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Regulation of zygotic Nvit cad expression. Wild-type zygotic Nvit cad expression (A,D). Nvit cad expression is de-repressed throughout embryo in Nvit otd RNAi embryos (B). Nvit cad is expressed in a complimentary pattern to Nvit otd (C). Nvit cad is a expressed with a duplicated stripe at the anterior on hb hl embryos (E,F). Wild-type expression of Dm cad (G). Dm hb –/– zygotic mutant embryos show a partial dorsal anterior ectopic stripe of Dm cad expression (H). Dm cad is ectopically activated in the presence of ventrally misexpressed Dm tll (I).
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One of the earliest steps of embryonic development is the establishment of polarity along the anteroposterior axis. Extensive studies of Drosophila embryonic development have elucidated mechanisms for establishing polarity, while studies with other model systems have found that many of these molecular components are conserved through evolution. One...
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... in abdominal segment 8 (A8) and sometimes A4, but are viable. Embryos lacking both maternal and zygotic Dm cad, however, show severe segmentation defects. Although the head and thorax are normal, the body is shortened owing to elimination of all anal structures and disruption of more anterior abdominal segments (Macdonald and Struhl, 1986) (Fig. ...
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... find that, in addition to an expansion of the posterior Dm cad stripe, hb zyg mutants show ectopic dorsal expression of Dm cad in the anterior of the embryo (Fig. 3H). Similarly, in the zygotic Nvit hb hl mutant ( Pultz et al., 2005), the posterior Nvit cad stripe is duplicated at the anterior of the embryo. Additionally, faint Nvit cad staining spans the region between the wild-type posterior Nvit cad stripe and the ectopic anterior Nvit cad stripe (Fig. 3D-F). This suggests that Nvit hb prevents ...
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... expression of Dm cad in the anterior of the embryo (Fig. 3H). Similarly, in the zygotic Nvit hb hl mutant ( Pultz et al., 2005), the posterior Nvit cad stripe is duplicated at the anterior of the embryo. Additionally, faint Nvit cad staining spans the region between the wild-type posterior Nvit cad stripe and the ectopic anterior Nvit cad stripe (Fig. 3D-F). This suggests that Nvit hb prevents Nvit cad expression in the anterior of the embryo. Furthermore, the ectopic anterior Dm and Nvit cad stripes are reminiscent of the duplication of the posterior Dm cad stripe at the anterior of bcd -mutant embryos (Mlodzik and Gehring, ...
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... Nvit otd-1 to a large extent plays a role similar to that of Dm bcd (Lynch et al., 2006a), we examined Nvit cad expression in otd- 1 RNAi embryos. Zygotic Nvit cad becomes derepressed from both poles, resulting in expression throughout the embryo. The area of Nvit cad derepression corresponds to the otd-1 expression domains ( Fig. ...
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... of Kr and tll in regulating cad, we next used ventral misexpression in Drosophila to examine the effect of the Dm gap genes tll, Kr, otd and hb on Dm cad expression. We used the snail (sna) promoter to drive ectopic expression in a ventral stripe (Andrioli et al., 2002). In sna>tll embryos, cad is activated in the ventral region of the embryo (Fig. 3I). Interestingly, Dm cad is not activated in the anterior ventral region of the embryo, suggesting that it is strongly repressed there. This activation by tll agrees with previous studies that showed a loss of the posterior Dm cad stripe in tll mutant embryos (Mlodzik and Gehring, ...
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... display variability that reflects their cuticular phenotypes. However, en stripes in the head always form normally. The most typical class of severely affected embryos displays all five normal head stripes but lacks 6 or seven trunk stripes. en trunk stripes also often display fusion, as is seen in the cuticles of both ho and cad RNAi embryos (Fig. 3H,I). These results are consistent with those previously reported for the ho mutant ( Pultz et al., 1999). We examined Nvit cad expression in ho embryos using a probe directed against the region encoding the homeodomain and 3UTR. Nvit cad expression is somewhat reduced at 28°C in ho mutants. However, when females are allowed to lay at 18°C ...
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... vitripennis, another long-germ insect lacking bcd, orthodenticle (Nv-otd1) mRNA is first localized to the oocyte posterior during early oogenesis and, then, from mid-oogenesis onward, Nv-otd1 mRNA is restricted to the oocyte anterior (Lynch et al. 2006). In addition to the posterior localization of are also localized to the oocyte posterior, with all contributing to form the posterior region in Nasonia (Lynch and Desplan 2010;Lynch et al. 2011;Olesnicky et al. 2006). Together, these observations suggest that different long-germ insects make use of distinct gene regulatory connections among conserved patterning factors for directing AP patterning. ...
Aphids are hemimetabolous insects that undergo incomplete metamorphosis without pupation. The annual life cycle of most aphids includes both an asexual (viviparous) and a sexual (oviparous) phase. Sexual reproduction only occurs once per year and is followed by many generations of asexual reproduction, during which aphids propagate exponentially with telescopic development. Here, we discuss the potential links between viviparous embryogenesis and derived developmental features in the pea aphid Acyrthosiphon pisum , particularly focusing on germline specification and axis determination, both of which are key events of early development in insects. We also discuss potential evolutionary paths through which both viviparous and oviparous females might have come to utilize maternal germ plasm to drive germline specification. This developmental strategy, as defined by germline markers, has not been reported in other hemimetabolous insects. In viviparous females, furthermore, we discuss whether molecules that in other insects characterize germ plasm, like Vasa, also participate in posterior determination and how the anterior localization of the hunchback orthologue Ap-hb establishes the anterior-posterior axis. We propose that the linked chain of developing oocytes and embryos within each ovariole and the special morphology of early embryos might have driven the formation of evolutionary novelties in germline specification and axis determination in the viviparous aphids. Moreover, based upon the finding that the endosymbiont Buchnera aphidicola is closely associated with germ cells throughout embryogenesis, we propose presumptive roles for B. aphidicola in aphid development, discussing how it might regulate germline migration in both reproductive modes of pea aphids. In summary, we expect that this review will shed light on viviparous as well as oviparous development in aphids.
... In the first five eve RNA stripes, Nv-cad RNA retracts across the anterior-posterior axis as Nv-opa RNA expands ( Fig. 3A-E, Olesnicky et al., 2006). Nv-cad RNA retracts from the anterior (30-65%EL) region before segmental expression of Nv-eve, and continues to retract towards the posterior as eve stripe45 splits ( Fig. 3A,B, Fig. S5). ...
Insect segmentation is a well-studied and tractable system with which to investigate the genetic regulation of development. Though insects segment their germband using a variety of methods, modelling work implies that a single gene regulatory network can underpin the two main types of insect segmentation. This means limited genetic changes are required to explain significant differences in segmentation mode between different insects. This idea needs to be tested in a wider variety of species, and the nature of the gene regulatory network (GRN) underlying this model has not been tested. Some insects, for example Nasonia vitripennis and Apis mellifera segment progressively, a pattern not examined in previous studies of this segmentation model, producing stripes at different times progressively through the embryo, but not from a segment addition zone.
Here we aim to understand the GRNs patterning Nasonia using a simulation-based approach. We found that an existing model of Drosophila segmentation (Clark, 2017) can be used to recapitulate Nasonia’s progressive segmentation, if provided with altered inputs in the form of expression of the timer genes Nv-caudal and Nv-odd paired. We predict limited topological changes to the pair rule network and show by RNAi knockdown, that Nv-odd paired is required for morphological segmentation. Together this implies that very limited changes to the Drosophila network are required to simulate Nasonia segmentation, despite significant differences in segmentation modes, implying that Nasonia use a very similar version of an ancestral GRN used by Drosophila, which must therefore have been conserved for at least 300 million years.
... First, we examined the expression of fkh relative to other terminal genes in wild-type embryos and in mutant genotypes in which tail or hindgut patterning is perturbed ( Figure Figure 21), as previously reported (Wu and Lengyel, 1998), contrasting with the tll and hkb domains in these embryos, which looked normal (Supplementary Figure 21; Wu and Lengyel, 1998;Olesnicky et al., 2006). In hkbmutants, the fkh domain was reduced in size, as previously reported (Weigel et al., 1990;Gaul and Weigel, 1991), which correlated with the reduced size of the tll domain and the posteriorly shifted wg and cad borders in this genotype ( Figure 7A third row, Supplementary Figure 21A). ...
In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the "timer" genes caudal, Dichaete and odd-paired, whose expression dynamics correlate with the mode of segmentation. In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small "tail" region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. We used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. We propose that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.
... This, together with the fact that, unlike Drosophila, Bombyx does not go through the syncytium blastoderm stage, warranting further scrutiny of mechanisms and evolution of Bombyx embryogenesis. More recently, it was found that RNAs encoding the anterior-organizing molecule orthodenticle (otd) showed localized distribution in the anterior of the germ anlage, followed by the localization of the posterior counterpart caudal (cad) in the posterior, as well as vas localization in the germ anlage within newly deposited egg periplasm (Fig. 2) (Nakao 1999;Nakao 2012;Copf et al. 2004;Lynch et al. 2006;Nakamura et al. 2010;Olesnicky et al. 2006;Wilson et al. 2010a;Wilson and Dearden 2011;Shinmyo et al. 2005) (also see below). Interestingly, cad mRNA appears to be localized in an anterior-posterior graded manner, although whether the in situ staining intensity faithfully reflects the concentration difference is uncertain (Nakao 2012). ...
... Thus, considering that the translation of these localized mRNAs occurs after cellularization to limit the diffusion of these products is more reasonable; this, in turn, suggests that the otd product effects on segmentation are limited to the anterior, since otd RNA localization does not appear to extend into the posterior region (Nakao 2012). Indeed, as cad RNAi embryo phenotype suggests, Bombyx segmentation preserves ancestral features as observed in Tribolium, the cricket Gryllus bimaculatus, and the "long germ" holometaboran wasp Nasonia vitripennis, exhibiting a strong posterior center activity (Copf et al. 2004;Shinmyo et al. 2005;Olesnicky et al. 2006) (see below). Also, the preliminary observation that Bombyx axin homolog maternal mRNA localized in the anterior part of the germ anlage in a similar manner to otd (unpublished), with pair-rule genes: eve, runt (run), and odd-skipped (odd) RNAi embryo posteriortruncation phenotypes, suggests further similarity to Tribolium segmentation (Nakao 2015;Choe et al. 2006). ...
Decades have passed since the early molecular embryogenesis of Drosophila melanogaster was outlined. During this period, the molecular mechanisms underlying early embryonic development in other insects, particularly the flour beetle, Tribolium castaneum, have been described in more detail. The information clearly demonstrated that Drosophila embryogenesis is not representative of other insects and has highly distinctive characteristics. At the same time, this new data has been gradually clarifying ancestral operating mechanisms. The silk moth, Bombyx mori, is a lepidopteran insect and, as a representative of the order, has many unique characteristics found in early embryonic development that have not been identified in other insect groups. Herein, some of these characteristics are introduced and discussed in the context of recent information obtained from other insects.
... Hox and gap gene expression is activated by posterior factors such as Cdx and Wnt [15,[161][162][163][164], and the individual genes also cross-regulate each other's expression. Thus Hox and gap gene expression depends partly on the state of the SAZ and partly on intrinsic dynamics [57,[165][166][167]. ...
Arthropod segmentation and vertebrate somitogenesis are leading fields in the experimental and theoretical interrogation of developmental patterning. However, despite the sophistication of current research, basic conceptual issues remain unresolved. These include: (i) the mechanistic origins of spatial organization within the segment addition zone (SAZ); (ii) the mechanistic origins of segment polarization; (iii) the mechanistic origins of axial variation; and (iv) the evolutionary origins of simultaneous patterning. Here, I explore these problems using coarse-grained models of cross-regulating dynamical processes. In the morphogenetic framework of a row of cells undergoing axial elongation, I simulate interactions between an ‘oscillator’, a ‘switch’ and up to three ‘timers’, successfully reproducing essential patterning behaviours of segmenting systems. By comparing the output of these largely cell-autonomous models to variants that incorporate positional information, I find that scaling relationships, wave patterns and patterning dynamics all depend on whether the SAZ is regulated by temporal or spatial information. I also identify three mechanisms for polarizing oscillator output, all of which functionally implicate the oscillator frequency profile. Finally, I demonstrate significant dynamical and regulatory continuity between sequential and simultaneous modes of segmentation. I discuss these results in the context of the experimental literature.
... Key evolutionary events are numbered on the tree: 1 and 3, loss of maternal hunchback (Hb); 2, loss of maternal Caudal (Cad) expression. The second column shows maternal morphogen gradients (Yoon et al., 2019), while column three depicts the relative position of gap gene expression domains along the A-P axis (Jaeger, 2011;Lemke et al., 2010;Lynch et al., 2006;Olesnicky et al., 2006;Pultz et al., 2005;Wilson & Dearden, 2011;Wilson et al., 2010). Column four shows the structure of the three reverse-engineered gap gene networks described in the text, with inhibitory interactions indicated by T-bars and self-activation by circular arrows (activation by maternal gradients is omitted for simplicity). ...
... The posterior morphogen of A. gambiae is unknown. Data is ambiguous regarding the ordering of posterior gt and kni in N. vitripennis (Olesnicky et al., 2006). ...
This manuscript is a chapter in the book "Evolutionary Systems Biology: Advances, Questions, and Opportunities" to be published with Springer-Nature.
... Indeed, segment number and segment 304 identity can be decoupled by various perturbations [19, 305 32, 158-160]. 306 Hox and gap gene expression is activated by poste-307 rior factors such as Cdx and Wnt [89,[161][162][163][164], and the 308 individual genes also cross-regulate each other's expres-309 sion. Thus Hox and gap gene expression depends partly 310 on the state of the SAZ and partly on intrinsic dynam-311 ics [56,[165][166][167]. ...
Arthropod segmentation and vertebrate somitogenesis are leading fields in the experimental and theoretical interrogation of developmental patterning. However, despite the sophistication of current research, basic conceptual issues remain unresolved. These include (1) the mechanistic origins of spatial organisation within the segment addition zone (SAZ); (2) the mechanistic origins of segment polarisation; (3) the mechanistic origins of axial variation; and (4) the evolutionary origins of simultaneous patterning. Here, I explore these problems using coarse-grained models of cross-regulating dynamical processes. In the morphogenetic framework of a row of cells undergoing axial elongation, I simulate interactions between an “oscillator”, a “switch”, and up to three “timers”, successfully reproducing essential patterning behaviours of segmenting systems. I also compare the output of these largely cell-autonomous models to variants that incorporate positional information. I find that scaling relationships, wave patterns, and patterning dynamics all depend on whether the SAZ is regulated by temporal or spatial information. I also identify three mechanisms for polarising oscillator output, all of which functionally implicate the oscillator frequency profile. Finally, I demonstrate significant dynamical and regulatory continuity between sequential and simultaneous modes of segmentation. I discuss these results in the context of the experimental literature.
... A more conserved interaction of cad is with Mex-3 as an anterior determinant that functions similarly to Bicoid (Schoppmeier et al., 2009). A role for Cad as a morphogen has been demonstrated in both hemimetabolous and holometabolous insects (Dearden and Akam, 2001;El-Sherif et al., 2014;Olesnicky et al., 2006;Shinmyo et al., 2005;Wilson et al., 2010), and in at least some cases this role is mediated by a gradient of mRNA, protein or both (Dearden and Akam, 2001;Olesnicky et al., 2006;Schulz et al., 1998;Shinmyo et al., 2005;Wolff et al., 1998;Xu et al., 1994). This morphogen is then responsible for the correct expression of early patterning genes, such as gap genes, along the main embryonic axis. ...
... A more conserved interaction of cad is with Mex-3 as an anterior determinant that functions similarly to Bicoid (Schoppmeier et al., 2009). A role for Cad as a morphogen has been demonstrated in both hemimetabolous and holometabolous insects (Dearden and Akam, 2001;El-Sherif et al., 2014;Olesnicky et al., 2006;Shinmyo et al., 2005;Wilson et al., 2010), and in at least some cases this role is mediated by a gradient of mRNA, protein or both (Dearden and Akam, 2001;Olesnicky et al., 2006;Schulz et al., 1998;Shinmyo et al., 2005;Wolff et al., 1998;Xu et al., 1994). This morphogen is then responsible for the correct expression of early patterning genes, such as gap genes, along the main embryonic axis. ...
The homeobox transcription factor Caudal has conserved roles in all Bilateria in defining the posterior pole and in controlling posterior elongation. These roles are seemingly similar and are difficult to disentangle. We have carried out a detailed analysis of the expression, function and interactions of the caudal ortholog of the milkweed bug, Oncopeltus fasciatus, a hemimetabolous insect with a conservative early development process, in order to understand its different functions throughout development. In Oncopeltus, caudal is not maternally deposited, but has a sequence of roles in the posterior of the embryos throughout early development. It defines and maintains a posterior-anterior gradient in the blastoderm and modulates the activity of segmentation genes in simultaneous segmentation during the blastoderm stage. It later defines the invagination site and the posterior segment addition zone (SAZ) in the germband. It maintains the posterior SAZ cells in an undifferentiated proliferative state, while promoting dynamic expression of segmentation genes in the anterior SAZ. We show that rather than being a simple posterior determinant, Caudal is involved in several distinct regulatory networks, each with a distinct developmental role.
... Both a specimen (top) and corresponding diagrammatic representation (bottom) are given ◂ genes in the anterior growth zone [24,25,56]. Where it has been examined, Wnt/cad signaling regulates the genes of the anterior growth zone [23,24,[57][58][59]. Our finding of anterior and posterior regionalization of cell behaviors in the growth zone that map to segmental gene expression is similar to what we found in Oncopeltus: the region of low cell division in the anterior of the growth zone is coincident with striped even-skipped (eve) and Delta expression, versus high cell division in the posterior coincident with cad and broad eve expression [25]. ...
Background:
Segmentation in arthropods typically occurs by sequential addition of segments from a posterior growth zone. However, the amount of tissue required for growth and the cell behaviors producing posterior elongation are sparsely documented.
Results:
Using precisely staged larvae of the crustacean, Thamnocephalus platyurus, we systematically examine cell division patterns and morphometric changes associated with posterior elongation during segmentation. We show that cell division occurs during normal elongation but that cells in the growth zone need only divide ~ 1.5 times to meet growth estimates; correspondingly, direct measures of cell division in the growth zone are low. Morphometric measurements of the growth zone and of newly formed segments suggest tagma-specific features of segment generation. Using methods for detecting two different phases in the cell cycle, we show distinct domains of synchronized cells in the posterior trunk. Borders of cell cycle domains correlate with domains of segmental gene expression, suggesting an intimate link between segment generation and cell cycle regulation.
Conclusions:
Emerging measures of cellular dynamics underlying posterior elongation already show a number of intriguing characteristics that may be widespread among sequentially segmenting arthropods and are likely a source of evolutionary variability. These characteristics include: the low rates of posterior mitosis, the apparently tight regulation of cell cycle at the growth zone/new segment border, and a correlation between changes in elongation and tagma boundaries.
... We did not observe cad expression in other structures; thus caudal serves as a marker for these tissues at this stage of development. cad, which functions in the anteriorposterior patterning network in embryogenesis (Macdonald and Struhl 1986;Rivera-Pomar et al. 1995;Olesnicky et al. 2006;Vincent et al. 2018), has been previously implicated in the development of the cercus and interacts with the genes Distaless (Dll) and brachyenteron (byn) in the L3 genital disc (Moreno and Morata 1999). ...
During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have found network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. Here, we performed RNA-seq in whole Drosophila melanogaster male terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the male terminalia are highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. Our results are housed online in a searchable database (https://flyterminalia.pitt.edu) as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.
