glycolipid [47] . Unlike pipe, windbeutel is expressed in all follicle cells around the oocyte. However, genetic data clearly show that Windbeutel like Pipe is involved in defining the ventral domain [41]. Windbeutel encodes a novel protein implicated in ER trafficking, and is required for proper localisation of Pipe to the Golgi apparatus [40,48]. Loss of Windbeutel function causes retention of Pipe in the ER. No connection has yet been established between Pipe and the protease Nudel. Nudel is expressed in all follicle cells, and the protein localises to the surface of the oocyte and early embryo [42,49]. It is a large protein (>300 kDa) with multiple domains, including a central 33 kDa protease domain. To be active, Nudel requires autocatalytic processing as well as cleavage by a yet unknown factor. The fragments segregate to different compartments within the embryo. The protease domain, for instance, which is crucial for generation of the ventralising signal, localises to vesicles in the embryo. Entry of this fragment into the embryo is dependent on its protease activity and temporally coincides with activation of the Toll receptor, but is not asymmetric along the dorsoventral axis. Moreover, carboxy- and amino-terminal fragments of Nudel have also been detected in the embryo, in another subcellular compartment. These domains of Nudel have motifs found in many proteins of the extracellular matrix, including three potential glycosaminoglycan addition sites in the amino-terminal portion. Modification of these sites is important for activation of the protease domain, but, interestingly, does not depend on Pipe function [49,50]. Besides autocatalytic processing, the target of the Nudel protease remains to be identified; it has been shown that the best candidate, GD, is processed independently of Nudel [51], but computational modelling of Nudel and GD suggests that Nudel may indeed bind and cleave GD [29]. Generation of the 33 kDa Nudel protease domain is also independent of Pipe and

Windbeutel function, as it is present in embryos from pipe and windbeutel mutant females and it occurs evenly throughout the circumference of the embryo. Hence, the follicular factors do not act in a linear pathway to activate the downstream protease cascade, but rather in parallel. Starting the Protease Cascade — the Role of gd How is the information provided by the ventral Pipe domain used to generate the ventralising signal? The protease cascade in the perivitelline fluid starts with GD, which encodes an unusual serine protease, structurally similar to mammalian complement factors C2 and B [52–54]. In the absence of GD, the downstream serine proteases show a basic level of activation, which however lacks dorsoventral polarity [52–54]. Thus, GD provides the critical link between pipe expression and local activation of the protease cascade. The gd mRNA is stored in the oocyte and the GD protein is secreted as an inactive precursor into the perivitelline space. GD acts prior to egg deposition during late stages of oogenesis. The GD precursor is proteolytically processed, giving rise to an amino-terminal propeptide and the carboxy-terminal catalytic chain. GD acts locally at the embryo surface [52,54,55]. Indeed, it has been shown in vitro that GD associates with heparin [51], suggesting that in vivo it might associate with sulfated glycoproteins of the extracellular matrix. Injection assays support the idea that GD is not freely diffusible as the ventralising effect of GD is restricted to the side of injection. Interestingly, Pipe appears not to be essential for GD localisation and activation as injections of gd RNA in the dorsal region of the embryo can initiate axis formation. However, the product of Pipe seems to facilitate GD activation as injection of GD into the ventral side of the embryo shows stronger and spatially more extended effects. Thus, Pipe may just provide a bias for the activation of the cascade. This would be sufficient to induce pattern formation if the system has self-organising properties [10]. What actually activates GD remains uncertain, although a direct interaction of GD with Nudel cannot be ruled out [29,56]. However, as stated above,

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Figure 3. The cytoplasmic events downstream of the Toll receptor. The activated Toll receptor recruits the membrane-localised dMyd88–Tube heterodimer to the intracellular domain of the activated Toll receptor. Subsequently, the kinase Pelle is recruited to the heterotrimeric Toll–dMyd88–Tube complex and undergoes autophosphorylation, thereby enhancing its kinase activity. By phosphorylation of Toll and Tube Pelle is released from the heterotetrameric complex. At this step, signalling is disrupted, and the Toll–Spätzle complex is presumably internalised. Next, Pelle transduces the signal to downstream factors, resulting in the degradation of Cactus. This allows binding of Dorsal to Tamo which regulates nuclear entry of Dorsal through a nuclear pore containing DNTF-2 and Mbo.

Spätzle Toll

Perivitelline fluid

Plasma membrane Embryo

dMyd88

dMyd88

2

2

Tube

Tube 1 1

Pelle

P 3 3

Tamo

P Pelle

DNTF-2 Cactus

sog rho

Mbo

Dorsal

sim snail twist

Nucleus Current Biology

exogenous GD can induce the Toll pathway in a nudel mutant background suggesting that Nudel is not absolutely required for GD activation [53]. Two mechanisms have been proposed for GD activation at the ventral side of the embryo [51]: either the GD precursor is processed dependent on the Pipe substrate and Nudel by a yet unknown factor and subsequently triggers the downstream cascade, or GD has a low protease activity as a precursor, which is inhibited by a yet unknown factor. As in other complement-like factors [57], a conformational change within GD induced by the Pipe substrate and Nudel may release GD from the suppressor. We would like to propose a third model of GD activation, which is in agreement with an interpretation put forward by Ellen LeMosy and colleagues [56]. This model is based on the observation that GD protease activity is enhanced in the presence of Snake, the downstream target of GD [51]. In addition, Snake like GD has been shown to associate with heparin [51]. In our model, activation of GD requires Snake to be locally concentrated in the vicinity of GD. This in turn would depend on the Pipe substrate and/or Nudel. As GD is bound to the embryo surface, the signal could not spread beyond the domain defined by Pipe, which would ensure that the dorsoventral asymmetry within the follicular epithelium is faithfully conveyed to the embryo. How does GD act on the downstream proteases? Interestingly, interallelic complementation occurs between mutations affecting the catalytic chain in trans to mutations within the amino-terminal polypeptide. Indeed, injection assays have shown that both polypeptides are important for signalling and have different roles during axis formation. The GD protease domain serves to transmit the ventralising signal, while the amino-terminal fragment appears to have several functions. It is required to link GD to the embryo surface, but it might also repress the ventralising signal [54]. Interestingly, both polypeptides provided in parallel have no effect on the mutant phenotype indicating that an intact unprocessed GD

protein is required to correctly transmit the signal. This requirement kinetically links the activation process to the formation of the potentially inhibitory amino-terminal fragment. In vitro data suggest that GD is subject to feedback regulation after activation of Snake [51]. First, processed GD is subject to autoproteolysis, and second, Easter, the target of Snake, cleaves GD. So far it has not been established whether these proteolytic processes lead to activation or inactivation of GD, but both possibilities are interesting from a theoretical point of view (see below). In case of negative feedback, they might, together with the generation of an inhibitory amino-terminal fragment, provide a first step by which the broad domain of activation defined by pipe is narrowed down towards the ventral midline. New Insights into the Role of Easter Recent work has put Easter into the centre of interest as a major factor responsible for the shape of the Dorsal gradient [58–63]. Easter codes for a serine protease that requires proteolytical processing by Snake in order to be active [51,63]. Regulation of Easter activity involves the serine protease inhibitor Serpin27A, which irreversibly binds to the catalytic centre of Easter once it is cleaved from the amino-terminal pro-domain [59–61]. Interestingly, the interaction of Easter with Serpin27A is subject to feedback regulation, as the amount of the Easter–Serpin27A complex being formed depends on downstream components of the pathway. In dorsalised embryos, the level of Easter–Serpin27A is increased, whereas in Cactus mutants it is decreased [62]. This signal dependence of Serpin levels is difficult to explain. One could imagine that the secretion of Serpin27A into the extracellular space is controlled by Toll signalling. At the ventral side of the embryo, secretion is inhibited, whereas at the lateral and dorsal sides outside the Pipe domain Serpin27A is secreted to inhibit Easter. An inverse correlation of Toll pathway activity and Serpin27A secretion would explain the observation

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Amnioserosa

Dorsal ectoderm

sog rho sim twist

Low nuclear Dorsal Intermediate nuclear Dorsal High nuclear Dorsal

Neuroectoderm

Mesectoderm Mesoderm Current Biology

Figure 4. Positional information generated by the Toll pathway. A schematic half cross-section of an early Drosophila embryo; graded activation of the Toll pathway in the ventral half leads to the subdivision of the embryo into three broad regions along the dorsoventral axis — the presumptive mesoderm, neuroectoderm and non-neurogenic ectoderm. Subsequent signalling processes lead to further subdivisions within these regions. Indicated here are the mesectoderm, the dorsal ectoderm and the amnioserosa.

that the amount of the Easter–Serpin27A complex is increased by dorsalising mutations, but decreased by ventralising ones. What is the biological significance of the Easter–Serpin27A interaction? Some dominant easter mutations within the conserved Serpin binding pocket cause partial ventralisation of the embryo without increasing the total amount of Spätzle indicating that Easter spreads around the embryo to ectopically activate Spätzle [61]. Dorsoventral polarity is maintained in these mutants, confirming that Easter is asymmetrically activated, but the slope of the Dorsal gradient is dramatically flattened [61]. Likewise, in embryos derived from Serpin27A mutant females ectopic Easter activity causes a complete ventralisation of the embryo. Hence, Serpin27A is required to repress signal transduction through the interaction with Easter outside the domain defined by Pipe. Additional insight into the mechanisms of regulation of the protease cascade comes from the analysis of the dominant mutation Easter5.13, which causes an inhibitor-independent but low protease activity sufficient to induce sog but not twist expression leading to a loss of polarity that results in lateralised embryos [61]. Easter5.13 mutant embryos have increased amounts of processed Spätzle. Injection of the perivitelline fluid from gastrulating ea5.13;spz double mutant embryos, in which Toll signalling has ceased, into embryos from pipe mutant females still

induced axis formation. The increased amount of processed Spätzle thus results from prolonged activity of Easter. This indicates that Easter–Serpin27A interaction is also required to temporally restrict Easter activity. The spatial and temporal regulation of Easter are thus both crucial for establishing a peak of the ventralising signal and a graded decrease of the signal towards the borders of the ventral domain. The Morphogen Spätzle Recent genetic and molecular findings underscore that Spätzle does not simply respond to the refined Easter activity, but assumes a more active role in gradient formation. Spätzle encodes a NGF-like protein with a cystine knot motif, found in many vertebrate growth factors, and alternative splicing creates multiple isoforms, which are present at different time points of development [64,65]. The isoforms acting during dorsoventral axis formation are processed into two active molecules, the constant carboxy-terminal C-106 ligand that dimerises to bind two monomers of Toll [65,66] and the variable amino-terminal prodomain [64]. Controlling the amount of Spätzle is crucial for correct pattern formation. Feedback regulation apparently acts to reduce Spätzle activity. This is notably observed in embryos derived from Toll mutant females [67], in which the amount of Spätzle is clearly increased, suggesting that degradation of Spätzle probably through internalisation of the receptor–ligand complex is important in the regulation of signalling [68–70]. How does Spätzle contribute to the generation of the dorsoventral gradient? As noted above, an increase of the amount of Spätzle does not affect the slope of the gradient, as assayed by sog expression, but rather causes the enlargement of the twist domain, which yet never exceeds the ventral region specified by Pipe [67]. This is even true for eggs laid by egfr mutant females, in which the pipe domain increases from 20% to 70% of the circumference. Reduction of the Spätzle dose by half causes a reduction of the twist domain from 20% to 15% of the embryo circumference without affecting the domain of sog expression [67]. In conclusion, Spätzle seems to respond stably to the slope of Serpin-regulated Easter activity. However, it appears that this stable transmission is not resulting from an exact copying of the Easter activity gradient. Evidence for a rather complicated mechanism of Spätzle activity comes from injection essays showing that increased local amounts of Spätzle cause axis duplication, a phenomenon that has been observed also in embryos laid by grk and egfr mutant females [24,25]. In these embryos, two ventral furrows are formed within the enlarged pipe domain (Figure 5D) [67]. However, embryos from Toll10b mutant females that induce twist expression around the embryo circumference do not exhibit a duplicated axis [25,30,67]. Hence, expanding the embryonic ventral domain per se is not sufficient to induce axis duplication. Rather, axis duplication

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Figure 5. The pipe expression domain and the Dorsal gradient. (A) Cross-section of a stage 10 egg chamber at the level of the oocyte nucleus (n) showing the distribution of pipe mRNA (blue). pipe is evenly expressed in ventral follicle cells (fc), encompassing 40% of the egg chamber circumference. (B) Cross-section of a syncytial blastoderm embryo showing the distribution of Dorsal protein (red). The nuclear Dorsal gradient is confined to a region corresponding to the pipe domain of the follicular epithelium. (C,D) Cross-sections of embryos at the beginning of gastrulation showing Twist protein distribution (brown). In wild-type (C), Twist is expressed in a group of ventral cells which have received high levels of Toll signalling and, therefore, accumulated high levels of nuclear Dorsal protein. These cells start to invaginate to form the mesoderm. In an embryo derived from gurken mutant (grkHK/grkWG) females (D), the reduction of Grk signalling leads to an expansion of the pipe domain. As a result, Toll signalling is active in a broader region. This results in the splitting of the peak of Toll activation and consequently in two regions accumulating high levels of nuclear Dorsal. Therefore, two separate groups of cells express twi and start to invaginate. (A and B) modified from [26].

occurs upstream of the Toll receptor, outside the embryo. Indeed, recently, using an elegant approach Morisato [67] has shown that axis duplication depends on Spätzle itself, as it is suppressed in embryos expressing high levels of a dominant version of Spätzle that enhances its interaction with the receptor Toll. This can be explained if the amino-terminal fragment of Spätzle that is formed by Eastermediated proteolysis acts as a negative regulator of signalling. Injections of this part of the Spätzle protein cause a dorsalisation of the embryo, thus antagonising the effect of the carboxyl terminus of Spätzle. However, the dorsalising effect of the amino-terminal fragment is weaker compared to the ventralising effect of the carboxy-terminal fragment. It has been proposed that the amino-terminal fragment of Spätzle directly or indirectly inhibits Easter. As Easter is also the substrate for inhibition by Serpin27A, it is attractive to propose a link between both types of inhibition. The amino-terminal fragment of Spätzle might either bind to Easter and facilitate the interaction with the Serpin or bind and activate the Serpin. However, irrespective of the biochemical details, the production of an inhibitory peptide linked to the activation process is likely to be essential in generating the Spätzle morphogen gradient. The more Easter activates Spätzle, the more the Spätzle amino-terminal fragment inactivates Easter. Assuming a higher diffusion rate for the amino-terminal Spätzle fragment than for the carboxy-terminal fragment, the amino-terminal fragment would gradually inhibit Easter at the borders of the position with high concentration of the

carboxy-terminal Spätzle fragment, thereby creating a Spätzle slope where sog expression is activated around a Spätzle peak that induces twist expression (Figure 6). Axis duplication upon injection of high amounts of Spätzle or after expansion of the pipe domain might result from perturbation of the delicate balance between the ligand (Spätzle) and its inhibitor (Spätzle amino-terminal fragment). Higher concentrations of unprocessed Spätzle or an expansion of the ventral domain where Spätzle is processed are likely to cause increased inhibitor concentrations at ventralmost positions. Easter inactivation would thus be maximal at this position and, consequently, the ventral maximum of Spätzle activity would split into two maxima at more lateral positions. In summary, generation of the ventralising signal comprises at least three potential pairs of activators and inhibitors: the carboxy- and amino-terminal fragments of GD, Easter and Serpin27A, and the carboxyand amino-terminal fragments of Spätzle. While Easter and Serpin27A physically interact to shape the borders of the Spätzle gradient, the role of the GD and Spätzle amino-terminal fragments is not entirely clear. Theoretical Approaches As the formation of the Spätzle gradient is due to activating and inhibiting interactions among components, which are diffusible within a fluid-filled space, the perivitelline space, it should be ideally suited for modelling based on reaction-diffusion kinetics. Pattern formation mechanisms based on reaction-diffusion kinetics can be divided into two groups [71,72]:

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(1) Mechanisms in which the final pattern is stable over time. After a transition period, a steady-state is reached in such systems at which dynamic interactions of the components stably maintain the pattern. Patterns formed under such steady-state conditions have been termed Turing structures. (2) Chemical wave mechanisms in which the pattern is transient and constantly changes its shape. It is important to decide whether the pattern to be modelled represents a steady-state or a non-steady state situation since this decision leads to certain constraints regarding molecular interactions and diffusion rates. As in Drosophila the Spätzle gradient appears to be stable during the relevant developmental stages, a steady-state approach was used for modelling [10,73]. Theoretical considerations show that the production of a stable pattern requires the link between a process of local self-activation and long-range or lateral inhibition [74,75]. The inhibition can be realized in two different ways either through production of an inhibitor, which spreads from the peak of local activation or by the depletion of a substrate, which is consumed by the activation process [75]. The biochemical data described above show that the formation of the Spätzle gradient involves a local activation process, which may have multiple positive feedback loops, as required by the theory. The selfactivation appears to be linked to the production of inhibitors — in this case the amino-terminal prodomains of the proteases and Spätzle — which might have higher diffusion rates than the catalytic domains of the proteases and the receptor-binding domain of Spätzle. This suggests lateral inhibition by diffusible inhibitors. However, simulations using this type of lateral inhibition are unable to reproduce a Spätzle gradient with one peak, but rather result in two peaks of high activation at the lateral borders of the pipe domain [73]. The correct pattern can be simulated only with the help of a substrate-depletion model. Because the substrate is depleted around the activation peak, the peak remains stably localised in the centre. Maybe the ECM components modified by Pipe lead to the production of a diffusible substrate for the proteolytic cascade [73]. Substrate depletion might also result if downstream proteases inactivate up-stream proteases, which are diffusible in the perivitelline space. More biochemical details, however, are required to reconstruct the full kinetic complexity for more realistic modelling approaches. In addition, some of the assumptions made in the simulations so far should be reconsidered. It is possible that the system never reaches a steady state. For instance, in a more primitive insect, the red-flour beetle Tribolium castaneum, the nuclear Dorsal gradient forms only transiently and undergoes progressive shape changes before it disappears [76]. Thus, modelling approaches should also consider presteady-state solutions or non-steady-state mechanisms. Interestingly, the blood coagulation cascade, which shares many organisational features with the dorsoventral pathway was successfully modelled using a nonsteady state travelling-wave mechanism [77–79].

Inside the Embryo The shape of the Dorsal gradient seems to be established mainly outside the embryo, but how does the cytoplasm respond to these cues (Figure 3)? The central factor for robust transmission of the ventralising information into the embryo appears to be the serine/threonine kinase Pelle [80–87]. Graded Pelle activity is sufficient to induce all zygotic genes specifying different regions of the dorsoventral axis, as has been shown by monitoring zygotic gene expression in response to ectopic activation of Pelle in absence of the ventralising signal [88]. How is this accuracy achieved? Pelle acts downstream of the two adaptor proteins Tube and Krapfen/dMyd88 [83,84,89]. These two factors associate through their DEATH domains and localise to the plasma membrane independently of the signal [84]. In the absence of signal, Pelle is distributed in the cytoplasm [90,91]. Upon ligand binding and Toll dimerisation, the intracellular domain of Toll recruits the Tube–Krapfen/dMyd88 complex through the association of the TIR (Toll and Interleukin Receptor) domains of Krapfen/dMyd88 and Toll. This complex then recruits Pelle that binds to the DEATH domain of Tube through its own DEATH domain [92]. The amount of Tube and Krapfen/dMyd88 at the ventral plasma membrane of the embryo is now two times higher than at the dorsal side. Multimerisation of the Toll receptor together with the formation of the tetrameric complex of Toll– Tube–Krapfen/dMyd88–Pelle leads to a local increase of Pelle concentration [83,85,93,94]. At high concentrations Pelle undergoes autophosphorylation, thus enhancing its kinase activity and phosphorylating Toll and Tube, thereby getting released from the complex to activate downstream targets [95]. Phosphorylation of Tube and Toll stops signal transduction, which is thought to prevent over-amplification of the signal and ensures that Pelle activity stably transmits the Spätzle gradient into the embryo. Consistently, lack of Pelle function increases the amount of Tube and Krapfen/dMyd88 at the ventral side of the embryo, indicating negative feedback regulation. How does Pelle activation lead to a robust response in the transcription of zygotic target genes? In vitro assays suggest that Cactus and Dorsal are direct targets of Pelle [95–97]. In the mammalian Toll pathway, however, the Pelle homologue IRAK (Interleukin 1 Receptor-Associated Kinase) does not phosκB (nor Nfκ κB, the phorylate the Cactus homologue Iκ κB is phosphoryDorsal homologue) [98,99]. Rather, Iκ κB-Kinase (IKK) complex to release Nfκ κB. lated by the Iκ Thus, it might be possible that in vivo IKK related kinases act between Pelle and Cactus in Drosophila. β exist in Drosophila, one of which, Indeed, two IKKβ Ird5, has an essential role in immune response [100–102]. In addition, Ird5 seems to be required for correct Toll signalling during dorsoventral axis formation, but only a very small portion of the embryos from ird5 mutant females display a dorsalised phenotype (0.5%), suggesting that redundant factors might act just upstream of Cactus and Dorsal. This redundancy may explain why these factors escaped identification

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Figure 6. Model for the generation of the Spätzle gradient. (A) Spatial distribution of Easter protease activity (green), initiated by GD in the ventral domain defined by Pipe, is tightly regulated by its inhibitor Serpin27A. Serpin27A activity (light blue) in turn may be under the control of the amino-terminal Spätzle fragment (dark blue), which is produced together with the carboxy-terminal Toll binding fragment by Easter (green). As a result of its presumably higher rate of diffusion, the Spätzle amino-terminal fragment is present in a broader domain than the carboxy-terminal fragment. In lateral regions, the amino-terminal fragment leads to an inhibition of Easter. The resulting Spätzle gradient (red) is stably copied into the embryo through the graded activation of Pelle (red) which regulates the nuclear transport of Dorsal (pink). Ventrally, high nuclear Dorsal concentrations initiate twist transcription, and at more lateral positions, lower nuclear Dorsal concentrations lead to sog transcription. (B) An expansion of the Pipe domain reveals the self-regulatory capacity of the system. The inhibition exerted by the aminoterminal fragment of Spätzle has only a certain range. If the region in which the protease cascade is activated expands beyond this range of inhibition the activation peak splits and two peaks form, one at each side of the ventral midline (see Figure 5D).

in genetic screens and it may contribute to the robustness of the Toll pathway during axis formation. α and IKKγγ homologues in Notably, a role for IKKα dorsoventral axis formation has been ruled out κB [101,102], but they act in the activation of the Nfκ homologue Relish, which activates expression of immune response factors. Furthermore, the Cactus κB kinase recognition protein, besides its canonical Iκ motif harbours another redundant motif that is responsible for signal dependent degradation [97,103]. Besides Cactus, also Dorsal has to be phosphorylated upon Toll signalling in order to translocate to the nucleus [96,97]. This is revealed by a mutant allele of Dorsal, DorsalS234P, which fails to interact with Cactus. Nuclear translocation of the mutant protein was abolished in a GD mutant background. Hence, nuclear translocation of Dorsal seems to be a three-step process. First, Cactus is phosphorylated releasing Dorsal into the cytoplasm. Then, Cactus is ubiquitinated and degraded by the proteasome [104,105]. Third, Dorsal is phosphorylated, dimerises and enters the nucleus [106]. It will be interesting to know whether Cactus and Dorsal are phosphorylated by the same kinase.

Surprisingly, there is yet another mechanism of controlling Dorsal nuclear import independent of cactus [107,108]. WntD (Wnt inhibitor of Dorsal) can block the nuclear import of Dorsal, even in the absence of Cactus. However, wntD is expressed only at the anterior and posterior termini of the early embryo and its deletion does not lead to embryonic patterning defects, suggesting that it does not play a major role in establishing the Dorsal gradient. WntD acts also as a feedback inhibitor in the Drosophila innate immune system where its expression is activated by Toll signaling and downregulates the immune response. This immune function of WntD appears to be the reason why this regulatory loop has been maintained in evolution. Regulation of Dorsal activity also occurs at the level of nuclear entry which is under the control of the nuclear transport machinery. Three factors modulating nuclear transport, Tamo, Drosophila Nuclear Transport Factor-2 (DNTF-2) and Members-only (Mbo), the Drosophila homologue of the nuclear pore protein Nup88, selectively interact with Dorsal [109,110]. Tamo attenuates nuclear translocation of Dorsal, whereas Members-only and DNTF-2 are

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essential for this process. However, these interactions do not depend on Toll signalling. While the question of how Cactus and Dorsal receive the signal allowing Dorsal to enter the nucleus is still not completely resolved, it seems that downstream of Pelle no further refinement takes place and that redundant factors regulate Dorsal to ensure a robust transcriptional response. However, the system shows plasticity at the level of the Dorsal target genes and thus is able to correct possible fluctuations of the ventralising signal. For instance, a reduction of the dose of Spätzle leads to a narrower Twist domain and shifted ventrolateral anlagen. However, such embryos develop into normal larvae [67]. It is not clear at what level the size regulation of the anlagen occurs. Twist itself might play an important role as it forms a gradient, which extends beyond the mesodermal region. It is not only required for mesoderm development, but also acts synergistically with Dorsal to regulate the expression of genes required for more lateral fates [88]. Dorsoventral Axis Formation and Immunity — Evolutionary Considerations In Drosophila, there are multiple connections between dorsoventral axis formation and immunity, both within the Toll pathway and in its activation [13,111]. The latter relates to an important difference in the way the Toll pathway acts in innate immunity in mammals and insects. The Toll receptors of mammals appear to be directly activated by microbial molecules [112], with individual classes of receptors being specific to particular pathogen-derived molecules. However, in Drosophila, which has nine Toll receptor genes, only Toll-1, the founding member involved in dorsoventral patterning, appears to be required for innate immunity [13]. Some of the other Toll receptors are required for particular developmental processes [113–119], suggesting that the diversity of Toll receptors is not related to the diversity to microbial patterns. Indeed Toll-1 is not activated by microbial molecules, but rather requires activation by Spätzle [13,111]. Microbial infections are sensed upstream of Spätzle through secreted peptidogylcan recognition proteins (PGRPs) βGRPs) [111,120, and βGlucan recognition proteins (β 121], which are believed to activate specific proteases that cleave Spätzle [122,123]. Activation of Toll-1 leads to the rapid and massive secretion of antimicrobial peptides, which contribute to the clearing of invading microorganisms from the hemolymph. This mechanism constitutes together with another signaling pathway responsible for the recognition of Gramnegative bacteria (the IMD pathway) the humoral branch of the pathogen defense [111,121]. While so far none of the upstream proteases acting during dorsoventral pattern formation have been found to be required for the humoral response, one important regulator of the protease cascade, Serpin27A, is required for another branch of the pathogen response, the melanisation reaction [124,125]. In arthropods, melanisation is required for wound healing, encapsulation and sequestration of microbes and production of cytotoxic reactive oxygen

species. Melanisation is controlled by a hemolymphborne protease cascade, the terminal step of which is inhibited by Spn27A. Why is there such a close relationship between pathogen defense and dorsoventral patterning in insects? Either axis formation or innate immunity is the ancestral function of the pathway. While there is no clear evidence that Toll signaling is required for dorsoventral patterning in vertebrates, the function in innate immunity appears to be conserved. Moreover, similar receptors are even involved in pathogen resistance in plants [126]. Thus, it is likely that the role of the pathway in axis formation in insects stems from its earlier role in pathogen defense. A possible evolutionary scenario can be envisaged from certain embryological features of phylogenetically more basal insects [127]. The adaptation to the terrestrial life-style is linked to the formation of large yolk-rich eggs, which may become the target of microbial infection. During early development, the blastoderm covers the yolk, but only a small portion gives rise to the embryo proper, while the remainder forms a protective extra-embryonic tissue, the serosa. Interestingly, there is evidence that the serosa of some insects has an immune function [76,128]. For instance, in beetles Dorsal protein is highly expressed in the serosa and transported to the nuclei after pathogen challenge [76]. By extension, the large serosa of more basal insects may also have an immune function which was acquired early in evolution to provide protection from infections and thus a selective advantage. We suggest that this was the reason why the Toll pathway and some components of the upstream activating cascade were expressed in the serosa during early embryonic development. From there, only a small shift in temporal and spatial expression would have been necessary to shift the function of the Toll pathway towards axis formation. Initially, it might have only been used to induce axis polarity, while most of the dorsoventral patterning was achieved by interactions of zygotic genes. In beetle embryos, which may reflect such an intermediate situation, the Dorsal gradient is more transient and influences its fewer target genes less directly compared to Drosophila [76] (S. Roth, unpublished). Thus, the high amount of spatial information encoded in the Dorsal gradient in Drosophila is likely to be a late product of insect evolution. Conclusions In Drosophila, the regulatory circuitry comprising the three serine proteases GD, Snake and Easter, as well as the Toll ligand Spätzle, accounts for the robustness of a pathway that establishes a delicate balance of pattern elements that organise the embryo (Figure 6). Stable entry of the signal into the embryo is mediated by the kinase Pelle. The intracellular segment of the axis-forming pathway is, hence, not contributing to formation of the Dorsal gradient, but rather to the stabilization and transmission of the spatial information generated outside the embryo. In turn, the Dorsal gradient converts this information into patterns of gene expression, which specify the cell fates of the embryo.

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