slow as molasses


The slam expression pattern was followed in wild-type embryos using in situ hybridization. In early embryos before zygotic transcription has started, no slam RNA could be detected, suggesting that the gene might not be maternally provided, although the possibility cannot be excluded of a very low maternal contribution. Transcripts rapidly increase during cycle 13 and peak during the slow phase of cellularization, consistent with the strong zygotic requirement for slam. Note that this is the developmental period during which membrane invagination defects are seen in slam(RNAi) mutant embryos. During fast phase, levels of the transcript rapidly decrease and reach low levels by the onset of gastrulation. In control deficiency embryos in which slam is deleted, no transcript is detected at any time during cellularization (Lecuit, 2002).

To gain insight into the function of Slam, its subcellular localization was examined during cellularization. The C terminus of full-length Slam was tagged with three tandem copies of the hemagglutinin (HA) epitope from influenza virus. Flies expressing slamHA under the control of the heterologous UAS/Gal4 promoter (UAS-SlamHA) were made. Embryos derived from mothers expressing the Gal4 transcriptional activator in the germline can induce the UAS-SlamHA transgene, like the endogenous slam gene, zygotically during cellularization. Anti-HA antibodies recognize a single 135 kDa band on Western blot analysis of embryonic extracts prepared from embryos expressing UAS-SlamHA. Membrane fractions were prepared using concanavalin A-coated beads that can concentrate membranes very efficiently. SlamHA is found in a membrane and a cytosolic/nuclear fraction. Under similar extraction conditions, Nrt is only found in the membrane fraction, and the transcription factor Engrailed (En) is only found in the cytosolic/nuclear fraction. Another cytosolic protein known to localize to the cell surface, β-catenin/Armadillo, is more tightly associated with the membrane than Slam under these extraction conditions (Lecuit, 2002).

UAS-SlamHA embryos were next stained with anti-HA antibodies. SlamHA is first detected in the presumptive furrow canal region as it forms. Later, during the slow phase of membrane invagination, Slam persists mostly at the furrow canal region but is also detected along the adjacent lateral membrane. SlamHA is found in punctate structures of the cytoplasm. Similar observations were made with a fusion protein of Slam and GFP. In conclusion, although not an integral membrane protein, Slam is associated with the plasma membrane. Note that the localization is polarized -- Slam is mostly found in the furrow canal and in the basal portion of the lateral membrane but not in the apical plasma membrane. Slam localization corresponds to the location where membrane organization is perturbed in the mutant (Lecuit, 2002).

To address how Slam accumulation in the FC and adjacent regions affects the organization and growth of the basal-lateral membrane, other proteins with similar subcellular localizations were examined. The PDZ domain-containing protein Discs-lost (Dlt; renamed Patj) accumulates at the FC and shows a very striking similar localization to that of Slam. Dlt controls the polarity of the newly formed epithelium during cellularization. In addition, Dlt is also required later in development, where it localizes to and is required for the maintenance of apical junctions. Slam's localization extends to the adjacent basal junction during cellularization, where it overlaps with Armadillo, the Drosophila ortholog of the junctional component β-Catenin. Interestingly, when slamHA's expression is artificially maintained in epithelial cells after cellularization using the Gal4/UAS system, SlamHA is detected in apical junctions together with Arm. These observations suggest that Slam's function may be associated with junctions (Lecuit, 2002).

This proposal is supported by the finding that Arm's typical accumulation at basal junctions is absent in slam(RNAi) mutant embryos. Instead, Arm appears in a diffuse apical pattern that most likely corresponds to a cytoplasmic protein pool. Likewise, Dlt's membrane accumulation is clearly compromised in slam(RNAi) mutant embryos. Although Dlt is still detected at the membrane in a regular pattern that corresponds to the membrane area between adjacent somatic buds, its levels are clearly reduced in comparison to those in wild-type control embryos (Lecuit, 2002).

Myosin accumulates at the most basal part of the furrow canal, where it overlaps with Slam. In slam mutant embryos, the levels of Myosin accumulation are slightly reduced in comparison to those in the wild-type. More strikingly, the prolonged expression of slam after cellularization modifies the localization of Myosin at junctions. In control embryos, Myosin is present at apical junctions, although the protein is less tightly localized than the apical junction marker Arm. When slamHA's expression is maintained, SlamHA accumulates at junctions with an asymmetry in the plane of the epithelium. In contrast to Arm, SlamHA does not localize in a regular 'honeycomb' pattern. Instead, SlamHA concentrates at higher levels along the anterior and posterior contacts of adjacent epithelial cells and at very low levels along the dorsal and ventral contacts. Although the significance of this localization is unclear, this situation causes an increase in the apparent levels of junctional Myosin, and a similar planar asymmetry is observed. In fact, SlamHA and Myosin show almost identical subcellular localization. Under these circumstances, the apolar localization of Arm and Dlt is, however, unaffected. This observation argues that SlamHA is able to recruit Myosin in a complex assembled at junctions. Together, these data suggest that Slam's requirement for polarized membrane growth also involves an interaction with Myosin (Lecuit, 2002).

Detailed analysis of the RNA expression pattern during embryogenesis revealed low levels of uniformly distributed slam RNA, which is most likely of maternal origin. During nuclear cycle 10, an increase in slam RNA is detected, presumably because of new zygotic transcription. During nuclear cycle 14 as cellularization begins, slam RNA is highly expressed and this expression is maintained throughout cellularization. slam RNA is localized basally within the cytoplasm surrounding each nucleus. Slam expression is due to transcription from the embryonic genome, since it is only observed in somatic nuclei but not in the germ cells, which are transcriptionally repressed during early embryogenesis. At the end of cellularization, slam RNA is limited to three stripes; the RNA levels then rapidly decline and slamRNA can no longer be detected after gastrulation (stage 8 onwards) (Stein, 2002).

To analyze the time course and distribution of Slam protein, an antibody was raised against a peptide of the Slam protein. Slam protein first can be detected at the plasma membrane of embryos at nuclear cycle 11, before cellularization begins, but after nuclei have migrated to the cortex. At this stage, Slam is also observed in the cytoplasm basal to the peripheral nuclei; the position of this staining suggests that Slam is detected in the Golgi. During cellularization, Slam is localized to the most advancing membrane as it invaginates between each nucleus. Confocal microscopy of embryos doubly labeled for Slam protein and Neurotactin, which outlines the cell membranes, reveals that Slam is localized to the furrow canal, which marks the most basal part of the growing membrane. The germ cells do not contain any Slam protein (Stein, 2002).

Once the embryo begins to gastrulate, Slam protein is not longer seen at the membrane of invaginating cells; however, it is maintained in the cell membranes of the rest of the epithelium. Slam staining was analyzed in thin sections, where localization to the basal-lateral membrane is clearly evident. Slam protein seems to be associated with the membrane. In thin sections of gastrulating embryos, Slam protein is observed to be first lost in the membranes of invaginating cells. Slam protein can no longer be detected in embryos by the end of gastrulation. The RNA and protein expression profile of slam correlate well with the slam cellularization phenotype. No RNA or protein was detected, however, during the second phase of Slam function (germ cell migration); this suggests that early Slam expression is not only necessary for cellularization but also later for germ cell migration (Stein, 2002).

Slam is a molecular marker for polarized cell behavior during germband extension

During convergent extension in Drosophila, polarized cell movements cause the germband to narrow along the dorsal-ventral (D-V) axis and more than double in length along the anterior-posterior (A-P) axis. This tissue remodeling requires the correct patterning of gene expression along the A-P axis, perpendicular to the direction of cell movement. A-P patterning information results in the polarized localization of cortical proteins in intercalating cells. In particular, cell fate differences conferred by striped expression of the even-skipped and runt pair-rule genes are both necessary and sufficient to orient planar polarity. This polarity consists of an enrichment of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 protein at the reciprocal D-V cell borders. Moreover, bazooka mutants are defective for germband extension. These results indicate that spatial patterns of gene expression coordinate planar polarity across a multicellular population through the localized distribution of proteins required for cell movement (Zallen, 2004).

Polarized cell movement during convergent extension ultimately derives from the asymmetric localization of proteins that direct cell motility. Interestingly, intercalating cells in the Drosophila germband display a polarized localization of the ectopically expressed Slam protein (Lecuit, 2002). Slam is present in a bipolar distribution that correlates spatially and temporally with intercalary behavior. These observations indicate that Slam can serve as a molecular marker for polarized cell behavior. Pair-rule patterning genes expressed in stripes along the A-P axis are necessary for Slam localization and, conversely, altering the geometry of their expression is sufficient to reorient Slam polarity. An endogenous planar polarity in intercalating cells has been shown to be manifested by the accumulation of nonmuscle myosin II at A-P cell borders and Bazooka/PAR-3 at D-V cell borders. Moreover, germband extension is defective in bazooka mutant embryos, supporting a model where molecular polarization of the cell surface is a prerequisite for polarized cell movement. Therefore, differences in gene expression along the A-P axis may direct planar polarity in intercalating cells through the creation of molecularly distinct cell-cell interfaces that differ in migratory potential (Zallen, 2004).

Cell movement during germband extension is oriented along the D-V axis, suggesting a mechanism that restricts the productive generation of motility to dorsal and ventral cell surfaces. Molecules that are asymmetrically localized during convergent extension may therefore contribute to the spatial regulation of cell motility. Interestingly, intercalating cells in the Drosophila germband display a polarized localization of the ectopically expressed Slam protein, a novel cytoplasmic factor required for cellularization in the early embryo (Lecuit, 2002). While proteins such as Armadillo/β-catenin are uniformly distributed at the cell surface, ectopic Slam is enriched in borders between neighboring cells along the A-P axis. This polarized Slam population is present in a punctate apical distribution, coincident with the adherens junction component Armadillo/β-catenin. Therefore, intercalating cells have distinct apical junctional domains that differ in their capacity for Slam association (Zallen, 2004).

Interestingly, the polarized distribution of ectopic Slam protein is spatially and temporally correlated with intercalary behavior. Slam polarity is not observed in Stage 6 embryos prior to the onset of intercalation. Slam accumulation at A-P cell borders first appears in late Stage 7, when cells of the germband initiate intercalation, and reaches its full extent during the period of sustained intercalation in Stage 8. In contrast, Slam is uniformly distributed in cells of the head region and the dorsal ectoderm, tissues which do not undergo intercalary movements. These results indicate that the polarized distribution of ectopic Slam protein is specific to intercalating cells and that Slam can therefore serve as a molecular marker for the visualization of polarized cell behavior (Zallen, 2004).

The enrichment of Slam at borders between neighboring cells along the A-P axis is consistent with two modes of localization: Slam could mark one side of each cell in a unipolar distribution, or Slam could localize to both anterior and posterior surfaces in a bipolar pattern. To distinguish between these possibilities, mosaic embryos were generated where Slam-expressing cells were juxtaposed with unlabeled cells, using the Horka mutation to induce sporadic chromosome loss in early embryos. Slam protein accumulates at anterior and posterior boundaries of mosaic clone, indicating that ectopic Slam protein is targeted to both anterior and posterior surfaces of intercalating cells in a symmetric, bipolar distribution. The bipolar localization of ectopic Slam corresponds well with the bidirectionality of cell movement during germband extension, where cells are equally likely to migrate dorsally or ventrally during intercalation. Bipolar motility is also observed during convergent extension in the presumptive Xenopus and Ciona notochords and in Xenopus neural plate cells in the absence of midline structures (Zallen, 2004).

To extend the spatial and temporal correlation between Slam polarity and cell movement, it was asked if this polarized Slam localization is achieved in mutants that are defective for intercalation. Cell intercalation is dependent on the transcriptional cascade that generates cell fates along the A-P axis, in the direction of tissue elongation and perpendicular to the migrations of individual cells. A-P patterning reflects the hierarchical action of maternal, gap, and pair-rule genes. Cell fate differences along the A-P axis are abolished in embryos maternally deficient for the bicoid, nanos, and torso-like genes (referred to as bicoid nanos torso-like mutants), and these mutant embryos do not exhibit intercalary behavior. Ectopic Slam is correctly targeted to the apical cell surface in bicoid nanos torso-like mutants, but fails to adopt a polarized distribution in the plane of the epithelium (Zallen, 2004).

Downstream of the maternal patterning genes, gap genes establish overlapping subdomains along the A-P axis. A quadruple mutant for the gap genes knirps, hunchback, forkhead, and tailless lacks A-P pattern within the germband while retaining terminal structures. This quadruple mutant exhibits severely reduced cell intercalation, and mutant embryos also display a loss of Slam polarity. The absence of planar polarity in A-P patterning mutants correlates with a more hexagonal appearance of germband cells, in contrast to the irregular morphology of wild-type intercalating cells (Zallen, 2004).

In response to maternal and gap genes, pair-rule patterning genes expressed in narrow stripes act in combination to assign each cell a distinct fate along the A-P axis. In particular, the even-skipped (eve) and runt pair-rule genes are essential for germband extension. This strong requirement for eve and runt during germband extension contrasts with the more subtle effects in mutants for other pair-rule genes such as hairy and ftz. Consistent with these defects in intercalation, eve and runt mutants also display aberrant Slam localization. These results establish a correlation between intercalary behavior and the polarized localization of the ectopic Slam marker (Zallen, 2004).

While eve and runt mutants fail to complete germband extension, they extend further than embryos lacking maternal and gap genes, suggesting that some intercalary behavior is retained. Consistent with this possibility, Slam polarity is only partially disrupted in eve and runt mutants. While some cells display an aberrant uniform Slam distribution, in other cells Slam is correctly enriched at A-P cell interfaces. Therefore, the residual intercalation in eve and runt mutants correlates with the establishment of planar polarity in a subset of germband cells (Zallen, 2004).

The disruption of Slam polarity in A-P patterning mutants demonstrates that proper gene expression along the A-P axis is required for planar polarity in intercalating cells. In particular, the Eve and Runt transcription factors are expressed in 7 stripes at the onset of germband extension and 14 stripes as intercalation proceeds. Each cell in the germband is assigned a fate distinct from its anterior and posterior neighbors through the graded and partially overlapping expression of these and other pair-rule genes. Slam preferentially accumulates at contacts between cells with different levels of pair-rule gene activity, suggesting a model where cells concentrate specific proteins at interfaces with neighbors that differ in A-P identity. To directly address this model, mosaic embryos were generated with altered patterns of pair-rule gene expression in order to artificially introduce differences between dorsal and ventral neighbors. It was then asked if Slam protein is aberrantly recruited to these ectopic juxtapositions between different cell types, even at interfaces that are perpendicular to the normal axis of polarity (Zallen, 2004).

The Horka mutation was used to generate embryos that ectopically express Eve or Runt in a mosaic pattern. When these genes are ubiquitously expressed, planar polarity is generally disrupted and Slam displays a more uniform localization. This disruption of Slam polarity correlates with defective germband extension in Eve and Runt overexpressing embryos. The effects of Eve and Runt overexpression are not mimicked by overexpression of other pair-rule proteins such as Paired, Odd-paired, or Sloppy-paired. Moreover, localized sources of Eve or Runt expression direct aberrant patterns of polarity in mosaic embryos. For example, mosaic embryos display circles of Slam polarity that are bordered by ectopic Eve clones. Similarly, Slam polarity in germband cells is diverted from its normal orientation to follow boundaries of Runt misexpression. These results demonstrate that ectopic sites of Eve and Runt expression can reorient Slam polarity at clone boundaries, even when these interfaces are perpendicular to the normal axis of polarity (Zallen, 2004).

In contrast to the reorientation of planar polarity at boundaries of Eve and Runt misexpression, cells distant from the clone often exhibited complex patterns of Slam localization. These patterns may arise from nonautonomous effects of pair-rule gene activity, as well as aberrant cell movements and ectopic folds that form at clone boundaries, suggestive of a disruption in cell adhesion. Therefore mosaic embryos were examined at Stage 7, prior to the onset of cell movement and ectopic fold formation. While early Stage 7 embryos do not normally exhibit Slam polarity, ectopic Eve induces a precocious accumulation of Slam at clone boundaries. In contrast, ectopic Runt only occasionally induces a subtle polarity at Stage 7. The more potent effect of the eve transgene may reflect higher levels of ectopic expression compared to the endogenous eve stripes. These mosaic experiments indicate that differences in gene expression play an instructive role in the generation of planar polarity in intercalating cells. While Eve and Runt are both sufficient for planar polarity, the absence of either gene alone disrupts polarity. However, the defects in eve or runt single mutants may result from a combined disruption of multiple pair-rule genes; loss of eve leads to altered runt expression and vice versa (Zallen, 2004).

Generation of planar polarity by ectopic Eve expression is subject to the same spatial requirements as in wild-type polarity: Eve clones in the head region failed to induce polarity, suggesting that these cells are resistant to Eve-dependent polarization. In contrast, ectopic Runt expression in the head led to a concentration of Slam at clone boundaries, despite the fact that these cells do not normally display Slam polarity or intercalary behavior. These results indicate that in contrast to Eve, Runt can induce planar polarity in head cells, raising the possibility of functional distinctions between Eve and Runt target genes (Zallen, 2004).

The Eve and Runt transcription factors ultimately direct Slam polarity and cell intercalation through the transcriptional regulation of target genes. To identify downstream effectors involved in this process, components of the noncanonical planar cell polarity (PCP) pathway, which is required for convergent extension in vertebrates, were examined. Germband extension occurs normally in the majority of embryos lacking the Frizzled and Frizzled2 receptors. Similarly, germband extension is unaffected in the absence of Dishevelled. Moreover, dishevelled mutants exhibit a normal polarization of the Slam marker. These results demonstrate that molecular and behavioral properties of planar polarity in the Drosophila germband do not require Frizzled or Dishevelled function (Zallen, 2004).

The polarized distribution of ectopic Slam in intercalating cells provides the first clue to a molecular distinction between D-V cell interfaces that generate productive cell motility and A-P interfaces that do not. However, endogenous Slam mRNA and protein are not detected during germband extension, indicating that Slam may not play a functional role in cell intercalation. Slam colocalizes with the Zipper nonmuscle myosin II heavy chain subunit during cellularization and when Slam is ectopically expressed at germband extension (Lecuit, 2002). Therefore, the endogenous distribution of myosin II was examined during germband extension in wild-type embryos. During cell intercalation, myosin II is present in a punctate distribution at the apical cell surface, colocalizing with the adherens junction component Armadillo/β-catenin. In Stage 8 embryos, apical myosin II protein accumulates at interfaces between cells along the A-P axis. Slam can enhance this polarized localization when ectopically expressed (Lecuit, 2002), suggesting that Slam and myosin II may associate with a common localization machinery. Myosin II polarity is not apparent in Stage 6 or early Stage 7 embryos that have not begun intercalation, indicating that the enrichment of myosin II at A-P interfaces is specific to intercalating cells (Zallen, 2004).

The localized distribution of myosin II is not as pronounced as that of ectopic Slam, suggesting that additional asymmetries contribute to the polarization of intercalating cells. To identify such proteins, the localization was examined of components implicated in cell polarity in other cell types. In particular, the PDZ domain protein Bazooka/PAR-3 participates in both apical-basal and planar polarity. Bazooka/PAR-3 also exhibits a polarized distribution in intercalating cells. Bazooka, like myosin II, is present in a punctate apical distribution, coincident with the adherens junction component Armadillo/β-catenin. However, in contrast to the accumulation of myosin II at A-P cell interfaces, Bazooka is enriched in the reciprocal D-V interfaces. Bazooka polarity is specific to intercalating cells, where it first appears at the onset of intercalary movements in late Stage 7. Bazooka polarity is not observed in cells of the head region, which do not undergo intercalation, nor is it observed in germband cells following the completion of germband extension at Stage 9 (Zallen, 2004).

To characterize the relationship between cell shape and the polarized localization of cortical proteins, the orientation of cell borders was measured as an angle relative to the A-P axis (with A-P interfaces closer to 90° and D-V interfaces closer to 0° and 180°). Interfaces from embryos stained for Bazooka and myosin II were ranked according to mean fluorescence intensity as a relative measure of protein distribution. These results illustrate that Bazooka and myosin II are enriched in distinct sets of cell-cell interfaces that adopt largely nonoverlapping orientations relative to the A-P axis. This quantitation confirms the visual impression from confocal images and demonstrates that the molecular composition of a cell surface domain is a reliable predictor of its orientation within the epithelial cell sheet (Zallen, 2004).

The polarized localization of Bazooka is abolished in the absence of A-P patterning information in bicoid nanos torso-like mutant embryos. A similar disruption of myosin II polarity is observed in A-P patterning mutants. The A-P patterning system may therefore mediate cell intercalation through the polarized accumulation of cell surface-associated proteins. Bazooka participates in a conserved protein complex containing the atypical PKC (DaPKC), and DaPKC is also enriched in D-V cell interfaces during germband extension (Zallen, 2004).

To determine whether the polarized Bazooka/PAR-3 protein is functionally required for germband extension, homozygous bazooka (baz) mutant embryos were examined. In zygotic baz mutants, residual Bazooka protein persists from maternal stores and is often, but not always, correctly distributed along the apical-basal and planar axes. Despite this maternal Bazooka contribution, loss of zygotic Bazooka disrupts germband extension. In wild-type embryos, the posterior end of the extended germband is located at 70% egg length from the posterior pole. Of the progeny of bazYD97/+ females and wild-type males, 72% were wild-type-like, 25% were partially defective, and 3% were strongly defective. These results demonstrate that Bazooka is required for normal germband extension (Zallen, 2004).

Bazooka/PAR-3 and the associated DmPAR-6 and DaPKC components also influence epithelial cell polarity along the apical-basal axis. To address the possibility that germband extension defects may occur indirectly as a result of disrupted apical-basal polarity, properties of apical-basal polarity were examined in zygotic baz mutants, where some functions are carried out by maternal gene products. Zygotic baz mutant embryos exhibit several signs of normal apical-basal polarity at gastrulation, including a monolayer epithelial morphology in the germband and the correct distribution of proteins to apical and lateral membrane domains. This is consistent with findings that zygotic baz mutants exhibit proper localization of the Armadillo/β-catenin adherens junction component prior to Stage 10 of embryogenesis. These results demonstrate that properties of apical-basal polarity are established correctly in baz mutant embryos during germband extension, consistent with a direct role for Bazooka in cell movements along the planar axis, independent of its later effects on apical-basal polarity (Zallen, 2004).

The local reorientation of planar polarity in response to Eve and Runt expression argues that planar polarity is generated by cell-cell interactions, rather than a distant polarizing cue. In addition to these local effects of Eve and Runt on planar polarity, Slam polarity frequently adopted a circular pattern in mosaic embryos, even when Eve and Runt were not present along the entire circumference of the circle. This unexpected configuration indicates that polarizing information can propagate from cell to cell downstream of an Eve-dependent signal. A similar relay mechanism is suggested by the swirling patterns of wing hair polarity that persist in Drosophila mutants defective for the PCP signaling pathway. Therefore, mechanisms of cell-cell communication may reinforce local polarizing events in the organization of a two-dimensional cell population (Zallen, 2004).

Planar polarity in Drosophila germband extension is locally established through the concentration of specific proteins at sites of contact between cells with different levels of Eve and Runt expression. Cells can monitor the identity of their neighbors through qualitative or quantitative differences in the activity of cell surface proteins, perhaps through ligand-receptor mediated signaling events or adhesion-based cell sorting. Transcriptional targets of Eve and Runt are therefore likely to include components that mediate intercellular signaling events involved in the transmission of polarizing information during multicellular reorganization (Zallen, 2004).

RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation: Requirement for Slow as molasses

The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitant with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. Both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. These results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane (Grosshans, 2005).

slow as molasses (slam) is required for timed formation of the furrow and invagination of the membrane in the first half of cellularisation. Like Dia and RhoGEF2 Slam protein localises to the furrow canal and localisation precedes furrow canal formation. Slam may act by recruiting MyoII to the furrow canal, but the biochemical activities of Slam have not been defined. Although the membrane does not invaginate initially in slam mutants, a complete F-actin array is visible. Thus despite the overlapping localisation of Slam, RhoGEF2 and Dia, their functions are clearly distinguishable (Grosshans, 2005).

Effects of Mutation or Deletion

The distinction between the slow and fast phases of cellularization can be followed using either DIC or a GFP/nonmuscle Myosin II fusion protein (hereafter called Myo-GFP) that labels the furrow canal (FC) located at the front of membrane invagination. At the beginning of cycle 14, the FC and Myo-GFP are not visible. During the first 10 min of cycle 14, the furrow canal becomes visible in DIC as a large contrasted membrane structure that persists during cellularization with high levels of Myo-GFP. During the initial slow phase of cellularization, the canal invaginates at a rate of about 0.3 μm/min. This period (35-40 min) is followed by a rapid (2 min) transition to fast phase, where membrane invaginates at a steady rate of 0.8 μm/min. During both slow phase and fast phase, other morphological transformations occur, e.g., the initially rounded nuclei elongate along their apical-basal axis and eventually reach 12 μm in length (Lecuit, 2002).

Using translocation crosses to generate embryos deficient for cytologically defined portions of the genome, two chromosomal regions on the left arm of the second chromosome (2L) were identified that are required for distinct phases of cellularization (Merrill, 1988). The more distal region is required specifically during slow phase and is defined by the translocations ME10 and J136, which produce the phenotype and break in 26C/D and 26F respectively, and H121, which does not produce the phenotype and breaks in 26B. Genomic mapping of the ME10 and H121 breakpoints by PCR allowed the region to be refined to a 100 kb interval containing 15 candidate genes. Candidate genes were then tested by RNA-mediated interference (RNAi). Double-stranded RNA probes were made and injected shortly after the egg was laid. A single gene, CG9506, was found for which RNA(i) produces membrane invagination defects identical to those seen in ME10 mutant embryos. The phenotype is highly penetrant, with 100% of the embryos showing a similar phenotype (Lecuit, 2002).

At the onset of cellularization, wild-type and CG9506(RNAi) embryos are indistinguishable. In the CG9506(RNAi) embryos, however, no membrane invagination is seen during the next 38 min, and the furrow canal remains in its original apical position. Other aspects of cellularization that occur during this period (e.g., nuclear elongation) proceed normally. The cellularization defects are stage specific. Later, when the speed of invagination rapidly increases to about 0.8 μm/min in the wild-type, invagination begins in CG9506(RNAi) embryos, albeit at a lower speed (0.4 μm/min). When cellularization would normally be completed, the membrane in CG9506(RNAi) embryos has not invaginated enough to enclose each nucleus. Cortical contractile movements severely disrupt the invaginated membrane and nuclei as well as gastrulation (Lecuit, 2002).

The phenotype observed in CG9506 mutant embryos could either be interpreted as a delayed onset of the entire developmentally controlled program of cellularization or as a specific block in membrane behavior that occurs in slow phase. In wild-type embryos, slow phase is characterized by the rapid induction and high expression levels of nullo (Hunter, 2000) and bottleneck, two genes zygotically required during slow phase but not during fast phase. During fast phase in wild-type embryos, nullo and bnk are no longer expressed, and the transcripts are undetectable. To test whether aspects other than membrane invagination are delayed in CG9506 embryos, the expression levels of nullo and bnk were monitored in J136 deficiency embryos as well as CG9506(RNAi) embryos. Both genes are normally expressed at the onset of cycle 14, and by the time membrane invagination has initiated, their expression has dropped to the low levels observed during fast phase in wild-type embryos. These data argue that the time window during which no invagination is seen in CG9506(RNAi) embryos has the molecular landmarks of a slow phase. It is concluded that CG9506 is not a timer controlling all aspects of slow phase, but, rather, that it encodes a regulator of the membrane growth that would normally occur during slow phase (Lecuit, 2002).

To gain insight into the mechanisms by which slam controls the growth of the plasma membrane during slow phase, the organization of the plasma membrane and the cytoplasm was examined in both wild-type and slam mutant embryos using electron microscopy. At the onset of slow phase, the plasma membrane of wild-type embryos is composed of two adjacent membrane regions. The first shows many villous projections (VP) and is located above each nucleus. The other lies adjacent to it and forms a smooth depression of the plasma membrane devoid of villous projections. This region will form the furrow canal during the first 15 min of slow phase, as the plasma membrane becomes curved and lateral regions of adjacent plasma membrane contact each other at the level of a basal junction (Hunter, 2000; Lecuit, 2000). Formation of the basal junction initiates an area of smooth membrane insertion that progressively separates the apical villous membrane from the furrow canal (Lecuit, 2000).

The most striking abnormality observed in slam mutant embryos is the failure to form a furrow canal whose membrane domain is structurally distinct from the apical region rich in villous projections. Although a mild depression of the plasma membrane can be seen between adjacent nuclei, no basal junction forms, villous projections extend into the flat area of membrane, and the organization of the basal-lateral surface is obviously impaired. Because the furrow canal and basal junction formation precedes the extension of the basal-lateral surface, these observations suggest that the early phases of membrane insertion may depend on the formation of the furrow canal (FC) and basal junction. Later, when membrane invagination resumes during fast phase, the furrow canal invaginates basally, although its structure is still abnormal; instead of being smooth, as seen in the wild-type, the plasma membrane extends villous projections (VPs) toward the engulfed area of extracellular milieu. The presence of VPs at the furrow canal is most likely a consequence of the earlier defect in the segregation between the FC and the adjacent membrane domains rich in VPs. The fact that membrane invagination still occurs in spite of the abnormal furrow canal organization observation supports the view that the mechanisms of membrane growth are different in slow and fast phases (Lecuit, 2002).

In vivo labeling experiments have shown that, when the surface of the embryo is labeled with fluorescent wheat germ agglutinin at the onset of cellularization, the plasma membrane of the somatic buds (i.e., rich in VPs) is rapidly replaced with unlabeled membrane, whereas the membrane located between adjacent somatic buds keeps a high level of fluorescence (Lecuit, 2000). This more stable membrane domain corresponds to the flat region of membrane that will form the furrow canal. As membrane invagination progresses through slow phase, the distinct polarized behavior of the plasma membrane is maintained; apically labeled plasma membrane is replaced with unlabeled membrane, whereas the basal-lateral membrane accumulates fluorescent membrane as it assembles and grows (Lecuit, 2000). This differential labeling behavior is not simply a consequence of membrane invagination itself, since it is observed in early wild-type embryos at stages before the membrane invagination begins (phase 1 of slow phase; Lecuit, 2000). Instead, the differential rates of membrane turnover in the VP-rich versus basal-lateral surfaces appear to be a property of the two regions. When membrane dynamics were examined in J136 deficiency embryos that remove the slam locus, the apically labeled membrane was found to remain unchanged during the entire period in which membrane growth is inhibited, and no obvious domains are observed. While different mechanisms could underlie this behavior, these experiments coupled with the EM studies described above argue that the inhibition of membrane growth observed in slam mutants is associated with and may be caused by a failure to establish defined sites of polarized membrane insertion (Lecuit, 2002).

To further characterize the defects in slam mutants, the intracellular transport and insertion was followed of Toll and Neurotactin (Nrt), a transmembrane protein synthesized from new zygotically supplied transcripts at the onset of cellularization. In wild-type embryos, Nrt is first detected in Golgi peripheral membranes basal to the nuclei (Lecuit, 2000) and is rapidly inserted in the apical plasma membrane before membrane invagination has started. The level of Nrt in the apical membrane continues to increase and, as slow phase proceeds, Nrt is also found progressively in the growing lateral membrane. In slam(RNAi) mutant embryos, Nrt is induced with the normal timing, i.e., ~15 min into cellularization, as judged by the relative elongation of nuclei and the pattern of even-skipped expression, two very dynamic events that provide an accurate timing method. However, the progressive accumulation at the plasma membrane is abnormal. As early as it can be detected, Nrt appears in a diffuse subapical pattern and can never be resolved as a distinct membrane accumulation, unlike wild-type embryos (Lecuit, 2002).

The localization was examined of a second transmembrane protein, Toll, which, in contrast to Nrt, is maternally provided and can already be detected at the plasma membrane before cellularization. In wild-type embryos prior to membrane invagination in cycle 14, Toll is found between somatic buds. As membrane invagination proceeds, Toll accumulates in the basal portion of the lateral membrane. The pattern of Toll also shows an abnormal localization in slam(RNAi) embryos. Instead of concentrating in the basal-lateral membrane, Toll, like Nrt, is found in the apical cytoplasm. Most of the protein, like Nrt, appears in a diffuse pattern, although occasional punctate accumulations persist (Lecuit, 2002).

Both Nrt and Toll accumulate in a diffuse pattern above the nuclei. Because the staining extends well below the reported depth of surface microvilli in wild-type embryos, this is thought to reflect a cytoplasmic accumulation of newly synthesized protein not incorporated into surface membrane. To test this hypothesis, the depth of the microvillous surface was determined in slam mutant embryos using phalloidin to highlight the cortical actin in microvilli. The comparison with Nrt and Toll indicates that, while these proteins may partly be inserted in the plasma membrane, a significant fraction also accumulates abnormally in the apical cytoplasm below the microvilli. This observation suggests that the defects in trafficking of transmembrane proteins and thus the failure to form basal-lateral membrane might be due to an altered pattern of membrane trafficking (Lecuit, 2002).

Slam is required cellularization

Germline clones homozygous mutant for slamwaldo were generated using the flp-FRT-ovoD system. Removal of maternal and zygotic slam (MZ slamwaldo) revealed a new and unexpected phenotype: MZ slamwaldo mutant embryos fail to cellularize during nuclear cycle 14. Interestingly, germ cells form normally during cycle 10. The germ cells have no soma to move through, so germ cells are found distributed throughout an otherwise unstructured embryo (Stein, 2002).

In wild type, nuclei migrate to the periphery of the embryo during the ninth nuclear cycle. During each of the following four nuclear divisions, these nuclei and their associated centrioles induce structural changes in the actin cytoskeleton (actin cap) and the cortical membrane. After the 14th nuclear division, the membrane grows and invaginates between each peripheral nucleus to create an epithelium of somatic cells. This membrane invagination proceeds first slowly for 35-40 minutes (slow phase) and then rapidly for 15-20 minutes (fast phase) until each nucleus is surrounded by a cell membrane (Stein, 2002).

Nuclei reach the periphery normally in MZ slamwaldo mutants, the primordial germ cells bud normally, and the somatic nuclei continue to divide until cycle 14. The phases of cellularization during cycle 14 were followed by live observation and by observing the progression of the cellularization front (furrow canal) using an anti-Myosin antibody in wild-type and MZ slamwaldo mutants. The first deviation from wild type is seen during the slow phase of cellularization. As the embryo proceeds through cycle 14, the nuclei elongate and the membrane invaginates. However, compared with wild type, membrane invagination in slam mutants is delayed. At the time when membranes normally enclose each nucleus basally, the incompletely invaginated membranes of MZ slamwaldo mutants entrap the nuclei as they pinch off basally. In wild type, after cellularization is complete, the complex movements of gastrulation lead to the invagination of the midgut primordia and the mesoderm and to the extension of the germ band. MZ slamwaldo mutants do attempt to gastrulate, but the vigorous movements of gastrulation disrupt the incompletely formed somatic cells; the embryos look like they fall apart and fail to develop further (Stein, 2002).

This cellularization phenotype is only seen in M–/–Z–/–slam mutants. A single zygotic copy of slam (M–/–Z+/– slam) restores both cellularization and germ cell migration, and a single maternal copy (M+/–Z–/–slam) rescues the cellularization defect, but not the germ cell migration defect. These data suggest that the cellularization defect is the null phenotype because it is the phenotype of embryos that lack maternal and zygotic slamwaldo alleles, embryos deficient for the genomic region containing slam and embryos injected with slam RNAi (Lecuit, 2002). The slamwaldo mutants are presumably hypomorphic alleles, such that maternally provided slam activity is sufficient for cellularization and thereby reveal a second function for Slam in germ cell migration (Stein, 2002).

Localization of RhoGEF2 during Drosophila cellularization is developmentally controlled by Slam

Essential for proper function of small GTPases of the Rho family, which control many aspects of cytoskeletal and membrane dynamics, is their temporal and spatial control by activating GDP exchange factors (GEFs) and deactivating GTPase-activating-proteins (GAPs). The regulatory mechanisms controlling these factors are not well understood, especially during development, when the organization and behaviour of cells change in a stage dependent manner. During Drosophila cellularization Rho signalling and RhoGEF2 are involved in furrow canal formation and the organization of actin and myosin. This study analyzed, how RhoGEF2 is localized at the sites of membrane invagination.The PDZ domain is necessary for localization and function of RhoGEF2, and Slam was identified as a factor that is necessary for RhoGEF2 localization. It was also demonstrated that Slam can recruit RhoGEF2 to ectopic sites. Furthermore the PDZ domain of RhoGEF2 can form a complex with Slam in vivo, and Slam transcripts and protein colocalize at the furrow canal and in basal particles. Based on these findings, it is proposed that accumulation of slam mRNA and protein at the presumptive invagination site provides a spatial and temporal trigger for RhoGEF2-Rho1 signalling (Wenzl, 2010).

RhoGEF2 is an essential regulator of Rho1 activity during many different stages of Drosophila development including cellularization. However, little has been known about the events and factors that control RhoGEF2 localization and subsequent Rho1 activation at the furrow canal. This study assigned a new function to the PDZ domain of RhoGEF2 in being sufficient and required for furrow canal localization. The pattern and the dynamics of furrow canal localization of different PDZRG2 containing constructs are very similar to that of endogenous RhoGEF2 thereby reflecting the behaviour of the full-length protein during cellularization. The domain could be used to effectively target other proteins like RFP, Myc or GST to the furrow canal. Thus despite being a multidomain protein, furrow canal localization depends ultimately only on residues that assure the structural integrity of the ligand recognition site of the PDZ domain. It was reported previously that the RhoGEF2 PDZ domain is involved in the subcellular localization of RhoGEF2 during apical constriction of mesodermal cells in gastrulation. It has been suggested that a direct interaction between the PDZ domain and the PDZ binding motive at the C-terminus of the apically localized transmembrane protein T48 is involved in the recruitment of RhoGEF2 to the apical site of the cells. However, it is clear that this interaction is not essential for apical RhoGEF2 localization, since this localization is lost only in T48/cta double mutants (Wenzl, 2010).

By using immunoprecipitations from staged embryonic extracts it was possible to show that a transgenic 4xPDZRG2-myc6 construct can physically interact with Slam in vivo. Of course this does not directly proof that Slam also interacts with full-length endogenous RhoGEF2. Nevertheless different arguments are presented that support the assumption that a physical interaction between Slam and RhoGEF2 underlies the observed functional relationship between these two factors in cellularizing embryos. The PDZ domain is the critical element that mediates the localization of RhoGEF2 at the furrow canal where it colocalizes with Slam. It was shown that this PDZ domain can form a complex with Slam in vivo. Further in vivo experiments confirmed that furrow canal localization of RhoGEF2 depends on slam in a dosage dependent manner which supports the biochemical findings. Moreover Slam can recruit RhoGEF2 to ectopic sites in embryos as well as in S2 cells and aspects of the RhoGEF2 mutant phenotype can be observed in slam deficient embryos. Overall it is reasonable to conclude that there may be a direct or indirect interaction between Slam and RhoGEF2 during formation of the cellular blastoderm. This interaction would be mediated by the PDZ domain of RhoGEF2. The data also demonstrate that slam acts upstream of RhoGEF2 (Wenzl, 2010).

The molecular function of slam has remained unknown, although the essential role of this gene in cellularization is well established (Merrill, 1988). It has been proposed that Slam is involved in membrane traffic, since in slam mutants the polarized insertion of membrane is disturbed. This study describes an additional cell biological function of slam in being a developmental switch that temporally and spatially controls Rho activity in blastoderm embryos by regulating the subcellular localization of the Rho1 activator RhoGEF2. Thus by proposing the existence of a protein complex containing RhoGEF2 and Slam, physiological and molecular function of Slam can be linked (Wenzl, 2010).

PDZ domains often interact with the C-termini of transmembrane proteins. There are different classes of PDZ binding motifs that can be classified according to their amino acid composition. Although not being a transmembrane but a membrane associated protein, Slam possesses a potential class II PDZ binding motif at its C-terminus. However, this motif seems to be dispensable for the recruitment of RhoGEF2 by Slam to ectopic sites. This is consistent with the fact that a slam allele with a mutated C-terminus rescues the cellularization phenotype of slam deficient embryos. In addition this allele is able to recruit RhoGEF2 to the furrow canal membrane. Furthermore RhoGEF2 to be still present although with reduced levels at the furrow canal in germline clones of a C-terminally truncated slam allele slamwaldo1 (Wenzl, 2010).

Besides the interaction between Slam and the PDZ domain of RhoGEF2, an interaction between Slam and Patj was observed in co-IPs from staged embryonic extracts. This is consistent with the fact that both proteins almost perfectly colocalize during cellularization at the furrow canal as well as in basal particles. Furthermore a functional relation between Slam and Patj is seen, since Patj levels at the furrow canal are reduced in embryos that are zygotically deficient for slam. Patj is a conserved protein that contains 4 PDZ domains and was previously reported to be able to interact with Crumbs in vitro and in vivo during epithelial polarity establishment later in development. However, the importance of this interaction remains unclear, since embryos that are maternally and zygotically mutant for Patj have been reported to develop until adulthood without obvious phenotypes. This would argue against an essential role of Patj during cellularization. As shown by another report, the mutants used in the study still expressed a truncated Patj protein that contained the first PDZ domain thus it is likely that residual Patj function was still present. Zygotic Patj null mutants, in which the coding sequence of Patj was removed completely, died during second instar larval stage, indicating that Patj is an essential gene. Therefore it would be worth to generate maternal Patj null mutants to investigate the role of this protein during cellularization in more detail. Nevertheless the interaction between Patj and Slam seems to depend mainly on the C-terminus of Slam, since in slamwaldo1 mutants Patj levels at the furrow canal are strongly reduced. Thus it is possible that the putative PDZ binding motif at the C-terminus of Slam is important for a direct interaction with one of the PDZ domains of Patj. The Slam Patj interaction also shows that besides controlling RhoGEF2 localization Slam has other independent functions, which could account for the strikingly stronger cellularization phenotype of slam mutants compared to the weaker phenotype of RhoGEF2 deficient embryos (Wenzl, 2010).

RhoGEF2 also functions in different epithelial invagination processes like salivary gland formation or in the establishment of the epithelium in the wing imaginal disc of Drosophila L3 larvae. It appears likely that the subcellular localization of the protein is controlled by genes encoding different receptors that are expressed during different developmental stages in a tissue specific manner like slam or T48 which would allow a very precise temporal and spatial regulation of Rho activity by employing the same ubiquitously expressed activating factor. RhoGEF2 also has a function in the maternally controlled formation of the metaphase furrows during the cleavage divisions 10-13 of the syncytial blastoderm stage and it was shown that localization of the protein to these furrows depends on maternal components of the recycling endosome. The start of zygotic slam expression at the onset of cellularization thus could assure that sufficient levels of RhoGEF2 and thus Rho activity become associated with the membrane tip during invagination. At the same time the metaphase furrows that have recently been shown to be rather active endocytic membrane domains are transformed into a domain forming the furrow canal, which were reported to be much more inactive and stable (Wenzl, 2010).

This study also shows that slam transcripts exhibit a new and unique mRNA localization pattern. A significant portion of slam mRNA is associated with the furrow canal membrane domain. Surprisingly the initial processes that ensure a local restriction of Rho activity would be the proper localization of the slam RNA/protein particles. The asymmetrical localization of transcripts within a cell often linked with localized translation is an important mechanism for the spatial regulation of gene activity. Apical localization of transcripts during cellularization has been described for a number of genes including wg, run and ftz. This study showed that the transcripts are transported to localize to the apical cytoplasm of the cells of the cellular blastoderm. However, little is known about the functional importance of this transcript localization. The localization of slam transcripts might also include a basal to apical transport step, since large basal particles were seen containing slam mRNA and protein in cellularizing embryos. It has been reported previously that apical Rho activity during posterior spiracle formation is mediated in part by RhoGEF64C. The transcript of this gene does localize to the apical membrane of the epithelial cells which undergo apical constriction and subsequent invagination. The mechanisms that ensure the association of transcripts with a specific membrane domain remain to be solved and slam would offer a good system to study this question. Future studies will show whether and how the localization of slam mRNA is involved in defining the sites for membrane invagination and what other functions are served by slam besides initiating Rho signalling (Wenzl, 2010).

Taken together, a model is proposed for the developmental control of Rho1 signalling at the furrow canal, in that the slam RNA-protein particles are targeted to the prospective site of membrane invagination at the onset of cellularization. Slam would have several functions, mainly initiating the formation of the furrow canal as a distinct membrane domain by regulating membrane traffic and at the same time it would recruit and restrict RhoGEF2 and maybe other factors to this domain. After reaching a critical concentration the GEF activity would be activated by a yet unknown mechanism. Rho1 would be converted into its GTP-bound form and downstream targets like Dia or Rho-kinase would be activated. Consistent with this model is the observation that the dose-dependent activity of Slam, both higher or lower than normal levels, directly corresponds to the amount of RhoGEF2 protein and the speed of cellularization as for example shown by the local injection of slam RNA (Wenzl, 2010).

Slam is required germ cell migration

Two alleles of slam were identified in an EMS mutagenesis screen for zygotically acting genes that affect germ cell migration. These two mutants are referred to as the waldo alleles of slam, or slamwaldo alleles, to indicate their origin. Both alleles have similar germ cell migration phenotypes. In slamwaldo mutant embryos, germ cells migrate correctly through the midgut epithelium and along the midgut towards the dorsal side of the embryo, but fail to enter the mesoderm to contact the SGPs. Entering the mesoderm at this stage is crucial for germ cells to become incorporated into the embryonic gonad later. Thus, slamwaldo mutant germ cells that fail to contact the SGPs at stage 11 will be located outside the gonads at stage 14 (Stein, 2002).

Detailed comparison between wild-type and mutant development suggests that germ cells in slamwaldo mutants are delayed in their movement from the gut to the mesoderm during stage 11. As a result, some germ cells remain on the midgut throughout embryogenesis and still are found on the midgut at stage 14. Other germ cells move off the midgut but seemingly too late to reach the most posterior cluster of SGPs. Because the germ band has begun to retract, delayed germ cells move into a more posterior region of the mesoderm, which lacks SGPs. These germ cells are found outside and posterior to the embryonic gonad at stage 14. The remaining germ cells are found associated with SGPs in the embryonic gonad at the end of embryogenesis. On average, 50% of germ cells are lost per embryo in slamwaldo1 and slamwaldo2, compared with fewer than 10% in the control (slam/+) embryos (Stein, 2002).

The germ cell migration phenotype in slam embryos could be due to a germ cell autonomous defect; however, Slam RNA and protein are not expressed in the germ cells. Alternatively, the phenotype could be due to a defect in either the specification or differentiation of the somatic tissues that contact germ cells during their migration, or more directly in the production or localization of a guidance cue. Since the migration defect is first observed when the germ cells leave the midgut, the development of the midgut and the gonadal mesoderm was analyzed in mutant embryos. In situ hybridization with RACE, an enzyme expressed in the posterior midgut primordium, reveals a delay in the transition from an epithelium to a mesenchyme in mutant embryos. At stage 11, when in wild-type embryos the midgut primordium has already started to flatten out and extend along the mesoderm, the midgut remains more compact in the mutant. The length of the posterior midgut is visibly shorter in the mutant. This defect is transient, since later midgut markers, such as dpp-lacZ, reveal that a continuous digestive tract is formed; however, in some embryos the second midgut constriction fails to form. FasIII staining reveals that the visceral mesoderm forms normally and surrounds the midgut. Most mutant embryos become crawling larvae and are capable of passing colored yeast normally through their digestive tracts. Even if a mild midgut defect exists, this cannot explain the germ cell migration defect of slamwaldo mutants. The midguts of embryos that lack maternal and zygotic integrin function also fail to undergo an epithelial to mesenchymal change at stage 11 and appear morphologically very similar to those of slamwaldo, but the integrin mutants have normal germ cell migration. This implies that timing of the epithelial to mesenchymal transition in the posterior midgut is not crucial for germ cells to move from the midgut into the mesoderm, and suggests that the slamwaldo mutant germ cell migration defect is not simply a result of the altered midgut morphology (Stein, 2002).

For the analysis of the mesoderm, two markers were used: RNA expression of the retrotransposon 412 and antibody staining for Zfh-1 protein, which mark the lateral mesoderm at stage 10-11 as well as the gonadal mesoderm, later. Both markers are expressed in slamwaldo mutants, and the number and distribution of lateral mesoderm cells in mutant embryos seems similar to that of wild type during stages 10-11. However, later when gonadal mesoderm cells move towards each other to align and eventually coalesce to form the embryonic gonad, some mutant embryos show a reduction in the number of SGPs. In abdA mutants, lateral mesoderm forms correctly, but no SGPs are specified. Germ cells in these mutants still migrate off the midgut into the lateral mesoderm, but are lost later in embryogenesis. This suggests that lateral mesoderm, not later SGP formation, is necessary for germ cells to move off the midgut into the mesoderm. Therefore the reduction in SGP number in slamwaldo mutants is not responsible for the slamwaldo germ cell migration defect. Taken together, these data suggest that the defect in slamwaldo mutants is more likely to be due to a defect in midgut signaling or guidance than to a failure in gonadal mesoderm specification or midgut development (Stein, 2002).

The phenotype of slamwaldo mutant embryos is strikingly similar to that of Hmgcr mutants. In both mutants, germ cells fail to move off the midgut and do not associate with SGPs in the mesoderm at stage 11-12. Some germ cells are also seen correctly migrating in both single mutants, even in null alleles of Hmgcr. An instructive role as a germ cell attractant has been demonstrated for Hmgcr by the finding that mis-expression of Hmgcr leads to attraction of germ cells to the ectopic site independent of SGP differentiation. The data make it less likely that Slam and Hmgcr act within the same pathway and favor the hypothesis that Hmgcr and slam act independently and provide separate guidance cues. This conclusion is further supported by the analysis of slamwaldo; Hmgcr double mutants. The germ cell migration phenotype in double mutant embryos is stronger than either single mutant. The fact that the double mutant has a novel phenotype suggests that, while the activity of Hmgcr and slam may not directly rely on each other’s function, the two genes may act in parallel and may regulate common downstream pathways (Stein, 2002).

The data strongly suggest that for germ cell migration, as for cellularization, Slam function is needed in the somatic cells and not in the germ cells. slam RNA and protein are detected only during blastoderm and the onset of gastrulation (stage 4-5, 2-3.5 hours after fertilization) but not later (stage 10-11, 5-7 hours after fertilization) during germ cell migration. No maternally loaded Slam protein was detected in germ cells and zygotic expression of slam RNA was absent from germ cells. Furthermore, the germ cell migration phenotype is only observed in homozygous mutant embryos; a paternal slam+ allele rescues both the cellularization and the germ cell migration defect. Since germ cells are transcriptionally repressed until they initiate migration, the genetics of the slam phenotype and the slam RNA and protein expression pattern argue against a role of slam in germ cells (Stein, 2002).

slam RNA and protein expression profiles led to the conclusion that expression of Slam in the soma during blastoderm formation must be necessary later in development for germ cell migration. One possible explanation for the role of slam in germ cell migration could be that proper cellularization is required for germ cell migration and that zygotic slamwaldo mutants have a subtle cellularization defect that later causes a germ cell migration defect. No obvious cellularization defects were detected in homozygous slamwaldo mutant embryos. Thus, as an alternative, it is proposed that slam-targeted membrane vesicles may not only accomplish rapid membrane growth but may also be used to insert a germ cell guidance signal into the growing membrane. This hypothetical guidance signal would be deposited before or during gastrulation, when Slam is present, but act after gastrulation when germ cells are migrating off the midgut. Mutant analysis suggests that Slam function in the midgut primordium is crucial for germ cell migration. A delay in germ cells migrating from the midgut to the mesoderm is observed as well as a delay in the epithelial to mesenchymal transition of the midgut primordium in mutant embryos. A possible model is that during blastoderm formation Slam is needed for the deposition of a germ cell guidance factor into the basal-lateral membrane. This factor could be present in all cells, or just in the posterior midgut. During gastrulation, the germ cells migrate through the posterior midgut primordium from the apical to the basal side. Once they reach the basal side of the epithelium, germ cells require this guidance factor to move quickly off the midgut and into the mesoderm. The nature of this guidance factor is unknown, it could be a factor very specific to germ cells or it could be a component of the basement membrane, which mediates cell migration in many systems. The guidance factor may be a germ cell repellant or a protein that promotes germ cell movement. It is likely to be independent of Hmgcr based on the slamwaldo;Hmgcr double mutant phenotype. It is also likely independent of wun and wun 2. The wun/wun 2-dependent germ cell reorientation on the midgut appears normal in slamwaldo mutant embryos, suggesting that slamwaldo mutations affect a later step in germ cell migration than wun/wun 2 (Stein, 2002).

Blastoderm-specific genes, like slam, nullo, bottleneck and serendipity-alpha affect formation of cells at only the blastoderm stage. These genes are not required for germ cell formation, which happens at the same developmental stage, and are not expressed during cytokinesis at other stages of development. This suggests that blastoderm cellularization is a highly specialized form of cytokinesis. Blastoderm formation has been used as a model system to study the generation of polarized epithelia. In support of this idea, blastoderm cells form apical junctions and contain proteins in a polarized distribution typical for epithelial cells. However, it has been difficult to establish a functional link between the polarity established during the blastoderm stage and the polarity observed in epithelial cells at later stages of differentiation, because embryos that fail to form cells in the first place cannot be analyzed later. slamwaldo mutations cause defects in germ cell migration and midgut morphology, but not in blastoderm cellularization. Nevertheless, the blastoderm-specific expression of Slam suggests that Slam function at this early stage is required for later development. These findings, therefore, provide evidence that Slam may not only generate the cells from which the embryo develops but may also establish spatial cues within those cells needed for later morphogenesis (Stein, 2002).


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Biological Overview | Developmental Biology | Effects of Mutation

date revised: 20 June 2014

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