The synthesis-diffusion-degradation model explains Bicoid gradient formation in unfertilized eggs.

Precise formation of morphogen gradients is essential to the establishment of reproducible pattern in development. Mechanisms proposed for obtaining the requisite precision range from simple models with few parameters to more complex models involving many regulated quantities. The synthesis-diffusion-degradation (SDD) model is a relatively simple model explaining the formation of the Bicoid gradient in Drosophila melanogaster, in which the steady-state characteristic length of the gradient is determined solely by the rates of diffusion and degradation of the morphogen. In this work, we test the SDD model in unfertilized D. melanogaster eggs, which contain a single female pronucleus and lack the nuclear division cycles and other zygotic regulatory processes seen in fertilized eggs. Using two-photon live imaging as well as a novel method for quantitative imaging based on decorrelation of photoswitching waveforms, we find that the Bicoid gradient is longer and shallower in unfertilized eggs as compared to the gradient at the same time points in fertilized eggs. Using a means of measuring the Bicoid lifetime by conjugation to a photoconvertible fluorophore, we find that the lifetime is correspondingly longer in unfertilized eggs, providing qualitative and quantitative agreement with the predictions of the SDD model.

Cell and developmental biology--a shared past, an intertwined future.

Cell and developmental biology are distinct disciplines with clear differences in emphasis and domains of interest, yet they also share a common historic origin and benefit from an increasingly productive exchange of insights and influences. Our goal in this commentary is to examine the common origin of cell and developmental biology, to explore ways in which they currently interact, and to consider the connections and differences that exist between these two fields.

Regulated expression of nullo is required for the formation of distinct apical and basal adherens junctions in the Drosophila blastoderm.

During cellularization, the Drosophila embryo undergoes a large-scale cytokinetic event that packages thousands of syncytial nuclei into individual cells, resulting in the de novo formation of an epithelial monolayer in the cortex of the embryo. The formation of adherens junctions is one of the many aspects of epithelial polarity that is established during cellularization: at the onset of cellularization, the Drosophila beta-catenin homologue Armadillo (Arm) accumulates at the leading edge of the cleavage furrow, and later to the apicolateral region where the zonula adherens precursors are formed. In this paper, we show that the basal accumulation of Arm colocalizes with DE-cadherin and Dalpha-catenin, and corresponds to a region of tight membrane association, which we refer to as the basal junction. Although the two junctions are similar in components and function, they differ in their response to the novel cellularization protein Nullo. Nullo is present in the basal junction and is required for its formation at the onset of cellularization. In contrast, Nullo is degraded before apical junction formation, and prolonged expression of Nullo blocks the apical clustering of junctional components, leading to morphological defects in the developing embryo. These observations reveal differences in the formation of the apical and basal junctions, and offer insight into the role of Nullo in basal junction formation.

armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila.

Cellularization of the Drosophila embryo results in the formation of a cell monolayer with many characteristics of a polarized epithelium. We have used antibodies specific to cellular junctions and nascent plasma membranes to study the formation of the zonula adherens (ZA) in relation to the establishment of basolateral membrane polarity. The same approach was then used as a test system to identify X-linked zygotically active genes required for ZA formation. We show that ZA formation begins during cellularization and that the basolateral membrane domain is established at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome, we have identified genes that are required for the formation of the ZA and the generation of basolateral membrane polarity. We show that embryos mutant for both stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to establish the ZA, the formation of the monolayered epithelium is disrupted after cellularization, resulting in formation of a multilayered cell sheet by mid-gastrulation. SEM analysis of mutant embryos revealed a conversion of cells exhibiting epithelial characteristics into cells exhibiting mesenchymal characteristics. To investigate how mutations that affect an integral component of the ZA itself influence ZA formation, we examined embryos with reduced maternal and zygotic supply of wild-type Arm protein. These embryos, like embryos mutant for both sdt and baz, exhibit an early disruption of ZA formation. These results suggest that early stages in the assembly of the ZA are critical for the stability of the polarized blastoderm epithelium.

Polarized insertion of new membrane from a cytoplasmic reservoir during cleavage of the Drosophila embryo.

Cellularization of the Drosophila embryo is a specialized form of cytokinesis that results in the formation of a polarized epithelium. The mechanisms of membrane growth during cytokinesis are largely unknown. It is also unclear whether membrane growth and polarization represent distinct processes that occur simultaneously or whether growth of the membrane is involved in the emergence of polarity. Using a combination of surface labeling and particles tracking techniques, we monitored the dynamics of marked membrane regions during cellularization. We find that the major source of membrane is intracellular, rather than in the form of a plasma membrane reservoir. Depolymerization of microtubules inhibits the export of a newly synthesized transmembrane protein from the Golgi apparatus to the plasma membrane and simultaneously blocks membrane growth. Membrane insertion occurs in a defined sequence at specific sites, first apical, then apical-lateral. Diffusion of the membrane appears insufficient to compete with the massive local insertion of new membrane. We thus identify a tightly regulated scheme of polarized membrane insertion during cellularization. We propose that such a mechanism could participate in the progressive emergence of apical-basal polarity.

Maternal effect mutations of the sponge locus affect actin cytoskeletal rearrangements in Drosophila melanogaster embryos.

In the syncytial blastoderm stage of Drosophila embryogenesis, dome-shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically required for the formation of actin caps and metaphase furrows. The specificity of the sponge phenotype permits dissection of both the process of actin cap formation and the functions of actin caps and metaphase furrows. Our data demonstrate that the distribution of actin binding protein 13D2 is unaffected in sponge embryos and suggest that 13D2 is upstream of actin in cortical cap assembly. Although actin caps and metaphase furrows have been implicated in maintaining the fidelity of nuclear division and the positions of nuclei within the cortex, our observations indicate that these structures are dispensible during the early syncytial blastoderm cell cycles. A later requirement for actin metaphase furrows in preventing the nucleation of mitotic spindles between inappropriate centrosomes is observed. Furthermore, the formation of actin caps and metaphase furrows is not a prerequisite for the formation of the hexagonal array of actin instrumental in the conversion of the syncytial embryo into a cellular blastoderm.

The vertebrate adhesive junction proteins beta-catenin and plakoglobin and the Drosophila segment polarity gene armadillo form a multigene family with similar properties.

Three proteins identified by quite different criteria in three different systems, the Drosophila segment polarity gene armadillo, the human desmosomal protein plakoglobin, and the Xenopus E-cadherin-associated protein beta-catenin, share amino acid sequence similarity. These findings raise questions about the relationship among the three molecules and their roles in different cell-cell adhesive junctions. We have found that antibodies against the Drosophila segment polarity gene armadillo cross react with a conserved vertebrate protein. This protein is membrane associated, probably via its interaction with a cadherin-like molecule. This cross-reacting protein is the cadherin-associated protein beta-catenin. Using anti-armadillo and antiplakoglobin antibodies, it was shown that beta-catenin and plakoglobin are distinct molecules, which can coexist in the same cell type. Plakoglobin interacts with the desmosomal glycoprotein desmoglein I, and weakly with E-cadherin. Although beta-catenin interacts tightly with E-cadherin, it does not seem to be associated with either desmoglein I or with isolated desmosomes. Anti-armadillo antibodies have been further used to determine the intracellular localization of beta-catenin, and to examine its tissue distribution. The implications of these results for the structure and function of different cell-cell adhesive junctions are discussed.

Dynein-mediated cargo transport in vivo. A switch controls travel distance.

Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Here, we use mutations in the dynein heavy chain gene to impair the motor's function, and employ biophysical measurements to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows us to quantitate dynein-mediated cargo motion in vivo, providing a framework for investigating how dynein's activity is controlled. We identify three distinct travel states whose general features also characterize plus end motion. These states are preserved in different developmental stages. We had previously provided evidence that for each travel direction, single droplets are moved by multiple motors of the same type (Welte et al. 1998). Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, we propose the existence of a process that ends runs before the motors fall off the microtubules. We find that this process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport.

A novel basic helix-loop-helix protein is expressed in muscle attachment sites of the Drosophila epidermis.

We have found that a novel basic helix-loop-helix (bHLH) protein is expressed almost exclusively in the epidermal attachments sites for the somatic muscles of Drosophila melanogaster. A Drosophila cDNA library was screened with radioactively labeled E12 protein, which can dimerize with many HLH proteins. One clone that emerged from this screen encoded a previously unknown protein of 360 amino acids, named delilah, that contains both basic and HLH domains, similar to a group of cellular transcription factors implicated in cell type determination. Delilah protein formed heterodimers with E12 that bind to the muscle creatine kinase promoter. In situ hybridization with the delilah cDNA localized the expression of the gene to a subset of cells in the epidermis which form a distinct pattern involving both the segmental boundaries and intrasegmental clusters. This pattern was coincident with the known sites of attachment of the somatic muscles to tendon cells in the epidermis. delilah expression persists in snail mutant embryos which lack mesoderm, indicating that expression of the gene was not induced by attachment of the underlying muscles. The similarity of this gene to other bHLH genes suggests that it plays an important role in the differentiation of epidermal cells into muscle attachment sites.

Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology.

In mutagenesis screens for recessive female sterile mutations on the second chromosome of Drosophila melanogaster 528 lines were isolated which allow the homozygous females to survive but cause sterility. In 62 of these lines early stages of oogenesis are affected, and these females usually do not lay any eggs. In 333 lines oogenesis proceeds apparently normally to stage 8 of oogenesis, but morphological defects become often apparent during later stages of oogenesis, and are visible in the defective eggs produced by these females whereas 133 lay eggs that appear morphologically normal, but do not support normal embryonic development. Of the lines 341 have been genetically characterized and define a total of 140 loci on the second chromosome. Not all the loci are specific for oogenesis. From the numbers obtained we estimate that the second chromosome of Drosophila contains about 13 loci that are relatively specific for early oogenesis, 70 loci that are specifically required in mid to late oogenesis, and around 30 maternal-effect lethals.

Female sterile mutations on the second chromosome of Drosophila melanogaster. I. Maternal effect mutations.

In mutagenesis screens for recessive female sterile mutations on the second chromosome of Drosophila melanogaster 529 chromosomes were isolated which allow the homozygous females to survive, but cause them to be sterile. In 136 of these lines, mutant females produce morphologically normal eggs which cannot support normal embryonic development. These "maternal-effect" mutations fall into 67 complementation groups which define 23 multiply hit and 44 singly hit loci. In eggs from 14 complementation groups development is blocked before the formation of a syncytial blastoderm. In eggs from 12 complementation groups development is abnormal before cellularization, 17 complementation groups cause abnormal cellularization, 12 complementation groups cause changes in cellular morphology in early gastrulation stages, and 12 complementation groups seem to affect later embryonic development.

Signaling activities of the Drosophila wingless gene are separately mutable and appear to be transduced at the cell surface.

The Drosophila segment polarity gene wingless encodes an intercellular signaling molecule that transmits positional information during development of the embryonic epidermis. We have explored the mechanism of wg signal transduction by perturbing cellular processes genetically and by performing structure/function analysis of the Wg protein. We present evidence that Wingless protein may transduce signal at the cell surface and that Wg may bind to its cell surface receptor without necessarily activating it. We demonstrate that two specific signaling activities of the Wg molecule can be disrupted independently by mutation. Sequence analysis indicates that these different signaling activities are not promoted by discrete functional domains, but rather than the overall conformation of the molecule may control distinct signaling functions. We conclude that wg signaling may involve complex interactions between the Wg ligand and its cell surface receptor molecule(s) and that some of this complexity resides within the Wg ligand itself.

Dominant maternal-effect mutations of Drosophila melanogaster causing the production of double-abdomen embryos.

Dominant mutations at two loci, BicaudalC (BicC) and BicaudalD (BicD), cause heterozygous females to produce double-abdomen embryos. These mutations cause the production of embryos with a range of defects extending from the anterior end of the differentiated embryo. The same array of defective embryos is caused by mutations at either locus and is similar to that produced by the original mutation at bicaudal (bic). The array of defective embryos suggests that these mutations cause the loss of positional values from the anterior end of the embryo, associated with a duplication of the posterior end if too few positional values remain. BicaudalD mutations appear to be antimorphic, gain-of-function mutations, whereas BicaudalC mutations are likely to be hypomorphic or amorphic mutations. Mutations at all these loci (bic, BicC and BicD) act as mutual enhancers of each other, and a number of other maternal-effect mutations also act to either enhance or suppress the expression of these dominant bicaudal mutations.

A cell marker system and mosaic patterns during early embryonic development in Drosophila melanogaster.

An embryonic cell marker system has been developed in Drosophila melanogaster that has enabled us to identify the genotype of cells as early as the cellular blastoderm stage of development. This system allows unambiguous detection of embryos homozygous for most X-linked lethal mutations at stages prior to when their first defects become obvious. By examining gynandromorphs at this stage, we have observed that the number of nuclei per unit area in male regions is about half that in female regions. An examination of early cleavage stage embryos whose DNA has been stained with Hoechst 33258 and whose actin has been stained with phalloidin suggests that this difference is due to a cell cycle delay in cells losing the ring-X. These experiments also demonstrate the existence of a mechanism which controls the timing of nuclear divisions in cycle 10-14 embryos.

Precise domain specification in the developing Drosophila embryo.

A simple morphogen gradient based on the protein bicoid is insufficient to explain the precise (i.e., similar in all embryos) setting of anteroposterior gene expression domains in the early Drosophila embryo. We present here an alternative model, based on quantitative data, which accounts for all of our observations. The model also explains the robustness of hunchback boundary setting in unnatural environments such as published recently [Luccheta, Nature 434, 1134 (2005)]. The model is based on the existence of a secondary gradient correlated to bicoid through protein degradation by the same agent.

Probing for gene specificity in epithelial development.

We surveyed a total of 228 random insertions of a P[GawB] element to determine the fraction of regulatory regions in the Drosophila genome that activate gene expression specifically in follicle cells versus producing more complex patterns of expression. We monitored the GAL4 expression encoded by this construct in the ovarian follicle cells by crossing the lines to a strain containing a lacZ reporter construct. Sixty four per cent of the insertions showed ovarian expression. To assess the specificity of this expression, 124 of the 228 lines were crossed to strains containing either an activated form of Armadillo, the Drosophila homolog of beta-catenin, or an activated form of Torpedo/Egfr, the Drosophila homolog of the Epidermal Growth Factor receptor, under the control of GAL4 target sites. The lethality and imaginal disc phenotypes observed in these crosses suggest that most random insertions cause GAL4 expression in a variety of tissues. Very few insertions appear to drive expression only in follicle cells. Although the activated form of Armadillo produced higher frequencies of lethality and disk phenotypes, expression in the follicle cell epithelium at later stages of oogenesis did not lead to a visible phenotype. This contrasts with the dorsalized phenotypes observed in the combination of the same GAL4 lines with the activated Torpedo construct.

Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila.

Nine maternal-effect loci Drosophila melanogaster were tested in germline mosaics to determine whether the wildtype gene activity is required in somatic or germline components of the maternal ovary. Mutations in these loci affect the anterior-posterior or dorso-ventral body pattern. In all nine loci (torso, trunk, exuperantia, vasa, valois, staufen, tudor, dorsal, Toll) a mutant genotype in the germ cells is sufficient to produce all aspects of the mutant embryonic phenotype, even when those germ cells are surrounded by wildtype somatic tissues.

Mutations in the Drosophila gene extradenticle affect the way specific homeo domain proteins regulate segmental identity.

We characterized a gene, extradenticle, which seems to interact with a specific subset of Drosophila homeo domain proteins, possibly affecting their target specificity. This interpretation is based on an examination of the zygotic and maternal effect phenotypes of extradenticle mutations. In embryos with reduced levels of extradenticle gene product, anterior and posterior segmental transformations occur. Segmental identity in Drosophila is mediated by the products of the Antennapedia and bithorax complexes. These homeo domain proteins are thought to regulate different target genes specifically in each segment, resulting in different morphologies. extradenticle alters segmental identity without affecting the pattern of expression of homeotic genes. Genetic tests demonstrate that in extradenticle mutants, the homeotic proteins are functional and act in their normal segmental domains, yet segmental identities are altered. Even when homeotic proteins are ectopically expressed under the control of a heterologous promoter, extradenticle mutations affect their consequences. Thus, in the absence of sufficient extradenticle product, altered segmental morphology results from alteration of the functional consequences of specific homeo domain proteins, possibly through alterations in their target gene specificity. extradenticle is also expressed maternally. Complete removal of extradenticle, maternally and zygotically, leads to specific alterations in segmentation, many of which result from failure to maintain the expression of the homeo domain protein engrailed.