Building proteomic pathways using Drosophila ventral furrow formation as a model

Ventral furrow formation is the first morphogenetic movement to occur during Drosophila gastrulation causing the internalization of mesodermal precursors. A previous proteomic screen for ventral-specific proteome changes identified a set of about forty "difference-proteins" that spanned many cellular functions. To understand the connections between these disparate proteins, we initiated a pathway-building scheme using cycles of protein expression manipulation and proteome analysis. This pathway-building exercise started with the proteasomal subunit, Pros35, one of three proteasome subunits found to be ventral-specific difference-proteins. Here we show that Pros35 is a key regulator in ventral furrow formation. Altering the level of Pros35 led to ventral furrow defects. Proteome analysis of the changes induced by Pros35 RNAi showed extensive overlap with the original set of ventral-specific difference-proteins. One of the most prominent changes was in the extracellular iron carrier, Transferrin (Tsf1). Tsf1 is normally less abundant in ventral cells relative to lateral cells; however, RNAi of Pros35 in ventralized embryos negated this ventral-specific difference. Increasing Tsf1 in wild-type embryos blocked ventral furrow formation and caused proteome changes that were similar to the previously seen ventral-specific difference-proteins, including Pros35, which indicates the existence of an unprecedented regulatory loop between the proteasome and iron homeostasis. Additionally, we show that the iron regulatory protein, Irp-1A, also plays an important role in ventral furrow formation. Together these three proteins are part of a regulatory loop that coordinately controls a large number of ventral-specific protein changes.     

The Drosophila gastrulation gene concertina encodes a G alpha-like protein

Gastrulation is a complex process requiring the coordination of cell shape changes and cell movements. In Drosophila, gastrulation begins immediately upon cellularization of the blastoderm stage embryo with the formation of the ventral furrow and posterior midgut. Cells that form both of these invaginations change their shape via apical constriction. Embryos from mothers homozygous for mutations in the concertina (cta) gene begin furrow formation by forming a zone of tightly apposed cells, constrict some cells, and then fail to constrict enough cells to form an organized groove. The cta gene has been cloned, and sequence analysis suggests that it encodes an alpha subunit of a G protein. G proteins have a role in cell-cell communication as mediators of signals between membrane-bound receptors and intracellular effectors. The phenotype of embryos from homozygous cta mothers suggests that the cta gene plays a role in a signal transduction pathway used during gastrulation.     

Drosophila gastrulation: analysis of cell shape changes in living embryos by three-dimensional fluorescence microscopy.

The first event of Drosophila gastrulation is the formation of the ventral furrow. This process, which leads to the invagination of the mesoderm, is a classical example of epithelial folding. To understand better the cellular changes and dynamics of furrow formation, we examined living Drosophila embryos using three-dimensional time-lapse microscopy. By injecting fluorescent markers that visualize cell outlines and nuclei, we monitored changes in cell shapes and nuclear positions. We find that the ventral furrow invaginates in two phases. During the first 'preparatory' phase, many prospective furrow cells in apparently random positions gradually begin to change shape, but the curvature of the epithelium hardly changes. In the second phase, when a critical number of cells have begun to change shape, the furrow suddenly invaginates. Our results suggest that furrow formation does not result from an ordered wave of cell shape changes, contrary to a model for epithelial invagination in which a wave of apical contractions causes invagination. Instead, it appears that cells change their shape independently, in a stochastic manner, and the sum of these individual changes alters the curvature of the whole epithelium.     

The secreted cell signal Folded Gastrulation regulates glial morphogenesis and axon guidance in Drosophila

During gastrulation in Drosophila, ventral cells change shape, undergoing synchronous apical constriction, to create the ventral furrow (VF). This process is affected in mutant embryos lacking zygotic function of the folded gastrulation (fog) gene, which encodes a putative secreted protein. Fog is an essential autocrine signal that induces cytoskeletal changes in invaginating VF cells. Here we show that Fog is also required for nervous system development. Fog is expressed by longitudinal glia in the central nervous system (CNS), and reducing its expression in glia causes defects in process extension and axon ensheathment. Glial Fog overexpression produces a disorganized glial lattice. Fog has a distinct set of functions in CNS neurons. Our data show that reduction or overexpression of Fog in these neurons produces axon guidance phenotypes. Interestingly, these phenotypes closely resemble those seen in embryos with altered expression of the receptor tyrosine phosphatase PTP52F. We conducted epistasis experiments to define the genetic relationships between Fog and PTP52F, and the results suggest that PTP52F is a downstream component of the Fog signaling pathway in CNS neurons. We also found that Ptp52F mutants have early VF phenotypes like those seen in fog mutants.     

Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations

The ventral furrow and posterior midgut invaginations bring mesodermal and endodermal precursor cells into the interior of the Drosophila embryo during gastrulation. Both invaginations proceed through a similar sequence of rapid cell shape changes, which include apical flattening, constriction of the apical diameter, cell elongation and subsequent shortening. Based on the time course of apical constriction in the ventral furrow and posterior midgut, we identify two phases in this process: first, a slow stochastic phase in which some individual cells begin to constrict and, second, a rapid phase in which the remaining unconstricted cells constrict. Mutations in the concertina or folded gastrulation genes appear to block the transition to the second phase in both the ventral furrow and the posterior midgut invaginations.     

Correlations between cell fate and the distribution of proteins that are synthesized before the midblastula transition in Xenopus

Summary The proteins synthesized before the 512-cell stage by Xenopus blastomeres with different fates were compared by one dimensional PAGE. Blastomeres that contributed more progeny to antero-dorsal axial structures produced proportionately more of two proteins of 225000 and 245000 daltons. Additionally, these proteins were reversibly increased in ventralized embryos and were decreased in dorsalized embryos. These observations indicate that some proteins that are synthesized during cleavage stages are expressed to different degrees in different regions of the embryo, that their expression can be correlated to cell fate in the normal embryo, and that their expression is altered quantitatively in dorsalized and ventralized embryos. The inverse relationship between the production of these proteins and the potential to produce dorsal structures in the normal and in dorsalized/ventralized embryos is consistent with a model in which cell fate is influenced by a gradient of particular proteins.Supported by NIH grants HD 06619 (SLK) and GM 33932 (MLK).     

Ventral cell rearrangements contribute to anterior-posterior axis lengthening between neurula and tailbud stages in Xenopus laevis

Studies of morphogenesis in early Xenopus embryos have focused primarily on gastrulation and neurulation. Immediately following these stages is another period of intense morphogenetic activity, the neurula-to-tailbud transition. During this period the embryo is transformed from the spherical shape of the early stages into the long, thin shape of the tailbud stages. While gastrulation and neurulation depend largely on active cell rearrangement and cell shape changes in dorsal tissues, we find that the neurula-to-tailbud transition depends in part on activities of ventral cells. Ventral explants of neurula lengthen autonomously as much as the ventral sides of intact embryos, while dorsal explants lengthen less than the dorsal sides of intact embryos. Analyses of cell division, cell shapes, and cell rearrangement by transplantation of labeled cells and by time lapse recordings in live intact embryos concur that cell rearrangements in ventral mesoderm and ectoderm contribute to the autonomous anterior-posterior axis lengthening of ventral explants between neurula and tailbud stages.     

Ventralization of the Drosophila embryo by deletion of extracellular leucine-rich repeats in the Toll protein.

Dorsoventral polarity of the Drosophila embryo is established by a signal transduction pathway in which the maternal transmembrane protein Toll appears to function as the receptor for a ventrally localized extracellular ligand. Certain dominant Toll alleles encode proteins that behave as partially ligand-independent receptors, causing embryos containing these proteins to become ventralized. In extracts of embryos derived from mothers carrying these dominant alleles, we detected a polypeptide of approximately 35 kDa in addition to full-length Toll polypeptides with antibodies to Toll. Our biochemical analyses suggest that the smaller polypeptide is a truncated form of Toll lacking extracellular domain sequences. To assay the biological activity of such a shortened form of Toll, we synthesized RNA encoding a mutant polypeptide lacking the leucine-rich repeats that comprise most of Toll's extracellular domain and injected this RNA into embryos. The truncated Toll protein elicited the most ventral cell fate independently of the wild-type Toll protein and its ligand. These results support the view that Toll is a receptor whose extracellular domain regulates the intrinsic signaling activity of its cytoplasmic domain.     

Abelson kinase (Abl) and RhoGEF2 regulate actin organization during cell constriction in Drosophila

Morphogenesis involves the interplay of different cytoskeletal regulators. Investigating how they interact during a given morphogenetic event will help us understand animal development. Studies of ventral furrow formation, a morphogenetic event during Drosophila gastrulation, have identified a signaling pathway involving the G-protein Concertina (Cta) and the Rho activator RhoGEF2. Although these regulators act to promote stable myosin accumulation and apical cell constriction, loss-of-function phenotypes for each of these pathway members is not equivalent, suggesting the existence of additional ventral furrow regulators. Here, we report the identification of Abelson kinase (Abl) as a novel ventral furrow regulator. We find that Abl acts apically to suppress the accumulation of both Enabled (Ena) and actin in mesodermal cells during ventral furrow formation. Further, RhoGEF2 also regulates ordered actin localization during ventral furrow formation, whereas its activator, Cta, does not. Taken together, our data suggest that there are two crucial preconditions for apical constriction in the ventral furrow: myosin stabilization/activation, regulated by Cta and RhoGEF2; and the organization of apical actin, regulated by Abl and RhoGEF2. These observations identify an important morphogenetic role for Abl and suggest a conserved mechanism for this kinase during apical cell constriction.     

Sequence of the twist gene and nuclear localization of its protein in endomesodermal cells of early Drosophila embryos.

The twist gene is involved in the establishment of germ layers in Drosophila embryos: twist homozygous mutant embryos fail to form the ventral furrow at gastrulation and lack mesoderm and all internal organs. We have determined the sequence of the twist gene, that contains 'CAX' repeats in its 5' moiety, and codes for a protein of 490 amino acids. We have raised anti-twist antibodies that were used to study the distribution of the twist protein in whole mounts and tissue sections of wild-type embryos. Twist protein appears to be a nuclear protein at all developmental stages. It is present over both poles and in the midventral region (endoderm and mesoderm anlagen) at cellular blastoderm stage; later in development, it is detected within the mesodermal layer until its differentiation into somatopleura and splanchnopleura in which some cells are still labelled by anti-twist antibodies.     

Proteomic analysis reveals CCT is a target of Fragile X mental retardation protein regulation in Drosophila

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that is required for the translational regulation of specific target mRNAs. Loss of FMRP causes Fragile X syndrome (FXS), the most common form of inherited mental retardation in humans. Understanding the basis for FXS has been limited because few in vivo targets of FMRP have been identified and mechanisms for how FMRP regulates physiological targets are unclear. We have previously demonstrated that Drosophila FMRP (dFMRP) is required in early embryos for cleavage furrow formation. In an effort to identify new targets of dFMRP-dependent regulation and new effectors of cleavage furrow formation, we used two-dimensional difference gel electrophoresis and mass spectrometry to identify proteins that are misexpressed in dfmr1 mutant embryos. Of the 28 proteins identified, we have identified three subunits of the Chaperonin containing TCP-1 (CCT) complex as new direct targets of dFMRP-dependent regulation. Furthermore, we found that the septin Peanut, a known effector of cleavage, is a likely conserved substrate of fly CCT and is mislocalized in both cct and in dfmr1 mutant embryos. Based on these results we propose that dFMRP-dependent regulation of CCT subunits is required for cleavage furrow formation and that at least one of its substrates is affected in dfmr1− embryos suggesting that dFMRP-dependent regulation of CCT contributes to the cleavage furrow formation phenotype.     

The program of protein synthesis during the development of the micromere-primary mesenchyme cell line in the sea urchin embryo

Changes in the pattern of protein synthesis were analyzed during the in vitro development of the micromere-primary mesenchyme cell line of the sea urchin embryo. Micromeres were isolated and cultured from 16-cell stage embryos, and primary mesenchyme cells were isolated and cultured from early gastrulae. Both cell isolates developed normally in culture with about the same timing as their in situ counterparts in control embryos. Newly synthesized proteins were labeled with [³H]valine at several stages of development and were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorgraphy. The electrophoretic pattern of labeled proteins changed dramatically during development. More than half of the analyzed proteins underwent qualitative or quantitative changes in their relative rates of valine incorporation and these changes were highly specific to this cell line. Almost all of the changes were initiated prior to gastrulation and many prior to hatching. The highest frequency of changes in the micromere pattern of protein synthesis occurred between hatching and the start of gastrulation. This peak of activity coincided with the normal time of ingression of the primary mesenchyme and preceded the differentiation of spicules by more than 30 hr. Most of the observed changes were characterized as either decreases in the synthesis of proteins that showed maximum incorporation at the 16-cell stage or increases in the synthesis of proteins that showed maxima in the fully differentiated cells. Very few proteins exhibited transient synthetic maxima at intermediate stages. Thus, the program of protein synthesis associated with the development of micromeres consists largely of a switch in emphasis from early to late proteins, with the primary time of switching being between hatching and the onset of gastrulation.     

A Small Genomic Region Containing Several Loci Required for Gastrulation in Drosophila

Genetic screens in Drosophila designed to search for loci involved in gastrulation have identified four regions of the genome that are required zygotically for the formation of the ventral furrow. For three of these, the genes responsible for the mutant phenotypes have been found. We now describe a genetic characterization of the fourth region, which encompasses the cytogenetic interval 24C3-25B, and the mapping of genes involved in gastrulation in this region. We have determined the precise breakpoints of several existing deficiencies and have generated new deficiencies. Our results show that the region contains at least three different loci associated with gastrulation effects. One maternal effect gene involved in ventral furrow formation maps at 24F but could not be identified. For a second maternal effect gene which is required for germ band extension, we identify a candidate gene, CG31660, which encodes a G protein coupled receptor. Finally, one gene acts zygotically in ventral furrow formation and we identify it as Traf4.     

Scanning electron microscopy of Drosophila melanogaster embryogenesis. II. Gastrulation and segmentation.

The sequence of gastrulation events in Drosophila melanogaster, starting with the cellular blastoderm and culminating in a segmented embryo, have been studied with scanning electron microscopy (SEM). Extensive use is made of dissected embryos to illustrate changes taking place within the embryo during gastrulation. During the first 15 min of gastrulation, the mesodermal portion of the germ band is established by the invagination of approximately 1000 cells through the ventral furrow. The primordia for the proctodeum and hindgut are shown to form during early gastrulation. Detailed examination of the surfaces of invaginating primordia shows similarities to other systems and suggests possible underlying mechanisms. Germ band elongation and the formation of the amnioserosa are described. At the time of segmentation, three pairs of rudimentary cephalic appendages develop posterior to the cephalic furrow. Tracheal pits invaginate on all eight abdominal segments and on the second and third thoracic segments. Modifications of the embryonic fate map are discussed.     

Protein synthesis in dorsal and ventral regions of Xenopus laevis embryos in relation to dorsal and ventral differentiation

Two-dimensional gel electrophoresis has been used to analyze protein synthesis in dorsal and ventral regions in embryonic stages of Xenopus laevis. Proteins specific either to dorsal or to ventral regions are synthesized for the first time at gastrulation, concomitant with morphological differentiation. The reliability of these proteins as markers of dorsal and ventral differentiation was tested by examining their synthesis in Uv-irradiated embryos, which have severely reduced capacity for dorsal development, reflected in reduced levels of the neuromuscular-specific enzyme acetylcholinesterase, but which continue to synthesize the great majority of proteins at normal rates. Synthesis of dorsal indicator proteins should be reduced or absent in these embryos, whereas ventral indicators should be synthesized at least to the same extent as in control embryos. Some of the putative dorsal and ventral indicators failed this test, but the majority were confirmed as reliable markers of dorsal and ventral differentiation, thus providing a connection between morphology and gene expression in the establishment of the dorsal-ventral axis in X. laevis.     

Rho mediates cytokinesis and epiboly via ROCK in zebrafish

To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3-exoenzyme (C3) into zebrafish embryos. We found that C3 inhibited cytokinesis during early cleavages. C3 inhibition appeared to be specific on RhoA, since the constitutively active RhoA could partially rescued the C3-induced defects. Distributions of actin and the cleavage furrow associated beta-catenin were disrupted by C3. Belbbistatin, a myosin II inhibitor, also caused blastomeres disintegration. It suggested that Rho mediates cytokinesis via cleavage furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements during epiboly and gastrulation as evident by the impairment on no tail and goosecoid expression in blastoderm front runner cells and the dorsal lip of blastopore, respectively. Y-27632, an antagonist of Rho-associated kinase (ROK/ROCK), had the similar inhibitory effects on zebrafish development as the C3 treatments. Taken together, these results suggest that Rho mediates cleavage furrow protein assembly during cytokinesis and cellular migration during epiboly and gastrulation via a ROK/ROCK-dependent pathway.     

Two-step induction of primitive erythrocytes in Xenopus laevis embryos: signals from the vegetal endoderm and the overlying ectoderm

Primitive blood cells differentiate from the ventral mesoderm blood islands in Xenopus embryos. In order to determine the tissue interactions that propagate blood formation in early embryogenesis, we used embryos that had the ventral cytoplasm removed. These embryos gastrulated normally, formed a mesodermal layer and lacked axial structures, but displayed a marked enhancement of alpha-globin expression. Early ventral markers, such as msx-1, vent-1 and vent-2 were highly expressed at the gastrula stage, while a dorsal marker, goosecoid, was diminished. Several lines of experimental evidence demonstrate the critical role of animal pole-derived ectoderm in blood cell formation: 1) Mesoderm derived from dorsal blastomeres injected with beta-galactosidase mRNA (as a lineage tracer) expressed alpha-globin when interfaced with an animal pole-derived ectodermal layer; 2) Embryos in which the animal pole tissue had been removed by dissection at the blastula stage failed to express alpha-globin; 3) Exogastrulated embryos that lacked an interaction between the mesodermal and ectodermal layers failed to form blood cells, while muscle cells were observed in these embryos. Using dominant-negative forms of the BMP-4 and ALK-4 receptors, we showed that activin and BMP-4 signaling is necessary for blood cell differentiation in ventral marginal zone explants, while FGF signaling is not essential. In ventralized embryos, inactivation of the BMP-4 signal within a localized area of the ectoderm led to suppression of globin expression in the adjacent mesoderm layer, but inactivation of the activin signal did not have this effect. These observations suggest that mesodermal cells, derived from a default pathway that is induced by the activin signal, need an additional BMP-4-dependent factor from the overlying ectoderm for further differentiation into a blood cell lineage.