Characterization of <Emphasis Type="Italic">twist</Emphasis> and <Emphasis Type="Italic">snail</Emphasis> gene expression during mesoderm and nervous system development in the polychaete annelid <Emphasis Type="Italic">Capitella</Emphasis> sp. I

To investigate the evolutionary history of mesoderm in the bilaterian lineage, we are studying mesoderm development in the polychaete annelid, Capitella sp. I, a representative lophotrochozoan. In this study, we focus on the Twist and Snail families as candidate mesodermal patterning genes and report the isolation and in situ expression patterns of two twist homologs (CapI-twt1 and CapI-twt2) and two snail homologs (CapI-sna1 and CapI-sna2) in Capitella sp. I. CapI-twt1 is expressed in a subset of mesoderm derivatives during larval development, while CapI-twt2 shows more general mesoderm expression at the same stages. Neither twist gene is detected before the completion of gastrulation. The two snail genes have very distinct expression patterns. At cleavage and early gastrula stages, CapI-sna1 is broadly expressed in precursors of all three germ layers and becomes restricted to cells around the closing blastopore during late gastrulation; CapI-sna2 expression is not detected at these stages. After gastrulation, both snail genes are expressed in the developing central nervous system (CNS) at stages when neural precursor cells are internalized, and CapI-sna1 is also expressed laterally within the segmental mesoderm. Based on the expression patterns in this study, we suggest a putative function for Capitella sp. I twist genes in mesoderm differentiation and for snail genes in regulating CNS development and general cell migration during gastrulation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dual functions of the heartless fibroblast growth factor receptor in development of the Drosophila embryonic mesoderm

The Drosophila embryonic mesoderm forms by invagination of the ventral-most blastoderm cells. Subsequent development of this germ layer involves the dorsolateral migration of the internalized cells and expansion by cell division, followed by the specification of particular cell fates through the coordinate actions of both intrinsic and extrinsic regulatory mechanisms. The latter include several intercellular signals that function across germ layers. These processes combine to generate a diversity of mesodermal subtypes, including the cardial and pericardial cells of the heart or dorsal vessel, a complete set of somatic muscle founders each with its unique identity, a population of cells that form the visceral musculature, and other cells that develop into hemocytes and the fat body. Here, we review recent evidence for the involvement of a fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene in early directional migration of the Drosophila mesoderm. In addition, we provide new data that 1) demonstrate a second role for Htl in promoting the specification of the precursors to certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its cell migration function, 2) suggest that Ras and at least one other signal transduction pathway act downstream of Htl, and 3) establish a functional relationship between the Ras pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some of the same dorsal mesodermal cells that are dependent on Htl. Finally, parallels between requirements for FGFR signaling in Drosophila and vertebrate mesoderm development are considered. Dev. Genet. 22:212–229, 1998. © 1998 Wiley-Liss, Inc.     

Unexpected functional redundancy between Twist and Slug (Snail2) and their feedback regulation of NF-κB via Nodal and Cerberus

A NF-κB-Twist-Snail network controls axis and mesoderm formation in Drosophila. Using translation-blocking morpholinos and hormone-regulated proteins, we demonstrate the presence of an analogous network in the early Xenopus embryo. Loss of twist (twist1) function leads to a reduction of mesoderm and neural crest markers, an increase in apoptosis, and a decrease in snail1 (snail) and snail2 (slug) mRNA levels. Injection of snail2 mRNA rescues twist's loss of function phenotypes and visa versa. In the early embryo NF-κB/RelA regulates twist, snail2, and snail1 mRNA levels; similarly Nodal/Smad2 regulate twist, snail2, snail1, and relA RNA levels. Both Twist and Snail2 negatively regulate levels of cerberus RNA, which encodes a Nodal, bone morphogenic protein (BMP), and Wnt inhibitor. Cerberus's anti-Nodal activity inhibits NF-κB activity and decreases relA RNA levels. These results reveal both conserved and unexpected regulatory interactions at the core of a vertebrate's mesodermal specification network.     

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     

Investigating divergent mechanisms of mesoderm development in arthropods: the expression of Ph-twist and Ph-mef2 in Parhyale hawaiensis

The evolution of mesoderm was important for the development of complex body plans as well as key organ systems. Genetic and molecular studies in the fruitfly, Drosophila melanogaster, have provided the majority of information concerning mesoderm development in arthropods. In Drosophila, twist is necessary for the specification and correct morphogenesis of mesoderm and myocyte enhancing factor 2 (mef2) is involved downstream of twist to activate muscle differentiation. In Drosophila, mesoderm is defined by positional cues in the blastoderm embryo, while in another arthropod group, the amphipod crustaceans, cell lineage plays a greater role in defining the mesoderm. It is not known how different mechanistic strategies such as positional information vs. cell-lineage-dependent development affect the timing and use of gene networks. Here we describe the development of the mesoderm in a malacostracan crustacean, Parhyale hawaiensis, and characterize the expression of Parhyale twist and mef2 orthologues. In Parhyale, the mesoderm of the post-mandibular segments arises mainly through the asymmetric division of mesoteloblasts as the germband elongates. Ph-twist expression is seen in a subset of segmental mesoderm during germband development, but not during early cleavages when the specific mesodermal cell lineages first arise. ph-mef2 expression starts after the segmental mesoderm begins to proliferate and persists in developing musculature. While the association of these genes with mesoderm differentiation appears to be conserved across the animal kingdom, the timing of expression and relationship with different mechanisms of mesoderm development may give us greater insight into the ancestral use of these genes during mesoderm differentiation.     

Embryogenesis of an embryonic lethal, ‘kidney-shaped egg’ in Bombyx mori,with special reference to mesoderm differentiation

Summary

Within the kidney-shaped egg laid by the homozygous female, the embryo develops only ectodermal organs, but no mesodermal organs. The early embryogenesis of this strain was examined with light and electron microscopy. Until the early germband stage no remarkable differences are distinguishable between the normal and the kidney-shaped eggs. Afterwards, in the kidney-shaped egg large amount of cytoplasmic masses and often several cells are extruded from the presumptive head region into the yolk at the ‘pyriform’ stage, resulting in inhibiting the formation of a normal wide head lobe and an invagination occurring in its central region, the first sign of the mesoderm differentiation. This phenomenon proceeds gradually toward the posterior part and thus the differentiation of mesoderm becomes impossible throughout the entire length of germband. The results are discussed in the context of the mesoderm differentiation in Bombyx.     

Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila

The homeobox gene tinman plays a key role in the specification of Drosophila heart progenitors and the visceral mesoderm of the midgut, both of which arise at defined positions within dorsal areas of the mesoderm. Here, we show that in addition to the heart and midgut visceral mesoderm, tinman is also required for the specification of all dorsal body wall muscles. Thus it appears that the precursors of the heart, visceral musculature, and dorsal somatic muscles are all specified within the same broad domain of dorsal mesodermal tinman expression. Locally restricted activities of tinman are also observed during its early, general mesodermal expression, where tinman is required for the activation of the homeobox gene buttonless in precursors of the “dorsal median” (DM) glial cells along the ventral midline. These observations, together with others showing only mild effects of ectopic tinman expression on heart development, indicate that tinman function is obligatory, but not sufficient to determine individual tissues within the mesoderm. Therefore, we propose that tinman has a role in integrating positional information that is provided by intersecting domains of additional regulators and signals, which may include Wingless, Sloppy Paired, and Hedgehog in the dorsal mesoderm and EGF-signaling at the ventral midline. Previous studies have shown that Dpp acts as an inductive signal from dorsal ectodermal cells to induce tinman expression in the dorsal mesoderm, which, in turn, is needed for heart and visceral mesoderm formation. In the present report, we show that Thickveins, a type I receptor of Dpp, is essential for the transmission of Dpp signals into the mesoderm. Constitutive activity of Tkv in the entire mesoderm induces ectopic tinman expression in the ventral mesoderm, and this results in the ectopic formation of heart precursors in a defined area of the ventrolateral mesoderm. We further show that Screw, a second BMP2/4-related gene product, Tolloid, a BMP1-related protein, and the zinc finger-containing protein Schnurri, are required to allow full levels of tinman induction during this process. It is likely that some of these functional and regulatory properties of tinman are shared by tinman-related genes from vertebrates that have similarly important roles in embryonic heart development. Dev. Genet. 22:187–200, 1998. © 1998 Wiley-Liss, Inc.     

<Emphasis Type="Italic">snail</Emphasis> expression during embryonic development of the coral<Emphasis Type="Italic"> Acropora</Emphasis>: blurring the diploblast/triploblast divide?

Although corals are nominally diploblastic, the early development of Acropora millepora involves a process that clearly resembles gastrulation in higher metazoans. This similarity at the morphological level led us to search for the Acropora equivalents of genes whose key roles in gastrulation are conserved across the higher Metazoa. We here report the characterisation of one such gene, snail, which in both Drosophila and the mouse is expressed in cells undergoing an epithelial-mesenchyme transition and/or morphogenetic movements. In addition to an N-terminal SNAG domain, the Acropora snail protein contains four zinc fingers with sequences diagnostic for members of the snail protein subfamily. In situ hybridisation reveals expression in epithelial tissue in the central portion of one side of the flattened pre-gastrulation embryo, which continues to express snail as it is engulfed by its opposite layer. Comparison to snail expression during gastrulation in bilaterians such as Drosophila reveals striking similarities and suggests mechanistic, and possibly evolutionary, links between the processes of mesoderm formation in bilaterians and endoderm formation in the Cnidaria.Edited by P. Simpson     

Cell lineage of flight muscle fibers in Drosophila: a fate map of the induced shibire phenotype in mosaics

Summary A blastoderm fate map has been prepared for Drosophila, using mosaics of a temperature-sensitive mutation, shibire (shi). The mutation can cause abnormal flight muscle morphology, inducible only by a short heat pulse in early metamorphosis. Thus muscle lineage and development are unperturbed until the heat pulse in the early pupa. The developmental focus of the shi muscle phenotype maps to the ventral thorax at the expected site of thoracic mesoderm, and probably indicates the blastoderm progenitors of the adult flight muscle. The fate map provides greater detail than previously available for the dorsolongitudinal fibers (DLM) of flight muscle, showing wide separation of the fibers of flight muscle. DLM fibers a and b map close together, and far anterior to fibers e and f, which also map together. On a fate map, common developmental focus indicates a common blastoderm origin. Thus, the observed pattern for DLM fibers suggests that the blastoderm progenitors for each of these syncytial fiber pairs (a, b; e, f) include only one or two cells. It follows that there is usually a single genotype within each fiber pair (a, b; e, f), and that this genotype is directly reflected in the fiber phenotype. In a large number of cases, DLM fibers a and b differ in phenotype from other DLM fibers, in parallel with their other differences (e.g., timing of development in pupa, innervation, motor activity). The separation of fate map locations of the developmental focus for DLM fibers within mesoderm suggests that specific fibers of flight muscle may, in normal development, originate in all three thoracic mesodermal parasegments.     

Lamprey <Emphasis Type="Italic">snail</Emphasis> highlights conserved and novel patterning roles in vertebrate embryos

snail genes mark presumptive mesoderm across bilaterian animals. In gnathostome vertebrates, snail genes are a multimember family that are also markers of premigratory neural crest (pnc) and some postmigratory neural crest derivatives in the pharyngeal arches. Previous studies of nonvertebrate chordates indicate that they have single snail genes that retain ancestral functions in mesoderm development and perhaps in specification of a pnc-like cell population. Lampreys are the most basal extant vertebrates, with well-defined developmental morphology. Here, we identify a single snail gene from the lamprey Petromyzon marinus that is the phylogenetic outgroup of all gnathostome snail genes. This single lamprey snail gene retains ancestral snail patterning domains present in nonvertebrate chordates. Lamprey snail is also expressed in tissues that are broadly equivalent to the combined sites of expression of all three gnathostome snail paralogy groups, excepting in embryonic tissues that are unique to gnathostomes. Importantly, while snail does not appear to demarcate an early neural crest population in lampreys as it does in gnathostomes, it may be involved in later neural crest development. Together, our results indicate that significant cis-regulatory innovation occurred in a single snail gene before the vertebrate radiation, and significant subfunctionalization occurred after snail gene duplications in the gnathostome lineages.     

An amphioxus snail gene: Expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo

 Homologs of the Drosophila snail gene have been characterized in several vertebrates. In addition to being expressed in mesoderm during gastrulation, vertebrate snail genes are also expressed in presumptive neural crest and/or its derivatives. Given that neural crest is unique to vertebrates and is considered to be of fundamental importance in their evolution, we have cloned and characterized the expression of a snail gene from amphioxus, a cephalochordate widely accepted as the sister group of the vertebrates. We show that, at the amino acid sequence level, the amphioxus snail gene is a clear phylogenetic outgroup to all the characterized vertebrate snail genes. During embryogenesis snail expression initially becomes restricted to the paraxial or presomitic mesoderm of amphioxus. Later, snail is expressed at high levels in the lateral neural plate, where it persists during neurulation. Our results indicate that an ancestral function of snail genes in the lineage leading to vertebrates is to define the paraxial mesoderm. Furthermore, our results indicate that a cell population homologous to the vertebrate neural crest may be present in amphioxus, thus providing an important link in the evolution of this key vertebrate tissue. Received: 11 May 1998 / Accepted: 2 August 1998     

Germ layer specification and axial patterning in the embryonic development of the freshwater planarian Schmidtea polychroa

Although patterning during regeneration in adult planarians has been studied extensively, very little is known about how the initial planarian body plan arises during embryogenesis. Herein, we analyze the process of embryo patterning in the species Schmidtea polychroa by comparing the expression of genes involved in the establishment of the metazoan body plan. Planarians present a derived ectolecithic spiralian development characterized by dispersed cleavage within a yolk syncytium and an early transient embryo capable of feeding on the maternally supplied yolk cells. During this stage of development, we only found evidence of canonical Wnt pathway, mostly associated with the development of its transient pharynx. At these stages, genes involved in gastrulation (snail) and germ layer determination (foxA and twist) are specifically expressed in migrating blastomeres and those giving rise to the temporary gut and pharyngeal muscle. After yolk ingestion, the embryo expresses core components of the canonical Wnt pathway and the BMP pathway, suggesting that the definitive axial identities are established late. These data support the division of planarian development into two separate morphogenetic stages: a highly divergent gastrulation stage, which segregates the three germ layers and establishes the primary organization of the feeding embryo; and subsequent metamorphosis, based on totipotent blastomeres, which establishes the definitive adult body plan using mechanisms that are similar to those used during regeneration and homeostasis in the adult.     

Mesoderm-specific B104 expression in the Drosophila embryo is mediated by internal cis-acting elements of the transposon

The Drosophila melanogaster genome contains about 100 copies of the B104 transposable element, which is strongly expressed during embryogenesis. Here we show that B104 expression is restricted to the esophageal and amnioproctodeal regions of the embryo and to the developing mesoderm. Mesoderm-specific B104 expression requires the activity of the mesoderm-determining factors twist and snail. Virtually the same expression patterns were observed in Drosophila yakuba, a species that a separated from D. melanogaster by some 15 million years of evolution. We show that B104 expression is directed by internal sequences of the retrotransposon that are capable of acting as a cis-acting regulatory element in front of a heterologous Drosophila promoter. Our findings suggest that retrotransposon insertions can affect the expression patterns of endogenous genes by adding and distributing specific cis-acting control elements throughout the host genome. We therefore propose that transposable elements in addition to reducing the fitness of their hosts may also provide a rich pool of cis-acting sequences that contribute to the long-term evolutionary potential of the population in a beneficial manner.