The cytoskeleton and morphogenesis of the early Drosophila embryo.

Drosophila embryogenesis begins with thirteen mitotic divisions that occur without cytokinesis. During these syncytial divisions, a series of stereotyped nuclear movements produce a syncytial blastoderm embryo that is characterized by a uniform monolayer of cortical nuclei. Inhibitor studies indicate that actin filaments and microtubules mediate the coordinated nuclear movements of the syncytial stages of embryogenesis. Recent genetic and cytological analyses provide new insight into the functions of specific microtubule and actin filament arrays in organizing the syncytial embryo, and these may lead to the identification of novel regulatory and structural components of the cytoskeleton.

DNA-replication/DNA-damage-dependent centrosome inactivation in Drosophila embryos.

During early embryogenesis of Drosophila melanogaster, mutations in the DNA-replication checkpoint lead to chromosome-segregation failures. Here we show that these segregation failures are associated with the assembly of an anastral microtubule spindle, a mitosis-specific loss of centrosome function, and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. The DNA-replication inhibitor aphidicolin and DNA-damaging agents trigger identical mitotic defects in wild-type embryos, indicating that centrosome inactivation is a checkpoint-independent and mitosis-specific response to damaged or incompletely replicated DNA. We propose that centrosome inactivation is part of a damage-control system that blocks chromosome segregation when replication/damage checkpoint control fails.

Centrosomes and the Scrambled protein coordinate microtubule-independent actin reorganization.

In Drosophila syncytial blastoderm embryos, centrosomes specify the position of actin-based interphase caps and mitotic furrows. Mutations in the scrambled locus prevent assembly of mitotic furrows, but do not block actin cap formation. The scrambled gene encodes a protein that localizes to the mitotic furrows and centrosomes. Sced localization, actin reorganization from caps into mitotic furrows, and centrosome-coordinated assembly of actin caps are not blocked by microtubule disruption. Our results indicate that centrosomes may coordinate assembly of cortical actin caps through a microtubule-independent mechanism, and that Scrambled mediates a second microtubule-independent process that drives mitotic furrow assembly.

Dynamic changes in microtubule configuration correlate with nuclear migration in the preblastoderm Drosophila embryo.

Drosophila embryogenesis is initiated by a series of syncytial mitotic divisions. The first nine of these divisions are internal, and are accompanied by two temporally distinct nuclear movements that lead to the formation of a syncytial blastoderm with a uniform monolayer of cortical nuclei. The first of these movements, which we term axial expansion, occurs during division cycles 4-6 and distributes nuclei in a hollow ellipsoid underlying the cortex. This is followed by cortical migration, during cycles 7-10, which places the nuclei in a uniform monolayer at the cortex. Here we report that these two movements differ in their geometry, velocity, cell-cycle dependence, and protein synthesis requirement. We therefore conclude that axial expansion and cortical migration are mechanistically distinct, amplifying a similar conclusion based on pharmacological data (Zalokar and Erk, 1976). We have examined microtubule organization during cortical migration and find that a network of interdigitating microtubules connects the migrating nuclei. These anti-parallel microtubule arrays are observed between migrating nuclei and yolk nuclei located deeper in the embryo. These arrays are present during nuclear movement but break down when the nuclei are not moving. We propose that cortical migration is driven by microtubule-dependent forces that repel adjacent nuclei, leading to an expansion of the nuclear ellipsoid established by axial expansion.

Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein.

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.

A protein kinase bound to the projection portion of MAP 2 (microtubule-associated protein 2).

In previous work we have demonstrated that the microtubule-associated protein 2 (MAP 2) molecule consists of two structural parts. One part of the molecule, referred to as the assembly-promoting domain, binds to the microtubule surface and is responsible for promoting microtubule assembly; the other represents a filamentous projection observed on the microtubule surface that may be involved in the interaction of microtubules with other cellular structures. MAP 2 is known to be specifically phosphorylated as the result of a protein kinase activity that is present in microtubule preparations. We have now found that the activity copurifies with the projection portion of MAP 2 itself. Kinase activity coeluted with MAP 2 when microtubule protein was subjected to either gel- filtration chromatography on bio-gel A-15m or ion-exchange chromatography on DEAE- Sephadex. The activity was released from microtubules by mild digestion with chymotrypsin in parallel with the removal by the protease of the MAP 2 projections from the microtubule surface. The association of the activity with the projection was demonstrated directly by gel filtration chromatography of the projections on bio-gel A-15m. Three protein species (M(r) = 39,000, 55,000, and 70,000) cofractionated with MAP 2, and two of these (M(r) = 39,000 and 55,000) may represent the subunits of an associated cyclic AMP- dependent protein kinase. The projection-associated activity was stimulated 10-fold by cyclic AMP and was inhibited more than 95 percent by the cyclic AMP-dependent protein kinase inhibitor from rabbit skeletal muscle. It appeared to represent the only significant activity associated with microtubules, almost no activity being found with tubulin, other MAPs, or the assembly-promoting domain of MAP 2, and was estimated to account for 7-22 percent of the total brain cytosolic protein kinase activity. The location of the kinase on the projection is consistent with a role in regulating the function of the projection, though other roles for the enzyme are also possible.

Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.

Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes.

Embryonic axis specification in Drosophila melanogaster is achieved through the asymmetric subcellular localization of morphogenetic molecules within the oocyte. The cappuccino and spire loci are required for both posterior and dorsoventral patterning. Time-lapse confocal microscopic analyses of living egg chambers demonstrated that these mutations induce microtubule reorganization and the premature initiation of microtubule-dependent ooplasmic streaming. As a result, microtubule organization is altered and bulk ooplasm rapidly streams during the developmental stages in which morphogens are normally localized. These changes in oocyte cytoarchitecture and dynamics appear to disrupt axial patterning of the embryo.

Cytoskeletal functions during Drosophila oogenesis.

Organismal morphogenesis is driven by a complex series of developmentally coordinated changes in cell shape, size, and number. These changes in cell morphology are in turn dependent on alterations in basic cytoarchitecture. Elucidating the mechanisms of development thus requires an understanding of the cytoskeletal elements that organize the cytoplasm of differentiating cells. Drosophila oogenesis has emerged as a versatile system for the study of cytoskeletal function during development. A series of highly coordinated changes in cytoskeletal organization are required to produce a mature Drosophila oocyte, and these cytoskeletal transformations are amenable to a variety of experimental approaches. Genetic, molecular, and cytological studies have shed light on the specific functions of the cytoskeleton during oogenesis. The results of these studies are reviewed here, and their mechanistic implications are considered.

Actin cytoskeleton. Through the bottleneck.

Like serendipity-alpha and nullo, the newly characterized gene bottleneck is involved in organizing the actin cytoskeleton of the Drosophila embryo to achieve the transition from a syncytium to a cellular blastoderm.

Oocyte differentiation: a motor makes a difference.

In a variety of developmental systems, asymmetric mitoses precede, and are essential for, cellular differentiation. Recent studies demonstrate a role for the motor protein cytoplasmic dynein in generating the mitotic asymmetries that lead to Drosophila oocyte differentiation.

Actin cytoskeleton: putting a CAP on actin polymerization.

Two recent studies have identified a Drosophila homolog of cyclase-associated protein (CAP) as a developmentally important negative regulator of actin polymerization that may also directly mediate signal transduction.

The Drosophila ATM homologue Mei-41 has an essential checkpoint function at the midblastula transition.

BACKGROUND: Drosophila embryogenesis is initiated by 13 rapid syncytial mitotic divisions that do not require zygotic gene activity. This maternally directed cleavage phase of development terminates at the midblastula transition (MBT), at which point the cell cycle slows dramatically, membranes surround the cortical nuclei to form a cellular blastoderm, and zygotic gene expression is first required. RESULTS: We show that embryos lacking Mei-41, a Drosophila homologue of the ATM tumor suppressor, proceed through unusually short syncytial mitoses, fail to terminate syncytial division following mitosis 13, and degenerate without forming cells. A similar cleavage-stage arrest is produced by mutations in grapes, which encodes a homologue of the Checkpoint-1 kinase. We present biochemical, cytological and genetic data indicating that Mei-41 and Grapes are components of a conserved DNA-replication/damage checkpoint pathway that triggers inhibitory phosphorylation of the Cdc2 kinase and mediates resistance to replication inhibitors and DNA-damaging agents. This pathway is nonessential during postembryonic development, but it is required to terminate the cleavage stage at the MBT. Cyclins are required for Cdc2 kinase activity, and mutations in cyclin A and cyclin B bypass the requirement for mei-41 at the MBT. These mutations do not restore wild-type syncytial cell-cycle timing or the embryonic replication checkpoint, however, suggesting that Mei-41-mediated inhibition of Cdc2 has an additional essential function at the MBT. CONCLUSIONS: The Drosophila DNA-replication/damage checkpoint pathway can be activated by externally triggered DNA damage or replication defects throughout the life cycle, and under laboratory conditions this inducible function is nonessential. During early embryogenesis, however, this pathway is activated by developmental cues and is required for the transition from maternal to zygotic control of development at the MBT.

Requiem for distributive segregation: achiasmate segregation in Drosophila females.

The segregation of achiasmate chromosome pairs at meiosis I is not brought about by a single 'distributive system' as previously thought, but rather by two separate mechanisms. One system uses the pairing of proximal heterochromatic sequences to mediate the segregation of achiasmate homologs-an observation that, at long last, defines a function for heterochromatin. The other system facilitates the segregation of heterologous chromosomes, by an as yet undiscovered mechanism.

Distribution of microtubule-associated protein 2 in the nervous system of the rat studied by immunofluorescence.

It has recently been reported that high molecular weight microtubule-associated proteins are differently distributed in dendrites and axons of neurons [ Matus Bernhardt and Hugh-Jones (1981), Proc. natn Acad. Sci. U.S.A. 78, 3010-3014; Vallee (1982), J. Cell Biol. 92, 435-442]. We have reported earlier in a preliminary form [Miller, Walter, Theurkauf , Vallee and De Camilli (1982), Proc. natn Acad. Sci. U.S.A. 79, 5562-5566] that an antiserum specific for microtubule-associated protein 2, one of the most prominent high molecular weight microtubule-associated proteins in brain and a major brain phosphoprotein, stains specifically neuronal dendrites and perikarya. We have now extended those observations by performing a detailed analysis of the distribution of microtubule-associated protein 2 throughout the nervous system of the rat. We found that microtubule-associated protein 2 is present at high concentrations in the great majority of neurons. Under our conditions of immunostaining microtubule-associated protein 2 was not detected in nonneuronal cells. In all neurons it was compartmentalized in perikarya and dendrites. In most cases, the latter were more heavily stained than perikarya. The pattern of staining (overall intensity, relative intensity in dendrites vs perikarya, and in proximal vs distal segments of the dendritic tree), varied in different classes of neurons but was identical for all neurons with similar geometry in the same brain region. Different patterns of staining were found in dendritic trees with dissimilar branching characteristics. In all cases staining for microtubule-associated protein 2 in dendrites was consistent with a localization of microtubule-associated protein 2 on dendritic microtubules. Neuronal processes clearly identifiable as axons or axon terminals were not immunostained. Afferent processes of primary sensory cells were also unstained. Our findings indicate that microtubule-associated protein 2 is a component of the vast majority, and possibly all, neurons. It is highly concentrated in "bona fide" dendrites, i.e. in processes specialized for the reception of synaptic inputs on their surface and highly dependent on such inputs for their growth. The location of microtubule-associated protein 2, a major target for second messenger-regulated protein kinases, in these processes, supports the hypothesis that its phosphorylation might participate in the transduction of neurotransmitter signals in target nerve cells.

rasiRNAs, DNA damage, and embryonic axis specification.

Drosophila repeat-associated small interfering RNAs (rasiRNAs) have been implicated in retrotransposon and stellate locus silencing. However, mutations in the rasiRNA pathway genes armitage, spindle-E, and aubergine disrupt embryonic axis specification, triggering defects in microtubule organization and localization of osk and grk mRNAs during oogenesis. We show that mutations in mei-41 and mnk, which encode ATR and Chk2 kinases that function in DNA damage signal transduction, dramatically suppress the cytoskeletal and RNA localization defects associated with rasiRNA mutations. In contrast, stellate and retrotransposon silencing are not restored in mei-41 and mnk double mutants. We also find that armitage, aubergine, and spindle-E mutations lead to germ-line-specific accumulation of gamma-H2Av foci, which form at DNA double-strand breaks, and that mutations in armi lead to Chk2-dependent phosphorylation of Vasa, an RNA helicase required for axis specification. The Drosophila rasiRNA pathway thus appears to suppress DNA damage in the germ line, and mutations in this pathway block axis specification by activating an ATR/Chk2-dependent DNA damage response that disrupts microtubule polarization and RNA localization.

Behavior of structurally divergent alpha-tubulin isotypes during Drosophila embryogenesis: evidence for post-translational regulation of isotype abundance.

Two major alpha-tubulin isotypes are present during Drosophila embryogenesis: an evolutionarily divergent maternal isotype that is synthesized only in the ovary and deposited in the oocyte and a highly conserved constitutive isotype that is both maternally supplied and zygotically synthesized. A maternal isotype-specific antibody and a monoclonal antibody that recognizes both the maternal and constitutive isotypes were characterized and used to determine the distribution and abundance of alpha-tubulins during embryogenesis. Both isotypes are abundant and assemble into all classes of microtubules from the syncytial blastoderm stage until completion of germ band retraction. During subsequent development, however, the maternal isotype is retained only in the developing CNS, and later in a subset of connective fibers within the CNS. In contrast, total alpha-tubulin levels remain high in essentially all tissues throughout embryogenesis, indicating that most tissues selectively accumulate the constitutive isotype. To determine if selective accumulation of the constitutive isotype requires zygotic synthesis of this protein, mutant embryos that do not contain functional constitutive alpha-tubulin genes were examined. In these embryos, as in wild type, the maternal isotype decreases to background levels in tissues that retain high levels of the constitutive isotype. The constitutive isotype therefore appears to be more stable than the maternal isotype in most tissues. Differences in isotype stability may play an important role in determining the developmental pattern of isotype accumulation in Drosophila embryos.