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Epithelial cell specialization at a limb segment boundary in the grasshopper embryo
DEVELOPMENTAL
BIOLOGY
118,.%9-402
(1986)
Epithelial Cell Specialization at a Limb Segment Boundary in the Grasshopper Embryo MICHAEL
CAUDY*
AND DAVID
BENTLEYt
*Biophysics Group and TDepartment of Zoology, University of Co&forma, Berkeley, California 94720 Received February 20, 1986;accepted in revised form July 9, 1986
Epithelial cells at the developing femur-trochanter limb segment boundary in the grasshopper embryo are specialized with respect to nonboundary cells. They are elongated, with the long axis oriented along the limb circumference. Some cells along the boundary preferentially bind an antibody (anti-HRP), and so are molecularly specialized as well. The specialized cells are the most proximal cells of the more distal segment. o 1986 Academic press, he. RESULTS
INTRODUCTION
The body surface of developing insects is divided into a variety of epithelial domains, including body segments, limb segments, compartments, and additional intrasegmental lines of lineage restriction which may or may not be compartmental (Lawrence, 1981; Brower, 1985). The differentiation of epithelial cells within each domain is relatively independent of differentiation of cells in other domains. Some domains have been shown to be bounded by specialized cells which differ in shape (Lawrence, 1975: Blennerhassett and Caveney, 1984), in junctional properties (Warner and Lawrence, 1982; Blennerhassett and Caveney, 1984), and/or in the frequency and orientation of cell divisions (O’Brochta and Bryant, 1985). Although limb segmentation is a major organizational feature of the insect body, specialized boundary cells have not been demonstrated in limb segments. In this paper, we describe cells at a limb segment boundary in the embryonic grasshopper. MATERIALS
AND METHODS
Embryos of the grasshopper, Schistocerca americana, were obtained from a colony maintained at Berkeley. Detailed staging of limbs by pioneer (Til) neuron morphogenesis, the names of afferent neurons (e.g., Trl), and orientation of limb axes are described in Caudy and Bentley (1986a). Embryos were labeled with anti-HRP rabbit serum, followed by rhodamine-conjugated goat anti-rabbit (IgG fraction), as described in Caudy and Bentley (1986a). Nuclei were stained with Hoechst 33258, as described in Caudy and Bentley (1986b). With antiHRP labeling, we examined approximately 500 embryos.
Morphological D#erentiation The differentiation of the femur-trochanter boundary is first evident at a gross morphological level as a circumferential constriction in the region of the prospective trochanter segment at the 33% stage (Caudy and Bentley, in press). At the distal end of this region, the femurtrochanter boundary becomes visible as an increasingly sharp discontinuity in the curvature of the epithelium at the 34-35% stage (Fig. 1). When the epithelium is stained with the DNA stain, Hoechst 33258, the boundary is also marked by a line of nuclear displacement and/or alignment (Fig. la). The epithelium comprises a single layer of cells, with each cell extending between the outer surface (apical endfoot) and the inner surface (basal endfoot). At the 34-35s stage, most apical endfeet are shaped as roughly equilateral polygons (Fig. 2). In contrast, the apical surfaces of cells along the femurtrochanter boundary are elongated, with the long axis parallel to the limb circumference (Fig. 2). Selective staining (see below) of boundary cells shows that circumferentially elongated apical endfeet are usually at least three times as long as wide (Figs. 3a, b), and can be as much as seven times as long as wide (Fig. 3~). Therefore, epithelial cells at the boundary are morphologically specialized with respect to nonboundary cells.
Molecular D@erentiation In some limbs, some cells at the boundary label selectively with anti-HRP antibody. Between the 33 and 42% stages of development, the labeling of epithelial cells with anti-HRP is sporadic, and in many embryos no cells label. When labeling does occur, epithelial cells at non399
0012-1606/86 $3.00 Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
400
DEVELOPMENTALBIOLOGY
VOLUME118,1986
CAUDY AND BENTLEY
Epithelial
boundary locations also occasionally bind anti-HRP; for example, scattered, nonelongated cells in the middle of the presumptive coxa sometimes label. However, distal to the coxa, cells which are not at, or very near, the femur-trochanter boundary almost never label. The axial position of the boundary is independently marked by the discontinuity of the epithelial invagination and by nuclear displacement or alignment (Fig. la). At or near the boundary, a row of several circumferentially elongated epithelial cells often label in the same limb (Fig. 4). Therefore, in addition to their morphological differentiation, some boundary cells are molecularly differentiated from nearby nonboundary cells by selective binding of anti-HRP antibodies. Anti-HRP antibodies selectively label insect neurons, and the binding site for this labeling is located at the cell surface (Jan and Jan, 1982). Boundary epithelial cells examined in the light microscope appear to show the strongest labeling at the apical surface (Fig. 5), with some labeling at the basal surface (Fig. 5a). (In addition, there is light labeling throughout the cytoplasm, while nuclei are distinctively free of label; cf. Figs. 5a-c). Therefore, some anti-HRP may be binding to surface molecules on epithelial cells, as in neurons. Specialized Boundary End of the Femur
Cells Lie at the Proximal
The specialized epithelial cells located at the femurtrochanter boundary could lie in the femur, in the trochanter, or in both segments. By the 34-35s stage, the dorsal edge of the epithelium where the boundary is marked by a discontinuity of curvature (Fig. la) is also the most common site for anti-HRP labeling. When the boundary discontinuity and a labeled epithelial cell are
401
Cells at a Segment Boundary
both visible in profile at the dorsal edge, the labeled cell always lies on the distal side of the boundary, i.e., at the proximal end of the femur (Figs. 5c, d). Moreover, the apical endfoot of the labeled cells is the most proximal apical endfoot in the femur; there is not sufficient space for a nonlabeled endfoot to lie between the boundary and the labeled endfoot (Figs. 5c, d). DISCUSSION
Segment Polarity and Segmental Identity Boundary Cells
of Segment
In the milkweed bug, Oncopeltus, segment boundaries in the abdominal body wall are marked by a groove in the cuticle, which results from the invagination of the underlying epithelium (Wright and Lawrence, 1981). An invagination also marks the developing femur-trochanter segment boundary in the grasshopper leg. When this invagination is viewed in profile in regions containing anti-HRP-stained boundary cells, these elongated cells are seen to be contained within the more distal limb segment at the boundary, and are the most proximal cells in that segment (Fig. 5). Specialized boundary cells of body segments are the most anterior cells of the more posterior segment (Lawrence, 1975; Blennerhassett and Caveney, 1984). These cells appear to lie at the peak of a gradient of positional information within the segment which increases in the posterior to anterior direction (Locke, 1959; Lawrence, 1981). The gradient has been documented by transplantation of epithelial cells to ectopic locations, and may reflect adhesive (or other) properties of epithelial cells. Transplantation and regeneration experiments in various insect appendages have provided evidence that
FIG. 1. (a) A 35% stage metathoracic limb with epithelial nuclei (DNA) labeled with Hoechst 33258. The developing femur-trochanter boundary (arrowheads) is marked by a circumferential line of nuclear displacement (or alignment) and by a sharp change in the curvature of the epithelium. Calibration: 100 pm. (b) The same leg photographed in the same focal plane through rhodamine optics (indirect labeling with anti-HRP antibody). The apical endfoot of an anti-HRP-labeled epithelial cell (arrow) is elongated circumferentially at the boundary. The Til pioneer neurons (open arrow) are seen out of the plane of focus on the inner surface of the epithelium. FIG. 2. SEM of the prospective femur-trochanter boundary (arrowhead) at the 33-34% stage. Within the constricted region, apical endfeet of boundary epithelial cells (thin arrows) regularly are circumferentially elongated, while endfeet which are not at the boundary (thick arrows) usually are not elongated. Calibration: 10 pm. FIG. 3. Anti-HRP-labeled apical endfeet of epithelial cells at the femur-trochanter boundary in 35% stage limbs are circumferentially elongated. In (a) a second very lightly labeled cell (left) which is not at the boundary is not elongated. In (c) arrows mark the ends of a highly elongated cell. Calibrations: 10 pm. FIG. 4. Several anti-HRP-labeled apical endfeet (arrows) of epithelial cells elongated circumferentially at the femur-trochanter segment boundary (arrowhead) in each of three different 35% stage limbs. Til pioneer neurons (open arrows) and Trl guidepost neurons (short arrows) are visible, although out of the plane of focus. Calibrations: 50 pm. FIG. 5. Optical sections through single, anti-HRP-labeled epithelial cells at the femur-trochanter boundary on the dorsal side of the limb. (a) 33% stage limb. The apical (upper arrow) and basal (lower arrow) endfeet label more intensely than the cytoplasm. (b) 34% stage limb. A labeled cell (arrow) is at the general location of the presumptive femur-trochanter boundary, which is just distal to the ventral (downward) turn of the pioneer axons (open arrow). (c) 35% stage limb. The location of the boundary (arrowhead) is marked by a change in curvature of the epithelium just distal to neuron Trl (short arrow) and the ventral turn of the pioneer axons (open arrow). A labeled epithelial cell (curved arrow) is adjacent to the boundary on the distal side. (d) 36% stage limb. A labeled epithelial cell (solid arrow) is adjacent to the boundary (arrowhead) on the distal side (the trochanter-coxa boundary, (open arrow) is now visible). Calibrations: (a, b) 10 Frn; (c, d) 25 pm.
402
DEVELOPMENTALBIOLOGY
the epithelium of appendages expresses a continuous gradient of positional information, which is believed to increase in the distal to proximal direction (Locke, 1966; Bohn, 1970;Nardi and Kafatos, 1976;French, 1976;Nardi, 1983). The location of specialized boundary cells at the proximal end of the femur suggests that limb segments in grasshoppers embryos are polarized in a distal to proximal direction. Increased spreading and apposition of pioneer neuron growth cones on the epithelium in more proximal regions of limb segments, with a peak in spreading at the boundary, also suggests a gradient of this polarity (Caudy and Bentley, 1986a). Consequently, specialized boundary cells may be located at the peaks of segmental gradients in both limb and body segments. Expression of the Anti-HRP Binding Site and Intercellular Adhesion All of the cell types in the limb which express the anti-HRP binding site appear to be high affinity substrates for the Til pioneer growth cones (Caudy and Bentley, 198613).Guidepost neurons, the highest affinity cells, always label strongly with anti-HRP (Bentley and Keshishian, 1982; Caudy and Bentley, 198633).Til growth cones also orient toward and make filopodial contact with certain labeled mesodermal cells (Caudy and Bentley, 198613).In addition, growth cone branches are extended along the femur-trochanter boundary after it has differentiated (Caudy and Bentley, in press; whether those branches interact specifically with the boundary cells that express the anti-HRP binding site has not been determined). Therefore, expression of either the antiHRP binding site, or an unidentified but coexpressed feature, seems to confer affinity for pioneer growth cones. The association of the anti-HRP binding site with affinity between neuronal growth cones and several cell types suggests that it may also be associated with affinity (or adhesion) between boundary epithelial cells. That boundary cells may have special adhesive properties has been inferred from the elongation of boundary cells (Lawrence, 1975), and from the invagination of epithelial sheets (Nardi, 1981). We thank Alma Toroian-Raymond for the SEM photomicrograph, and Dr. David Weisblat and Dr. Lily-Yeh Jan for criticizing the manuscript. Support provided by NIH Training Grant 2T32-GM07379, by a University of California Einstein Fellowship in Developmental Neu-
VOLUME1181986
robiology (M.C.), and by a Javits Neuroscience Investigator Award (NIH NS 09074-16)(D.B). REFERENCES BENTLEY,D., and KESHISHIAN, H. (1982). Pathfinding by peripheral pioneer neurons in grasshoppers. Science 218.1082-1088. BLENNERHASSETT, M. G., and CAVENEY,S. (1984). Separation of developmental compartments by a cell type with reduced junctional permeability. Nature (London) 309,361-364. BOHN,H. (1970). Interkalare Regeneration und segmentale Gradienten bei den Extremitaten von Leucophaea-Larven (Blattaria) I. Femur und Tibia. Wilhelm Roux’s Arch. Dev. Bid 165,303-341. BROWER,D. L. (1985). The sequential compartmentalization of Drosophilu segments revisited. Cell 41.361-364. CAUDY,M., and BENTLEY,D. (1986a). Pioneer growth cone morphologies reveal proximal increases in substrate affinity within leg segments of grasshopper embryos. J. Neurosci 6,364-379. CAUDY, M., and BENTLEY, D. (1986b). Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different atllnites. J. Neurosci. 6,1781-1795. CAUDY,M., and BENTLEY,D. Pioneer growth cone behavior at a differentiating limb segment boundary in the grasshopper embryo. In press. FRENCH,V. (1976). Leg regeneration in the cockroach, Blattella germania, II. Regeneration from a non-congruent tibia1 graft/host junction. J. Embrgol Exp. MorphaL 35,267-301. JAN, L. Y., and JAN, Y. N. (1982). Antibodies to horseradish peroxidase as specific neuronal markers in DrosuphiZu and grasshopper embryos. Proc NatL Acad Sci USA 79.2700-2704. LAWRENCE,P. A. (1975).The structure and properties of a compartment border: The intersegmental boundary in Oncope.Jtus.In “Cell Patterning.” Ciba Foundation Symposium 29, pp. 3-23. Elsevier, Amsterdam/New York. LAWRENCE,P. A. (1981). The cellular basis of segmentation in insects. CeU,26, 3-10. LOCKE,M. (1959). The cuticular pattern in an insect, Rhodniusprolbus Stal. J. Exp. Biol. 36,459-477. LOCKE,M. (1966). The cuticular pattern in an insect: The behaviour of grafts in segmented appendages. J. Insect PhysioL 12,397-402. NARDI, J. B. (1981). Epithelial invagination: Adhesive properties of cells can govern position and directionality of epithelial folding. Di~erenti4ktion 20.97-103. NARDI, J. B. (1983). Neuronal pathfinding in developing wings of the moth Manduca sexta Dev. Biol 95,X3-174. NARDI, J. B., and KAFATOS,F. (1976). Polarity and gradients in lepidopteran wing epidermis. II. The differentail adhesiveness model: Gradient of a non-diffusible cell surface parameter. J. EmbrgoL Ezp. MwrphoL 36,489-512. O’BROCHTA,D., and BRYANT,P. J. (1985). A zone of non-proliferating cells at a lineage restriction boundary in Drosophila Nature (Londun) 310,138-141. WARNER,A. E., and LAWRENCE,P. A. (1982). Permeability of gap junctions at the segmental border in insect epidermis. Cell 28,243-252. WRIGHT,D., and LAWRENCE,P. A. (1981). Regeneration of the segment boundary in 0nwpeltus Dev. BioL 85,317-327.
BIOLOGY
118,.%9-402
(1986)
Epithelial Cell Specialization at a Limb Segment Boundary in the Grasshopper Embryo MICHAEL
CAUDY*
AND DAVID
BENTLEYt
*Biophysics Group and TDepartment of Zoology, University of Co&forma, Berkeley, California 94720 Received February 20, 1986;accepted in revised form July 9, 1986
Epithelial cells at the developing femur-trochanter limb segment boundary in the grasshopper embryo are specialized with respect to nonboundary cells. They are elongated, with the long axis oriented along the limb circumference. Some cells along the boundary preferentially bind an antibody (anti-HRP), and so are molecularly specialized as well. The specialized cells are the most proximal cells of the more distal segment. o 1986 Academic press, he. RESULTS
INTRODUCTION
The body surface of developing insects is divided into a variety of epithelial domains, including body segments, limb segments, compartments, and additional intrasegmental lines of lineage restriction which may or may not be compartmental (Lawrence, 1981; Brower, 1985). The differentiation of epithelial cells within each domain is relatively independent of differentiation of cells in other domains. Some domains have been shown to be bounded by specialized cells which differ in shape (Lawrence, 1975: Blennerhassett and Caveney, 1984), in junctional properties (Warner and Lawrence, 1982; Blennerhassett and Caveney, 1984), and/or in the frequency and orientation of cell divisions (O’Brochta and Bryant, 1985). Although limb segmentation is a major organizational feature of the insect body, specialized boundary cells have not been demonstrated in limb segments. In this paper, we describe cells at a limb segment boundary in the embryonic grasshopper. MATERIALS
AND METHODS
Embryos of the grasshopper, Schistocerca americana, were obtained from a colony maintained at Berkeley. Detailed staging of limbs by pioneer (Til) neuron morphogenesis, the names of afferent neurons (e.g., Trl), and orientation of limb axes are described in Caudy and Bentley (1986a). Embryos were labeled with anti-HRP rabbit serum, followed by rhodamine-conjugated goat anti-rabbit (IgG fraction), as described in Caudy and Bentley (1986a). Nuclei were stained with Hoechst 33258, as described in Caudy and Bentley (1986b). With antiHRP labeling, we examined approximately 500 embryos.
Morphological D#erentiation The differentiation of the femur-trochanter boundary is first evident at a gross morphological level as a circumferential constriction in the region of the prospective trochanter segment at the 33% stage (Caudy and Bentley, in press). At the distal end of this region, the femurtrochanter boundary becomes visible as an increasingly sharp discontinuity in the curvature of the epithelium at the 34-35% stage (Fig. 1). When the epithelium is stained with the DNA stain, Hoechst 33258, the boundary is also marked by a line of nuclear displacement and/or alignment (Fig. la). The epithelium comprises a single layer of cells, with each cell extending between the outer surface (apical endfoot) and the inner surface (basal endfoot). At the 34-35s stage, most apical endfeet are shaped as roughly equilateral polygons (Fig. 2). In contrast, the apical surfaces of cells along the femurtrochanter boundary are elongated, with the long axis parallel to the limb circumference (Fig. 2). Selective staining (see below) of boundary cells shows that circumferentially elongated apical endfeet are usually at least three times as long as wide (Figs. 3a, b), and can be as much as seven times as long as wide (Fig. 3~). Therefore, epithelial cells at the boundary are morphologically specialized with respect to nonboundary cells.
Molecular D@erentiation In some limbs, some cells at the boundary label selectively with anti-HRP antibody. Between the 33 and 42% stages of development, the labeling of epithelial cells with anti-HRP is sporadic, and in many embryos no cells label. When labeling does occur, epithelial cells at non399
0012-1606/86 $3.00 Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
400
DEVELOPMENTALBIOLOGY
VOLUME118,1986
CAUDY AND BENTLEY
Epithelial
boundary locations also occasionally bind anti-HRP; for example, scattered, nonelongated cells in the middle of the presumptive coxa sometimes label. However, distal to the coxa, cells which are not at, or very near, the femur-trochanter boundary almost never label. The axial position of the boundary is independently marked by the discontinuity of the epithelial invagination and by nuclear displacement or alignment (Fig. la). At or near the boundary, a row of several circumferentially elongated epithelial cells often label in the same limb (Fig. 4). Therefore, in addition to their morphological differentiation, some boundary cells are molecularly differentiated from nearby nonboundary cells by selective binding of anti-HRP antibodies. Anti-HRP antibodies selectively label insect neurons, and the binding site for this labeling is located at the cell surface (Jan and Jan, 1982). Boundary epithelial cells examined in the light microscope appear to show the strongest labeling at the apical surface (Fig. 5), with some labeling at the basal surface (Fig. 5a). (In addition, there is light labeling throughout the cytoplasm, while nuclei are distinctively free of label; cf. Figs. 5a-c). Therefore, some anti-HRP may be binding to surface molecules on epithelial cells, as in neurons. Specialized Boundary End of the Femur
Cells Lie at the Proximal
The specialized epithelial cells located at the femurtrochanter boundary could lie in the femur, in the trochanter, or in both segments. By the 34-35s stage, the dorsal edge of the epithelium where the boundary is marked by a discontinuity of curvature (Fig. la) is also the most common site for anti-HRP labeling. When the boundary discontinuity and a labeled epithelial cell are
401
Cells at a Segment Boundary
both visible in profile at the dorsal edge, the labeled cell always lies on the distal side of the boundary, i.e., at the proximal end of the femur (Figs. 5c, d). Moreover, the apical endfoot of the labeled cells is the most proximal apical endfoot in the femur; there is not sufficient space for a nonlabeled endfoot to lie between the boundary and the labeled endfoot (Figs. 5c, d). DISCUSSION
Segment Polarity and Segmental Identity Boundary Cells
of Segment
In the milkweed bug, Oncopeltus, segment boundaries in the abdominal body wall are marked by a groove in the cuticle, which results from the invagination of the underlying epithelium (Wright and Lawrence, 1981). An invagination also marks the developing femur-trochanter segment boundary in the grasshopper leg. When this invagination is viewed in profile in regions containing anti-HRP-stained boundary cells, these elongated cells are seen to be contained within the more distal limb segment at the boundary, and are the most proximal cells in that segment (Fig. 5). Specialized boundary cells of body segments are the most anterior cells of the more posterior segment (Lawrence, 1975; Blennerhassett and Caveney, 1984). These cells appear to lie at the peak of a gradient of positional information within the segment which increases in the posterior to anterior direction (Locke, 1959; Lawrence, 1981). The gradient has been documented by transplantation of epithelial cells to ectopic locations, and may reflect adhesive (or other) properties of epithelial cells. Transplantation and regeneration experiments in various insect appendages have provided evidence that
FIG. 1. (a) A 35% stage metathoracic limb with epithelial nuclei (DNA) labeled with Hoechst 33258. The developing femur-trochanter boundary (arrowheads) is marked by a circumferential line of nuclear displacement (or alignment) and by a sharp change in the curvature of the epithelium. Calibration: 100 pm. (b) The same leg photographed in the same focal plane through rhodamine optics (indirect labeling with anti-HRP antibody). The apical endfoot of an anti-HRP-labeled epithelial cell (arrow) is elongated circumferentially at the boundary. The Til pioneer neurons (open arrow) are seen out of the plane of focus on the inner surface of the epithelium. FIG. 2. SEM of the prospective femur-trochanter boundary (arrowhead) at the 33-34% stage. Within the constricted region, apical endfeet of boundary epithelial cells (thin arrows) regularly are circumferentially elongated, while endfeet which are not at the boundary (thick arrows) usually are not elongated. Calibration: 10 pm. FIG. 3. Anti-HRP-labeled apical endfeet of epithelial cells at the femur-trochanter boundary in 35% stage limbs are circumferentially elongated. In (a) a second very lightly labeled cell (left) which is not at the boundary is not elongated. In (c) arrows mark the ends of a highly elongated cell. Calibrations: 10 pm. FIG. 4. Several anti-HRP-labeled apical endfeet (arrows) of epithelial cells elongated circumferentially at the femur-trochanter segment boundary (arrowhead) in each of three different 35% stage limbs. Til pioneer neurons (open arrows) and Trl guidepost neurons (short arrows) are visible, although out of the plane of focus. Calibrations: 50 pm. FIG. 5. Optical sections through single, anti-HRP-labeled epithelial cells at the femur-trochanter boundary on the dorsal side of the limb. (a) 33% stage limb. The apical (upper arrow) and basal (lower arrow) endfeet label more intensely than the cytoplasm. (b) 34% stage limb. A labeled cell (arrow) is at the general location of the presumptive femur-trochanter boundary, which is just distal to the ventral (downward) turn of the pioneer axons (open arrow). (c) 35% stage limb. The location of the boundary (arrowhead) is marked by a change in curvature of the epithelium just distal to neuron Trl (short arrow) and the ventral turn of the pioneer axons (open arrow). A labeled epithelial cell (curved arrow) is adjacent to the boundary on the distal side. (d) 36% stage limb. A labeled epithelial cell (solid arrow) is adjacent to the boundary (arrowhead) on the distal side (the trochanter-coxa boundary, (open arrow) is now visible). Calibrations: (a, b) 10 Frn; (c, d) 25 pm.
402
DEVELOPMENTALBIOLOGY
the epithelium of appendages expresses a continuous gradient of positional information, which is believed to increase in the distal to proximal direction (Locke, 1966; Bohn, 1970;Nardi and Kafatos, 1976;French, 1976;Nardi, 1983). The location of specialized boundary cells at the proximal end of the femur suggests that limb segments in grasshoppers embryos are polarized in a distal to proximal direction. Increased spreading and apposition of pioneer neuron growth cones on the epithelium in more proximal regions of limb segments, with a peak in spreading at the boundary, also suggests a gradient of this polarity (Caudy and Bentley, 1986a). Consequently, specialized boundary cells may be located at the peaks of segmental gradients in both limb and body segments. Expression of the Anti-HRP Binding Site and Intercellular Adhesion All of the cell types in the limb which express the anti-HRP binding site appear to be high affinity substrates for the Til pioneer growth cones (Caudy and Bentley, 198613).Guidepost neurons, the highest affinity cells, always label strongly with anti-HRP (Bentley and Keshishian, 1982; Caudy and Bentley, 198633).Til growth cones also orient toward and make filopodial contact with certain labeled mesodermal cells (Caudy and Bentley, 198613).In addition, growth cone branches are extended along the femur-trochanter boundary after it has differentiated (Caudy and Bentley, in press; whether those branches interact specifically with the boundary cells that express the anti-HRP binding site has not been determined). Therefore, expression of either the antiHRP binding site, or an unidentified but coexpressed feature, seems to confer affinity for pioneer growth cones. The association of the anti-HRP binding site with affinity between neuronal growth cones and several cell types suggests that it may also be associated with affinity (or adhesion) between boundary epithelial cells. That boundary cells may have special adhesive properties has been inferred from the elongation of boundary cells (Lawrence, 1975), and from the invagination of epithelial sheets (Nardi, 1981). We thank Alma Toroian-Raymond for the SEM photomicrograph, and Dr. David Weisblat and Dr. Lily-Yeh Jan for criticizing the manuscript. Support provided by NIH Training Grant 2T32-GM07379, by a University of California Einstein Fellowship in Developmental Neu-
VOLUME1181986
robiology (M.C.), and by a Javits Neuroscience Investigator Award (NIH NS 09074-16)(D.B). REFERENCES BENTLEY,D., and KESHISHIAN, H. (1982). Pathfinding by peripheral pioneer neurons in grasshoppers. Science 218.1082-1088. BLENNERHASSETT, M. G., and CAVENEY,S. (1984). Separation of developmental compartments by a cell type with reduced junctional permeability. Nature (London) 309,361-364. BOHN,H. (1970). Interkalare Regeneration und segmentale Gradienten bei den Extremitaten von Leucophaea-Larven (Blattaria) I. Femur und Tibia. Wilhelm Roux’s Arch. Dev. Bid 165,303-341. BROWER,D. L. (1985). The sequential compartmentalization of Drosophilu segments revisited. Cell 41.361-364. CAUDY,M., and BENTLEY,D. (1986a). Pioneer growth cone morphologies reveal proximal increases in substrate affinity within leg segments of grasshopper embryos. J. Neurosci 6,364-379. CAUDY, M., and BENTLEY, D. (1986b). Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different atllnites. J. Neurosci. 6,1781-1795. CAUDY,M., and BENTLEY,D. Pioneer growth cone behavior at a differentiating limb segment boundary in the grasshopper embryo. In press. FRENCH,V. (1976). Leg regeneration in the cockroach, Blattella germania, II. Regeneration from a non-congruent tibia1 graft/host junction. J. Embrgol Exp. MorphaL 35,267-301. JAN, L. Y., and JAN, Y. N. (1982). Antibodies to horseradish peroxidase as specific neuronal markers in DrosuphiZu and grasshopper embryos. Proc NatL Acad Sci USA 79.2700-2704. LAWRENCE,P. A. (1975).The structure and properties of a compartment border: The intersegmental boundary in Oncope.Jtus.In “Cell Patterning.” Ciba Foundation Symposium 29, pp. 3-23. Elsevier, Amsterdam/New York. LAWRENCE,P. A. (1981). The cellular basis of segmentation in insects. CeU,26, 3-10. LOCKE,M. (1959). The cuticular pattern in an insect, Rhodniusprolbus Stal. J. Exp. Biol. 36,459-477. LOCKE,M. (1966). The cuticular pattern in an insect: The behaviour of grafts in segmented appendages. J. Insect PhysioL 12,397-402. NARDI, J. B. (1981). Epithelial invagination: Adhesive properties of cells can govern position and directionality of epithelial folding. Di~erenti4ktion 20.97-103. NARDI, J. B. (1983). Neuronal pathfinding in developing wings of the moth Manduca sexta Dev. Biol 95,X3-174. NARDI, J. B., and KAFATOS,F. (1976). Polarity and gradients in lepidopteran wing epidermis. II. The differentail adhesiveness model: Gradient of a non-diffusible cell surface parameter. J. EmbrgoL Ezp. MwrphoL 36,489-512. O’BROCHTA,D., and BRYANT,P. J. (1985). A zone of non-proliferating cells at a lineage restriction boundary in Drosophila Nature (Londun) 310,138-141. WARNER,A. E., and LAWRENCE,P. A. (1982). Permeability of gap junctions at the segmental border in insect epidermis. Cell 28,243-252. WRIGHT,D., and LAWRENCE,P. A. (1981). Regeneration of the segment boundary in 0nwpeltus Dev. BioL 85,317-327.