Pathfinding by pioneer neurons in isolated, opened and mesoderm-free limb buds of embryonic grasshoppers

DEVELOPMENTAL

BIOLOGY

(1987)

119,466-480

Pathfinding by Pioneer Neurons in Isolated, Opened and Mesoderm-Free Limb Buds of Embryonic Grasshoppers FRANCES LEFCORT* AND DAVID BENTLEY*+ *Neurobiology

Group

and TDepartment

Received

January

of Zoology,

22, 1986;

accepted

University

of California,

in revised

form

September

Berkeley,

California

94720

18, 1986

The Til afferent neurons are the first neurons to undergo axonogenesis in limb buds of embryonic grasshoppers. Their growth cones pioneer a stereotyped pathway through the limb which becomes the route of one of the major leg nerve trunks. The growth cones appear to be oriented by several kinds of guidance cues, including guidepost neurons, a developing limb segment boundary, and an additional proximally orienting cue(s). In the experiments reported here, we have investigated the possible nature and source of proximally orienting and segment boundary cues by surgical manipulations of the limb. Before the onset of pioneer axonogenesis, limbs were (i) isolated from the body, (ii) opened longitudinally and pinned out flat, or (iii) stripped of mesoderm. Pioneer axon routes in cultured, surgically manipulated limb buds were compared to routes in cultured control limbs. The results indicate that proximal extension of pioneer growth cones along the limb axis does not require (during the period of growth) tissue extrinsic to the limb, contact guidance by the limb contour, an axial electrical field, a diffusion gradient generated by a localized source, mesodermal cells, or guidepost neurons; adequate guidance information for proximal growth apparently can be provided by the limb epidermal epithelium (including the basal lamina) and/or by internal polarity of the pioneer neurons. Adequate guidance information for the segment boundary portion of the pioneer route apparently can be provided by the limb epithelium. 0 1987 Academic

Press, Inc.

the limb axis where the pioneer growth cones are not being guided by guidepost neurons. One such region is the initial segment of pioneer growth. Pioneer growth cones emerge from the proximal side of the cell bodies before filopodial contact with the first guidepost neuron has been established (Bentley and Caudy, 1983b; Berlot and Goodman, 1984). In limbs which are relatively undifferentiated at the time of pioneer axonogenesis, and in which pioneer growth cones migrate before guidepost neurons have differentiated, the growth cones still emerge from the proximal side of the cell bodies and extend proximally in the limb (Caudy and Bentley, 1986a). Therefore, there must be an additional cue, or cues, which can guide growth cones proximally. One possible cue is internal polarity of the pioneer neurons (Bentley and Caudy, 1983b; Berlot and Goodman, 1984). These cells undergo their final division with the cleavage plane perpendicular to the limb axis, and they appear to maintain this orientation when they emerge from the limb epithelium (Keshishian, 1980). Thus the cytoskeleton, or other internal factors, might cause growth cones to emerge from the proximal pole of the cell. Several other kinds of cues might orient growth cones proximally along the limb axis, both at the limb tip, and also more proximally in the region between (guidepost) neurons Fe1 and Trl (Fig. 1). There is evidence from growth cone morphologies in different regions of the

INTRODUCTION

The Til afferent neurons (hereafter termed the pioneers) are the first to undergo axonogenesis in limb buds of embryonic grasshoppers (Bate, 1976; Keshishian, 1980). The pioneer axons extend through the limb to the CNS. Axons arising later fasciculate with the pioneers, so that the pioneer trajectory becomes the route of one of the major limb nerve trunks (Ho and Goodman, 1982; Bentley and Keshishian, 1982; Keshishian and Bentley, 1983). We are interested in the guidance cues which direct the pioneer growth cones along their normal trajectory through the limb. There is some variation in the degree of differentiation of limb buds when the pioneer growth cones extend through them. In relatively well differentiated limb buds, the growth cones appear to be steered by a series of different cues as they traverse different regions of the limb (Fig. 1; and Caudy and Bentley, 1986b). The most prominent cues are the cell bodies of certain immature neurons (hereafter termed guidepost neurons), which have not begun axonogenesis (Fig. 1). When approaching growth cones are within approximately 30 pm of these neurons, they apparently contact them with filopodia and then grow directly to them. This process evidently leaves a sharp bend in the pioneer axons at the site where the growth cones reoriented. Judging by the locations of these reorientations, there are several regions along 0012-160618’7 Copyright All rights

$3.00

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

466

LEFCORT

AND

BENTLEY

PathJinding

limb for a proximally increasing gradient of affinity within limb segments for the growth cones (Caudy and Bentley, 1986a). Growth cones might migrate proximally up such a gradient. Whether this gradient is derived from the epidermal epithelium or from the mesodermal layer has not been determined. Alternatively, growth cones might be guided by a diffusible tropic factor emanating from tissue extrinsic to the limb or from a source within the limb (cf. Gunderson and Barnett, 1980). Another possibility is an oriented electrical field generated by transepithelial current (cf. Jaffe, 1981; Pate1 and Poo, 1982; McGinnis and Vanable, 1986). Finally, the growth cones could be oriented along the limb axis simply by contact guidance due to the internal curvature of the limb itself (cf. Weiss, 1959; Brunette, 1986). Near the base of the limb, in the region of the prospective trochanter, pioneer growth cones reorient from axial to circumferential growth, and grow from the dorsal to the ventral region of the limb, where they contact the Cxl neurons (Fig. 1). Usually, the growth cones do not grow directly toward the Cxl neurons, but instead

,,- -----,

Fe ,

FIG. 1. A composite diagram of a 33 to 35% stage limb bud showing a typical path of the Til pioneer neurons in a well differentiated limb (adapted from Caudy and Bentley, 1986b). From the cell bodies (Til), the axons extend proximally along the axis (shown in each segment by the interrupted line) of the tibia (Ti). After crossing the tibia/ femur (Fe) boundary, they reorient toward the Fe1 guidepost neuron and extend straight to it. Leaving the Fe1 neuron, the axons usually reorient dorsally toward the ml and m2 mesodermal cells (possible muscle pioneers). In the vicinity of the prospective femur/trochanter (TR) boundary, the axons reorient toward the Trl guidepost neuron, and extend straight to it. Leaving the Trl neuron, the axons extend circumferentially and ventrally along the prospective trochanter/coxa (Cx) boundary. From the boundary, they reorient toward and grow straight to the Cxl neurons. Leaving the Cxl neurons, the axons extend proximally to the central nervous system (CNS): they sometimes can be seen to be growing along a series of cells (interrupted outlines) which label with anti-HPR. Ta, tarsus; FCO, presumptive femoral chordotonal organ; ef, growth cones of efferent neurons; e, specialized epithelial boundary cells at the proximal end of the femur which sometimes label with anti-HRP (Gaudy and Bentley, submitted). Ventral, down; proximal, to left.

by Pioneer

467

Neurons

extend along the prospective trochanter/coxa segment boundary; they appear to turn proximally when they establish filopodial contact with the Cxl neurons (Bentley and Caudy, 1983a, 1983b; Caudy and Bentley, 1986a, 1987a). The information which orients growth cones along the boundary could be provided by either the epithelium or the mesoderm (or both). Many of these possible sources of guidance information potentially could be tested by surgical manipulation of the limb. Such manipulations potentially could isolate the limb from extrinsic tissue, could alter the contour of the internal surface, could short-circuit possible transepithelial currents, could expose possible diffusion gradients to a large volume of bathing medium, and could remove the mesoderm. We report here the effect on pioneer neuron guidance of a series of manipulations of this type (abstracted in Lefcort and Bentley, 1985). MATERIALS

AND

METHODS

Microsurgery and embryo culture. Embryos of Schistocerca americana at the 29-30% stage (Bentley et ah,

19’79; Caudy and Bentley, 1986a) were obtained from a culture at Berkeley. Eggs were sterilzed in 0.02% benzethonium chloride (in 70% ethanol), and dissected in normal saline (Bentley et al., 1979). Three surgical procedures were performed: (i) limb isolation-with a glass needle, limb buds were separated from the thorax between the Cxl neurons and the CNS; (ii) limb openingwith a glass needle, the epithelium (and mesoderm if present) of the posterior side of the limb was split open from the limb base to the tip; the tip and the two flaps of epithelium at the limb base were then pinned out on sylgard with glass needles; (iii) mesoderm removaldissected embryos were pinned to Sylgard-coated 35-mm petri dishes and bathed in Ca2’ and M$+ free saline to facilitate the dissociation of mesodermal cells from each other and from the inner surface of the epithelium. Firepolished glass micropipettes (i.d. 25-55 pm) were mounted on a micromanipulator and inserted through the dorsal closure of the embryo into the limb lumen. Mesodermal cells were visualized with transmitted light and removed by suction. All operations were performed on metathoracic limb buds; limb buds which were contralateral to operated limbs served as controls for the culture conditions (therefore, each experimental limb bud was from a different embryo). Either one (Table 1) or two (Table 2) manipulations were performed on each experimental limb. Embryos and limbs were cultured at 30 + 1°C in a COZincubator at pH 7.0 in the following medium: RPM1 1640 (Gibco, N.Y.), 0.2% sodium carbonate, lwrn oxaloacetic acid, 0.45 mM sodium pyruvate, 2 mM glutamine, 0.45% D(S) glucose, 0.02 i.u./ml insulin, lo-’ M b-ecdys-

468

DEVELOPMENTAL BIOLOGY

terone (Sigma), and 50 pg/ml gentamicin. The period of culture ranged from 40 to 65 hr, and varied for sets of embryos subjected to different manipulations (therefore, manipulated limbs were compared to the contralateral control limbs). Histology and cell counting. At the end of the culture period, embryos were fixed and labeled with serum antibodies against horseradish peroxidase (protocol in Caudy and Bentley, 1986a). These antibodies selectively label insect neurons (Jan and Jan, 1982). Pioneer neurons, and other neurons, were visualized and photographed with fluorescence optics in embryos (or limbs) whole-mounted under glass cover slips with 40-pm wire spacers. Additional whole-mounted limbs were photographed in transmitted light with interference contrast optics. Cell names and features of the pioneer pathway are labeled in Fig. 1, and described in more detail in Caudy and Bentley, 1986a,b. For transmission electron microscopy, embryos were fixed in 2% glutaraldehyde and 2.2% paraformaldehyde in pH 7.5 sodium cacodylate buffer, post-fixed in 2% Os04, embedded in araldite, sectioned, and viewed in a JEOL-lOO-CX EM. For scanning electron microscopy, embryos were fixed as above, critical point dried, coated in gold palladium, and viewed in a ISI-DS-130 SEM. Four limb buds were fixed in 2% paraformaldehyde, embedded in araldite, sectioned at 4 pm, and stained with 1% toluidine blue. One limb was an unoperated control; a second limb was fixed immediately after mesoderm removal, and two additional limbs were examined after the culture period. In the operated limbs, each remaining adepithelial cell was traced through all sections in which it appeared. In the control limb, the number of adepithelial cell profiles in each section was counted. Selected sections were taken from two additional (mesoderm-free) opened and pinned out limbs to provide information on the contour of the epithelium (cf. Fig. 9C). Assessment of pioneer axOn growth. Fluorescently labeled pioneer neurons were examined in whole-mounted embryos, and photomicrographs were made of manipulated limbs and their contralateral controls. The growth of pioneer axons in experimental limbs was compared to that in control limbs and scored according to four criteria. The criteria were selected because they could be scored unequivocally, and because they evaluated regions or aspects of the pioneer axon trajectory where the growth cones might be guided by the cues being evaluated. The criteria were (1) Did the pioneer neurons undergo axonogenesis? (2) Did the axons extend proximally along the limb axis? (3) In the vicinity of the prospective trochanter/coxa boundary, did the axons reorient from axial to circumferential growth and extend from the dorsal to the ventral region of the limb? (4)

VOLUME 119,1987

How far along this route did the axons extend? The results from 48 experimental limbs and 46 control limbs are scored in Tables 1 and 2. RESULTS

Pioneer Neuron Behavior in Isolated Limb Buds In isolated limb buds, pioneer neurons extended axons proximally along the limb axis (Figs. 2A, B). In the vicinity of the trochanter/coxa segment boundary, the axons reoriented from axial to circumferential growth and grew into the ventral region of the limb (Figs. 2A, B). Some axons left the segment boundary and extended further proximally to contact the Cxl neurons (Fig. 2B).

A

B

FIG. 2. Pioneer axon routes in isolated limb buds. (A, B) Anti-HRP labeled, whole-mounted metathoracic limb buds isolated from embryos at the 29-30% stage (before the onset of axonogenesis) and cultured for 48 hr. The epithelium at the opened (proximal) end of the limb heals to form a continuous, smooth surface. (A) The pioneer neurons (open arrow) extend proximally along the limb axis to the vicinity of the trochanter/coxa boundary (arrowhead) where they turn and grow circumferentially into the ventral region of the limb. (B) The pioneer neurons (open arrow) extend to the vicinity of the trochanter/coxa boundary, grow circumferentially into the ventral region, and then turn proximally to contact the Cxl neurons (solid arrow). Curved arrows (A, B) indicate lightly labeled Fe1 guidepost neurons: the pioneer axons have not contacted these neurons (see text). Ventral, down; proximal, to left. Calibration bar: 50 Wm.

LEFCORT

AND

Pathjinding

BENTLEY

Neuron

Behavior

in Opened Limb Buds

Embryonic limb buds at the 29-30% stage are roughly tubular toward the base and conical toward the tip. In experimental embryos, the metathoracic limb bud was cut open along the posterior face from the base to the tip; the apex of the cone (limb tip) and the free edges at the base were then pinned out flat, and the embryo was

TABLE PIONEER

NEURON

GROWTH

1 IN SINGLY

OPERATED

LIMBS

Pioneer

Treatment

Number of limb buds

469

Neurons

maintained in culture during the period of pioneer growth (the contralateral limb served as a control). In interference contrast optics, the inner surface of the pinned out limb appeared to be quite flat (cf. Fig. 9A). From two embryos (which also had the mesoderm removed; see below), the experimental limbs were fixed at the end of the culture period, labeled with anti-HRP, and whole-mounted; the pioneer neurons were examined (cf. Fig. 9B), and the limbs were then embedded, cross sectioned, stained, and reexamined (cf. Fig. 9C). The sections confirmed that the inner contour of the epithelium under these conditions was quite flat. Pioneer neurons growing on opened limb buds initiated axonogenesis and extended axons proximally along the limb axis (Figs. BB, 9B). In the vicinity of the trochanter/coxa boundary, axons turned and crossed from the limb region which would be dorsal in an intact limb to the limb region which would be ventral (Figs. BB, 9B). Some axons made a second, proximal turn from the segment boundary region and contacted the Cxl neurons (Fig. 9B). Consequently, the major features of the pioneer route can be expressed on an opened limb bud. Pioneer axon routes in 12 experimental limbs were compared to routes in 12 contralateral metathoracic control limbs. All neurons initiated axonogenesis, and no neurons extended processes in abnormal directions. By the criteria scored (Table l), experimental and control routes were the same: all neurons reached the trochanter/coxa boundary, and about half (‘7/12) of both experimental and control neurons extended into the region of ventral epithelium. As in most isolated limb buds (Fig. 2) and culture control limb buds (Fig. ‘7A), the initial proximally oriented portions of the axons were quite straight and did not appear to be growing between guidepost neurons (Fig. BB).

Thus, the major elements of the normal pioneer route were expressed. Axon route in 16 experimental limbs were compared to those in 16 contralateral control limbs (which remained attached to the thorax; Table 1). Axons in experimental and control limbs were similar (except that more axons in experimental limbs reached the ventral region of the segment boundary): all neurons in experimental limbs initiated axonogenesis and extended axons ventrally along the segment boundary; in control limbs, most axons (14/16) reached the segment boundary, and about half (606) extended into the ventral region of the limb. No axons in experimental or control limbs extended in abnormal directions. Although pioneer axons in isolated limbs grew proximally along the limb axis, they extended in a generally straight route (Figs. 2A, B) rather than the “zigzag” route which is seen when the growth cones are responding to the distal guidepost neurons (Fel, Trl; Fig. 1). As estimated by the acquisition of anti-HRP labeling, differentiation of the distal guidepost neurons (Trl, Fel) appeared to be delayed in culture. Light anti-HRP labeling of the most distal (Fel) guidepost neuron in some isolated limbs confirmed that the pioneer axons had not contacted the cell (Figs. 2A, B). These observations suggest that in many isolated, cultured limbs, the distal guideposts neurons differentiated too late to guide the proximal migration of pioneer growth cones. Pioneer

by Pioneer

axon

Femur (midpoint)

Trochanter (dorsal)

elongation

to: Tr/Coxa boundary (ventral)

Limb isolated (controls)

16 16

16/16 E/16

16/16 14/16

16/16 6/16

Limb opened (controls)

12 12

12/12 12/12

12/12 12/12

?A2 7/12

8 8

X/8 8/8

8/8 818

7/8 ‘7/8

Mesoderm (controls)

removed

Note. The number of axons reaching each experiment should be compared

the ventral region of the Tr/Cx to its own controls.

boundary

depended

on time in culture

(which

varied

between

experiments);

470 Arrangement

DEVELOPMENTAL

of Mesoderm

BIOLOGY

in Limb Buds

To evaluate the successof the mesoderm removal procedure, the disposition of mesoderm in normal limb buds first was assessed. At the 31% stage of development, when the pioneer axons are initiating axonogenesis, the limb bud comprises two concentric layers of cells (Fig. 3A). The outer layer is a tightly packed epithelium, with each epithelial cell extending from the outer (apical) surface to the inner (basal) surface. Apposed to the basal surface of the epithelium is the second, mesodermal layer of cells. This layer is also a single cell thick (Figs. 3A, C, 4C), with the outer surfaces of mesodermal cells ap-

VOLUME

119, 198’7

posed to the basal surface of the epithelium, and the inner surfaces bounding the limb lumen. A basal lamina lies between the epithelium and the mesoderm (Wigglesworth, 1953; Ashhurst, 1965,1982; Anderson, 1985). The mesodermal layer is complete around the entire circumference of the limb, and also extends from the tip of the limb to its base. In a limb sectioned at the 30% stage, making a conservative assumption that every mesodermal cell appears in four (4-pm) sections, results in a mesodermal cell count of at least 100 cells between the limb tip and the location of the Cxl neurons in the coxa (Fig. 5A); in a single cross-section (Fig. 3A), 26 mesodermal cell profiles are seen around the limb circum-

n

FIG. 3. Cross sections of metathoracic limb buds before and after mesoderm extraction. (A, B, D) Toluidine blue-stained 4-pm sections. Arrows in all panels indicate the normal position of the interface between mesoderm and epithelium. (A) Normal 30% stage limb bud sectioned near the Cxl neurons. The mesoderm forms a circumferentially continuous layer a single cell thick. (B) A 30% stage limb bud after removal of the mesoderm. The epithelium remains intact with a smooth basal (inner) surface. (C) Transmission EM photomicrograph of a cross section near the trochanter of a 40% stage limb bud. An axon fascicle (longer arrow) composed of the two pioneer axons and three additional axons lies between the epithelial cell (e) layer and the mesodermal (m) layer. (D) Section near the tip of a 30% stage limb bud with the mesoderm removed. The pair of pioneer neurons (open arrow) remain in their normal location. Calibration bars (A, B, D) 25 Gm; (C) 5 Fm.

LEFC~RT

AND

BENTLEY

Pnth&ndin,g

lq~ Pioneer

Narcms

FIG. 4. Morphology of metathoracic limb buds v&h and without mesoderm. (A, B, D) whole-mounted limb buds viewed with interference contrast optics. Arrows in each panel indicate the normal position of the interface between mesoderm and epithelium. (A) 32% stage limb bud showing the mesodermal layer (asterisk) in surface view. (B) 32% stage limb bud after removal of the mesoderm. The relatively fine-grained basal surface of the epithelium (asterisk) is now visible. (C) Scanning EM photomicrograph of a 34% stage limb bud fractured to show the apposition of mesodermal cells (m) to the inner surface of the epithelial cell (e) layer. A basal lamina (arrow) lies between the two layers. (D) Tip of a 31% stage limb with the mesoderm removed. The pioneer neurons (open arrow) remain in their normal location and with their normal polarity (with the cleavage plane between the two pioneers perpendicular to the limb axis). Ventral, down; proximal, to left. Calibration bars: (A,B)5O~m;(C)lO~m;(D)25~m.

ference. The mesodermal layer consists of several cell types, including myoblasts and muscle pioneers (Ho et al, 1983; Ball et al., 1985). The pioneer neurons extend axons between the epithelial and mesodermal layers (Fig. 3C). Numerous filopodia from pioneer growth cones extend between epithelial cells, reaching close to the apical surface, and also between mesodermal cells, reaching close to the luminal surface (Caudy and Bentley, 1986a). Identified efferent pioneer neurons are dependent on mesodermal muscle pioneer cells for guidance information (Ball et al., 1985), and the afferent pioneer neurons also appear to respond to putative muscle pioneers at certain locations in the limb (Caudy and Bentley, 1986b). Mesoderma1 cells are therefore a potential source of guidance information for afferent growth cones migrating through the limb.

Removal

of the Mesoderm

In intact limb buds, mesodermal cells can be visualized (Fig. 4A), and can be removed with a suction pipette (Methods). After the removal procedure, the remaining epithelium appears as a smooth, relatively fine-grained surface comprised of the basal endfeet of epithelial cells (Fig. 4B). The efficacy of the removal procedure was evaluated in limb buds fixed and cross sectioned either immediately after the extraction, or after an intervening culture period. Most sections contained no mesodermal cells (Figs. 3B, D; 5). Between the pioneer cell bodies and the location of the Cxl neurons in the coxa, two sectioned limbs contained a single adepithelial cell, and one limb contained two adjacent adepithelial cells (Fig. 5B). The positions of the remaining cells varied from limb to limb, so that there was no axial location between the pioneer

472

BIOLOGY

DEVELOPMENTAL

cell bodies and the Cxl neurons which always retained a cell (Fig. 5B); conversely, no specific adepithelial cell remained in every limb. Consequently, in at least some embryo(s) in the experimental set, each position in the limb would have to be crossed by pioneer growth cones in the absence of any adepithelial cell. Guidepost neurons or neuron pairs are found at three locations along the limb (Fig. 1; Bentley and Keshishian, 1982). These cells lie against the basal surface of the epithelium (Keshishian and Bentley, 1983, Fig. 1E). The adepithelial cells found in limbs sectioned after mesoderm removal could be either mesodermal cells or

A.

CONTROL

(mesoderm

intact)

25-

10

5

01

: z g

B.

EXPTL. (mesodermal

cells

34. i

left after

extraction)

1

II)

k

TIN SECTION (IO) I

I 0

40

POSITION

ALONG

NUMBER (20) 1 El0

LIMB

(30) I I 120

AXIS

I

VOLUME

119. 1987

guidepost neurons. In two limbs, a single adepithelial cell was found between the pioneer cell bodies and the location of the Cxl neurons. Since two guidepost neurons (Fel, Trl, Fig. 1) are located in this region of the limb, at least one of them had to be missing in each of the experimental limbs. In the third sectioned limb (Fig. 5, L-2), two adepithelial cells remained; these cells were adjacent, were each over 50 pm in length, and extended into the region of the Cxl cells. Since guidepost neurons are about 15 pm in diameter in fixed material (Keshishian and Bentley, 1983, Fig. 1E; Bentley and Caudy, 1983a, Fig. 3A), and do not extend into the region of the Cxl neurons, neither of these remaining cells appears to be a guidepost neuron. Therefore, in this limb both the Fe1 and Trl guidepost neurons appear to be missing. In summary, at least one of the two distal guidepost neurons was missing in every sectioned limb and, evidently, both were missing in one limb. The efficacy of removal of adepithelial cells was also assessed by viewing limbs in the scanning electron microscope (Fig. 6). After extraction of the mesoderm, embryos were fixed immediately, critical point dried, and mounted for scanning. Under a dissecting microscope, the posterior side of the limb was chipped away with a sharpened tungsten needle. This exposed the inner surface of the anterior side of the limb where the pioneer growth cones migrate, and where the distal (Trl and Fel) guidepost neurons are located. In two limbs prepared by this method, no adepithelial (mesodermal or neuronal) cells remained on the inner surface between the pioneer cell bodies and the trochanter/coxa boundary. Following extraction of the mesoderm, the epithelium remains in good condition. The normal shape of the limb bud is retained, and in cross sections, the epithelium remains tightly packed with a smooth basal surface (Fig. 3B). The pioneer neurons are left in their normal location at the limb tip (Figs. 3D, 4D, 6) and, at least in cases where it can be observed, with their normal polarity (with the cleavage plane perpendicular to the limb axis; Fig. 4D). After a period in culture, the limb bud becomes reduced in cross-sectional area (Figs. 7B, D), possibly due to the loss of support normally provided by mesoderm.

OJrn)

FIG. 5. Adepithelial cell counts in a normal limb and in limbs from which the mesoderm has been removed. (A, B) Number of profiles of adepithelial cells in each 4-pm section is shown. Section order starts at limb tip (to left). (A) The control limb contains more than 100 cells if each cell appears in four sections. (B) Experimental limbs: L-l, fixed immediately after removal of mesoderm; L-2, mesoderm removed and limb cultured (Fig. 7B); L-3, mesoderm removed, limb isolated, opened, and cultured. Til, position of the pair of Til neurons. Cxl, position of a pair of neurans at the axial location of the Cxl neurons. The number of adepithelial cells between the Til and the Cxl positions is shown in parentheses.

Pioneer

Neuron

Behavior

in the Absence of Mesoderm

In limb buds from which the mesoderm (and other adepithelial cells) had been extracted, pioneer neurons extended axons proximally along the limb axis (Figs. 7B, D). In the vicinity of the trochanter/coxa boundary, axons crossed from the dorsal to the ventral region of the limb, and frequently contacted the Cxl neurons (Fig. 7D). Therefore, the major elements of the pioneer axon route were established under these conditions.

LEFCORT

AND

BENTLEY

Path&ding

by Piower

Pioneer Neuron Limb Buds

FIG. 6. Scanning electron micrographs of a :32-X:‘% stage limb fixed immediately after removal of the mesoderm. The posterior side of the limb has heen dissected to show the inner aspect of the anterior side of the limb (along which the pioneer growth curies migrate). No mesodermal cells or guidepost neurons remain along the dorsal routr which the growth cones will follow to the vicinity of the trochanter/ coxa boundary (arrows). Arrowhead: pioneer axons extending into the basal lamina. (B) Higher gain view of the pioneer neuron cell bodies (open arrows) from (A). Ventral, down; proximal, to left. Calibration bars (A) 20 pm; (Bi 5 pm.

In eight embryos, pioneer routes in the experimental metathoracic limb were compared to routes in contralateral control metathoracic limbs (Table 1). Experimental and control neurons were indistinguishable by the criteria scored (Table 1). No neurons failed to undergo axonogenesis and no axons were extended in abnormal directions. All experimental and control neurons grew as far as the trochanter/coxa boundary, and most axons (7/8) extended along the boundary into the ventral region of the limb. As in isolated and in opened limb buds, axons initially extended in a generally straight proximal direction and did not display the abrupt changes in direction associated with growth to the distal guidepost neurons (Figs. ?‘A, B). The presence of these neurons also was not indicated at this stage either by filopodial wrapping (Caudy and Bentley, 1986b) or by anti-HRP labeling.

473

New-ens

Behavior in Doubly Operated

To test whether or not some cues might serve as alternatives for each other (seeDiscussion), two operations were performed on three sets of limbs (Table 2). Four replications of each permutation of paired operations were performed (isolation and opening; isolation and mesoderm removal; opening and mesoderm removal). Contralateral limbs again served as culture controls. Pioneer neurons in all classesof doubly operated limbs extended axons proximally along the limb axis (limb isolated and opened, Fig. 8B; limb isolated and mesoderm removed, Fig. 8A; limb opened and mesoderm removed, Fig. 9B). In the vicinity of the troehanter/coxa boundary, axons in all classes turned and crossed into the limb region which would be ventral in an intact limb (Figs. 8, 9B). Therefore, these major features of the pioneer route were expressed under all doubly operated conditions. In all classes of doubly operated limbs, performance of pioneer neurons in experimental limbs was indistinguishable from that in controls (Table 2). With the exception of one limb (limb isolated/mesoderm removed), neurons in all limbs initiated axonogenesis. No neurons extended axons in abnormal directions. Most neurons in isolated/mesoderm removed limbs (3/4) and their controls (2/3) extended into the ventral region of the limb, as did all neurons in other operations and their controls (Table 2). DISCIJSSION

The Til afferent pioneer neurons extend the first axons through developing grasshopper limb buds (Bate, 1976). Pioneer growth cones migrate along a sterotyped trajectory which establishes the route of a major nerve (Ho and Goodman, 1982; Bentley and Keshishian, 1982; Keshishian and Bentley, 1983). A set of guidepost neurons which arise at particular locations in the limb are guidance cues for pioneer growth cones along part of their route (Ho and Goodman, 1982; Bentley and Keshishian, 1982; Bentley and Caudy, 1983a; Caudy and Bentley, 198613).However, along other parts of their route, and in limbs in which guidepost neurons have not differentiated, pioneer growth cones appear to be guided by other cues (Bentley and Caudy, 1983b; Berlot and Goodman, 1984; Caudy and Bentley, 1986a,b). The experiments described here (Fig. 10) provide an assessment of several types of additional potential guidance cues. Cue Extrinsic

to the Limb

A prominent feature of pioneer axonogenesis is the characteristic emergence of the growth cones from the

474

DEVELOPMENTAL

BIOLOGY

VOLUME

119, 1987

FIG. 7. Pioneer axon routes in mesoderm-free limb buds (and in contralateral control limb buds). (A-D) anti-HRP labeled. whole-mounted metathoracic limb buds operated and/or placed in culture at the 29-30% stage. (A) Contralateral control limb from the same embryo as (B). (cultured 42 hr) The pioneer neurons (open arrow) have extended axons proximally whose growth cones are near the prospective trochanter. (B) Limb from which the mesoderm has been removed (disposition of remaining adepithelial cells shown in Fig. 5, L-2), and which is contralateral to limb (A). Removal of mesoderm has reduced the cross-sectional area of the limb. The pioneer neurons (open arrow) have extended axons proximally whose growth cones are near the prospective trochanter. The route and appearance of the axons are similar in the control (A) and experimental (B) limbs. (C) Contralateral control limb from the same embryo as (D). (cultured 48 hr) The pioneer neurons (open arrow) have extended axons proximally in the dorsal region of the limb. In the vicinity of the prospective trochanter, the axons have crossed to the ventral side of the limb and have contacted the Cxl neurons (solid arrow). (D) Limb from which the mesoderm has been removed. The cross-sectional area of the limb is reduced relative to the control (C) limb. The pioneer neurons (open arrow) have extended axons proximally in the dorsal region of the limb. In the vicinity of the prospective trochanter, the axons have crossed to the ventral side of the limb and have contacted the Cxl neurons (solid arrow). The pioneer axons are similar in the control (C) and experimental (D) limbs. Ventral, down; proximal, to left. Calibration bars: 50 pm.

proximal face of the cells, and the subsequent proximal extension of axons along the limb axis. Since the axons are growing toward the thorax and the central nervous system, it is possible that they are oriented by information emanating from tissue extrinsic to the limb. To evaluate this possibility, limbs were isolated from the thorax before the onset of pioneer axonogenesis, and axon growth in these limbs was compared to growth in

PIONEER

NEURON

contralateral control limbs which remained attached to the thorax (Figs. 2, 10B; Table 1). The performance of pioneer axons in experimental and control limbs was very similar. We conclude that during the period of axon growth, the presence of tissue extrinsic to the limb is not required for expression of the aspects of pioneer neuron growth which were monitored. Growth of afferent pioneer axons has been examined in other insect

TABLE 2 GROWTH IN DOUBLY

OPERATED

LIMBS

Pioneer Number of limb buds

Treatment Limb

isolated

and opened

(controls)

Femur (midpoint)

axon

Trochanter (dorsal)

elongation

to: Tr/Coxa boundary (ventral)

4 3

4/4 313

4/4 3/3

4/4 313

Limb isolated and mesoderm removed (controls)

4 3

3/4 3/3

3/4 3/3

3/4 213

Limb opened and mesoderm removed (controls)

4 4

414 414

4/4 4/4

414 4/4

LEFCORT

AND

BENTLEY

Pathfinding

by

Pioneer

Neurcms

475

leg discs, afferent pathways also are formed normally (Jan et al., 1985). Therefore, afferent pathfinding does not appear to require information extrinsic to the limb (during the period of axon growth).

sophila

Contact

FIG. 8. Pioneer axon routes in doubly operated limb buds. (A, B) Anti-HRP-labeled whole-mounted metathoracic limb buds operated and placed in culture at the 29-30’70 stage. (A) A mesoderm-free, isolated limb bud (cultured 42 hr). The pioneer neurons (open arrow) extended axons proximally along the limb axis and across the prospective femur/trochanter boundary (marked at this stage by a sharp invagination encircling the limb; small arrows). Proximal to this boundary, the axons ceased axial growth and extended processes circumferentially. A ventrally directed process (arrowhead) appears to be extended straight along the prospective trochanter/coxa boundary (which lies parallel to and 15-25 pm proximal to the femur/trochanter boundary at this stage). (8) An isolated and opened limb bud (cultured 45 hr). The pioneer neurons (open arrow) extended axons proximally along the limb axis to the region of the prospective trochanter/coxa boundary (arrowhead). Here, the axons made a sharp turn and grew circumferentially along a straight route into the region of the ventral limb epithelium. Ventral, down; proximal, to left. Calibration bars: 50 pm.

appendages which have been isolated. In Drosophila wing discs which have been isolated from the body, afferent pioneer axons also extend along their normal route (Edwards et ab, 1978; Blair and Palka, 1985; Blair et al., 1985). Berlot and Goodman (1984) demonstrated proximal growth of afferent pioneer neurons in isolated antennae of grasshopper embryos, and in isolated Dro-

Guidance

A possible guidance cue for migrating growth cones or cells is stereotropism or contact guidance (Weiss, 1959; Singer et ab, 1979; reviewed in Dunn, 1982). The alignment or the curvature of the substrate may bias the direction of growth cone or cell migration. Fibroblasts, for example, orient in the direction of least curvature along micromachined grooves with a variety of crosssectional shapes, and with dimensions of 24-68 pm (Brunette, 1986). These dimensions are similar to the interior diameter or a grasshopper limb bud (cf. Fig. 6A). Pioneer growth cones are initially extended along the long axis of a cone or tube formed by the limb epithelium and mesoderm; this geometric configuration might bias growth cones in the proximal direction (along the path of least curvature). The limb curvature might be detected by filopodia and branches extended circumferentially from the growth cone; in limbs in which growth cones are migrating in the absence of guidepost neurons, such branches can occupy approximately 90” of arc along the limb circumference (Caudy and Bentley, 1986a). To test for a possible contribution of the limb curvature to growth cone guidance, the limb epithelium and mesoderm were split open from the limb base to the limb tip and pinned out flat before the pioneers began axonogenesis (Figs. 8B, 9,lOC; Tables 1,2). The embryos were maintained in culture to allow pioneers to extend axons, and axonogenesis in experimental limbs was then compared with axonogenesis in control limbs (which were the limbs contralateral to experimental limbs). Axon orientation and extension were very similar in experimental and control limbs. We conclude that the normal contour of the limb is not required for these aspects of pioneer growth. wing discs, the channels which will form In Drosophila wing veins have been considered as a possible source of stereotropic or contact guidance (Murray et al., 1984). However, the geometrical constraint imposed by channels has been eliminated as an essential guidance cue by correctly oriented pioneer growth in wing fragments after separation of the dorsal and ventral wing epithelia (Blair and Palka, 1985). Consequently, the tested aspects of epithelial contour do not appear to play an essential role in guidance of afferent growth cones in either of these cases. Electrical Fields Migrating growth cones are sensitive to electrical fields and can be highly oriented by current densities of a few pA per pm2 (Pate1 and Poo, 1982; Pate1 et al, 1985;

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DEVELOPMENTAL

BIOLOGY

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Robinson, 1985). Electrical potentials of several tens of millivolts are maintained across the transepithelial resistance of some epithelia (cf. Jaffe, 1981). In regenerating newt limbs, a gradient of transepithelial potential of sufficient magnitude to influence cell migration extends proximally along the limb axis from the tip (McGinnis and Vanable, 1986). Therefore, orientation of pioneer growth cones along the limb axis by an electrical field generated across the limb epithelium is a possibility. A field of this type would be short-circuited by opening the epithelium along the length of the limb bud and pinning it out, bathing the internal and external surfaces in the same solution. Therefore, the similarity of pioneer axon growth in opened and in control limb buds also indicates that the monitored aspects of growth are independent of such extracellular electrical fields. Diflusion

Gradients

In culture, migrating growth cones can be oriented by a diffusion gradient of a soluble molecule (Gunderson and Barrett, 1980). Therefore, it is possible that pioneer growth cones could be oriented along the limb axis by a diffusion gradient on the scale of the whole limb generated by a source localized at the tip or the base or other axial site along the limb. It seems likely that a gradient of this type would be disturbed by opening the limb and exposing it to a large volume of medium (particularly when the mesoderm is also removed). The absence, in opened limb buds, of changes in pioneer growth cone routes or morphology argues against this type of large-scale gradient derived from a localized source (a distributed source, such as graded release by the epithelium, is not excluded). Internal

FIG. 9. Pioneer axon routes in a mesoderm-free, opened limb bud. The mesoderm was removed from a metathoracic limb bud of a 2930% embryo (before the onset of pioneer axonogenesis). The limb epithelium was then cut open from the limb base to tip, pinned out flat, and cultured for 42 hr. (A) Interference contrast photomicrograph of the opened epithelium fixed at the end of the culture period. (B) AntiHRP labeled, whole-mounted limb (same limb as (A)). The pioneer neurons (open arrow) have extended axons proximally along the limb axis to the vicinity of the trochanter/coxa boundary (arrowhead) where they have crossed circumferentially to the region of ventral epithelium, and then turned proximally to contact the Cxl neurons (solid arrow). (C) Toluidine blue-stained 4-pm cross section of the limb epithelium at the level of the arrowhead in (B). The dorsal region of the epithelium is to the left, and the inner surface is up. The epithelium is flattened out, and no mesodermal or other adepithelial cells remain apposed to the inner surface. (A, B) ventral, down; proximal, to left; asterisks, nin hole. Calibration bar (for nanels): ~~~~-I- --50 r.... rim \-~- all ~~~ _~

Polarity

The initial direction of growth cone emergence from the pioneer cell bodies might be determined by internal polarization of the cell bodies themselves (cf. Solomon, 1981; Bentley and Caudy, 1983b; Berlot and Goodman, 1984; Blair and Palka, 1985). Growth cones emerge from the proximal side of the cell bodies before filopodial contact with the nearest guidepost neuron is established (Bentley and Caudy, 198313). Once started proximally, the growth cones might maintain proximal growth through the tendency of axons to continue relatively straight growth under uniform conditions (Katz, 1985). The possibility that intrinsic polarization of the pioneer cell bodies could provide adequate guidance information for proximal growth remains untested (however, intrinsic factors do not appear able to account for the circumferential turn at the trochanter/coxa boundary; Caudy and Bentley, 1986a,b). Mesoderm Pioneer growth cones migrate between a layer of limb epithelium and a layer of limb mesoderm, and filopodia

LEFCORT

AND

BENTLEY

P&finding

by Pioneer

Neurons

477

FIG. 10. A schematic diagram summarizing operations on limb buds and subsequent routes of pioneer neurons. (A) A cutaway view of an unoperated limb bud showing the apposition of mesoderm and epithelium. (B) A limb bud isolated from the thorax. d, Dorsal side of limb; p, posterior side of limb. (C) A limb bud isolated from the thorax, cut open along the posterior side, and pinned out flat. (D). A limb bud which has been opened and pinned out flat following removal of the mesoderm (the epithelium remains connected to the thorax along the interface indicated by the bracket arrows). Tr/Cx, Trochanter/coxa limb segment boundary (also shown in (A-C); Til, pioneer neuron pair; Cxl, pair of guidepost neurons (neurons shown by broken lines in (A-C). In the intact limb, the pioneer neurons extend proximally along the basal surface of the epithelium (and lamina) until they reach the trochanter/coxa boundary. They then reorient circumferentially along the boundary, cross to the ventral region of the limb, and turn to contact the Cxl neurons. Following each of the limb operations, this pattern continues to arise.

are in frequent contact with mesodermal cells (Caudy and Bentley, 1986a). Efferent growth cones have been shown to be guided by certain mesodermal cells (Ho et nl., 1983; Ball et ab, 1985), and afferent growth cones also appear to respond selectively to certain mesodermal cells (Caudy and Bentley, 198613). To evaluate the role of the mesoderm in pioneer guidance, we attempted to remove all mesodermal cells from the limb bud before the onset of pioneer axonogenesis (Fig. 10D). Embryos with mesoderm-free limbs were then cultured for the period during which pioneer growth cones would normally extend through most of the limb. Pioneer neuron behavior

in mesoderm-free limbs was then compared to behavior in control limbs. Mesoderm was removed by inserting a suction pipette through the dorsal surface of the embryonic thorax and into the limb lumen. In transmitted light, the mesoderma1 layer could be clearly visualized and it was evident when most of the cells had been extracted (Fig. 4). Three extracted limbs were serially sectioned (4-pm sections) and examined for remaining adepithelial cells. Two limbs had a single remaining cell, and one had two cells; these cells were in different locations in the different limbs (Figs. 3, 5). Two additional limbs examined in

478

DEVELOPMENTAL BIOLOGY

scanning EM had no adepithelial cells remaining distal to the trochanter/coxa boundary (cf. Fig. 6). We conclude that the mesoderm removal procedure was effective in that almost all cells were removed, no particular adepithelial cell remained in each limb, and every axial location was unoccupied by an adepithelial cell in at least one of the sectioned limbs. The effect of mesoderm removal on pioneer growth was evaluated by viewing labeled neurons in wholemounted limbs (Fig. 7), and by scoring several aspects of growth in experimental and control limbs (Table 1). The aspects were (i) whether the pioneers underwent axonogenesis, (ii) whether axons extended in the normal direction (proximally along the long axis of the limb), (iii) whether axons reoriented from proximal to circumferential growth in the region of the trochanter/coxa boundary and crossed from the dorsal to the ventral side of the limb, and (iv) how far along the route the axons extended. By each of these criteria, the pioneer axons in experimental limbs were indistinguishable from those in controls. We conclude that during the period of axon extension, the mesoderm is not required for these aspects of pioneer growth. Guidepost Neurons In normal, well-differentiated embryos, guidepost neurons appear to direct the pioneer growth cones for about two-thirds of the distance from the pioneer cell bodies to the most proximal (Cxl) neurons (Caudy and Bentley, 198613).This estimate is based on the observation that pioneer axons and growth cones approaching the vicinity of guidepost neurons sharply reorient toward them when they are approximately 30-35 pm away (this distance corresponds to the distance at which filopodial contact appears to be made). The inference is that before the reorientation, the pioneer growth cones were not being directed by the guidepost neurons. This inference is supported by the demonstration that when guidepost neurons are absent, pioneer growth cones are still able to grow proximally in the distal regions of the limb where, in well differentiated limbs, they do not appear to be directed by guidepost neurons (Caudy and Bentley, 1986a). Therefore, proximally orienting cue(s) in addition to guidepost neurons must be present. What role do guidepost cells play in proximal guidance of growth cones in cultured limbs from which mesoderma1 (and other adepithelial) cells have been removed? If all guidepost cells remained in these limbs and differentiated at the normal time, pioneer growth cones would still, as described above, have to be guided in certain regions (i.e., when not in contact with guideposts) by other cues. These cues could not be provided by mesoderm. Thus the observed normal orientation of growth cones indicates that mesodermal cells (as well as guide-

VOLUME 119,1987

post cells) are not required for proximal growth through these regions. Moreover, several observations suggest that in cultured, mesoderm-free limbs, guidepost neurons either have been removed along with the mesodermal cells or have failed to differentiate in time to influence passing pioneer growth cones: (i) in cultured, mesoderm-free limbs, labeling with anti-HRP antibodies usually does not indicate that guidepost neuron Fe1 or Trl is present (Figs. 7B, D, 8A); (ii) in normal limbs, the presence of guidepost cells which have not yet acquired their antiHRP binding sites is often revealed by selective wrapping by pioneer growth cone filopodia and branches (Caudy and Bentley, 198613).In cultured, mesoderm-free limbs, selective wrapping does not indicate that unlabeled Fe1 or Trl cells are present (Figs. 7B, D, 8A); (iii) in cultured, mesoderm-free limbs, the “zigzag” reorientations of pioneer axons, which indicate growth to guidepost cells (Caudy and Bentley, 1986b) are not seen (Figs. 7B, D, 8A); (iv) serial sectioning of limbs reveals that one or both of the distal guidepost cells typically are missing after removal of the mesoderm (Fig. 5); (v) scanning EM of the internal surface of the epithelium (and basal lamina) of mesoderm-free limbs reveals that no adepithelial cells remain in the distal region (Fig. 6): These results are consistent with either guidepost cell removal or delayed differentiation; (vi) observation of cultured limbs with the mesoderm intact indicates that even when guidepost cells are not removed, their differentiation is delayed (Figs. 2A, B, 7C, 8B). When isolated limbs are maintained in culture until growth cones reach the ventral region of the limb, distal (Fel) guidepost neurons sometimes begin to label with anti-HRP (Figs. 2A, B). Usually, these guidepost neurons have not been contacted by the pioneer axons (Figs. 2A, B). This configuration suggests that the pioneer growth cones passed the distal guidepost neurons before they differentiated (Caudy and Bentley, 1986a). Therefore, the process of placing embryos in culture just at the time when the distal guidepost neurons are differentiating may delay that differentiation sufficiently so that the neurons are ineffectual in orienting passing pioneer growth cones. These results indicate that in many cultured, mesodermfree limbs, guidepost neurons did not orient proximally migrating pioneer growth cones in the distal region of the limb. Therefore, these growth cones could still migrate proximally in regions where they were not provided orienting information by either guidepost neurons or by mesodermal cells. In Drosophila wing imaginal discs, the cells bodies of pre-axonogenesis neurons are also located in positions where they might provide proximal guidance for pioneer neuron growth cones. Whether or not these cells are essential for proximal guidance has been tested by removing them by genetic (Schubiger and Palka, 1985) or

LEFC~RT

AND

BENTLEY

Path&ruling

surgical (Blair and Palka, 1985; Blair et ah, 1985) manipulations. While neural routes along the wing margin (Ll) may be at least partially dependent upon the placement of immature neurons, the routes of pioneer neurons located more centrally in the wing blade (L3) are not dependent upon such cues. Since few, if any, mesodermal cells are found in wing discs, this guidance must also be independent of mesoderm. Epithelium

and Basal Lamina

Guidance information for both proximal migration of growth cones and for circumferential extension along the trochanter/coxa segment boundary might be provided by the epithelium and basal lamina. When pioneer growth cones which are migrating proximally along the limb axis of well differentiated grasshopper limbs reach the prospective trochanter/coxa boundary, they cease axial growth and extend circumferential processes dorsally and ventrally along the boundary (Bentley and Caudy, 198313; Caudy and Bentley, 1986a,b, 1987a). This response is clearly related to an extrinsic factor, the location and degree of differentiation of the segment boundary, and not to internal properties of the pioneer neurons (Caudy and Bentley, 1986b). These circumferential processes are not growing directly toward, and do not appear to be circumferentially aligned by, any guidepost cell (Bentley and Caudy, 1983b; Caudy and Bentley, 1986a). The guidance information for this circumferential growth could reside in either the mesodermal or the epithelial layer at the segment boundary. In limbs from which the mesoderm has been removed, growth cone processes still extend circumferentially at the location of the trochanter/coxa boundary (cf. Fig. 8A). We conclude that the epithelium itself (and/or its basal lamina) can provide adequate guidance information for this circumferential portion of the pioneer growth cone route. Circumferential extension of growth cone processes occurs in mesoderm-free limbs which have been isolated from the body (Fig. 8A), and in mesoderm-free limbs which have been opened and pinned out flat (Fig. 9). As discussed previously, these operations appear to eliminate several types of cues. Taken together, these observations indicate that the guidance information provided by the epithelium (and/or lamina) is highly localized along the segment boundary. The information might reside in the physical configuration of the substrate (on a microscale), in local release of soluble material, or in substrate-bound guidance molecules. Pioneer neuron branching, extension of lamellae, axon spreading, and the general level of membrane apposition to the substrate increase proximally within limb segments and reach a peak at the proximal boundary of the segment (Caudy and Bentley, 1986a). Within each segment, therefore, there appears to be a proximally in-

by Pioneer

479

Neurons

creasing gradient (either smooth or stepped) of affinity for the growth cones along the epithelium/mesoderm interface. The demonstration that the presence of the mesoderm is not required for the growth cone response at the location of the peak of this gradient (at the segment boundary), provides support for a hypothesis that the affinity gradient throughout the segment resides in the epithelium (and/or lamina). This possibility is consistent with previous work on insect appendages. In Manduca wings, Clever (1959) proposed that sensory axons might orient proximally along a gradient and transplantation of epithelial patches indicates that there is an adhesion gradient on the epithelium which is recognized by neurons (Nardi and Kafatos, 19’76; Nardi, 1983). Transplantation and regeneration experiments suggest that within segments there is a proximally increasing gradient of positional information (Locke, 1966; Bohn, 1970; French, 1976; French et ab, 1976). Genetic and surgical manipulations of wing discs indicate that cues in the epithelium (and/ or lamina) are responsible for important elements of afferent neuron pathfinding in Drosophila (Palka et al., 1983; Murray et al, 1984; Blair et al., 1985; Blair and Palka, 1985; Schubiger and Palka, 1985). Multiple

(Redundant)

Guidance

Cues

The operations described above were designed to evaluate the roles of particular potential guidance cues. But it is possible that guidance is achieved by any one of two (or more) cues, so that eliminating one at a time would have no effect (Harris, 1984). For example, it might be that either mesoderm, or tissue extrinsic to the limb is necessary for guidance, and that either would suffice. To evaluate this possibility, paired combinations of all operations were performed. Pioneer neurons growing under each of these conditions oriented and extended at levels very similar to controls (Table 2). We conclude that for these aspects of growth, cues eliminated by limb isolation, limb opening, or mesoderm removal are not alternative sources of guidance. In grasshopper limb buds, the explanted, opened, mesoderm-free epithelium (and lamina) can provide guidance information for migrating pioneer neuron growth cones. This preparation appears advantageous for analysis of growth cone guidance and steering. We thank Alma Toroian-Raymond for providing SEM and TEM material and for sectioning limb buds, and Dr. David Weisblat, Dr. Mark Cooper, and Dr. Ray Keller for criticizing the manuscript. Support provided by NIH 2T32-GM07379 to F.L., and by NIH Jacob Javits Award (NS09074-17) to D.B. REFERENCES ANDERSON,

velopment ASHHURST,

H. (1985). Basement membranes and nervous system dein the grasshopper embryo. Sot. Neurosci A&r. 11,335. D. E. (1965). The connective tissue sheath of the locust

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nervous system: Its development in the embryo. Quart. J. Micr. Sot. 106,61-‘73. ASHHURST, D. E. (1982). The structure and development of insect connective tissues. In “Insect Ultrastructure” (R. C. King, H. Akai, Eds.), pp. 313-350. Plenum, New York. BALL, E. E., Ho, R. K., and GOODMAN, C. S. (1985). Development of neuromuscular specificity in the grasshopper embryo: Guidance of motoneuron growth cones by muscle pioneers. J. Neurosci 5,18081819. BATE, C. M. (1976). Pioneer neurons in an insect embryo. Nature (London)260,54-56.

BENTLEY, D., KESHISHIAN, MOND. (1979). Quantitative grasshopper, Schistocercu

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74.

BENTLEY, D., and KESHISHIAN, H. (1982). Pathfinding by peripheral pioneer neurons in grasshoppers. Science 218,1082-1088. BENTLEY, D., and CAUDY, M. (1983a). Pioneer axons lose directed growth after selective killing of guidepost cells. Nature (London) 304, 6265.

BENTLEY, D., and CAUDY, M. (1983b). Navigational substrates for peripheral pioneer growth cones: limb-axis polarity cues, limb-segment boundaries, and guidepost neurons. Cold Spring Harbor Symp. Quark Biol. 48, 573-585. BERLOT, J., and GOODMAN, C. S. (1984). Guidance of peripheral pioneer neurons in the grasshopper: Adhesive hierarchy of epithelial and neuronal surfaces. Science 223,493-495. BLAIR, S. S., and PALKA, J. (1985). Axon guidance in cultured wing discs and disc fragments of Drosophila. Dev. Biol. 108, 411-420. BLAIR, S. S., MURRAY, M. A., and PALKA, J. (1985). Axon guidance in cultured epithelial fragments of Drosophila wing. Nature (London) 315,406-408.

BOHN, H. (1970a). Interkalare Regeneration und segmental Gradienten bei den Extremitaten von Leucophaea-Larven (Blattaria) I. Fermur und Tibia. Wilhelm Roux’s Arch. 165, 303-341. BRUNETTE, D. M. (1986). Fibroblasts on micromachined substrata orient hierarchically to grooves of different dimensions. &p. Cell Res. 164, 11-26.

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. (198613). Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different affinities. J. Neurosci. 6, 1781-1795. CAUDY, M., and BENTLEY, D. (1987a). Pioneer growth cone behavior at a differentiating limb segment boundary in the grasshopper embryo. Dev. Biol. 119,454-465. CAUDY, M., and BENTLEY, D. (198713). Epithelial cell specialization at a limb segment boundary in the grasshopper embryo. Dev. Biol., submitted. CLEVER, U. (1959). Uber experimentelle Modifikationen des Geaders und die Beziehungen zwischen den Versorgungssystemen im Schmetterlingsflugel. Untersuchungen an Galleria mellonella L. Wilhelm Roux’s Arch. 151, 242-279. DUNN, G. A. (1982). Contact guidance of cultured tissue cells: A survey of potentially relevant properties of the substratum. In “Cell Behavior” (R. Bellairs, A. Curtis and G. Dunn, Eds.), pp. 247-280. Cambridge Univ. Press, Cambridge. EDWARDS, J. S., MILNER, M. J., and CHEN, S. W. (1978). Integument and sensory nerve differentiation of Drosophila leg and wing imaginal discs in vitro. Wilhelms Roux k Arch. 185, 59-78. FRENCH, V. (1976). Leg regeneration in the cockroach, Blattella germania. II. Regeneration from a non-congruent tibia1 graft/host junction. J Embryo1 Exp. Mcwphol. 35, 267-301. FRENCH, V., BRYANT, P., and BRYANT, S. (1976). Pattern regulation in epimorphic fields. Science 193,969-981.

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GUNDERSON, R. W., and BARREN, J. N. (1980). Characterization of the turning response of dorsal root neurites toward nerve growth factor. J. Cell Biol. 87, 546-554. HARRIS, W. A. (1984). Axonal pathfinding in the absence of normal pathways and impulse activity. J. Neurosci. 4, 1153-1162. Ho, R. K., and GOODMAN, C. S. (1982). Peripheral pathways are pioneered by an array of central and peripheral neurones in grasshopper embryos. Nature (London) 297,404-406. Ho, R. K., BALL, E. E., and GOODMAN, C. S. (1983). Muscle pioneers: Large mesodermal cells that erect a scaffold for developing muscles and motoneurones in grasshopper embryos. Nature (London) 301, 66-69.

JAFFE, L. F. (1981). The role of ionic current in establishing developmental pattern. Philos. Trans. R. Sot. London Ser. B. 295, 553-566. JAN, L. Y., and JAN, Y. N. (1982). Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and grasshopper embryos. Proc. Natl. Acad. Sci. USA 79,2700-2704. JAN, Y. N., GHYSEN, A., CHRISTOPH, I., BARBEL, S., and JAN, L. Y. (1985). Formation of neuronal pathways in the imaginal discs of Drosophila melanogaster. J. Neurosci. 5,2453-2464. KATZ, M. J. (1985). How straight do axons grow? J. Neurosci. 5, 589595. KESHISHIAN, H. (1980). The origin and morphogenesis of pioneer neurons in the grasshopper metathoracic leg. Dev. Biol. 80,388-397. KESHISHIAN, H., and BENTLEY, D. (1983). Embryogenesis of peripheral nerve pathways in grasshopper legs. Dev. Biol. 96,98-124. LEFCORT, F., and BENTLEY, D. (1985). Neuronal guidance in dissected limb buds, in cell culture, and at ectopic locations in host limb buds. Sot. Neurosci. Abstr. 11,334. LOCKE, M. (1966). The cuticular pattern in an insect: the behavior of grafts in segmented appendages. J. Insect Physiol. 12,397-402. MCGINNIS, M. E., and VANABLE, J. W. (1986). Voltage gradients in newt limb stumps. In “Ionic Currents in Development” (R. Nuceitelli, Ed.), pp. 231-238. Liss, New York. MURRAY, M. A., SCHUBIGER, M., and PALKA, J. (1984). Neuron differentiation and axon growth in the developing wing of Drosophila melnnogaster. Dw. Biol. 104, 259-274. NARDI, J. B., and KAFATOS, F. (1976). Polarity and gradients in lepidopteran wing epidermis. II. The differential adhesiveness model: Gradient of a non-diffusible cell surface parameter. J. Embryo1 Exp. Morph,ol. 36, 489-512. NARDI, J. B. (1983). Neuronal pathfinding in developing wings of the moth Munduca sexta. Dem. Biol. 95,163-174. PALKA, J., SCHUBIGER, M., and ELLISON, R. L. (1983). The polarity of axon growth in the wings of Drosophila melanogaster. Dev. Biol. 98, 481-492. PATEL, N., and POO, M-M. (1982). Orientation of neurite outgrowth by extracellular electric fields. J. Neurosci 2, 483-496. PATEL, N. B., XIE, Z-P., YOUNG, S. H., and Poo, M-M. (1985). Response of nerve growth cones to focal electric currents. J. Neurosci. Res. 13, 245-256.

ROBINSON, K. R. (1985). The responses of cells to electric fields: A review. Cell 101,2023-2027. SCHUBIGER, M., and PALKA, J. (1985). Genetic suppression of putative guidepost cells: Effect on establishment of nerve pathways in Dro sophila wings. Dev. Biol. 108,399-411. SINGER, M., NORDLANDER, R. H., and EGAR, M. (1979). Axonal guidance during embryogenesis and regeneration in the spinal cord of the newt: The blueprint hypothesis of neuronal pathway patterning. J. Comp. Neural. 185, l-22. SOLOMON, F. (1981). Specification of cell morphology by endogenous determinants. J. cell Biol. 90, 547-553. WEISS, P. (1959). Cellular dynamics. Rev. Mod. Phys. 31,11-20. WIGGLESWORTH, V. B. (1953). The origin of sensory neurones in an insect, Rhodnius prolixus (Hemiptera). Quart. J. Microscop. Sci. 94, 93-112.