Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling

Abstract

Wiskott-Aldrich syndrome proteins, encoded by the Wiskott-Aldrich syndrome gene family, bridge signal transduction pathways and the microfilament-based cytoskeleton. Mutations in the Drosophila homologue, Wasp (Wsp), reveal an essential requirement for this gene in implementation of cell fate decisions during adult and embryonic sensory organ development. Phenotypic analysis of Wsp mutant animals demonstrates a bias towards neuronal differentiation, at the expense of other cell types, resulting from improper execution of the program of asymmetric cell divisions which underlie sensory organ development. Generation of two similar daughter cells after division of the sensory organ precursor cell constitutes a prominent defect in the Wsp sensory organ lineage. The asymmetric segregation of key elements such as Numb is unaffected during this division, despite the misassignment of cell fates. The requirement for Wsp extends to additional cell fate decisions in lineages of the embryonic central nervous system and mesoderm. The nature of the Wsp mutant phenotypes, coupled with genetic interaction studies, identifies an essential role for Wsp in lineage decisions mediated by the Notch signaling pathway.

Figure 1

(A) Conservation of primary sequence and domain structure in the Drosophila WASP homologue. The primary protein sequence encoded by Drosophila Wasp (wsp) was compared and aligned with bovine N-WASP (nwasp) using PileUp and PrettyBox (Genetics Computer Group). Residue numbers are indicated on the right and identical residues are shaded black. The major structural and functional domains of WASP proteins are boxed. These include: the NH₂-terminal WH1 membrane-interacting domain; the CDC42 GTPase binding domain (GBD); the proline-rich SH3-binding domain (PR); monomeric actin-binding domains homologous to yeast verprolin (VH1 and VH2); and two COOH-terminal domains: a cofilin-homologous domain (CH) and an acidic tail (A) that are responsible for Arp2/3 complex binding. Domain structure generally follows Miki et al. 1996 and Symons et al. 1996. The GBD is defined as the minimal CDC42 high-affinity binding fragment (Rudolph et al. 1998). Arrows mark the positions of the frameshift mutations in the three Wsp alleles. (B) Drosophila Wsp binds the activated form of CDC42 in a blot overlay assay. A bacterially expressed recombinant fragment of Wsp (residues 96–526) was blotted onto nitrocellulose filters and incubated with [γ-³²P]GTP labeled recombinant mammalian GTPases (bottom). A strong interaction between Wsp and GTP-CDC42 and weak binding to GTP-Rac are observed. Binding to GTP-Rho could not be detected. This profile resembles that determined for mammalian WASP proteins (Aspenstrom et al. 1996; Kolluri et al. 1996; Symons et al. 1996). Reprobing of the filters with anti-Wsp antibodies was performed to ensure that equal amounts of the Wsp fragment were used in the assay (top).

Figure 1

(A) Conservation of primary sequence and domain structure in the Drosophila WASP homologue. The primary protein sequence encoded by Drosophila Wasp (wsp) was compared and aligned with bovine N-WASP (nwasp) using PileUp and PrettyBox (Genetics Computer Group). Residue numbers are indicated on the right and identical residues are shaded black. The major structural and functional domains of WASP proteins are boxed. These include: the NH₂-terminal WH1 membrane-interacting domain; the CDC42 GTPase binding domain (GBD); the proline-rich SH3-binding domain (PR); monomeric actin-binding domains homologous to yeast verprolin (VH1 and VH2); and two COOH-terminal domains: a cofilin-homologous domain (CH) and an acidic tail (A) that are responsible for Arp2/3 complex binding. Domain structure generally follows Miki et al. 1996 and Symons et al. 1996. The GBD is defined as the minimal CDC42 high-affinity binding fragment (Rudolph et al. 1998). Arrows mark the positions of the frameshift mutations in the three Wsp alleles. (B) Drosophila Wsp binds the activated form of CDC42 in a blot overlay assay. A bacterially expressed recombinant fragment of Wsp (residues 96–526) was blotted onto nitrocellulose filters and incubated with [γ-³²P]GTP labeled recombinant mammalian GTPases (bottom). A strong interaction between Wsp and GTP-CDC42 and weak binding to GTP-Rac are observed. Binding to GTP-Rho could not be detected. This profile resembles that determined for mammalian WASP proteins (Aspenstrom et al. 1996; Kolluri et al. 1996; Symons et al. 1996). Reprobing of the filters with anti-Wsp antibodies was performed to ensure that equal amounts of the Wsp fragment were used in the assay (top).

Figure 4

Excess of neurons and reduction of support cells in the PNS of Wsp ^mat/zyg embryos. Structure of the PNS of wild-type and Wsp ^mat/zyg embryos is revealed by staining with informative markers. In all panels the embryonic anterior is to the left and the dorsal aspect is up. Arrows in A point to the segmentally reiterated pattern of PNS sensory organs in a stage 14 wild-type embryo (WT) stained for Couch Potato (Cpo), which is expressed in all mature sensory organ cells (Bellen et al. 1992). The brain and ventral nerve cord are highlighted by this marker as well. Staining for the proneural marker Ac (B) shows the initial phases of SOP selection (arrows) in a stage 11, germband extended wild-type embryo. Magnified views of the Ac pattern in wild-type (C) and Wsp ^mat/zyg (D) embryos suggest that SOP selection proceeds normally in the mutant embryos. Excess numbers and ectopic positions of neurons in magnified portions of the mature (stage 15/16) Wsp ^mat/zyg PNS is revealed by staining with the neuronal nuclear marker Elav (E and F), and with the cytoplasmic/cell surface neuronal marker mAb 22C10 (G and H). A corresponding reduction in the number of Wsp ^mat/zyg PNS nonneuronal support cells is detected with the A1-2-29 (β-galactosidase–based) shaft/socket cell marker (I and J). Staining with the sheath cell marker Prospero reveals only a mild effect on sheath cell numbers in Wsp ^mat/zyg embryos (K and L).

Figure 2

The bristle-loss phenotype of Wsp mutant flies. Panels show select portions of the external cuticle of adult wild-type (left) and Wsp (right) flies, which manifest large differences in bristle number. The mutant genotype in this and subsequent figures showing pupal and adult phenotypes is Wsp ¹/Df(3R)3450. Comparative views of head capsules (A and B) and of the dorsal aspect of two abdominal segments (C and D) reveal cuticular regions that are nearly devoid of bristles in the mutant flies. Comparison of magnified portions of abdominal segments (E and F) demonstrates the loss of both bristle shafts and sockets (smooth cuticle phenotype), and the occasional appearance of duplicated bristles (arrow) in Wsp mutants. The strong interommatidial bristle-loss phenotype in the eye is readily apparent (G and H). Note that the mutation does not affect the ordered spatial pattern of the hexagonal eye facets, in keeping with the general normal morphology of tissues and organs of Wsp flies.

Figure 3

Abnormal differentiation pattern and spatial arrangement of sensory organ cells in the Wsp mutant retina. Confocal micrographs reveal the staining patterns of informative nuclear markers in sensory organ cells of developing wild-type (WT, A–E) and Wsp (F–J) pupal retinas. Most markers are also expressed by photoreceptor cell nuclei, which are at a slightly different focal plane and may appear at the edges and corners of the panels. The enhancer trap marker A101 drives β-galactosidase expression in SOP nuclei. A similar pattern of evenly spaced SOP nuclei is found in wild-type and mutant tissue at 3 h APF (A and F). In contrast, in mature sensory organs at 30 h APF (B and G) the wild-type four-cell formations (visualized with anti-Ct) give way to abnormal clusters in the mutant. Double labeling with Ct (green) and Sv (red), which stains sheath and bristle shaft cell nuclei, reveals minimal Sv staining in the mutant tissue (C and H), whereas double labeling with Ct (green) and Elav (yellow), which stains neuronal nuclei (D and I), demonstrates that nearly all sensory organ cells in the mutant retina express the neuronal marker. Triple labeling with the differentiation markers Sv (blue), Elav (green), and Su(H) (red), a socket cell–specific marker, underscores the preponderance of neurons and paucity of other cell types in mutant tissue (E and J). (K) A schematic representation of the cell division pattern within the adult sensory organ lineage (following Hartenstein and Posakony 1989; Gho et al. 1999; Reddy and Rodrigues 1999). Arrows point to the divisions (orange bars) where the mutant phenotypes indicate a requirement for Wsp function in specifying distinct cell fates, as discussed in the text. Bars: (F) 5 μm; (G) 9 μm; and (I) 10 μm.

Figure 3

Abnormal differentiation pattern and spatial arrangement of sensory organ cells in the Wsp mutant retina. Confocal micrographs reveal the staining patterns of informative nuclear markers in sensory organ cells of developing wild-type (WT, A–E) and Wsp (F–J) pupal retinas. Most markers are also expressed by photoreceptor cell nuclei, which are at a slightly different focal plane and may appear at the edges and corners of the panels. The enhancer trap marker A101 drives β-galactosidase expression in SOP nuclei. A similar pattern of evenly spaced SOP nuclei is found in wild-type and mutant tissue at 3 h APF (A and F). In contrast, in mature sensory organs at 30 h APF (B and G) the wild-type four-cell formations (visualized with anti-Ct) give way to abnormal clusters in the mutant. Double labeling with Ct (green) and Sv (red), which stains sheath and bristle shaft cell nuclei, reveals minimal Sv staining in the mutant tissue (C and H), whereas double labeling with Ct (green) and Elav (yellow), which stains neuronal nuclei (D and I), demonstrates that nearly all sensory organ cells in the mutant retina express the neuronal marker. Triple labeling with the differentiation markers Sv (blue), Elav (green), and Su(H) (red), a socket cell–specific marker, underscores the preponderance of neurons and paucity of other cell types in mutant tissue (E and J). (K) A schematic representation of the cell division pattern within the adult sensory organ lineage (following Hartenstein and Posakony 1989; Gho et al. 1999; Reddy and Rodrigues 1999). Arrows point to the divisions (orange bars) where the mutant phenotypes indicate a requirement for Wsp function in specifying distinct cell fates, as discussed in the text. Bars: (F) 5 μm; (G) 9 μm; and (I) 10 μm.

Figure 5

Cell fate transformations in the CNS and mesoderm of Wsp ^mat/zyg embryos. Panels show matched portions of the embryonic CNS (A and B) and mesoderm (C–F) of wild-type (left) and Wsp ^mat/zyg (right) embryos stained with informative markers. The pattern of stage 16 embryonic ventral nerve cord neuroblasts expressing the Eve marker appears in A and B. Anterior is up. Arrows point to the RP2 neuroblasts, which are duplicated in the mutant (B). In the dorsal mesoderm of stage 15 embryos, Eve expression in pericardial (PC) and muscle DA1 founders is observed in wild-type (C), whereas expression at the PC position predominates in the mutant (D). Correspondingly, the number of Kr-expressing DA1 cells in the wild-type dorsal mesoderm (E) is significantly reduced in Wsp ^mat/zyg embryos (F).

Figure 6

Enhancement and suppression of the Wsp bristle-loss phenotype by the N pathway. (A) Thorax of an N ^ts1 fly raised at 25°C, showing a wild-type bristle pattern of both the larger macrochaetae (M) and the smaller and more numerous microchaetae (m). Reduction in microchaetae number on the thorax of a Wsp ¹/Df(3R)3450 mutant fly (B) is readily apparent, whereas the macrochaete pattern is only mildly affected. The thoracic Wsp bristle-loss phenotype is strongly enhanced in an N ^ts1; Wsp ¹/Df(3R)3450 double mutant fly raised at 25°C (C). (D) Scanning electron microscope image of a wild-type eye. The eye of a Wsp ¹/Df(3R)3450 mutant fly (E) is almost devoid of interommatidial bristles. Nearly full suppression of the Wsp phenotype is observed in a scanning electron microscope image of the eye of an H ³; Wsp ¹/+; Df(3R)3450 fly (F). Significant restoration of the abdominal wild-type bristle pattern (G) is observed when comparing abdomens of a Wsp ³/Df(3R)3450 fly (H) to that of an N ^int.hs; Wsp ³/Df(3R)3450 fly, raised at 29°C (I).

Figure 7

The unequal segregation of Numb and Pon-GFP is unaffected in Wsp mutant pI cells. Dissected nota from wild-type (WT, A–D) or Wsp ³/Df(3R)3450 (E–H) mutant pupae were stained to reveal the localization of Numb (red throughout), Pon-GFP (green in B, D, F, and H), the orientation of the mitotic spindle (green in A, C, E, and G; visualized with antibodies to α-tubulin), and the condensation of the chromatin (blue throughout; visualized with DAPI). Anterior is up throughout. In both wild-type and mutant tissue, Pon-GFP colocalizes with endogenous Numb at the anterior cortex of pI from late prophase (not shown) to anaphase (B and F) and the two proteins segregate to the anterior pIIb cells at telophase (D and H). The mitotic spindles of wild-type (A and C) and Wsp mutant pupae (E and G) are similarly positioned within the plane of the epithelium and along the antero-posterior axis of the fly, and are aligned with the Numb/Pon-GFP crescent, enabling the strictly unequal segregation of Numb and Pon-GFP into the pIIb cell.

Figure 9

Wsp is epistatic to numb. Portions of the adult head cuticle adjacent to the eye from wild-type (WT, A), Wsp ¹/Df(3R)3450 (B), ey-FLP; numb ² FRT40A/l(2)cl-L3 FRT40A (C), and ey-FLP; numb FRT40A/l(2)cl-L3 FRT40A; Wsp ¹/Df(3R)3450 (D) animals. The wild-type bristle pattern (A) gives way to multiple sockets when a numb clone is induced (arrow in C), whereas the smooth cuticle phenotype of Wsp mutants (arrow in B) is unaffected by such clones (D). The irregular eye facet pattern (C and D) is characteristic of numb head capsule clones and ensures that a large clone was indeed induced in the head of the Wsp mutant fly.

Figure 8

Dynamics of asymmetric cell division within the microchaete lineage, as revealed by the distribution of Pon-GFP. Time-lapse analysis of the first three divisions of this lineage are shown in living wild-type pupae (neu-GAL4/UAS-Pon-GFP [A–I]) and Wsp mutant pupae (neu-GAL4; Df(3R)3450/UAS Pon-GFP; Wsp ³ [K–S]). Details are described in the text. Anterior is up throughout. Cell types indicated include pI (SOP); pIIa and pIIb, the progeny of the pI division; g, the glial cell progeny derived from the division of pIIb; sh (bristle shaft), and so (socket), progeny of the pIIa division (see also the legend to Fig. 3 K for description of this lineage). The positions of the sensory organs that were followed by time-lapse microscopy are denoted by dashed circles on images of cuticular preparations of the eclosed wild-type (J) and Wsp mutant (T) adults. The socket and shaft cells have differentiated normally in the wild-type fly (J), whereas both socket and shaft are missing at the recorded position in the mutant (T).