Cellular Behaviors Underlying Pattern Formation and Evolution

Cellular Behaviors Underlying Pattern Formation and Evolution CM Mizutani, Case Western Reserve University, Cleveland, OH, USA r 2016 Elsevier Inc. All rights reserved.

Glossary Morphogenetic gradients Gradients of chemicals that inform cells about their position in the body in a dosagedependent fashion. Pattern formation The spatial and temporal organization of cells of distinct fates during embryonic and organ development.

Introduction One of the greatest wonders in nature is the diversity of body shapes, sizes, textures, and color patterns seen across different species. To understand how this diversity is generated and evolves over time, it is necessary to uncover and compare the underlying cellular mechanisms in different species. This type of research is the subject of the multidisciplinary field of Evolution and Development, or Evo-Devo. This maturing field has been propelled by the establishment of emerging models that encompass different branches of the phylogenetic tree, and is providing us a new perspective about how genes pattern organisms and how they can be modified to generate the diversity seen in nature (Sommer, 2009; Müller, 2007). This article discusses the importance of cell behaviors for the evolution of pattern formation. It first introduces the concept of morphogenetic gradients and cell fate specification during embryonic development. The focus given is on the fruit fly Drosophila melanogaster, a reference model that lent support to the theoretical idea that tissues are patterned by gradients of form-generating substances or morphogens, and allowed the discovery of main developmental mechanisms with broad implications to other invertebrate and vertebrate models alike (Veraksa et al., 2000). There are two relevant aspects that challenge the classical notion on how morphogenetic gradients are formed and exert their function, which will be presented here. The first regards to the problem of scaling to size during evolution, and the second regards the cell proliferation and movements during morphogenetic pattern formation. Significant advances to address those problems are being achieved by a combination of comparative evolutionary studies, mathematical modeling, and by revisiting wellestablished morphogenetic systems in flies and vertebrates using novel tools for live-imaging and single cell analysis.

Morphogenetic Gradients and the French Flag Model Embryonic Axis Formation Is Initiated by Maternal Information The D. melanogaster embryo is an iconic textbook example for understanding how pattern formation arises from a field of undifferentiated cells. Its development begins as a syncytial

Encyclopedia of Evolutionary Biology, Volume 1

Pleiotropic Requirement of same genes in different developmental stages or tissue types.

blastoderm and is characterized by rapid cycles of nuclear divisions, followed by the migration of the nuclei to the embryo surface (Wolpert et al., 2007). After the thirteenth cleavage cycle, about 6000 nuclei are arranged within a single layer, and cell membrane growth ensues without further nuclear divisions (Edgar et al., 1986). By the end of the cellular blastoderm stage, the embryo is subdivided into several gene expression domains, prior to any major gastrulation movement. These domains specify cells into antero-posterior (AP) fates that contribute to the head, thorax, abdomen, and tail; and dorso-ventral (DV) fates that contribute to the major tissue types of the body, i.e., the mesoderm, neuroectoderm, and ectoderm (Ip et al., 1992). Several mutations that disrupt AP and DV patterning were isolated in genetic screenings in the fly (Nusslein-Volhard and Wieschaus, 1980; Nusslein-Volhard et al., 1984). These screenings also identified mutations in two maternal genes, bicoid (bcd) and dorsal (dl), which encode transcription factors distributed in a graded fashion, with higher to lower levels of Bcd from anterior to posterior side, and higher to lower levels of Dl from ventral to dorsal side (Figure 1(b)) (Frohnhöfer and NüssleinVolhard, 1986; Driever and Nüsslein-Volhard, 1988a,b; Roth et al., 1989). These gradients provide a system of coordinates that inform cells about their position along the axes. In an embryo mutant for Dl, for instance, ventral and lateral fates giving rise to the mesoderm and neuroectoderm are abolished. As a result, the embryo became dorsalized and assumes an ectodermal fate along the entire DV axis (Nusslein-Volhard et al., 1980; Anderson et al., 1985; Nusslein-Volhard et al., 1984). Studies on Bcd and Dl provided solid experimental evidence for the early theoretical ideas of pattern generation by morphogenetic gradients proposed by Alan Turing (Turing, 1952), and the French Flag model of positional information proposed by Wolpert (Figure 1(a)) (Wolpert, 1969). According to this model, cells can discern their relative positions by sensing different threshold levels of morphogens, and therefore acquire distinct fates. This patterning mechanism was found to be general, and shared by invertebrates and vertebrates. Additional morphogens were identified, including decapentaplegic/Bone Morphogenetic Protein (dpp/BMP-4), wingless/Wnt (wg/Wnt), and Hedgehog/Sonic Hedgehog (Hh/Shh), all of which play roles in embryonic, limb, and organ patterning (Affolter and Basler, 2007; Neumann and Cohen, 1997).

doi:10.1016/B978-0-12-800049-6.00132-3

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Figure 1 Morphogenetic gradients organize the embryonic axes. (a) A schematic representation of the classical French flag model. Graph shows the concentration levels of a morphogen, which establish different thresholds that are read by undifferentiated cells and cause their differentiation in three fates represented by the colors blue, white, and red. (b) Representation of the Drosophila maternal gradients Bicoid (Bcd) along the AP axis (left panel), and Dorsal (Dl) along the DV axis (right panel). The Bcd gradient establishes the expression patterns of segmentation genes (blue stripes), while the Dl gradient defines the main tissue types of the embryo (MES, mesoderm in red; NE, neuroectoderm in blue; ECT, ectoderm in yellow).

Over the several past years, our understanding of gradient formation and interpretation has greatly advanced. Seminal work found evidence that morphogens diffuse from localized sources (e.g., Bcd gradient (Berleth et al., 1988), and the Shh and BMP gradients in vertebrate neural tube (Liem et al., 2000); reviewed in (Cohen et al., 2013). In Drosophila, gradients established by the same morphogen Dpp can be formed through distinct mechanisms in limbs and embryos. During wing formation, Dpp is secreted from an organizer region that is stabilized by a compartment boundary between the anterior and posterior regions of the developing wing (Basler and Struhl, 1994). In contrast, the Dpp gradient in the embryonic ectoderm is formed by a more unusual mechanism that involves the transportation of Dpp/BMP-4 to dorsal regions by extracellular antagonists (Eldar et al., 2002; Mizutani et al., 2005; Shimmi et al., 2005). Furthermore, the identification of biochemical interactions involved in gradient formation and cell specification, along with improved methods in confocal microscopy and fluorescent protein tags, have allowed the acquisition of precise quantitative data, and spurred a series of mathematical models that expand our ability to test new predictions of pattern generation (Tomlin and Axelrod, 2007).

Evolutionary Modifications of Gradient Formation and Interpretation The most general explanatory models of gradient formation and interpretation were first demonstrated in the Drosophila blastoderm, owing to its simple two-dimensional organization of a single cell layer and the genetic identification of Bcd and its target genes (Driever and Nüsslein-Volhard, 1988a,b). In the source–sink model (Crick, 1970), a morphogen is released from a localized source, and is asymmetrically distributed as a function of diffusion and degradation away from its source (sink). In the affinity-binding model, the interpretation of the gradient by cells is explained as a result of a differential sensitivity in Bcd DNA binding sites in the regulatory regions of its target genes (Driever et al., 1989). Although useful to define the basic mechanism of morphogen action, the models above are simplifications that do

not fully account for the observed temporal and spatial scales in which gradients are formed. They also do not explain the formation of very precise and sharp borders of gene expression domains (Coppey et al., 2007). Follow-up studies suggested alternative mechanisms to explain morphogen diffusion rather than simple passive diffusion (Müller et al., 2013), including facilitated transport (Eldar et al., 2002; Mizutani et al., 2005; Shimmi et al., 2005), or cell-based mechanisms such as dispersal through filopodial extensions called cytonemes (Fairchild and Barna, 2014). In addition, regulatory gene networks of target genes are necessary to refine and reinforce the expression borders initially established by gradients (Balaskas et al., 2012; Stathopoulos and Levine, 2005). The gradient mechanics also raise important issues of relevance to evolutionary biology. While direct experimentation in D. melanogaster has provided valuable insights into the mechanics of gradient formation and interpretation, this single species approach usually falls short of explaining how gradients define robust responses during evolution, or are reshaped to generate novel patterns. To address these issues and gain a more complete understanding of morphogenetic patterning, it is necessary to investigate other species. These species comparisons, coupled with experimentation in D. melanogaster, allow us to ask and test ideas that cannot be done in a single species. One example is how gradients can be established over variable distances to specify cell fates in relatively similar positions in embryos from related species that vary in egg size (Figure 2) (Gregor et al., 2005; Chahda et al., 2013). Another fascinating issue regarding the mechanism by which morphogens instruct cells about their fate is the fact that they act on a field where the position of cells changes as a function of time. This observation challenges the positional information models, which assume that cells remain in a fixed position in relation to the gradient in order to acquire a given fate. Although this has been known for a long time, it is difficult to reconcile this observation with the current models of morphogenetic pattern (Affolter and Basler, 2007; Cayuso and Martí, 2005). It is possible that evolutionary changes in cell proliferation and movements could refine or even modify the

Cellular Behaviors Underlying Pattern Formation and Evolution

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Figure 2 Hypothetical scenarios of scaled and non-scaled patterns of segmentation genes during the evolution of Diptera. An embryo from Drosophila melanogaster expressing a pair-rule gene is represented on top. The left panel represents the lack of segmentation scaling in small eggs (Drosophila busckii) and large eggs (Calliphora vicina). In this case, D. busckii would miss some stripes, while C. vicina would have additional stripes. The right panel represents the presence of scaling to size, as observed in actual experiments. The numbers and relative positions of the segmentation stripes would be preserved in all species.

overall pattern information provided by gradients, providing an alternative source for pattern changes across species. However, these possibilities remain largely untested. On a broader view, the fact that tissue patterning arises in a dynamic field, and that different species can pattern tissues differently, has direct relevance to regenerative medicine as it may provide important insights into the scaling of organs during growth. Recent advances in applying quantitative methods to address the problem of scaling of gradients in a variety of system has been discussed elsewhere (Umulis and Othmer, 2013; Ben-Zvi et al., 2011).

Scaling of Morphogenetic Gradients during Evolution The AP System Employs Different Scaling Strategies within and across Diptera Species Despite extensive work on maternal gradients in D. melanogaster, some questions regarding gradient dynamics can be best addressed using a combination of studies within and across species. One fascinating observation is the relatively proportional body pattern seen across different fly species. This scaling can be best illustrated by the embryonic position of stripes of various segmentation genes that respond to Bcd gradient, such as hunchback, giant, paired, and even-skipped. In embryos that significantly vary in size, not only the same number of stripes of these genes is conserved, but each individual stripe is located at relatively similar positions (Figure 2) (Gregor et al., 2005a; Lott et al., 2007; Hare et al., 2008; Fowlkes et al., 2011; Blechert et al., 2011). This remarkable finding could be either explained by the ability of the Bcd gradient to scale to embryo length or by a differential sensitivity to Bcd levels caused by changes in the cis-regulatory sequences of target genes.

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The quantitative measurement of the Bcd gradient in Drosophila busckii, a species with eggs twice as small as D. melanogaster, and in Calliphora vicina, a species with eggs three times larger than D. melanogaster, revealed that the Bcd gradient profile is similar in all species after normalization by embryo length (Gregor et al., 2005). In this study, Gregor et al. show that the amplitude of the gradient scales with its constant length and reach farther distances, suggesting that the gradient threshold information is adjusted to size and that the generation of conserved expression domains may not necessarily involve the evolution in cis-regulation of Bcd target genes (Gregor et al., 2005). Considering one of the gradient formation models that involves the diffusion of Bcd from the anterior region, the scaling of the Bcd gradient in the different species could be explained by either changes in the Bcd effective diffusion constant or a modified time scale for the gradient formation, or alternatively, through an increased stability of the protein. The first two possibilities were ruled out given that all species tested display similar developmental rates, and the measured passive diffusion of an inert molecule injected in small and large embryos reveals no significant difference (Gregor et al., 2005). The possibility that Bcd may have acquired distinct lifetimes during evolution remains open. In experiments in which the Bcd proteins from C. vicina and Lucilia sericata (two species with large embryos) were expressed in D. melanogaster revealed that the exogenous protein gradients assume the same shape of the host species gradient (Gregor et al., 2008). Although these experiments indicate that the degradation of Bcd from C. vicina and L. sericata is the same as in D. melanogaster, an exhaustive test of Bcd degradation in speciesspecific environment has not been performed. In any case, it appears that the scaling of Bcd gradient is likely to depend on other species-specific factors that affect the gradient formation. Another relevant question is whether scaling is achieved by similar or different mechanisms within same species. To answer this question, Cheung et al. used artificially selected strains of D. melanogaster with significantly different egg sizes (Cheung et al., 2011; Miles et al., 2011). They found that the Bcd gradients in strains with large eggs reach larger distances by having the production rate of Bcd scaled with embryo volume. The authors confirm that net amounts of maternally deposited Bcd RNA in the ovary increases in the strains with large eggs (Cheung et al., 2011). Given that variation in embryo size is a fast evolving trait determined by multiple independent loci (Markow et al., 2009; Warren, 1924), the increase of bcd RNA deposit provides a simple solution to buffer variations in embryo size and achieve gradient scaling within the same species, without requiring modifications in the Bcd protein sequence or its degradation pathways that would not have sufficient time to undergo selection.

The DV Axis Is Patterned by Two Opposing Gradients: One Regulated by a Fast Evolving Pathway, and Another That Is Highly Conserved across Evolution The DV patterning system has also provided insights about how tissues scale during evolution. As mentioned before, the main tissue types are initially established along the DV axis in

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Figure 3 Scaling properties of the DV system. (a) Representation of a DV cross-section of a Drosophila embryo (left panel). The nuclei from ventral to dorsal side can be modeled as a single strip of cells. Right panel shows the Toll (Tl) signaling pathway. Partial differential equations representing the relationships between the cytoplasmic components of this pathway were used in a mathematical model to test the formation of the Dl gradients, and its scaling properties across species (see text for details). (b) Cross-section of related Drosophila embryos that vary in size, stained for a nuclear dye (blue), and markers for the mesoderm (snail, in green), and neuroectoderm (short gastrulation, red). Note variation in the mesodermal domains, indicated by arrowheads. (c) Computational modeling of the Dl gradient. Representation of Dl nuclear concentration levels quantified from different Drosophila species (left panel). Blue line, D. melanogaster and D. sechellia gradients; yellow line, D. busckii gradient; red line, D. simulans gradient. By adjusting parameters of the Tl pathway (e.g., K[b], binding rate between Dl and Cactus), it is possible to reproduce the flattened gradient of D. simulans species. Panel shown in (a) is reproduced from Ambrosi, P., Chahda, J.S., Koslen, H., Chiel, H.J., Mizutani, C.M., 2014. Modeling of the dorsal gradient across species reveals interaction between embryo morphology and Toll signaling pathway during evolution. PLoS Computational Biology 10 (8), e1003807. Available at: http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=4148200&tool= pmcentrez&rendertype=abstract (accessed 27.05.15); Panel in (b) is reproduced from Belu, M., Mizutani, C.M., 2011. Variation in mesoderm specification across Drosophilids is compensated by different rates of myoblast fusion during body wall musculature development. PLoS One 6 (12), e28970. Available at: http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=3237579&tool= pmcentrez&rendertype=abstract (accessed 21.12.11).

response to the maternal Dl gradient. Dl is an NFκ-B transcription factor that is transported into the nucleus following the activation of Toll signaling pathway (Figure 3(a)) (Anderson et al., 1985). Unlike the Bcd gradient, which is thought to be established by the protein diffusion from tightly localized RNAs in the anterior region of the embryo (Gregor et al., 2007) or arise as a result of a RNA gradient (Spirov et al., 2009), both Dl and Toll (Tl) receptor are deposited ubiquitously in the embryo. The asymmetric distribution of nuclear Dl along the DV axis is achieved through a localized ventral source of the ligand Spätzle, which activates Tl in a graded fashion along the DV axis. The activation of Tl triggers the degradation Cactus, a cytoplasmic inhibitor of Dl. Once Dl is free from binding to Cactus, it is transported into the nucleus (Belvin et al., 1995). High nuclear levels of Dl in the ventral side of the embryo activate mesodermal genes, whereas moderate nuclear levels activate neuroectodermal genes (Hong et al., 2008). Finally, in the dorsal side of the embryo, the absence or low levels of nuclear Dl are insufficient to repress the expression of the secreted morphogen dpp/BMP-4, which in turn establishes an opposing dorsal-to-ventral gradient that induces the amnioserosa and ectodermal fates, and represses the expression of neural genes (Mizutani and Bier, 2008). It is noteworthy that the Tl pathway, which ancestrally played a role in innate immune response against pathogens in both invertebrates and vertebrates (Hoffmann and Reichhart, 2002), became co-opted for DV patterning in derived dipterans like Drosophila (Lemaitre, 2004; Hoffmann and Reichhart, 2002; Belvin and Anderson, 1996). Components of the Tl pathway are fast evolving, likely due to the constant contact to new pathogens in the environment (Clark et al., 2007; Jiggins and Kim, 2007; Sousa-Neves and Rosas, 2010; Obbard et al., 2009; Schlenke and Begun, 2003). As discussed later, the fact that the DV patterning is regulated by some fast evolving genes shared by the Tl pathway appears to produce a high rate of variability capable to create diverse Dl gradient shapes, which might have been an important feature to adapt to the fast evolution of egg size. In contrast to the newly acquired role of Dl in DV patterning, the dpp/BMP-4 is the most ancestral signaling pathway responsible for DV patterning among metazoans (Mizutani and Bier, 2008). This pathway plays a pivotal and conserved role in the formation of a centralized nervous system and in the establishment of neural domains of equivalent neural cell types within the neuroectoderm of organisms from the major branches of invertebrates (ecdyzoa and lophotrocozoa) and in the neural tube of vertebrates (Liem et al., 2000; Mizutani et al., 2006; Denes et al., 2007). Thus, in Drosophila, cells along the DV axis receive information from a highly conserved DV signaling pathway (Dpp/BMP), and from a newly co-opted fast evolving pathway (Dl/NF-κB).

The Domains of Neural Identity Genes along the DV Axis Are Highly Constant during Evolution One striking feature regarding the neuroectoderm described several years ago is the highly conserved numbers and types of neuroblasts (neural stem cell-like cells) across divergent

Cellular Behaviors Underlying Pattern Formation and Evolution

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Figure 4 Developmental stages leading to the formation of a conserved neuroblast map. During blastoderm stage, three non-overlapping domains of neural identity genes subdivide the DV axis of the Drosophila neuroectoderm. Later in development, neuroblasts delaminate from the neuroectoderm. About 30 neuroblasts of unique identity are formed per hemisegment. This neuroblast map is highly conserved among insects.

insects. Early studies that defined the ‘neuroblast map’ in divergent insects, including grasshopper and D. melanogaster, found that each hemisegment generates about 30 neuroblasts of unique identity in these species (Figure 4) (Doe, 1992; Whitington, 1996; Bate, 1976). These neuroblasts give rise to homologous neuronal lineages that establish connectivity patterns of remarkable similarity in these species (Thomas et al., 1984). The degree of conservation of neuroblast identity can even be observed to a certain extent in the crustacean branch (Ungerer and Scholtz, 2008). After the description of neuroblast maps, studies in D. melanogaster identified three key homeobox transcription factors, ventral nervous system defective (vnd)/Nkx2.2, intermediate neuroblast defective (ind), and muscle segment homeobox/Drop (msh)/Msx, which are expressed in three non-overlapping domains in the ventral, intermediate, and dorsal regions of the neuroectoderm, respectively (McDonald et al., 1998; Weiss et al., 1998; Chu et al., 1998; Isshiki et al., 1997; Jimenez et al., 1995). These genes were called neural identity genes because they define the types of neurons that are formed at distinct DV positions, such as motorneurons, serotonergic and secretory neurons (Figure 4). The neural identity genes and mesodermal genes are part of a DV self-regulatory gene network, in which genes expressed ventrally repress genes expressed more dorsally in what is referred to as ‘ventral dominance’ (Cowden and Levine, 2003). In the ventral dominance model, the initial DV gene expression domains established by the Dl gradient are reinforced by the repression of neural genes by snail (sna), a mesodermal gene expressed in the ventral most region of the embryo, followed by the repression of ind and msh by vnd, and repression of msh by ind. This cross-regulation assures that the borders of neuroectodermal genes are reinforced. In experiments that either induced expansions or retractions of the blastoderm domains of neural identity genes, there is a shift in ventral to dorsal neuroblast identity, or viceversa, caused by an invasion of expression of the adjacent genes (McDonald et al., 1998; Cornell and Ohlen, 2000; Weiss et al., 1998; Isshiki et al., 1997; Jimenez et al., 1995). As a consequence, after neuroblast delamination from these altered domains, entire neural lineages are duplicated at the expense of other lineages that are eliminated altogether. These experiments, along with the observation of neuroblast map conservation, suggest that there is a strong selective pressure to precisely maintain the neuroectodermal domains in the early blastoderm. However, the underlying molecular mechanism that imposes this selective pressure has only recently begun to be understood.

The Germ Layers Formed along the DV Axis Are Unequally Scaled during Evolution The remarkable constancy in the neuroectodermal expression domains occurs despite significant changes in embryo size in insect species that diverged over 300 MYA (i.e., grasshopper and Drosophila), and raises the outstanding question of how the remaining DV germ layers are specified. It turns out that while the neuroectoderm remains constant in size, the mesodermal and ectodermal domains can either retract or expand across Drosophila species (Belu and Mizutani, 2011). For instance, there is a significant reduction of the mesoderm in small eggs from D. busckii, and an expansion in large eggs from Drosophila sechellia (Figure 3(b)). The variation in mesoderm is not always correlated to egg size, since Drosophila simulans, a species more closely related to D. sechellia, but with similar egg size as D. melanogaster, also display an expansion in the mesoderm (Belu and Mizutani, 2011). The variation in the mesodermal size versus a more conserved neural patterning was also verified in the artificially selected D. melanogaster strains with variable egg sizes described above (Garcia et al., 2013; Miles et al., 2011). The changes in the mesodermal domain across species appear to be directly caused by a lack of scaling in the Dl gradient since the quantification of the nuclear Dl distribution across species revealed striking modifications in the shape of the gradient varying from sharp to flat profiles (Chahda et al., 2013). Furthermore, in experiments using hybrid embryos between sibling species D. sechellia, D. simulans, and D. melanogaster, it was possible to show that the mesodermal genes are activated by same Dl threshold levels, ruling out the possibility that the changes in their mesodermal domains are due to evolution in cis-regulation (Chahda et al., 2013). This result is significant because it provides clear alternatives to affinitybinding models of pattern evolution and also reveals how the requirement of a signaling pathway that participates in both immune response and DV patterning can lead to extremely fast changes in the embryonic organization. The experiments above also reveal the underlying mechanisms that restrict the neuroectoderm layer. Unlike what is seen with the Bcd gradient, the Dl gradient does not scale to size in the different species (Chahda et al., 2013; Gregor et al., 2008). Since mesodermal genes maintained their sensitivity to Dl activation, the ventral border between the mesoderm and neuroectoderm, and the dorsal border between the neuroectoderm and the ectoderm, are expected to be determined by a relatively constant slope of the Dorsal gradient across

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species. Thus, the neuroectoderm can be shifted as a single block to a new DV position in response to novel Dl gradient shapes. The ability to shift the position of the neuroectoderm as a single block would ensure that correct numbers of neuroectodermal cells are formed in small and large embryos (Chahda et al., 2013). One might ask whether the expansions and retractions of the mesoderm could lead to a loss or gain of muscles in larvae. In experiments that addressed this question, it was shown that variations in mesodermal size are corrected during later developmental stages. The stereotyped pattern of the muscle body wall, for instance, is preserved due to a modified regulation in myoblast fusion during formation of individual muscle fibers (Belu and Mizutani, 2011). Together, these results highlight the extreme versatility of the DV self-organizing system. This appears to be a very robust system capable of adjusting itself to a significant number of challenges imposed by embryo size and pleiotropic effects of the Tl pathway to produce fully viable individuals.

A Mathematical Modeling of Dl Gradient Indicates that Scaling Is Achieved by Changes in the Embryo Anatomy and Tl Signaling Pathway Different factors could influence the modified shapes of the Dl gradient during evolution. One of them is the evolution of the Tl signaling pathway. Indeed, analyses of different species reveal that the ranges Tl signaling are modified and these changes are accompanied by variations in the size of mesodermal domains (Figure 3(b)). However, there are noticeable changes in embryo length and circumference, number and size of nuclei, and in nuclear density patterns across species that could in principle modify this gradient. This possibility was tested in experiments that manipulated the number and density of nuclei in D. melanogaster. Interestingly, these manipulations cause the sharpening or flattening of the Dl gradient as predicted, indicating that nuclear density and size contribute to the shape of the gradient (Chahda et al., 2013). To tease apart the relative contributions of anatomical changes and Tl signaling pathway evolution, a mathematical model originally developed for the Dl gradient formation in D. melanogaster (Kanodia et al., 2009) was modified in order to identify which parameters are required to reproduce the different gradients observed from related Drosophila species (Ambrosi et al., 2014). This model is based on partial differential equations that represent the interactions of the Tl pathway components (Figure 3(a)) (Belvin et al., 1995; Kanodia et al., 2009). The model also takes into consideration the anatomical features of the embryo, and was shown to correctly reproduce the dynamics of gradient formation in liveembryos expressing Dl tagged with green fluorescent protein (Kanodia et al., 2009; DeLotto et al., 2007). By testing different parameter sets, the model indicates that changes in anatomy alone are not sufficient to recreate the gradients from the species, and it is necessary to include modifications in the Tl pathway (Figure 3(c)) (Ambrosi et al., 2014). This modeling experiment led to three important conclusions. First, pairs of very closely related species that diverged only B0.5 MYA and produce similar changes in the mesoderm, but underwent

separate speciation events (i.e., D. sechellia and D. simulans; and Drosoplila santomea and Drosophila yakuba), share similar modifications in the Tl pathway that correctly reproduce their gradients in model simulations. For instance, D. simulans has a flattened gradient in relation to the gradient of D. sechellia. Yet, the model can simulate these very different shapes after similar manipulations of Tl signaling parameters, such as increasing the rates of Cact degradation or decreasing the binding rate between Cactus and Dl (Figure 3(c)) (Ambrosi et al., 2014). Furthermore, the modifications in the Tl pathway predicted by the model are supported by changes in amino acid sequences of Dl and Cactus protein domains related to these functions in these species. The second conclusion that can be drawn from this modeling is that the changes in anatomy and Tl pathway interact with one another. In order words, changes in Tl pathway are exacerbated by the changes in anatomy. Finally, the model also predicts that small additive changes in the Tl pathway are more likely to occur in nature, since more drastic changes of a single parameter affecting this pathway renders the model more unstable. This prediction is supported by the amino acid changes seen in many Tl pathway components from these species (Sousa-Neves and Rosas, 2010; Clark et al., 2007; Jiggins and Kim, 2007; Obbard et al., 2009).

Contribution of Cell Migration during Morphogenetic Gradient Patterning The information for cell specification provided by gradients rarely occurs in a stable environment, and it is complicated by the constant proliferation and migration of cells that takes place during growth, such what is seen during the patterning of the vertebrate neural tube (Gouti et al., 2015). As mentioned at the beginning of this article, the Drosophila blastoderm has been a highly successful model to understand pattern formation due to its simple arrangement of cells. Furthermore, the blastoderm cells seemed to be a close approximation to static fields of cells as they receive morphogen signals, since they are no longer dividing and appear mostly immobile (Edgar et al., 1986). However, recent studies show that the nuclei actually move from one to three nuclear diameters in distance during blastoderm stage, and follow stereotyped trajectories that ultimately lead to species-specific nuclear densities that are polarized along the AP and DV axis (Keranen et al., 2006; Meyer et al., 2010; Fowlkes et al., 2011). These stereotyped movements are abrogated in mutant embryos that eliminate either the Bcd or Dl gradients, and appear to contribute to a shift in the position of the stripes of the segmentation gene fushi tarazu in mutant versus wildtype embryos (Figure 5(a)) (Keranen et al., 2006). These novel observations are fascinating at several levels. First, they indicate that Drosophila might have remnants of the ancestral and more dramatic nuclear blastoderm movements that occur in the embryogenesis of short-germ band insects, as it has been shown in crickets (Nakamura et al., 2010). Second, evidence mounts that species-specific cell movements in Drosophilids may produce some slight differences observed in the positioning of stripes of segmentation genes along the AP axis (Keranen et al., 2006; Lott et al., 2007; Gregor et al., 2008; Fowlkes et al., 2008; Meyer et al., 2010; Blechert et al., 2011). It

Cellular Behaviors Underlying Pattern Formation and Evolution

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Cell sorting and migration

Figure 5 Contribution of cell sorting to pattern formation. (a) The Drosophila blastoderm embryo displays stereotyped nuclear movements. Top panel: Trajectories of nuclear migration are represented by arrows, which show a nuclear flow toward the dorsal midline. By the end of the blastoderm stage, the nuclear density becomes polarized, with high density levels in the dorsal side (red to yellow shades) and low density levels in the ventral side (green to blue shades). Bottom panel: Nuclear density patterns (left) and gene expression patterns (left) are disrupted in mutants that eliminate the Dl gradient. The new locations of the segmentation stripes (red) in relation to wild type stripes (blue) is predicted to be partly caused by disruptions in nuclear flow. (b) Revised version of the French flag model according to findings reported by Xiong et al., 2013. The authors found that cells of the neural tube of zebrafish first acquire fate in a salt and pepper fashion while the Shh gradient is noisy. Over time, the gradient becomes sharp, and these cells maintain their fate and correctly sort to their final positions.

has also been reported that within the same species D. melanogaster, there is a dynamic shift in the positioning of stripes that occurs over time during the blastoderm stage, which was initially thought to be due to gene regulation (Jaeger et al., 2004). However, in experiments that identify all individual nuclei in cohorts of fixed embryos of different stages, it was possible to develop a model for the observed nuclear flow and predict that the nuclear flow contributes to the shift in stripe positions (Keranen et al., 2006). Together, these findings suggest cell sorting as a significant patterning mechanism that can sharpen the borders of AP segmentation, and that is most likely linked to morphogen regulation and distribution. Another significant role for cell sorting was uncovered in a recent study that revisited the DV patterning formation of the neural tube in zebrafish. This well-established system is patterned similarly to the Drosophila neuroectoderm, and is subdivided by homologous DV neural patterning genes that cross-regulate each other (Cohen et al., 2013). These neural genes are regulated by the dpp/BMP-4 gradient that emanates from the dorsal neural plate and by a ventral source of Shh gradient from the notochord, instead of the Dl gradient as seen in flies (reviewed in Mizutani and Bier (2008)). By tracking the formation of early ventral neural progenitor cells, Xiong et al. discovered that cell specification occurs in a salt and pepper

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pattern early in development, while the Shh gradient is still too shallow and noisy to provide reliable positional information (Xiong et al., 2013). Unexpectedly, these specified cells apparently maintain their fate, and migrate to their final positions as the Shh gradient sharpens (Figure 5(b)). The directional movement of these cells is disrupted by the removal of the cell-adhesion molecule Cadherin-2, but it is not influenced by Shh (Xiong et al., 2013). Although the exact molecular mechanisms for this cell sorting remain unclear, it is plausible that these progenitor cells respond to cues provided by different cell adhesion molecules that are asymmetrically distributed along the DV axis, which may in turn be regulated by other gradients. A candidate is the BMP-4 gradient, which has been shown to regulate the expression of Cadherin-2 and coordinate cell movements during dorsal convergence during early embryonic development of zebrafish (von der Hardt et al., 2007). Models for cell sorting mechanisms that integrate morphogens to generate patterns from initially scattered cell fates have been proposed during the patterning of the slime mold Dictyostelium (Vasiev and Weijer, 1999; Kay and Thompson, 2009). While there is no evidence that the Drosophila blastoderm development involves any massive salt and pepper patterns of specified cells that migrate to their final domains, there is evidence for a more subtle, but quantifiable, cell sorting mechanism that can contribute to the formation of gene expression borders (Keranen et al., 2006). This mechanism could be relevant to achieving the final numbers of cells within the neuroectoderm across Drosophila species, since the nuclei within the lateral regions of the embryo that give rise to the neuroectoderm actively move toward the dorsal midline (Figure 5(a)) (Keranen et al., 2006). Using Drosophila genetics, it may be possible to isolate candidate genes such as cell adhesion molecules that may be involved in cell sorting and test how this mechanism is integrated with morphogenetic gradients.

Acknowledgment The author is grateful for funds provided by the National Science Foundation (grant number IOS-1051662 to CMM).

See also: Developmental Mechanisms Controlling Cell Fate, Evolution of. Gene Networks Driving Development, Conservation and Evolution of. Novel Structures in Animals, Developmental Evolution of. Robustness and Evolvability in Molecular Evolution

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Umulis, D.M., Othmer, H.G., 2013. Mechanisms of scaling in pattern formation. Development 140 (24), 4830–4843. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/24301464 (accessed 12.03.14). Ungerer, P., Scholtz, G., 2008. Filling the gap between identified neuroblasts and neurons in crustaceans adds new support for Tetraconata. Proceedings Biological sciences/The Royal Society 275 (1633), 369–376. Available at: http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=2596827&tool=pmcentrez&render type=abstract (accessed 1.08.12). Vasiev, B., Weijer, C.J., 1999. Modeling chemotactic cell sorting during Dictyostelium discoideum mound formation. Biophysical Journal 76 (2), 595–605. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid= 1300066&tool=pmcentrez&rendertype=abstract (accessed 27.05.15). Veraksa, A., Del Campo, M., McGinnis, W., 2000. Developmental patterning genes and their conserved functions: From model organisms to humans. Molecular Genetics and Metabolism 69 (2), 85–100. Available at: http://www.ncbi.nlm.nih. gov/pubmed/10720435 (accessed 27.05.15). von der Hardt, S., et al., 2007. The Bmp gradient of the zebrafish gastrula guides migrating lateral cells by regulating cell cell adhesion. Current Biology: CB 17 (6), 475–487. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17331724 (accessed 4.05.15). Warren, D.C., 1924. Inheritance of Egg Size in DROSOPHILA MELANOGASTER. Genetics 9 (1), 41–69. Available at: http://www.pubmedcentral.nih.gov/article render. fcgi?artid=1200770&tool=pmcentrez&rendertype=abstract (accessed 2.04.15). Weiss, J.B., et al., 1998. Dorsoventral patterning in the Drosophila central nervous system: The intermediate neuroblasts defective homeobox gene specifies intermediate column identity. Genes & Development 12 (22), 3591–3602. Available at: http://www.ncbi.nlm.nih.gov/pubmed/9832510 Whitington, P., 1996. Evolution of neural development in the arthropods. Seminars in Cell & Developmental Biology 7 (4), 605–614. Available at: http://dx.doi.org/ 10.1006/scdb.1996.0074 (accessed 1.08.12). Wolpert, L., 1969. Positional information and the spatial pattern of cellular differentiation. Journal of Theoretical Biology 25 (1), 1–47. Available at: http:// www.ncbi.nlm.nih.gov/pubmed/4390734 (accessed 24.05.15). Wolpert, L., Jessell, T., Lawrence, P., et al., 2007. Development of the Drosophila body plan. In: Wolpert, J., Jessell, L., Lawrence, T., et al. (Eds.), Principles of Development. London: Oxford University Press, pp. 31–88.

Xiong, F., et al., 2013. Specified neural progenitors sort to form sharp domains after noisy Shh signaling. Cell 153 (3), 550–561. Available at: http://www. pubmedcentral.nih.gov/articlerender.fcgi? artid=3674856&tool=pmcentrez&rendertype=abstract (accessed 20.05.15).

Further Reading Ambrosi, P., Chahda, J.S., Koslen, H., Chiel, H.J., Mizutani, C.M., 2014. Modeling of the dorsal gradient across species reveals interaction between embryo morphology and Toll signaling pathway during evolution. PLoS Computational Biology 10 (8), e1003807. Available at: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=4148200&tool=pmcentrez&rendertype=abstract (accessed 27.05.15). Chahda, J.S., Sousa-Neves, R., Mizutani, C.M., 2013. Variation in the dorsal gradient distribution is a source for modified scaling of germ layers in Drosophila. Current Biology 23 (8), 710–716. Available at: http://www.ncbi.nlm. nih.gov/pubmed/23583556 (accessed 23.01.14). Gregor, T., et al., 2005. Diffusion and scaling during early embryonic pattern formation. Proceedings of the National Academy of Sciences of the United States of America 102 (51), 18403–18407. Keranen, S.V., et al., 2006. Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution II: dynamics. Genome Biology 7 (12), R124. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd= Retrieve&db=PubMed&dopt=Citation&list_uids=17184547 (accessed 24.08.15). Lott, S.E., et al., 2007. Canalization of segmentation and its evolution in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 104 (26), 10926–10931. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/17569783 (accessed 24.08.15). Xiong, F., et al., 2013. Specified neural progenitors sort to form sharp domains after noisy Shh signaling. Cell 153 (3), 550–561. Available at: http://www.pubmed central.nih.gov/articlerender.fcgi?artid=3674856&tool=pmcentrez&render type=abstract (accessed 20.05.15).