Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae

The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. We wish to understand how changes in this network produce evolutionary diversity in insect gastrulation. The present study focuses on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene.     

Zebrafish bmp4 functions during late gastrulation to specify ventroposterior cell fates

Bone morphogenetic proteins (BMPs) are key mediators of dorsoventral patterning in vertebrates and are required for the induction of ventral fates in fish and frogs. A widely accepted model of dorsoventral patterning postulates that a morphogenetic BMP activity gradient patterns cell fates along the dorsoventral axis. Recent work in zebrafish suggests that the role of BMP signaling changes over time, with BMPs required for global dorsoventral patterning during early gastrulation and for tail patterning during late gastrulation and early somitogenesis. Key questions remain about the late phase, including which BMP ligands are required and how the functions of BMPs differ during the early and late gastrula stages. In a screen for dominant enhancers of mutations in the homeobox genes vox and vent, which function in parallel to bmp signaling, we identified an insertion mutation in bmp4. We then performed a reverse genetic screen to isolate a null allele of bmp4. We report the characterization of these two alleles and demonstrate that BMP4 is required during the later phase of BMP signaling for the specification of ventroposterior cell fates. Our results indicate that different bmp genes are essential at different stages. In addition, we present genetic evidence supporting a role for a morphogenetic BMP gradient in establishing mesodermal fates during the later phase of BMP signaling.     

Postgastrular zen expression is required to develop distinct amniotic and serosal epithelia in the scuttle fly Megaselia

The amnioserosa is an extraembryonic epithelium that evolved in higher cyclorrhaphan flies from distinct serosal and amniotic epithelia. The underlying genetic mechanism of this evolutionary transition is unknown. Amnioserosa development of Drosophila correlates with novel expression characteristics of the homeobox gene zerknüllt (zen), including a broad zen expression domain in the syncytial blastoderm and the complete absence of postgastrular zen expression. Here we examine the functional significance of these features by altering the activity profile of zen in Megaselia (a lower cyclorrhaphan fly with distinct serosal and amniotic epithelia) and Drosophila, and by examining in Megaselia the function of u-shaped group (ush-group) genes, which in Drosophila maintain the amnioserosa after gastrulation when zen is no longer expressed. In Megaselia, loss of postgastrular zen expression abrogates serosa development but allows amnion development. Ectopic expression of zen in early Megaselia embryos allows serosa formation but perturbs amnion development. Megaselia homologues of u-shaped group genes are not essential for serosa formation but mediate germband retraction and dorsal closure. Finally, ectopic postgastrular zen expression in Drosophila causes an enlargement of amnioserosa cells and interferes with the morphogenetic functions of the amnioserosa. Our results suggest that the origin of the amnioserosa involved the loss of postgastrular zen expression from extraembryonic tissue, that the early broad expression domain of Drosophila zen evolved afterwards, and that the ush-group genes ancestrally played a role in morphogenetic functions of the amnion.     

Tailbud-derived Bmp4 drives proliferation and inhibits maturation of zebrafish chordamesoderm

In zebrafish, BMP signaling establishes cell identity along the dorsoventral (DV) axis during gastrulation. Owing to the early requirements of BMP activity in DV patterning, it has been difficult to assign later roles in cell fate specification to specific BMP ligands. In this study, we have taken advantage of two follistatin-like genes (fstl1 and fstl2), as well as a transgenic zebrafish line carrying an inducible truncated form of the BMP-type 1 receptor to study the role of Bmp4 outside of the context of DV specification. Characterization of fstl1/2 suggests that they exert a redundant role as BMP antagonists during late gastrulation, regulating BMP activity in axial mesoderm. Maintenance of appropriate levels of BMP signaling is crucial for the proper development of chordamesoderm, a subset of axial mesoderm that gives rise to the notochord, but not prechordal mesoderm, which gives rise to the prechordal plate. Bmp4 activity in particular is required during a crucial window beginning at late gastrulation and lasting through early somitogenesis to promote chordamesoderm proliferation. In the absence of Bmp4, the notochord precursor pool is depleted, and the notochord differentiates prematurely. Our results illustrate a role for Bmp4 in the proliferation and timely differentiation of axial tissue after DV axis specification.     

Antagonism of Nodal signaling by BMP/Smad5 prevents ectopic primitive streak formation in the mouse amnion

The strength and spatiotemporal activity of Nodal signaling is tightly controlled in early implantation mouse embryos, including by autoregulation and feedback loops, and involves secreted and intracellular antagonists. These control mechanisms, which are established at the extra-embryonic/embryonic interfaces, are essential for anterior-posterior patterning of the epiblast and correct positioning of the primitive streak. Formation of an ectopic primitive streak, or streak expansion, has previously been reported in mutants lacking antagonists that target Nodal signaling. Here, we demonstrate that loss-of-function of a major bone morphogenetic protein (BMP) effector, Smad5, results in formation of an ectopic primitive streak-like structure in mutant amnion accompanied by ectopic Nodal expression. This suggests that BMP/Smad5 signaling contributes to negative regulation of Nodal. In cultured cells, we find that BMP-activated Smad5 antagonizes Nodal signaling by interfering with the Nodal-Smad2/4-Foxh1 autoregulatory pathway through the formation of an unusual BMP4-induced Smad complex containing Smad2 and Smad5. Quantitative expression analysis supports that ectopic Nodal expression in the Smad5 mutant amnion is induced by the Nodal autoregulatory loop and a slow positive-feedback loop. The latter involves BMP4 signaling and also induction of ectopic Wnt3. Ectopic activation of these Nodal feedback loops in the Smad5 mutant amnion results in the eventual formation of an ectopic primitive streak-like structure. We conclude that antagonism of Nodal signaling by BMP/Smad5 signaling prevents primitive streak formation in the amnion of normal mouse embryos.     

The BMP signaling gradient patterns dorsoventral tissues in a temporally progressive manner along the anteroposterior axis

Patterning of the vertebrate anteroposterior (AP) axis proceeds temporally from anterior to posterior. How dorsoventral (DV) axial patterning relates to AP temporal patterning is unknown. We examined the temporal activity of BMP signaling in patterning ventrolateral cell fates along the AP axis, using transgenes that rapidly turn "off" or "on" BMP signaling. We show that BMP signaling patterns rostral DV cell fates at the onset of gastrulation, whereas progressively more caudal DV cell fates are patterned at progressively later intervals during gastrulation. Increased BMP signal duration is not required to pattern more caudal DV cell fates; rather, distinct temporal intervals of signaling are required. This progressive action is regulated downstream of, or in parallel to, BMP signal transduction at the level of Smad1/5 phosphorylation. We propose that a temporal cue regulates a cell's competence to respond to BMP signaling, allowing the acquisition of a cell's DV and AP identity simultaneously.     

Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation of BMP4 and X-delta-1 activity

In Xenopus, ectodermal patterning depends on a mediolateral gradient of BMP signaling, higher in the epidermis and lower in the neuroectoderm. Neural crest cells are specified at the border between the neural plate and the epidermis, at intermediate levels of BMP signaling. We recently described a novel secreted protein, Tsukushi (TSK), which works as a BMP antagonist during chick gastrulation. Here, we report on the Xenopus TSK gene (X-TSK), and show that it is involved in neural crest specification. X-TSK expression accumulates after gastrulation at the anterior-lateral edges of the neural plate, including the presumptive neural crest region. In gain-of-function experiments, X-TSK can strongly enhance neural crest specification by the dorsolateral mesoderm or X-Wnt8 in ectodermal explants, while the electroporation of X-TSK mRNA in the lateral ectoderm of embryos after gastrulation can induce the expression of neural crest markers in vivo. By contrast, depletion of X-TSK in explants or embryos impairs neural crest specification. Similarly to its chick homolog, X-TSK works as a BMP antagonist by direct binding to BMP4. However, X-TSK can also indirectly regulate BMP4 mRNA expression at the neural plate border via modulation of the Delta-Notch signaling pathway. We show that X-TSK directly binds to the extracellular region of X-delta-1, and modulates Delta-dependent Notch activity. We propose that X-TSK plays a key role in neural crest formation by directly regulating BMP and Delta activities at the boundary between the neural and the non-neural ectoderm.     

Axis specification in the spider embryo: dpp is required for radial-to-axial symmetry transformation and sog for ventral patterning

The mechanism by which Decapentaplegic (Dpp) and its antagonist Short gastrulation (Sog) specify the dorsoventral pattern in Drosophila embryos has been proposed to have a common origin with the mechanism that organizes the body axis in the vertebrate embryo. However, Drosophila Sog makes only minor contributions to the development of ventral structures that hypothetically correspond to the vertebrate dorsum where the axial notochord forms. In this study, we isolated a homologue of the Drosophila sog gene in the spider Achaearanea tepidariorum, and characterized its expression and function. Expression of sog mRNA initially appeared in a radially symmetrical pattern and later became confined to the ventral midline area, which runs axially through the germ band. RNA interference-mediated depletion of the spider sog gene led to a nearly complete loss of ventral structures, including the axial ventral midline and the central nervous system. This defect appeared to be the consequence of dorsalization of the ventral region of the germ band. By contrast, the extra-embryonic area formed normally. Furthermore, we showed that embryos depleted for a spider homologue of dpp failed to break the radial symmetry, displaying evenly high levels of sog expression except in the posterior terminal area. These results suggest that dpp is required for radial-to-axial symmetry transformation of the spider embryo and sog is required for ventral patterning. We propose that the mechanism of spider ventral specification largely differs from that of the fly. Interestingly, ventral specification in the spider is similar to the process in vertebrates in which the antagonism of Dpp/BMP signaling plays a central role in dorsal specification.     

Twisted gastrulation promotes BMP signaling in zebrafish dorsal-ventral axial patterning

In vertebrates and invertebrates, the bone morphogenetic protein (BMP) signaling pathway patterns cell fates along the dorsoventral (DV) axis. In vertebrates, BMP signaling specifies ventral cell fates, whereas restriction of BMP signaling by extracellular antagonists allows specification of dorsal fates. In misexpression assays, the conserved extracellular factor Twisted gastrulation (Tsg) is reported to both promote and antagonize BMP signaling in DV patterning. To investigate the role of endogenous Tsg in early DV patterning, we performed morpholino (MO)-based knockdown studies of Tsg1 in zebrafish. We found that loss of tsg1 results in a moderately strong dorsalization of the embryonic axis, suggesting that Tsg1 promotes ventral fates. Knockdown of tsg1 combined with loss of function of the BMP agonist tolloid (mini fin) or heterozygosity for the ligand bmp2b (swirl) enhanced dorsalization, supporting a role for Tsg1 in specifying ventral cell fates as a BMP signaling agonist. Moreover, loss of tsg1 partially suppressed the ventralized phenotypes of mutants of the BMP antagonists Chordin or Sizzled (Ogon). Our results support a model in which zebrafish Tsg1 promotes BMP signaling, and thus ventral cell fates, during DV axial patterning.     

The ventralized ogon mutant phenotype is caused by a mutation in the zebrafish homologue of Sizzled,a secreted Frizzled-related protein

The BMP signaling pathway plays a key role during dorsoventral pattern formation of vertebrate embryos. In zebrafish, all cloned mutants affecting this process are deficient in members of the BMP pathway. In a search for factors differentially expressed in swirl/bmp2b mutants compared with wild type, we isolated zebrafish Sizzled, a member of the secreted Frizzled-related protein family and putative Wnt inhibitor. The knockdown of sizzled using antisense morpholino phenocopied the ventralized mutant ogon (formerly also known as mercedes and short tail). By sequencing and rescue experiments, we demonstrate that ogon encodes sizzled. Overexpression of sizzled, resulting in strongly dorsalized phenotypes, and the expression domains of sizzled in wild type embryos, localized in the ventral side during gastrulation and restricted to the posterior end during segmentation stages, correlate with its role in dorsoventral patterning. The expanded expression domain of sizzled in ogon and chordino together with its downregulation in swirl suggests a BMP2b-dependent negative autoregulation of sizzled. Indicating a novel role for a secreted Frizzled-related protein, we show that, in addition to the BMP pathway, a component of the Wnt signaling pathway is required for dorsoventral pattern formation in zebrafish.     

The amnioserosa is an apomorphic character of cyclorrhaphan flies

In developing insect eggs the cells of the blastoderm adopt either an embryonic or an extraembryonic fate. The extraembryonic tissue consists of epithelia, termed amnion and serosa, which wrap the germ band embryo. The serosa develops directly from part of the blastoderm and surrounds the embryo as well as the yolk. The amnion develops from the margins of the germ band and in most insect species generates a transient ventral cavity for the developing embryo. The amniotic cavity and the serosa have been reduced in the course of dipteran evolution. The insect order of Diptera includes the paraphyletic Nematocera, including gnats and mosquitoes, and the more derived monophyletic Brachycera, the true flies. Nematocera develop within an amniotic cavity and the surrounding serosa, whereas cyclorrhaphan Brachycera do not. This observation implies that the amnion and serosa have been reduced before the radiation of the monophyletic cyclorrhaphan flies. Here I show that an amniotic cavity is formed during embryogenesis of the horsefly Haematopota pluvialis (Tabanidae) and the dancefly Empis livida (Empididae). The results suggest that extraembryonic tissue was reduced in the stem lineage of cyclorrhaphan flies, with consequences for the molecular basis of pattern formation along the anterior-posterior axis of the embryo. Received: 21 October 1999 / Accepted: 17 January 2000     

Temporal and spatial action of tolloid (mini fin) and chordin to pattern tail tissues

In vertebrates, a bone morphogenetic protein (BMP) signaling pathway patterns all ventral cell fates along the embryonic axis. BMP activity is positively regulated by Tolloid, a metalloprotease, that can eliminate the activity of the BMP antagonist Chordin. A tolloid mutant in zebrafish, mini fin (mfn), exhibits a specific loss of ventral tail tissues. Here, we investigate the spatial and temporal requirements for Tolloid (Mfn) in dorsoventral patterning of the tail. Through chimeric analyses, we found that Tolloid (Mfn) functions cell non-autonomously in the ventral-most vegetal cells of the gastrula or their derivatives. We generated a tolloid transgene under the control of the inducible hsp70 promoter and demonstrate that tolloid (mfn) is first required at the completion of gastrulation. Although tolloid is expressed during gastrulation and dorsally and ventrally within the tail bud, our results indicate that Tolloid (Mfn) acts specifically in the ventral tail bud during a approximately 4 h period extending from the completion of gastrulation to early somitogenesis stages to regulate BMP signaling. Examination of the temporal requirements of Chordin activity by overexpression of the hsp70-tolloid transgene indicates that Chordin is required both during and after gastrulation for proper patterning of the tail, contrasting Tld's requirement only during post-gastrula stages. We hypothesize that the gastrula role of Chordin in tail patterning is to generate the proper size domains of cells to enter the ventral and dorsal tail bud, whereas post-gastrula Chordin activity patterns the derivatives of the tail bud. Thus, fine modulation of BMP signaling levels through the negative and positive actions of Chordin and Tolloid, respectively, patterns tail tissues.     

The Integrator Complex Subunit 6 (Ints6) Confines the Dorsal Organizer in Vertebrate Embryogenesis

Dorsoventral patterning of the embryonic axis relies upon the mutual antagonism of competing signaling pathways to establish a balance between ventralizing BMP signaling and dorsal cell fate specification mediated by the organizer. In zebrafish, the initial embryo-wide domain of BMP signaling is refined into a morphogenetic gradient following activation dorsally of a maternal Wnt pathway. The accumulation of β-catenin in nuclei on the dorsal side of the embryo then leads to repression of BMP signaling dorsally and the induction of dorsal cell fates mediated by Nodal and FGF signaling. A separate Wnt pathway operates zygotically via Wnt8a to limit dorsal cell fate specification and maintain the expression of ventralizing genes in ventrolateral domains. We have isolated a recessive dorsalizing maternal-effect mutation disrupting the gene encoding Integrator Complex Subunit 6 (Ints6). Due to widespread de-repression of dorsal organizer genes, embryos from mutant mothers fail to maintain expression of BMP ligands, fail to fully express vox and ved, two mediators of Wnt8a, display delayed cell movements during gastrulation, and severe dorsalization. Consistent with radial dorsalization, affected embryos display multiple independent axial domains along with ectopic dorsal forerunner cells. Limiting Nodal signaling or restoring BMP signaling restores wild-type patterning to affected embryos. Our results are consistent with a novel role for Ints6 in restricting the vertebrate organizer to a dorsal domain in embryonic patterning.     

Tribolium embryogenesis: a SEM study of cell shapes and movements from blastoderm to serosal closure

Embryogenesis in the beetle Tribolium is of increasing interest to both molecular and evolutionary biology because it differs from the Drosophila paradigm by its type of segment specification (short- vs. long-germ) and by the extensive epithelial envelopes – amnion and serosa – that are typical of most insects but not of higher dipterans. Using scanning electron microscopy of DAPI staged embryos we document development in Tribolium castaneum from blastoderm to completion of the envelopes, recording many details not otherwise accessible; we also provide a time table of the respective stages at 30°C. The nascent blastoderm cells remain basally confluent with the yolksac until after the 13th (=last synchronous) mitotic cycle. The cells in the prospective serosa – the first domain to segregate visibly from the uniform blastoderm – carry surface protrusions likely to contact the overlying vitelline envelope. The embryonic rudiment, the other (and larger) blastodermal domain, gives rise to amnion and germ anlage. In the latter, visible differentiation begins with a ”primitive pit” reminiscent of the posterior midgut rudiment of Drosophila. The subsequent invagination of the mesoderm resembles Drosophila gastrulation, except in the head region where the median groove extends through the entire preoral region. The prospective amnion starts differing visibly from the germ anlage during early gastrulation. It then folds underneath the spreading serosa and, advancing with the latter, closes the amniotic cavity at the ventral face of the germband. The largest (=posterior) amniotic fold covers a crestlike protrusion of the yolksac. Together with marked changes in the shape and arrangement of the amnion cells, this protrusion may contribute to the fold’s elevation and early progress. Received: 12 August 1999 / Accepted: 4 November 1999     

A new molecular logic for BMP-mediated dorsoventral patterning in the leech Helobdella

Bone morphogenetic protein (BMP) signaling is broadly implicated in dorsoventral (DV) patterning of bilaterally symmetric animals [1-3], and its role in axial patterning apparently predates the birth of Bilateria [4-7]. In fly and vertebrate embryos, BMPs and their antagonists (primarily Sog/chordin) diffuse and interact to generate signaling gradients that pattern fields of cells [8-10]. Work in other species reveals diversity in essential facets of this ancient patterning process, however. Here, we report that BMP signaling patterns the DV axis of segmental ectoderm in the leech Helobdella, a clitellate annelid (superphylum Lophotrochozoa) featuring stereotyped developmental cell lineages, but the detailed mechanisms of DV patterning in Helobdella differ markedly from fly and vertebrates. In Helobdella, BMP2/4s are expressed broadly, rather than in dorsal territory, whereas a dorsally expressed BMP5-8 specifies dorsal fate by short-range signaling. A BMP antagonist, gremlin, is upregulated by BMP5-8 in dorsolateral, rather than ventral territory, and yet the BMP-antagonizing activity of gremlin is required for normal ventral cell fates. Gremlin promotes ventral fates without disrupting dorsal fates by selectively inhibiting BMP2/4s, not BMP5-8. Thus, DV patterning in the development of the leech revealed unexpected evolutionary plasticity of the conserved BMP patterning system, presumably reflecting its adaptation to different modes of embryogenesis.     

The maternal NF-kappaB/dorsal gradient of Tribolium castaneum: dynamics of early dorsoventral patterning in a short-germ beetle

In the long-germ insect Drosophila melanogaster dorsoventral polarity is induced by localized Toll-receptor activation which leads to the formation of a nuclear gradient of the rel/ NF-kappaB protein Dorsal. Peak levels of nuclear Dorsal are found in a ventral stripe spanning the entire length of the blastoderm embryo allowing all segments and their dorsoventral subdivisions to be synchronously specified before gastrulation. We show that a nuclear Dorsal protein gradient of similar anteroposterior extension exists in the short-germ beetle, Tribolium castaneum, which forms most segments from a posterior growth zone after gastrulation. In contrast to Drosophila, (i) nuclear accumulation is first uniform and then becomes progressively restricted to a narrow ventral stripe, (ii) gradient refinement is accompanied by changes in the zygotic expression of the Tribolium Toll-receptor suggesting feedback regulation and, (iii) the gradient only transiently overlaps with the expression of a potential target, the Tribolium twist homolog, and does not repress Tribolium decapentaplegic. No nuclear Dorsal is seen in the cells of the growth zone of Tribolium embryos, indicating that here dorsoventral patterning occurs by a different mechanism. However, Dorsal is up-regulated and transiently forms a nuclear gradient in the serosa, a protective extraembryonic cell layer ultimately covering the whole embryo.