Mathematical models of dorsal closure.

Dorsal closure is a model cell sheet movement that occurs midway through Drosophila embryogenesis. A dorsal hole, filled with amnioserosa, closes through the dorsalward elongation of lateral epidermal cell sheets. Closure requires contributions from 5 distinct tissues and well over 140 genes (see Mortensen et al., 2018, reviewed in Kiehart et al., 2017 and Hayes and Solon, 2017). In spite of this biological complexity, the movements (kinematics) of closure are geometrically simple at tissue, and in certain cases, at cellular scales. This simplicity has made closure the target of a number of mathematical models that seek to explain and quantify the processes that underlie closure's kinematics. The first (purely kinematic) modeling approach recapitulated well the time-evolving geometry of closure even though the underlying physical principles were not known. Almost all subsequent models delve into the forces of closure (i.e. the dynamics of closure). Models assign elastic, contractile and viscous forces which impact tissue and/or cell mechanics. They write rate equations which relate the forces to one another and to other variables, including those which represent geometric, kinematic, and or signaling characteristics. The time evolution of the variables is obtained by computing the solution of the model's system of equations, with optimized model parameters. The basis of the equations range from the phenomenological to biophysical first principles. We review various models and present their contribution to our understanding of the molecular mechanisms and biophysics of closure. Models of closure will contribute to our understanding of similar movements that characterize vertebrate morphogenesis.

Emergent properties during dorsal closure in Drosophila morphogenesis.

Dorsal closure is an essential stage of Drosophila development that is a model system for research in morphogenesis and biological physics. Dorsal closure involves an orchestrated interplay between gene expression and cell activities that produce shape changes, exert forces and mediate tissue dynamics. We investigate the dynamics of dorsal closure based on confocal microscopic measurements of cell shortening in living embryos. During the mid-stages of dorsal closure we find that there are fluctuations in the width of the leading edge cells but the time-averaged analysis of measurements indicate that there is essentially no net shortening of cells in the bulk of the leading edge, that contraction predominantly occurs at the canthi as part of the process for zipping together the two leading edges of epidermis and that the rate constant for zipping correlates with the rate of movement of the leading edges. We characterize emergent properties that regulate dorsal closure, i.e., a velocity governor and the coordination and synchronization of tissue dynamics.

Upregulation of forces and morphogenic asymmetries in dorsal closure during Drosophila development.

Tissue dynamics during dorsal closure, a stage of Drosophila development, provide a model system for cell sheet morphogenesis and wound healing. Dorsal closure is characterized by complex cell sheet movements, driven by multiple tissue specific forces, which are coordinated in space, synchronized in time, and resilient to UV-laser perturbations. The mechanisms responsible for these attributes are not fully understood. We measured spatial, kinematic, and dynamic antero-posterior asymmetries to biophysically characterize both resiliency to laser perturbations and failure of closure in mutant embryos and compared them to natural asymmetries in unperturbed, wild-type closure. We quantified and mathematically modeled two processes that are upregulated to provide resiliency--contractility of the amnioserosa and formation of a seam between advancing epidermal sheets, i.e., zipping. Both processes are spatially removed from the laser-targeted site, indicating they are not a local response to laser-induced wounding and suggesting mechanosensitive and/or chemosensitive mechanisms for upregulation. In mutant embryos, tissue junctions initially fail at the anterior end indicating inhomogeneous mechanical stresses attributable to head involution, another developmental process that occurs concomitant with the end stages of closure. Asymmetries in these mutants are reversed compared to wild-type, and inhomogeneous stresses may cause asymmetries in wild-type closure.

Role of myosin-II phosphorylation in V12Cdc42-mediated disruption of Drosophila cellularization.

Microinjection of constitutively active Cdc42 (V12Cdc42) disrupts the actomyosin cytoskeleton during cellularization (Crawford et al., Dev. Biol., 204, 151-164 (1998)). The p21-activated kinase (PAK) family of Ser/Thr kinases are effectors of GTP-bound forms of the small GTPases, Cdc42 and Rac. Drosophila PAK, which colocalizes with actin and myosin-II during cellularization, concentrates at sites of V12Cdc42-induced actomyosin disruption. In vitro biochemical analyses demonstrate that PAK phosphorylates the regulatory light chain (RLC) of Drosophila nonmuscle myosin-II on Ser21, a site known to activate myosin-II function. Although activated PAK does not disrupt the actomyosin cytoskeleton, it induces increased levels of Ser21 phosphorylated RLC. These findings suggest that increased levels of RLC phosphorylation do not contribute to disruption of the actomyosin hexagonal array.

Protein kinase C phosphorylates nonmuscle myosin-II heavy chain from Drosophila but regulation of myosin function by this enzyme is not required for viability in flies.

Conventional myosins (myosin-IIs) generate forces for cell shape change and cell motility. Myosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be important in metazoans. To investigate this regulation in a complex eukaryote, we purified the Drosophila myosin-II tail expressed in Escherichia coli and showed that it was phosphorylated in vitro by protein kinase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece. These sites are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosin-IIs. If the two serines are mutagenized to alanine or aspartic acid, phosphorylation no longer occurs. Using a 341 amino acid tail fragment, we show that there is no difference in the salt-dependent assembly of wild-type phosphorylated and mutagenized polypeptides. Thus, the nonmuscle myosin heavy chain in Drosophila, which is encoded by the zipper gene, appears to be similar to rabbit nonmuscle myosin-IIA. In vivo, we generated transgenic flies that expressed the various myosin heavy chain variants in a zipper null or near-null genetic background. Like their wild-type counterparts, such variants are able to completely rescue the lethal phenotype due to severe zipper mutations. These results suggest that while the myosin-II heavy chain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila. Conservation during 530-1000 million years of evolution suggests that regulation by heavy chain phosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way.

zipper Nonmuscle myosin-II functions downstream of PS2 integrin in Drosophila myogenesis and is necessary for myofibril formation.

Nonmuscle myosin-II is a key motor protein that drives cell shape change and cell movement. Here, we analyze the function of nonmuscle myosin-II during Drosophila embryonic myogenesis. We find that nonmuscle myosin-II and the adhesion molecule, PS2 integrin, colocalize at the developing muscle termini. In the paradigm emerging from cultured fibroblasts, nonmuscle actomyosin-II contractility, mediated by the small GTPase Rho, is required to cluster integrins at focal adhesions. In direct opposition to this model, we find that neither nonmuscle myosin-II nor RhoA appear to function in PS2 clustering. Instead, PS2 integrin is required for the maintenance of nonmuscle myosin-II localization and we show that the cytoplasmic tail of the beta(PS) integrin subunit is capable of mediating this PS2 integrin function. We show that embryos that lack zygotic expression of nonmuscle myosin-II fail to form striated myofibrils. In keeping with this, we demonstrate that a PS2 mutant that specifically disrupts myofibril formation is unable to mediate proper localization of nonmuscle myosin-II at the muscle termini. In contrast, embryos that lack RhoA function do generate striated muscles. Finally, we find that nonmuscle myosin-II localizes to the Z-line in mature larval muscle. We suggest that nonmuscle myosin-II functions at the muscle termini and the Z-line as an actin crosslinker and acts to maintain the structural integrity of the sarcomere.

Drosophila stretchin-MLCK is a novel member of the Titin/Myosin light chain kinase family.

Members of the titin/myosin light chain kinase family play an essential role in the organization of the actin/myosin cytoskeleton, especially in sarcomere assembly and function. In Drosophila melanogaster, projectin is so far the only member of this family for which a transcription unit has been characterized. The locus of another member of this family, a protein related to Myosin light chain kinase, was also identified. The cDNA and genomic sequences published explain only the shorter transcripts expressed by this locus. Here, we report the complete molecular characterization of this transcription unit, which spans 38 kb, includes 33 exons and accounts for transcripts up to 25 kb in length. This transcription unit contains both the largest exon (12,005 nt) and the largest coding region (25,213 nt) reported so far for Drosophila. This transcription unit features both internal promoters and internal polyadenylation signals, which enable it to express seven different transcripts, ranging from 3.3 to 25 kb in size. The latter encodes a huge, titin-like, 926 kDa kinase that features two large PEVK-rich repeats, 32 immunoglobulin and two fibronectin type-III domains, which we designate stretchin-MLCK. In addition, the 3' end of the stretchin-MLCK transcription unit expresses shorter transcripts that encode 86 to 165 kDa isoforms of stretchin-MLCK that are analogous to vertebrate Myosin light chain kinases. Similarly, the 5' end of the Stretchin-Mlck transcription unit can also express transcripts encoding kettin and Unc-89-like isoforms, which share no sequences with the MLCK-like transcripts. Thus, this locus can be viewed as a single transcription unit, Stretchin-Mlck (genetic abbreviation Strn-Mlck), that expresses large, composite transcripts and protein isoforms (sequences available at http://www.academicpress.com/jmb), as well as a complex of two independent transcription units, the Stretchin and Mlck transcription units (Strn and Mlck, respectively) the result of a "gene fission" event, that encode independent transcripts and proteins with distinct structural and enzymatic functions.

Genetic analysis demonstrates a direct link between rho signaling and nonmuscle myosin function during Drosophila morphogenesis.

A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. We have explored the regulation of myosin activity by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, we screened for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. We determined that a single missense mutation in the zipper(Ebr) allele gives rise to its sensitivity to second-site noncomplementation. We then identify the Rho signal transduction pathway as necessary for proper myosin function. First we show that a lethal P-element insertion interacts genetically with zipper. Subsequently we show that this second-site noncomplementing mutation disrupts the RhoGEF2 locus. Next, we show that two EMS-induced mutations, previously shown to interact genetically with zipper(Ebr), disrupt the RhoA locus. Further, we have identified their molecular lesions and determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, we show that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. Our studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan.

Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila.

The molecular and cellular bases of cell shape change and movement during morphogenesis and wound healing are of intense interest and are only beginning to be understood. Here, we investigate the forces responsible for morphogenesis during dorsal closure with three approaches. First, we use real-time and time-lapsed laser confocal microscopy to follow actin dynamics and document cell shape changes and tissue movements in living, unperturbed embryos. We label cells with a ubiquitously expressed transgene that encodes GFP fused to an autonomously folding actin binding fragment from fly moesin. Second, we use a biomechanical approach to examine the distribution of stiffness/tension during dorsal closure by following the response of the various tissues to cutting by an ultraviolet laser. We tested our previous model (Young, P.E., A.M. Richman, A.S. Ketchum, and D.P. Kiehart. 1993. Genes Dev. 7:29-41) that the leading edge of the lateral epidermis is a contractile purse-string that provides force for dorsal closure. We show that this structure is under tension and behaves as a supracellular purse-string, however, we provide evidence that it alone cannot account for the forces responsible for dorsal closure. In addition, we show that there is isotropic stiffness/tension in the amnioserosa and anisotropic stiffness/tension in the lateral epidermis. Tension in the amnioserosa may contribute force for dorsal closure, but tension in the lateral epidermis opposes it. Third, we examine the role of various tissues in dorsal closure by repeated ablation of cells in the amnioserosa and the leading edge of the lateral epidermis. Our data provide strong evidence that both tissues appear to contribute to normal dorsal closure in living embryos, but surprisingly, neither is absolutely required for dorsal closure. Finally, we establish that the Drosophila epidermis rapidly and reproducibly heals from both mechanical and ultraviolet laser wounds, even those delivered repeatedly. During healing, actin is rapidly recruited to the margins of the wound and a newly formed, supracellular purse-string contracts during wound healing. This result establishes the Drosophila embryo as an excellent system for the investigation of wound healing. Moreover, our observations demonstrate that wound healing in this insect epidermal system parallel wound healing in vertebrate tissues in situ and vertebrate cells in culture (for review see Kiehart, D.P. 1999. Curr. Biol. 9:R602-R605).

Wound healing: The power of the purse string.

Recently, Xenopus oocytes have been shown to repair wounds using a contractile system composed of actin and myosin-II. The work underscores the importance of actin-based myosin-II contractility in cellular and supracellular 'purse strings' that function in diverse biological processes.

Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein.

Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein. These FN-green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.

Cellularization in Drosophila melanogaster is disrupted by the inhibition of rho activity and the activation of Cdc42 function.

Regulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis. Drosophila melanogaster embryos offer a well-defined system for observing alterations in the cytoskeleton during the process of cellularization, a specialized form of cytokinesis. During cellularization, the actomyosin cytoskeleton forms a hexagonal array and drives invagination of the plasma membrane between the nuclei located at the cortex of the syncytial blastoderm. Rho, Rac, and Cdc42 proteins are members of the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenance of the actin cytoskeleton throughout phylogeny and in D. melanogaster. To investigate how Rho subfamily activity affects the cytoskeleton during cellularization stages, embryos were microinjected with C3 exoenzyme from Clostridium botulinum or with wild-type, constitutively active, or dominant negative versions of Rho, Rac, and Cdc42 proteins. C3 exoenzyme ADP-ribosylates and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound state to activate downstream effectors. Dominant negative mutations likely inhibit endogenous small G protein activity by sequestering exchange factors. Of the 10 agents microinjected, C3 exoenzyme, constitutively active Cdc42, and dominant negative Rho have a specific and indistinguishable effect: the actomyosin cytoskeleton is disrupted, cellularization halts, and embryogenesis arrests. Time-lapse video records of DIC imaged embryos show that nuclei in injected regions move away from the cortex of the embryo, thereby phenocopying injections of cytochalasin or antimyosin. Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is disrupted in a dose-dependent fashion. Additionally, DNA stain reveals that nuclei in the microinjected embryos aggregate in regions that correspond to actin disruption. These embryos halt in cellularization and do not proceed to gastrulation. We conclude that Rho activity and Cdc42 regulation are required for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning.

Volume changes induced by osmotic stress in freshly isolated rat hippocampal neurons.

The degree to which osmotic stress changes the volume of mammalian central neurons has not previously been determined. We isolated CA1 pyramidal cells and measured cell volume in four different ways. Extracellular osmolarity (pio) was lowered by omitting varying amounts of NaCl and raised by adding mannitol; the extremes of pio tested ranged from 134 to 396 mosm/kg. When pio was reduced, cell swelling varied widely. We distinguished three types of cells according to their response: "yielding cells" whose volume began to increase immediately; "delayed response cells" which swelled after a latent period of 2 min or more; and "resistant cells" whose volume did not change during exposure to hypo-osmotic solution. When pio was raised, most cells shrank slowly, reaching minimal volume in 15-20 min. We observed neither a regulatory volume decrease nor an increase. We conclude that the water permeability of the membrane of hippocampal CA1 pyramidal neurons is low compared to that of other cell types. The mechanical support of the plasma membrane given by the cytoskeleton may contribute to the resistance to swelling and protect neurons against swelling-induced damage.

Drosophila betaHeavy-spectrin is essential for development and contributes to specific cell fates in the eye.

The spectrin membrane skeleton is a ubiquitous cytoskeletal structure with several cellular roles, including the maintenance of cell integrity, determination of cell shape and as a contributor to cell polarity. We have isolated mutations in the gene encoding &bgr ;Heavy-spectrin in Drosophila, and have named this essential locus karst. karst mutant individuals have a pleiotropic phenotype characterized by extensive larval lethality and, in adult escapers, rough eyes, bent wings, tracheal defects and infertility. Within karst mutant eyes, a significant number of ommatidia specifically lack photoreceptor R7 alongside more complex morphological defects. Immunolocalization of betaHeavy-spectrin in wild-type eye-antennal and wing imaginal discs reveals that betaHeavy-spectrin is present in a restricted subdomain of the membrane skeleton that colocalizes with DE-cadherin. We propose a model where normal levels of Sevenless signaling are dependent on tight cell-cell adhesion facilitated by the betaHeavy-spectrin membrane skeleton. Immunolocalization of betaHeavy-spectrin in the adult and larval midgut indicates that it is a terminal web protein, but we see no gross morphological defects in the adult apical brush border in karst mutant flies. Rhodamine phalloidin staining of karst mutant ovaries similarly reveals no conspicuous defect in the actin cytoskeleton or cellular morphology in egg chambers. This is in contrast to mutations in alpha-spectrin, the molecular partner of betaHeavy-spectrin, which affect cellular structure in both the larval gut and adult ovaries. Our results emphasize the fundamental contribution of the spectrin membrane skeleton to normal development and reveals a critical interplay between the integrity of a cell's membrane skeleton, the structure of cell-cell contacts and cell signaling.

Second-site noncomplementation identifies genomic regions required for Drosophila nonmuscle myosin function during morphogenesis.

Drosophila is an ideal metazoan model system for analyzing the role of nonmuscle myosin-II (henceforth, myosin) during development. In Drosophila, myosin function is required for cytokinesis and morphogenesis driven by cell migration and/or cell shape changes during oogenesis, embryogenesis, larval development and pupal metamorphosis. The mechanisms that regulate myosin function and the supramolecular structures into which myosin incorporates have not been systematically characterized. The genetic screens described here identify genomic regions that uncover loci that facilitate myosin function. The nonmuscle myosin heavy chain is encoded by a single locus, zipper. Contiguous chromosomal deficiencies that represent approximately 70% of the euchromatic genome were screened for genetic interactions with two recessive lethal alleles of zipper in a second-site noncomplementation assay for the malformed phenotype. Malformation in the adult leg reflects aberrations in cell shape changes driven by myosin-based contraction during leg morphogenesis. Of the 158 deficiencies tested, 47 behaved as second-site noncomplementors of zipper. Two of the deficiencies are strong interactors, 17 are intermediate and 28 are weak. Finer genetic mapping reveals that mutations in cytoplasmic tropomyosin and viking (collagen IV) behave as second-site noncomplementors of zipper during leg morphogenesis and that zipper function requires a previously uncharacterized locus, E3.10/J3.8, for leg morphogenesis and viability.

Intragenic duplication and divergence in the spectrin superfamily of proteins.

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)-spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.

GFP-moesin illuminates actin cytoskeleton dynamics in living tissue and demonstrates cell shape changes during morphogenesis in Drosophila.

Moesin, ezrin, and radixin (MER) are components of the cortical actin cytoskeleton and membrane processes such as filopodia and microvilli. Their C-terminal tails contain an extended region that is predicted to be helical, an actin binding domain, and a region(s) that participates in self-association. We engineered an in vivo fluorescent actin binding protein (GFP-moe) by joining sequences that encode the jellyfish green fluorescent protein (GFP) to sequences that encode the C-terminal end of the sole Drosophila MER homolog, moesin [Moesin-like gene product, referred to previously as the D17 MER-like protein; Edwards et al., 1994, Proc. Natl. Acad. Sci. USA 91, 4589], and Dmoesin [McCartney and Fehon, 1996, J. Cell Biol. 133, 843]. Transgenic flies expressing this fusion protein under control of the hsp70 promoter were generated and used for analysis of cell shape changes during morphogenesis of various developmental stages and tissues. Following heat shock, high levels of stable fusion protein are produced by all somatic tissues. GFP-moe localizes to the cortical actin cytoskeleton, providing a strong in vivo marker for cell shape and pattern during epithelial morphogenesis. The protein also becomes highly enriched in pseudopods, microvilli, axons, denticles, the border cell process, and other membrane projections, potentially by binding to endogenous moesin as well as actin. We show that GFP-moe can be used to examine the development and behavior of these dynamic structures in live specimens. We observe a bright green fluorescent, presumably actin-rich, polar cell proboscis that inserts itself into the forming micropyle and appears to maintain an opening for sperm passage around which the chorion is formed. We also confirm the existence of an actin-rich purse string at the leading edge of the lateral epidermis and provide a dynamic analysis of its behavior as it migrates during dorsal closure. Observations of embryos, larvae, and pupae show that GFP-moe is also useful for labeling the developing nervous system and will be a good general marker of dynamic cell behavior during morphogenesis in live tissues and demonstrate that fusion of a subcellular localization signal to GFP greatly increases its utility as a cell marker.

The role of HOM-C genes in segmental transformations: reexamination of the Drosophila Sex combs reduced embryonic phenotype.

Homeotic genes in the Antennapedia Complex of Drosophila specify identity of the posterior head segments; the labial segment requires Sex combs reduced (Scr) for proper development, Deformed (Dfd) specifies maxillary and mandibular identity, and labial is necessary for intercalary segment identity. Although mutations in these genes cause homeotic transformations during imago development, the only obvious homeotic transformation during embryonic head development is found in Scr mutants, where a partial transformation of the labial segment to a more anterior, maxillary identity has been reported. This transformation is unusual because DFD protein does not accumulate in the labial cells of Scr mutants, although DFD is required for development of maxillary structures. Here, we present evidence that casts doubt on whether the labial to maxillary transformation actually exists in embryos lacking Scr. The observed morphological characteristics and gene expression patterns of various mutant embryos indicate a loss of segmental identity rather than a transformation.