Drosophila SCAR colocalizes with actin structures during embryonic development
A search of the sequenced Drosophila genome identified a single Scar/WAVE homologue (SCAR, corresponding to transcription unit CG4636) that maps to cytogenetic band 32C4-5 on the second chromosome. We determined the structure of the SCAR transcript by sequencing three ESTs from the Berkeley Drosophila Genome Project database (). The 2,184 nucleotide SCAR transcript is predicted to encode a 613 amino acid protein possessing the major hallmarks of Scar/WAVE proteins ().
To examine SCAR protein expression and subcellular localization, we generated a polyclonal antibody to the unique SCAR NH2-terminal domain (see Materials and methods). We found that SCAR protein is present in early blastoderm embryos and in the embryonic CNS () consistent with its mRNA expression (unpublished data). In the blastoderm, SCAR protein colocalizes with filamentous actin structures that are dynamically regulated during the cell cycle (; see below). In the CNS, SCAR protein is specifically localized to axons (). This pattern of SCAR protein expression in the embryo provided us with an initial indication of the potential sites of SCAR gene activity.
Figure 2. SCAR protein expression in embryos. Anti-SCAR polyclonal antibody (top row); filamentous actin labeled with phalloidin (bottom row). In wild-type embryos at cycle 12, SCAR protein colocalizes with actin in interphase (A, SCAR; G, actin) (more ...)
Isolation of mutations in the Drosophila SCAR gene
To investigate SCAR function, two mutant alleles of SCAR
were identified and characterized. The recessive lethal P-element insertion mutation l(2)k13811
(Spradling et al., 1999
) lies within the 5′ UTR of the SCAR
transcript, 208 nucleotides upstream of the translation start codon ( A) (Berkeley Drosophila
Genome Project; unpublished data). Chromosomes bearing precise excisions of this insertion complemented the lethality of l(2)k13811
and were homozygous viable. In addition, ubiquitous expression of a full-length SCAR
cDNA rescued the lethality of the l(2)k13811
insertion. These data demonstrate that the zygotic recessive lethality is due to disruption of the SCAR
gene by the l(2)k13811
insertion, and we refer to this allele as SCARk13811
. Moreover, embryos that are maternally and zygotically mutant for the SCARk13811
allele display a strong reduction in staining with the anti-SCAR antibody (), confirming that this insertion disrupts SCAR
To obtain deletions in the SCAR
locus, we generated imprecise excision alleles of the SCARk13811
insertion, all of which were homozygous lethal and failed to complement the lethality of SCARk13811
. The homozygous lethality of the Δ37
excision allele was rescued by ubiquitous expression of the full-length SCAR
cDNA, and we refer to this allele as SCARΔ37
was molecularly characterized and removes all SCAR
sequences downstream of the insertion site ( A). This excision event also removes portions of the neighboring piwi
transcription unit. Since piwi
function is restricted to maintenance and proliferation of germline stem cells (Cox et al., 1998
), the SCARΔ37
phenotypes described below, in distinct tissues, are likely to represent consequences of disrupting SCAR
function alone. Developmental defects were considerably weaker in SCARk13811
, indicating that the insertion allele retains partial SCAR
In addition to SCAR and Wasp (Ben-Yaacov et al., 2001
), the sequenced Drosophila
genome contains predicted homologs of the seven members of the Arp2/3 complex (Fyrberg et al., 1994
; Goldstein and Gunawardena, 2000
). Mutations have been recovered in two Arp2/3 complex components, Arp3 (Rørth, 1996
; Berkeley Drosophila
Genome Project) and Arpc1 (Hudson and Cooley, 2002
). This set of mutations provides an opportunity to analyze the role of Arp2/3-based signaling in different contexts within a multicellular organism and to ascertain the physiological contributions of the SCAR and Wasp activators.
SCAR and Arpc1 are required for cytoplasmic organization in the blastoderm embryo
Homozygous mutations in either SCAR
allele result in zygotic lethality, which can occur during late embryogenesis, larval, and early pupal stages. However, maternally provided SCAR
gene products may compensate for loss of zygotic gene function and mask an essential requirement during embryogenesis. To interfere with the maternal gene contribution, we used FLP-mediated recombination to produce homozygous mutant clones within the germline of heterozygous females (Chou and Perrimon, 1996
). Strong disruption of maternal SCAR
in this manner results in developmental arrest during oogenesis (see below). However, germline clones homozygous for the weaker SCARk13811
alleles give rise to fertilizable eggs, enabling study of functional requirements for SCAR and the Arp2/3 complex during embryogenesis. These embryos are designated SCARmat
, respectively. To compare the roles of SCAR and Wasp, we examined embryos derived from germline clones for the strong loss of function Wsp3
embryos). The Wsp3
frameshift mutation truncates the protein before the highly conserved WA domain that is required for Arp2/3 activation and is a probable null allele (Ben-Yaacov et al., 2001
The early blastoderm embryo undergoes 13 nuclear divisions without accompanying cytokinesis, producing a multinucleate syncytium. The majority of nuclei migrate to the surface by cycle 10, where they undergo four synchronous rounds of division before their compartmentalization into individual cells during interphase of cycle 14 (Zalokar and Erk, 1976
). Surface nuclei maintain a uniform distribution throughout these final syncytial divisions (). Examination of the spatial distribution of nuclei revealed a requirement for SCAR and Arpc1, but not Wasp, during these cortical division cycles (, C–H). SCARmat
mutants exhibited defects in the uniform spacing of interphase nuclei beginning in cycle 11, whereas Wspmat
mutants displayed wild-type nuclear organization. By cycles 12 and 13, increased defects in nuclear spacing in SCAR
were accompanied by the appearance of abnormal nuclear morphologies, including large or elongate DNA masses that are likely to represent the fusion of adjacent nuclei.
Figure 3. Defects in nuclear arrangement and morphology in SCAR and Arpc1 mutant embryos. (A–H) Surface views of syncytial embryos. Cortical nuclei are uniformly distributed in wild-type cycle 12 (A) and 13 (B) embryos. Nuclei exhibit abnormal spacing and (more ...)
In wild-type syncytial embryos, nuclei are maintained in two separate populations: a uniform layer of surface nuclei and an internal mass of yolk nuclei. In SCARmat and Arpc1mat embryos, displacement of surface nuclei into the interior was first observed at cycle 12 and increased in severity by cycle 14 () (96% of cycle 14 SCARmat embryos, n = 24; 100% of cycle 14 Arpc1mat embryos, n = 24). The severity of these defects was strongly correlated with increased division cycles, demonstrating a late onset progressive defect (for SCARmat p = 10−17, n = 76 embryos, and rsp = 0.80; for Arpc1mat p = 10−21, n = 69 embryos, and rsp = 0.87; rsp is the Spearman rank correlation coefficient). Nuclear displacement was rarely observed in wild-type and Wspmat embryos () (3% of cycle 14 wild-type embryos, n = 33; 0% of cycle 14 Wspmat embryos, n = 32).
SCAR and Arpc1 are required for actin polymerization and regulation of dynamic actin structures in the blastoderm embryo
The syncytial blastoderm contains well-defined filamentous actin structures that exhibit dynamic cell cycle regulation (Karr and Alberts, 1986
). Actin is organized into caps overlying individual nuclei during interphase of cortical cycles 10–14 (). During mitosis, actin is redistributed into a network of metaphase furrows that separate adjacent spindles (). Genetic and drug interference studies demonstrate that organization of the actin cytoskeleton is crucial for the uniform arrangement of blastoderm nuclei (Foe et al., 1993
; Schejter and Wieschaus, 1993
). The nuclear defects in SCAR
mutants, and SCAR protein colocalization with filamentous actin (), raise the possibility that SCAR may function in the regulation of actin structures in the blastoderm embryo.
Figure 4. Interphase actin structures in SCAR and Arpc1 mutant embryos. Organization of interphase actin caps (first and third columns) and corresponding nuclei (second and fourth columns) are shown. All embryos were fixed, stained, and imaged under identical conditions (more ...)
Figure 5. Metaphase actin furrows are defective in SCAR and Arpc1 embryos. Filamentous actin structures (phalloidin in green) and mitotic spindles (tubulin in red and DNA in blue) during cycles 11 (columns 1, 2, 5) and 12 (columns 3 and 4). Actin and spindle staining (more ...)
In SCARmat embryos, actin caps appeared largely intact during interphase, although some defects were observed. In particular, SCARmat actin caps were consistently smaller and less rounded than in wild type ( E, 22/35 embryos). In a subset of embryos, primarily in later syncytial divisions 12 and 13, actin caps appeared less discrete and gaps were observed in regions where nuclei were abnormally clustered ( G, 13/35 embryos). In contrast to the relatively mild defects in interphase caps, metaphase furrows were completely absent in the majority of SCARmat embryos undergoing mitosis, and actin accumulated in aberrant structures positioned above rather than between individual spindles (, 21/28 embryos). A partial and discontinuous metaphase furrow network was observed in a minority of SCARmat embryos (, and J, 7/28 embryos). These two mitotic phenotypes were mutually exclusive and consistent across the entire embryo surface. These observations suggest an abnormal reorganization of interphase actin as SCARmat embryos enter mitosis, indicating that the transition between interphase caps and metaphase furrows requires SCAR function.
Major defects in cortical actin structures were also observed in Arpc1mat embryos, where interphase actin caps were abnormal and actin appeared to be depleted from the regions above individual nuclei (, 37/37 embryos). This depletion is apparent most readily in cross-section ( Q). As in SCAR, metaphase furrows failed to form in Arpc1mat embryos ( M, 12/12 embryos). However, unlike SCAR, metaphase actin exhibited a diffuse localization to the broad region between spindles ( R). These results demonstrate that the Arp2/3 complex component Arpc1 is required for the formation of both interphase actin caps and metaphase actin furrows. The greater severity of the Arpc1 phenotype compared with SCAR could reflect a difference in residual gene activity of these partial loss of function alleles.
SCAR and Arpc1, therefore, provide functions that are critical for proper formation of cortical actin structures. In contrast, actin caps and furrows formed normally in Wspmat embryos (27 interphase and 5 metaphase embryos) (). It is worth emphasizing in this context that both the SCAR and Arpc1 phenotypes result from a partial loss of gene function, whereas early embryogenesis can proceed normally despite complete lack of Wsp gene activity.
In addition to defects in organization of microfilament structures, overall actin levels in Arpc1mat and SCARmat embryos appeared consistently lower than in wild type. To rigorously assess differences in actin levels, we quantitated surface filamentous actin in syncytial embryos using a phalloidin fluorescence assay. We found that both Arpc1mat and SCARmat mutants exhibited significantly reduced levels of surface actin compared with control embryos (). The more severe loss of actin in Arpc1mat correlates with the greater disruption of actin structures in this mutant. These results indicate that Arpc1 and SCAR are both required for the generation of bulk filamentous actin in the blastoderm and suggest a common basis for the defects in cortical actin structures of Arpc1 and SCAR mutant embryos.
Figure 6. Reduced filamentous actin in SCAR and Arpc1 mutant embryos. Fluorescence intensity is indicated in arbitrary units on the y axis. Error bars represent the standard error of the mean and depict the variability between embryos. Embryos were staged according (more ...)
SCAR and Arp2/3 complex components are essential for embryonic CNS axon morphology
The striking enrichment of SCAR expression in the CNS () prompted us to examine CNS morphology in SCAR
mutants using the axon-specific BP102 monoclonal antibody (Fujita et al., 1982
). In wild-type embryos, CNS axons travel in two longitudinal bundles that flank the midline and two commissural bundles that cross the midline in each segment ( A). Although no apparent defects were observed in homozygous SCAR
embryos (), maternal contribution of SCAR
transcript or protein may provide sufficient wild-type SCAR activity to mask a functional requirement in the CNS. Partial maternal SCAR function provided by the weak SCARk13811
allele at lower temperature (20–22°C rather than 25°C) is sufficient to produce embryos that develop normally through the blastoderm stages described above, allowing an examination of later CNS development.
Figure 7. CNS axon morphology is disrupted by mutations in SCAR, Wasp, and the Arp2/3 complex components Arp3 and Arpc1. CNS axons were visualized with the axon-specific BP102 antibody. (A–H) Ventral view of stage 14/15 embryos; three to four segments (more ...)
Reduction of SCAR function achieved in this manner indeed caused dramatic CNS defects (). The phenotypes observed in these mutants (designated SCARmat/zyg embryos) required disruption of zygotic SCAR function (unpublished data). In SCARmat/zyg embryos, frequent breaks occurred in longitudinal and commissural bundles (93% of segments, n = 242). In extreme cases, a severe depletion of all axon bundles was observed ( C, 46% of segments). At a lower frequency, SCARmat/zyg embryos exhibited defects in commissure fasciculation and separation (18% of segments), and medial (13%) or lateral (9%) displacement of axons.
Since SCAR is essential for normal CNS axon morphology, we also examined the zygotic effect of mutations in two members of the Arp2/3 complex, Arp3 and Arpc1. Arp3 zygotic mutant embryos exhibited a partially penetrant defect in CNS axon morphology with a range of phenotypes that strongly resemble SCARmat/zyg mutants (). In particular, the majority of Arp3 mutants displayed breaks in the longitudinal and commissural axon bundles ( E) (48% of segments in mutant embryos, n = 115 segments). A subset of Arp3 mutants exhibited defects such as commissure defasciculation or fusion (28% of segments) and medially or laterally displaced axons (3% of segments). Arp3 heterozygotes also exhibited a low penetrance of axon defects ( J). The CNS morphology of zygotic Arpc1 single mutants appeared normal, perhaps due to the continued presence of maternal gene products. However, combining zygotic Arpc1 mutations with an Arp3 heterozygous background produced defects that were significantly more severe than in Arp3 heterozygotes alone (). These phenotypes demonstrate a similar functional requirement for SCAR and Arp2/3 complex components during CNS development.
The contribution of Wasp function to CNS axon morphology is more difficult to assess, since complete removal of maternal and zygotic Wsp
using the strong Wsp3
embryos) produces cell fate defects in CNS lineages (Ben-Yaacov et al., 2001
). An apparent thickening of commissural bundles suggestive of an increase in neuronal number was observed in a majority of Wspmat/zyg
embryos ( D). In addition, most Wspmat/zyg
embryos contained one to two segments with axon bundles collapsed at the midline ( D) (73% of embryos, n
= 41). Despite these phenotypes, Wspmat/zyg
embryos did not exhibit the severe defects in axon morphology present in SCAR
mutants. Although removal of zygotic SCAR
function alone did not disrupt CNS morphology (), zygotic reduction of SCAR
together produced significant defects () that resemble the strong SCARmat/zyg
phenotype. Therefore, although loss of Wasp function alone does not cause the significant axon defects produced by loss of SCAR, Wasp can influence axon morphology in situations where SCAR function is compromised.
SCAR, and not Wasp, is required with the Arp2/3 complex for egg chamber morphology during oogenesis
Although the partial reduction of SCAR function associated with the SCARk13811
insertion allele is sufficient for normal egg production, the more severe SCARΔ37
excision allele produces small and abnormally shaped eggs indicative of a defect in oogenesis. Drosophila
ovaries house a series of egg chambers that each contain 16 cells (the oocyte and a 15-cell nurse cell complex) interconnected by cytoplasmic bridges (ring canals) that arise from incomplete cytokinesis during mitosis (Spradling, 1993
). Morphological defects are apparent in SCARΔ37
germline clones during the final phases of oogenesis (). In particular, nurse cells become multinucleate, as many of the actin-lined nurse cell membranes are absent ( B). The morphological abnormalities extend to additional structures, including the actin-rich ring canals, which are significantly smaller than in wild-type and aberrantly shaped ().
Figure 8. Abnormal oogenesis in SCAR and Arpc1 mutants. (A–D) Single egg chambers stained to reveal nuclear arrangement (green, visualized with OliGreen) and nurse cell membranes (red, visualized with phalloidin). (A) Nurse cell (nc) nuclei in a late stage (more ...)
The defects observed in SCAR
mutant egg chambers closely resemble phenotypes described in mutants for the Arp2/3 complex subunits Arpc1
(Hudson and Cooley, 2002
), resulting in late stage deterioration of the nurse cell complex ( C). In marked contrast to the Arpc1
, and SCAR
phenotypes, oogenesis in germline clones for the strong loss of function Wsp3
allele appears wild type. No apparent morphological abnormalities were observed in Wsp3
late stage egg chambers ( D), which can support embryonic development after fertilization (Ben-Yaacov et al., 2001
). This phenotypic analysis indicates that SCAR, rather than Wasp, is the major mediator of Arp2/3 function during Drosophila
oogenesis, much as was observed in the blastoderm and the embryonic CNS.
SCAR and Wasp are required for distinct aspects of Arpc1 function in the adult eye
The above phenotypic analyses identify several Arp2/3-dependent morphological processes that rely on SCAR but are largely independent of Wasp. We therefore asked whether the reciprocal situation exists, namely, are there Arp2/3-mediated events that rely on Wasp but are independent of SCAR? Wasp provides an essential contribution to cell fate decisions in several neural lineages in the Drosophila
embryo and adult (Ben-Yaacov et al., 2001
). Furthermore, the Arp2/3 complex component Arpc1 is required for Wasp-dependent cell fate changes during sensory organ development, and association with Arp2/3 is essential for Wasp function in this context (Tal et al., 2002
). This requirement provides an opportunity to examine whether developmental events dependent on Wasp also require SCAR function.
In the adult peripheral nervous system, a primary consequence of mutations in Wsp
is the excessive differentiation of sensory organ neurons at the expense of nonneuronal cell types, resulting in a marked absence of mechanosensory bristles (). We used the ey-
FLP-FRT system (Newsome et al., 2000
) to generate mosaic SCAR
heterozygous flies in which head capsule structures and cuticle are derived from homozygous mutant clones induced in the eye imaginal disc. Arpc1
mosaic heads like, Wsp
, display a pronounced bristle loss phenotype ( C), which results from cell fate defects similar to those present in Wsp
mutants (Tal et al., 2002
). In contrast, the sensory organ pattern in mosaic heads of strong SCAR
alleles appears wild type ( D), suggesting that SCAR does not play an essential role in lineage decisions mediated by Wasp and the Arp2/3 complex.
Figure 9. Distinct head bristle patterns and eye morphologies of Wsp, Arpc1, and SCAR mutants. Scanning electron microscope images of head cuticle (A–D) and enlarged portions of compound eyes (E–H). Nearly all bristles present on the dorsal aspect (more ...)
In addition to loss of sensory organ structures, Arpc1
mosaics display abnormalities in eye structure, including a reduction in the overall size of the eye, irregularly shaped ommatidia, and a distinct loss of lens material in most eye facets (). Mosaics for the SCARΔ37
excision allele present a very similar eye phenotype (), with the exception that interommatidial bristles are largely present. As noted previously (Ben-Yaacov et al., 2001
), mutations in Wsp
have no discernible effect on eye morphology apart from the bristle loss phenotype (). This analysis provides a striking example of the distinct requirements for SCAR and Wasp, which mediate separate aspects of Arp2/3 complex function during adult development.