Chromophore-assisted laser inactivation of patched protein

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Sep 24, 1993 - (4-7). In addition, specific neuronal expression patterns of segment-polarity genes .... ulate to form a pioneer nerve, termedBolwig nerve (BN),.
Proc. Natd. Acad. Sci. USA Vol. 91, pp. 2664-2668, March 1994 Genetics

Chromophore-assisted laser inactivation of patched protein switches cell fate in the larval visual system of Drosophila DIETMAR SCHMUCKER*, ANNE L. Sut, ANKE BEERMANNt, HERBERT JACKLE*, AND DANIEL G. JAYt *Max-Planck-Institut fOr Biophysikalische Chemie, Abteilung Molekulare Entwicklungsbiologie, Postfach 2841, D-37018 G6ttingen, Germany; and 1Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA 02138

Communicated by Peter Starlinger, December 22, 1993 (received for review September 24, 1993)

ABSTRACT The Drosophila segment-polarity gene patched (l) is an integral component of the segmentation gene cascade acting In the early embryo. At later stages of embryogenesis, pe is expressed In the primordia of epithellal pacodes of a specific portion of the brain, the optic lobes. Mutant analysis shows that the lack of ptc activity alters the fate of optic-lobe primordia precursors. In plc mutants they give rise to supernumerary neurons in the larval light-sensory system, termed Bolwig organ, which is derived from precursor cells next to the optic-lobe anlagen. We specifically eliminated pkc protein by chromophore-assisted laser inactivation (CALI) in late wild-type embryos. Such embryos show a normal segment pattern, but they develop phenocopies equivalent to the phenotype of ple mutant Bolwg organs. Our results demonstrate that the CALI technique can be applied to separate genetic functions at different developmental stages of a living organism and that the seet-polarity gene plc is redeployed to functionally d minate between distinct developmental pathways in adjacent pools of precursor cells.

MATERIALS AND METHODS Mutant Analysis and Antibody Staining. Embryos were collected from Drosophila melanogaster Oregon-R for wild type or from ptcIN)08 for ptc mutants (12). The mutant chromosome is kept over a CyO "Blue-balancer" that bears a lacZ marker gene driven by a hunchback promoter insertion and allows us to unambiguously determine homozygous mutant embryos by anti-(3-galactosidase antibody staining (see below). The disco enhancer trap line C50.151 [insertion on the X chromosome (13)] was introduced into the ptc background by crossing C50.151 females with ptcN)08ICyO males. Virgins and males with wild-type wings from the F1 generation were mated for egg collections. After /3-galactosidase and antibody 22C10 double staining (see below), embryos which showed -galactosidase expression and the ptc mutant phenotype were analyzed. Antibody staining of whole-mount embryos was carried out (14) using the Vectastain ABC Elite system (Vector Laboratories) with modifications (15). In the doublestaining experiments, embryos were treated as described (3). Anti-Kriuppel antibodies (16) were used at 1:1000, monoclonal antibody 22C10 (17) at 1:20, 4A11 anti-ptc protein antibodies (18) at 1:5, alkaline phosphatase-coupled secondary antibody at 1:1000, and horseradish peroxidasecoupled secondary antibody (Vectastain ABC-Elite kit) at 1:1000. Embryos were mounted in Araldite (Fluka), sucked into borosilicate capillaries (19), and analyzed by differential interference contrast (DIC) microscopy using a weak DIC setting to optimize the information on the photographs. Incubation with the anti-ptc protein antibodies had to be done in the presence of 0.5% Triton (18). Chromophore-Aiisted Las Inacivation (CALI) of ptc Protein. One-hour egg collections were dechorionated with 50% bleach (Clorox) and transferred onto a sticky coverslip, dried in a desiccator, and covered with halocarbon oil as described for embryo injections (12). The embryos were arranged in pairs to allow laser irradiation of two embryos at a time. Malachite green-labeled 4A11 anti-ptc protein antibodies (0.2 mg/ml; dye/antibody weight ratio, 1:6) was injected into the yolk of early stage 10 embryos. Embryos were staged by phasecontrast microscopy and those with clearly visible intersegmental furrows (stage 11) (20) were laser irradiated for 1 min each [Nd-yttrium/aluminum garnet (YAG)-driven dye laser, GCR11 model, Spectra-Physics; wavelength, 630 nm generated with the fluorescent dye; pulse energy, 15 mJ, pulse width, 3.5 ns; frequency, 10 Hz; spot size, 2 mm (21)]. After laser irradiation, embryos were allowed to develop further until stage 16 (20) and then were fixed and devitellinized (22). Kriippel and 22C10 immunohistochemistry was done as described (3). To control the specific action of CALI against ptc protein, we injected the Malachite green-labeled 4A11 antibody into embryos before cellular blastoderm (20) and laser irradiated them during stage 7/8 (20). Embryos were allowed to develop until they hatched

Segmentation in Drosophila is controlled by a cascade of segmentation genes which subdivides the embryo into a series of segmental units along the anterior-posterior axis of the cellular blastoderm (1, 2). Members of the different classes of segmentation genes have been demonstrated to be essential for pattern-forming processes in the developing nervous system, including the gap gene Krlppel (3) and the pair-rule genes even-skipped, fushi tarazu, runt, and hairy (4-7). In addition, specific neuronal expression patterns of segment-polarity genes, such as gooseberry (8) and engrailed (9), have been observed, but the functional significance of their activities is not yet established, because the segmentpolarity genes, with the exception of the neurospecific transcript of the gooseberry locus (8), are expressed in the neuroectoderm of gastrulating embryos at the time when it contains precursor cells giving rise to both epidermis and central nervous system (9). Thus, segmentation and a possible neural function of those genes cannot be separated by conventional mutant analysis. To establish a possible neural function for a representative of the segment polarity genes, we analyzed the late expression patterns of patched (ptc). ptc encodes a transmembrane protein that is an integral component of a cell-cell signaling system which mediates positional information during the segmentation process (10, 11). We found that ptc is expressed in the anlagen of a specific brain portion, the optic lobes. ptc mutant analysis and stage-specific laser inactivation of ptc protein indicate that ptc activity is functionally redeployed after the segmentation phenocritical period to discriminate between neural and epithelial cell fates. The publication costs of this article were defrayed in part by page charge

payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ยง1734 solely to indicate this fact.

Abbreviations: BO, Bolwig organ; BN, Bolwig nerve; CALI, chromophore-assisted laser inactivation.

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FiG. 1. Expression of ptc protein and analysis of BOs of ptc mutant embryos as shown by optical sections through the head region of embryos stained with anti-ptc protein antibody (18) (a), or anti-KrUippel antibody (b and e), or with both the anti-Krfippel and 22C10 antibodies (c, d, andf). (a) Wild-type embryo at stage 11(20). ptc protein expression is seen in the groove in the center of the optic-lobe primordium (olp), in a position anterior to the dorsal ridge (dr), and in a position dorsal to the maxillary segment (ms). (b) BO (bo) of a wild-type embryo contains 12 tightly clustered cells which can be visuaized by nuclear anti-Krkppel antibody staining (see ref. 3). (c) Nervous system of a wild-type embryo stained with 22C10 antibodies, a neuro-specific molecular marker (17). BO (bo) is stained by both anti-KnIppel and 22C10 antibodies (dark brown due to the mixed staining of anti-Krdppel antibodies in blue and 22C10 in brown). The peripheral nervous system, including the antennomaxillary complex (amc), is stained by the 22C10 only (lighter brown). (d) Ventrolateral optical section through a ptc"'N'0 mutant embryo stained with 22C10. In this view (BO is out of focal plane), the splitting of the BN into several fascicles can be seen (white box). In the wild-type embryo, the BO axons would form a single nerve which follows a stereotype projection pathway into a central portion of the brain (for details see ref. 24; see also Fig. 2a) and a splitting of the BN as shown in the white box is never observed. (e) ptcIN1N mutant embryo (24) stained with anti-Krappel antibodies showing the most extreme BO phenotype-i.e., a vast excess ofsupernumery BO cells (upto6 nuclei insteadof the normal 12nuclei; compare with b). Note that theptc phenotype is variable with respect to the shape and the cell number of BO. (f) A less severe defect in the BO in a ptcuv'08 mutant embryo. In this ptc mutant embryo (stained with anti-Krtppel and 22C10 antibodies as in c) the BO is elongated and it forms a split organ structure containing a total of '30 cells on top of the amc (out of focus). The dorsolateral view of the wild-type embryo in c as compared with the ventrolateral view shown inf is to visualize the split BO organ structure. br, Brain hemisphere; vc, ventral cord. (a, x600; b-f, x380.)

and then collected to make cuticle prep

ns (12) for the

investigation of possible cuticle defects. Loss and disorganiza-

tion of denticle rows, highly similar to the phenotype of weak alleles as in ptcU12 (11) and ptc'w (D.S., unpublished observations), indicated the specific ablation of ptc protein by CALL. RESULTS AND DISCUSSION With the beginning of gastrulation, the segment-polarity gene ptc is expressed in a series of stripes along the anteriorposterior axis of the embryo (10, 11). At the time when segmentation becomes morphologically distinct (stage 11; staging according to ref. 20), we observed ptc protein expression in the optic-lobe primordia which are adjacent to the precursor cells of the pair of larval light-sensory organs termed the Bolwig organs (BOs) (3, 23) (Fig. la). Thus, ptc expression serves as a molecular marker for precursor cells that give rise to the optic lobe, an epithelial placode (25). Cells adjacent to the optic lobe anlagen lack ptc expression and give rise to a constant number of 12 sensory neurons representing the BO in wild-type embryos (23). BO axons fasciculate to form a pioneer nerve, termed Bolwig nerve (BN), which contacts the optic lobe and subsequently projects into a central brain region for synaptic targeting (3, 23). To see whether ptc activity is required for optic lobe development, we examined ptc mutant embryos by the expression of various marker genes. The different precursors

which give rise to an epithelial placode (25) and light-sensory neurons of BO (23) can be distinguished by the specific expression of marker genes: disco expression serves as a molecular marker for a portion of the optic-lobe epithelium (13), whereas Krilppel (3), glass (26), chaoptin (27), and a cell surface molecule common to many neurons, which can be visualized by 22C10 staining (17), define the development of BO neurons. Thus, on the basis of marker gene expression, optic-lobe and BO development can be unambiguously distinguished. The optic lobe of ptc mutants is significantly reduced (see below). The BO of ptc mutant embryos contains up to 60 instead of the 12 neurons in wild-type embryos (Fig. 1 b and e), and the BN has defasciculated into several separate axon bundles (Fig. ld). The reduced size of the optic lobe in theptc mutant embryos could be a consequence of cell death caused by the absence of ptc activity in the optic lobe precursor cells and thus, the defects observed in the visual system of ptc mutant embryos could be a secondary consequence of the earlier segmentation function of ptc. To rule out these possibilities as a cause of the ptc mutant phenotype, we intended to use a temperature-sensitive ptc mutant allele that would allow us to specifically reduce late ptc activity. However, the only temperature-sensitive allele of ptc, ptc'lF (28), is embryonic lethal and shows hypomorphic phenotypes for both segmentation and BO formation at permissive temper-

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Table 1. Effect of CALI of ptc protein on BO development

No. of embryos with phenotype (%) No. of embryos BN normal BN e BO enlarged BO gigantic d BO normal examined 0 (0) 0 (0) 0 (0) 100 (0) 100 100 (100) None 0 (0) 0 (0) 0 (0) 12 (100) 12 12 (100) MG-BSA, laser 3 (5) 0 (0) 61 (95) 64 (100) 0 (0) 64 Anti-ptc protein, no ler 55 (86) 64 0 (0) 55 (86) 9(14) 9(14) MG-anti-ptc protein, no laser 7 (14) 23 (44) 22 (42) 22 (42) 52 30(58) MG-anti-ptc protein, laer 2. as desnbdin the andlaser-treated with wee fiIected Afteriqection, only those legend ofFig. indicatedproteins Wilkype Drosophila embryos intactwee examined. BOandBNdevelopmentwexamined by22C10 antibody stainingand anti-Krppel embryoswhich appeared anti~ody staining as d in the legend of Fig. 1. In wild type the BO has rougbly half of the size of the dorsal organ of the ar complex (see Fig 2 a and b). BO enlarged, BO is sificayl than in wild type but is smaller or as large (see Fig 2c) as the dorsal organ of the antennomay complex serving as an internal control; BO gigantic, BO is larger than the dorsal organ of the antennxiary complex. *MG, malachite green; BSA, bovine serum albumin. Treatment of embryos*

Embryos were then allowed to develop the intersegmental furrows indicating that ptc-dependent segmentation was completed normally. After laser irradiation at stage 11 (20), the embryos developed a normal segment pattern, but BOs of these embryos strongly resembled those observed in ptc mutant embryos (Fig. 2). The CALI-dependent phenocopies d embryos ofptc-related defects in BOs of normally se indicate that the functions of ptc in peripheral neurogenesis and in segmentation are indeed unlinked. Interestingly ptc is not expressed in the BO precursors but rather in the adjacent optic-lobe primordia (see above). In ptc mutant embryos the optic lobes are significantly reduced in size (Fig. 3). This reduction and the finding of supernumerary

atures (data not shown). Thus, this allele cannot serve as an experimental tool to discriminate between the ptc functions that derive from early or late ptc expression. We therefore specifically eliminated ptc activity after the sensitive period for segmentation (28) by in vivo CALI of ptc protein (Table 1; Fig. 2) CALI specifically inactivates proteins of interest by targeting laser energy to specific proteins, using malachite green-labeled antibodies (21). To apply this technique to living embryos, as it had previously been used for laser inactivation of fasciclin I in grasshopper embryos (29), we injected malachite green-labeled 4A11 anti-ptc protein anti-

bodies (18) into the yolk region of early stage 10 embryos (20).

b

a bo amc

C

d

Fio. 2. CALI of ptc protein causes an overproduction of BO neurons. Optical sections through embryos (stage 15) stained with monoclonal antibody 22C10 (a and b) or with 22C10 and anti-Krnppel antibodies (c and d) are shown. (a) BO of a wild-type embryo (lateral view). Typically the BO (bo) is smaller than the dorsal cell cluster of the antennmaxiary complex (amc). Note that a single nerve is projecting to the brain (arrowhead). (b) Control embryo injected with malachite green-labeled anti-ptc protein antibodies. No laser treatment had been applied. Note that the size of the BO is indistnishable from wild type. This embryo (stage 15) is shown in a ventrolateral view to visualize the terminal and ventral organs (to/vo) of the mc, which are out of focus in a position below the amc in a. (c) After CALI of the ptc protein, the number of BO neurons has increased, and the BO is enlared to the size of the amc or split into two adjacent organ structures as shown in d. The slightly

darker staining of BO is due to the costaining of the BO nuclei by anti-Krfippel antibodies to unambiguously identify the BO. (d) Dorsal view of a CALI-treated embryo showing the pair of BOs in the head region of the embryo. Note that each of the BOs (slightly out of focus) forms split organ uctues composed of a total of about 30 BO cells (for comparison see the split BO structure of a ptc mutant embryo shown in a lateral view in Fig. f. The second constant defect in CALI-treated embryos is that the BN is defasciculated (arrowheads). Note also the branching of the BN; the larger branch (arrow) is out of focus. It is elongated toward the initial indentation in optic-lobe formation. See text for details and Table 1 for the frequency of CALI-induced ptc phenocopies and control experiments. (x360.)

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d

FIG. 3. Optic-lobe precursor cells are recruited into the BO of ptc mutant embryos. Optical sections of embryos containing the lacZ gene of the disco enhancer-trap C50.151 (13) in a wild-type (a and b) or ptc- (c and d) genetic background, stained with 22C10 (brown cell surface staining) and anti-p-galactosidase (blue nuclear staining) antibodies. (a) Example of the head region of a stage 16 embryo from the marker strain C50.151. The BO neurons (bo) express the 22C10 antigen. 3Galactosidase is expressed in epithelial cells anterior to the BO and in the antennomaxillary complex (amc) (arrowhead in a and arrows in b). (b) P-Galactosidase-expressing cells in the anterior part of the brain and optic-lobe primordia (ao) of a stage 15 C50.151 embryo, highlighting the domain of disco expression. Note the size of the unstained optic-lobe primordium and brain area (po) that is located posterior to the stained "disco domain" (indicated by the dashed line separating stained and unstained brain portions). Additional staining is seen in the visceral mesoderm (m). (c and d) An example of a ptc mutant embryo in two different focal planes. Cells of the enlarged mutant BO express 3-galactosidase (arrows, blue nuclei) and show the inappropriate expression of the optic-lobe marker in the BO of the mutant embryo (c; see also a and b), which reflects the aberrant recruitment of primordial optic lobe cells into the BO. The (3-galactosidase expression in the brain of ptc mutant embryos is frequently seen in two dispersed regions (ol-a and ol-b). Region ol-b (30) is not attached to the brain and it may be a part of the optic-lobe primordia that neither invaginated nor was recruited into the BO. d shows -Galactosidase expression in a ptc mutant embryo at stage 16 (13). Note ,8-galactosidase expression in the posterior part of the brain (arrow) of the mutant which corresponds to a group of unstained brain cells in the wild-type embryo (po in b). The BN (arrowheads) in the ptc mutant embryo is aberrantly projecting to the ol-b region (see c) that is not attached to the brain. Orientation of embryos: anterior left, dorsal up. (X600.)

neurons in BOs of ptc mutant or CALI-treated embryos suggest that one role for ptc activity is to prevent a fraction of the optic-lobe precursor cells from adopting BO cell fate. To test whether the supernumerary BO neurons are derived from the pool of optic-lobe precursor cells, we made use of an "enhancer-trap line" (13, 29) which causes f3-galactosidase expression in the optic lobe but not in the BO precursors of wild-type embryos (Fig. 3 a and b). Due to the stability of 3-galactosidase, cells derived from the pool of optic-lobe precursor cells can be traced at later stages by P-galactosidase activity. -3-Galactosidase-expressing BO neurons were observed in ptc mutant embryos (Fig. 3c), indicating that they had been recruited from the optic-lobe primordium. In addition, these neurons expressed chaoptin (Fig. 4), a molecular marker specific for photoreceptor cells (3, 27). This finding suggests that ptc activity normally suppresses neuronal fate in the optic-lobe precursor cells. Elimination of ptc activity in these precursors, either due to a mutation or by CALI treatment, then caused a switch of cell fate from an epithelial into a neuronal pathway. Thus, the function of ptc is reminiscent of that of Notch in the central nervous system (31). However, since no general neurogenic effect has been found to be associated with ptc mutants as observed in the absence of Notch activity (31-33), the role of ptc appeared to be restricted to a single neuronal pathway. Therefore, we speculate that, like Notch, the transmembrane protein encoded by ptc may act as a mediator of cell-cell interactions,

to signal specific cell fate decisions-i.e., optic lobe instead of BO development.

CONCLUSIONS Our results show the potential of CALI to efficiently eliminate a stage-specific gene function in a living organism, as its application to ptc protein results in specific phenocopies equivalent to the ptc mutant phenotype. As the ptc protein represents a minor fraction of the protein required in various precursor pools of cells of the developing embryo, the CALI assay may be useful for analysis of other Drosophila developmental genes in separating their specific functions at different stages of embryogenesis and possibly also the analysis of homologous gene functions in organisms lacking well-characterized genetic systems. The early expression and function of ptc, compared to Krfippel, in BO formation violate the hierarchy of segmentation genes during early embryogenesis, as has already been demonstrated by pair-rule genes. In neurogenesis of the central nervous system, the pair-rule genes even-skipped, fushi tarazu, and runt affect cell fate independently in a manner unrelated to their ordered role in the early segmentation process (3, 4, 7, 10). Taken together, these results argue that the combinatorial interactions of the segmentation genes during early development are not applicable to neural patterning. The expression patterns of segmentation gene

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b

FIG. 4. Optic-lobe precursor cells of ptc mutant embryos differentiate into photoreceptor neurons expressing the chaoptin antigen. (a) Close-up microphotograph of the head region of a wild-type embryo (stage 16) showing the BO and BN as visualized by the anti-chaoptin antibody 24B10. The 24B10 antibody staining is specific for photoreceptor neurons (27). Arrow points to a portion of the BN which is in the appropriate focal plane. (b) The corresponding region in a ptc mutant embryo (stage 16; lateral view). Note that the BO has significantly increased (see also Fig. 1) and that additional BO neurons expressing photoreceptor-specific chaoptin are observed. The BN (out of focal plane) extends towards the inside of the embryonic head at the position marked by an arrow. Thus the additional BO neurons in ptc mutant embryos which are derived from optic-lobe precursor cells (see Fig. 3) differentiate into photoreceptor neurons as judged by their ability to express the chaoptin marker gene.

homologues in organisms other than Drosophila (21) suggest that these genes may have been co-opted from a role in neurogenesis and have been deployed to execute the rapid and almost simultaneous development of all body segments during blastoderm and early gastrulation in the Drosophila embryo. The different roles of ptc and Krfippel in segmentation and BO development support this hypothesis. We thank A. Xu for technical assistance, P. Ingham and F. Pelegri for supplying antibodies, K. Lee for providing the disco enhancertrap line, and R. Lehmann, W. Driever, H. Keshishian, and T. N. Changforhelpful discussion. We thank M. Hflskamp, L. Crosby, M. Gonzalez-Gaitan, M. Hoch, and M. Pankratz for critical reading of the manuscript. D.G.J. is a Lucille P. Markey Scholar, D.S. is a fellow of the Studienstiftung des Deutschen Volkes. This work was supported by the National Institutes of Health and the Human Frontier Science Program (D.G.J.), a special award by the German Society of Zoology (D.S.), and by the Max Planck Society and the Fonds der Chemischen Industrie (H.J.).

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