projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the...

Projection Pattern of Sensory Neurons in the Central Nervous System of a Homeotic Mutation of the Moth Manduca sexta

Ronald Booker* and Carol 1. Miles

Section of Neurobiology and Behavior, Division of Biological Sciences, Cornell University, Ithaca, New York 14853

SUMMARY

Octopod (Octo) is a mutation of the moth Munducu senfa, which transforms the first abdominal segment (A1 ) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on Al . We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Munduca. Me- chanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral

surface of Al , resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: a n anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmen- tal identity influences the intersegmental projection pattern of the sensory neurons of Munducu, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. o 1995

Keywords: insect, sensory neurons, projections, mutant. John Wiley & Sons, Inc.

INTRODUCTION

Among insects, one of the best examples of a somatotopic sensory map is that created by the projections of the mechanosensory hair neurons on the body surfaces of larvae of the moth Munducu sextu (Levine et al., 1985; Kent and Levine, 1988). In tile fifth larval instar, there are 500 to 700 thin hairs with tightly fitting sockets typical of the insect bristle sensilla covering each segment. Each sensory

Received August 22, 1994, accepted June 19, 1995 Journal ofNeurobiology, Vol. 28, No. 3, pp. 28 1-296 ( 1995) 0 1995 John Wiley & Sons, Inc. CCC 0022-3034/95 /03028 1-16

* To whom correspondence should be addressed.

hair is innervated by a single neuron that projects to the segmental ganglion. This projection joins those of other sensory neurons to create a precise somatotopic map of the animal’s tactile environment. The rules governing the formation of the sensory map ofManduca are rather simple and will be described briefly. For more detailed descriptions of the somatotopic projections of the mechanosensory neurons of Munduca, we refer you to the work ofLevineetal.,( 1985);andKent andLevine,(1988).

The projection pattern of a sensory neuron depends on the position of its sensillum on the body surface (Fig. 1 ). In both the abdomen and thorax, axons from sensory neurons located on the posterior portion of a segment branch primarily in the posterior half of the ventral medial neuropil of

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282 Booker and Miles

B Thoracic Bodywall

A Abdominal Bodywall

C Thoracic Leg

Figure 1 Drawings summarizing the projection patterns of sensory neurons located on the body wall of a typical abdominal ( A ) and thoracic ( B ) segment, as well as those located on the thoracic legs (C) . In all cases sensory neurons located in the anterior (Ant.; bottom drawings) and posterior (Post.; top drawings) region of the segment project to the anterior and posterior region of the ventral neuropil, respectively. Many of the sensory neurons located on the anterior surface of an abdominal segment (A , bottom) also possess an anteriorly directed intersegmental projection. In the thorax it is the posteriorly located sensory neurons that have the intersegmental projections (B, top), projecting to the next most posterior segment. Sensory neurons located on the proximal segments of the thoracic legs (C, top) also have posteriorly directed intersegmental projections. The sensory neurons located on the leg share two other leg-specific features: terminal arbors in the dorsal lateral leg neuropil (large arrows) and projections from the more proximal leg segments that approach and often cross the midline of the thoracic ganglia through both the anterior and posterior ventral commissures (small arrows).

their respective segmental ganglion. Anteriorly located sensory neurons project to the anterior region of the ventral medial neuropil. Sensory neurons located between these extremes project in an orderly anterior-to-posterior gradient within the ganglion that corresponds to the position they occupy on the body surface.

Although this somatotopic projection pattern within the ventral medial neuropil is similar in all thoracic and abdominal segments, there is one feature that varies in a segment-specific manner. In each larval segment, a subset of the sensory neurons project intersegmentally (Levine et al., 1985; Kent and Levine, 1988). In the abdominal segments, the sensory neurons located on the anterior

surface send projections intersegmentally, terminating in the next anterior ganglion (Fig. 1 ). In the thoracic segments, it is the posteriorly positioned sensory neurons that project intersegmentally to the next posterior ganglion. As a result, the first abdominal and metathoracic ganglia each receive an extra set of sensory afferents.

The projections of the sensory neurons innervating the bristle sensilla of the thoracic legs are in- tegrated into the somatotopic maps of the thoracic ganglia (Kent and Levine, 1988). The projection patterns of the leg sensilla in the ventral medial neuropil are generally the same as those followed by the neurons located on the general body surface of the thoracic segments, but with two important

Sensory Projections in a Homeotic Mutant 283

differences: The projections of the sensory neurons located on the more proximal leg segments typically will cross the midline of the thoracic ganglia through the anterior and posterior ventral commissures. In addition, all of the leg sensory neurons have a unique arborization in a more lateral and dorsal region of the thoracic neuropil referred to as the lateral leg neuropil [Fig. 1 ( C ) ] . The leg sensory neurons share the lateral leg neuropil with projections from the leg motoneurons.

Several possible mechanisms for generating the projection patterns of insect sensory neurons have been suggested, including the position of the sensory neuron (Walthall and Murphey, 1986; Kamper and Murphey, 1987), competitive in- teractions with other neurons (Murphey, 1986) and segmental determination (Ghysen et al., 1983). In this study we used the transformed segments of the moth mutant Octopod (Octo) t o eval- uate the effect of this mutation on the projection patterns of the larval sensory neurons in Manduca. In Octo animals, the first abdominal segment ( A 1 ) is transformed in the anterior direction, resulting in the appearance of homeotic thoracic legs on A 1 (Booker and Truman, 1989). W e examined the structure, function, and projection patterns of the sensilla that were located on A l of mutant larvae. We conclude that both the sensory neuron’s segmental identity and its position on the body surface play a role in determining its projection pattern within the central nervous system (CNS).

METHODS

Experimental Animals

Larvae of the tobacco hornworm, M. sexta, were reared on an artificial diet under conditions of 26°C and a 16L: 8D photoperiod as described by Bell and Joachim ( 1978). Under these conditions approximately 18 days elapsed between hatching and pupal ecdysis. All the animals used in these studies were in their fifth (final) larval instar.

The Octo mutation originated as a spontaneous mutation first observed in the breeding colony maintained at the University of Washington, Seattle, in 1984 (Booker and Truman, 1989). Normally, the ventral surfaces of the first and second abdominal segments (A1 and A2, respectively) are devoid of appendages. Octo animals are characterized by the appearance of homeotic appendages on the first abdominal segment. While the penetrance of the mutant phenotype is complete, the expression is variable, ranging from a slight deformation of the ventral cuticle of A 1 to the presence of a well-formed but miniature larval thoracic leg. All of the animals used

in this study had at least a well-formed coxa, the most proximal segment of the leg. As a control, we used fifth instar animals from a wild-type colony we maintain in the laboratory.

Histological Techniques

After carbon dioxide anesthesia, we placed the larvae in an airtight container filled with paraffin. The animals were restrained using 1 / 2 inch staples which were placed around the body in several locations and pressed firmly into the wax. A solution of 100 mMcobalt chloride con- taining 0.7% (w/v) low melt agarose (Sigma) was used to label the sensory neurons. The cobalt mixture was melted in a microwave oven just prior to use and kept warm by placing it in a heating block. Using a syringe, a well of petroleum jelly was built around an individual hair or group of hairs. A drop of the liquefied cobalt solution was placed in the well, and before the agarose so- lidified, the hair was cut using a modified pair of forceps. For multiple hair fills, several hairs were cut. To prevent dehydration, several moist tissues were placed around the animal before sealing the container. The animals were stored at 4’C for 3 to 5 days. Following incubation, the larvae were sacrificed, their ganglia removed and rinsed in saline. The cobalt was precipitated using saline saturated with hydrogen sulfide (bubbled into the saline for 5 minutes) for 10 to 15 min. After rinsing in saline, the ganglia were fixed in Carnoy’s fixative for 1 to 2 h. The ganglia were then rehydrated and the cobalt intensified using a modified Timm’s technique (Bacon and Alt- man, 1977). The ganglia were dehydrated through a graded ethanol series, cleared using methyl salicylate, and mounted in Canada balsam. The filled sensory arbors were drawn with the aid of a drawing tube attachment on a compound microscope. The total length ofthe arbors of the sensory neuron was estimated by projecting an image of the arbors using a drawing tube and tracing the length of individual processes using a map measurer ( K and R Instruments). The readout of the map measurer was then multiplied by the appropriate scaling fac- tor to derive the length of the measured arbor.

For examining projection patterns in cross-section, intensified whole mounts were embedded in paraffin and sectioned at 15 pm. The dendritic arbors of the filled neurons were then reconstructed with the aid of a drawing tube attached to a compound microscope.

Physiological Techniques

Larvae were anesthetized with carbon dioxide and dis- sected by cutting along the dorsal midline and removing the gut. This exposed the Al ganglion, which was subse- quently isolated from the remainder of the ventral nervous system by cutting the anterior and posterior con- nectives. One of the homeotic legs was selected for study, and the nerves and tracheae on the opposite side of the ganglion were also cut, leaving the ganglion both neu-


Figure 2 Scanning electron micrograph of a (A) metathoracic leg and (B) homeotic leg on the ventral surface of A 1 of an Octopodlarva. Although the homeotic leg is distorted, it has a claw and several large bristle sensilla on its surface, which are characteristic of sensilla located on thoracic legs. Scale bar == 10 pm.

rally and physically connected only to the side of the segment that included the chosen homeotic leg. The homeotic leg and some surrounding body wall was excised from the rest of the animal, bringing with it the A 1 ganglion, attached by its nerves. The ganglion-body wall preparation was placed in a Sylgard ( Dow Corning) lined dish, with the homeotic leg and surrounding body wall facing upward and the ganglion positioned to the side. The ganglion was oriented with either its dorsal or ventral side up, depending on which motor neurons were to be recorded. It was treated with a solution of 0.5 mg/ml collagenase/dispase ( Boehringer Mannheim) for 5 min, bathed in physiological saline (Tnmmer and Weeks, 1989) and desheathed using fine forceps. Intracellular recordings were made from motoneuron cell bodies, using glass microelectrodes filled with 100 m A4 hexammine- cobalt 111 chloride (resistance, 80 to 90 MQ).

To test for connectivity between the motoneurons and sensory hairs, individual or groups of sensory hairs were mechanically stimulated using a hand-held glass probe while recording from the motoneuron. The motoneuron’s response was recorded (Vetter model 420) for subsequent playback in real time using a high-speed chart recorder ( Astro-Med Model MT95000). Follow- ing the characterization of a motoneuron’s response to stimulation of the sensilla, it was stained, by ionto- phoresing cobalt into the cell for 3 to 8 min using 800 ms pulses of 10 nA of positive current at a rate of I Hz. The ganglion was processed as described for whole mount cobalt chloride back-fills. The neuron was identified by comparing the position of its cell body, aborization pattern, and the nerve through which its axon projected with published descriptions ofthe motor neurons ofManducu (Levine and Truman, 1985).

RESULTS

External Morphology of Sensilla Located on A1 of Mutant Animals

The bristle sensilla that cover the general body surface of the three thoracic segments and A 1 range in length from 50 to 200 pm with diameters of 10 to 15 pm at their base. The body surface also possesses a small number of larger filiform sensilla, which we did not examine. The bristle sensilla located on the thoracic leg are much larger than those located on the general body surface, measuring 400 to 600 pm in length and 20 to 30 pm in diameter at their base [Fig. 2( A)]. All of the hairs on the surface of the homeotic legs of Octo animals were morphologi- cally indistinguishable from those found on the thoracic legs [Fig. 2(B)]. We never observed the smaller hairs typical of the general body surface associated with the homeotic legs. The thoracic larval legs normally possess three dome-shaped campaniform sensilla on the intersegmental membrane between the trochanter and the femur. We have not found this class of receptor on the homeotic legs of mutant animals.

In addition to the thoracic legs, several other pattern elements differ between A1 and the thoracic segments in larvae. In wild-type animals, the cuticle of A 1 has characteristic lateral and dorsal pigmented stripes, and like all of the abdominal segments, A I possesses a pair of spiracles that are

Sensory Projections in u Ilomeotic Mutant 285

absent from T2 and T3. None of these pattern elements were transformed in mutant animals.

Responses of Octopod Animals to Sensory Neuron Stimulation

In wild-type animals, stimulation of bristle sensilla located on the ventral surface of the thoracic and abdominal segments elicits several different responses (Levine et al., 1985 ). We were interested in whether the presence of the Octo mutation al- tered the responses elicited with stimulation of the body wall sensilla. Stimulation of the general body wall sensilla on A 1 of mutant animals had the same effect as stimulating sensilla at the same location in wild-type animals. Responses ranged from dim- pling of the cuticle with a weak stimulation to arch- ing the body away from the source of the stimulus as the intensity increased. In a few cases the larvae responded by rapidly whipping their bodies away from the source of a strong stimulus. The range of behaviors exhibited by mutant animals was indistinguishable from that displayed by wild-type animals.

Stimulation of the large bristle sensilla located on the thoracic legs reliably elicits specific reflexive movements of the legs independent of the movement of the body wall (Kent and Levine, 1988). In general, stimulation of sensilla on the lateral leg surface results in the extension of the leg, whereas stimulation of medial sensilla causes the legs to flex inward. Mutant animals with well-developed homeotic legs are capable of moving these legs, using functional homeotic muscles that appear homologous to muscles associated with the thoracic larval legs (Miles and Booker, 1993). We were interested in determining whether stimulation of the sensilla located on the homeotic legs could elicit specific reflexive movements of the homeotic legs similar to those of the thoracic leg. For these experiments, we examined more than 100 Octo animals with well-developed homeotic legs. In general, the responses of the animals to stimulation of the homeotic sensilla were similar to those elicited by stimulation of the general body wall sensilla. Typically, the animals responded to the stimulation of their homeotic sensilla by moving away from the source of the stimulus. There were no obvious differences in either the strength or frequency of the responses elicited by stimulation of the homeotic sensilla compared to those located on the ventral body wall. In most instances the elicited movements of the body wall were not accompanied by movement of the homeotic legs. Only 5% of the mutant ani-

mals examined responded to stimulation of the homeotic sensilla with a reflexive movement of the homeotic leg. When a response was elicited, it was weak and only involved simple bending motions of the coxa and in a few instances flexion of the leg at the femoral-tibia1 joint. The elicited movements of the homeotic leg did not occur independently of the body wall but were always coincident with movements of the ventral body wall. The movements of the homeotic leg elicited by stimulation of the sensilla on the homeotic leg did not differ qualitatively from those elicited by stimulation of sensilla located on the general ventral body surface ofAl.

These observations suggested that the sensory neurons on the homeotic leg formed functional connections within the A1 ganglion. To examine this further, we stimulated a sensory hair or hairs located on the homeotic leg, while recording intra- cellularly from individual motoneurons located in A 1. A sampling of both dorsal and ventral motor neurons revealed that a number of them received input from the sensory neurons associated with the homeotic leg sensilla. These included several previously described neurons with cell bodies located along the dorsal midline of the ganglion (Levine and Truman, 1985). Figure 3 presents some examples of the responses of representative motoneurons to stimulation of the sensilla located on the homeotic leg. In general, the responses of the intersegmental muscles’ motoneurons to stimulation of the homeotic leg sensilla were similar to their responses to stimulation of sensilla located on the ventral body wall (Levine, et al., 1985). Stimula- tion of homeotic leg sensilla led to an inhibition of activity in motoneurons innervating dorsal intersegmental muscles, and increase in the activities of motoneurons innervating ventral intersegmental muscles. For example, motoneuron 6, which in- nervates the dorsally located pleural muscle, received inhibitory input from stimulation of the homeotic sensilla [Fig. 3 (A) ] . In contrast, the motoneurons innervating the ventrally located ventral internal medial (VIM) and ventral internal lateral (VIL) muscles received excitatory inputs [Fig. 3 (B,C)] . Not all ventral muscle motoneurons received excitatory inputs from stimulation of the homeotic leg sensilla. For example, motoneurons innervating the external muscles ventral external oblique (VEO) and ventral lateral external (VLE) were inhibited by stimulating these sensilla. In an earlier study, we described supernumerary muscles that were associated with the homeotic legs of Octo animals (Miles and Booker, I993 ) . These muscles


A

I

BI C

I

A

A A

Figure 3 lntracellular recordings of the responses of three motoneurons to mechanical stimulation of homeotic leg hairs. ( A ) MN-6. Arrowheads indicate time at which several homeotic hairs were deflected. Scale bar = 10 mV. (B) VIM. At the arrowheads a single homeotic leg hair was gently deflected. The heavy bar marks a stroking of the homeotic leg that deflected several hairs. Scale bar = 5 mV. (C) VILl. Arrowheads indicate de- flections of a single homeotic leg sensillum. Scale bar = 10 mV. Time scale bar = 5 s.

were always innervated by the VLE motoneuron which also innervated its normal target. We recorded from the VLE motoneuron in mutant animals and found that it was inhibited by stimulation of either the ventral body wall hairs [Fig. 4( A)] or the homeotic leg hair sensilla [Fig. 4(B)] . Al- though stimulation of a single homeotic hair could produce a response in any of the motoneurons just described, we did not attempt to determine whether the connections between the sensory neuron and the motoneuron was monosynaptic, as has been shown for the sensory and motor neurons of the abdominal prolegs (Weeks and Jacobs, 1987).

Projections of Sensory Neurons Located on A1 of Octo Animals

The results illustrate that the sensory neurons located on the homeotic legs could form functional connections within the CNS. However, the physiological data did not tell us whether the projections of the sensory neurons located on the homeotic legs were transformed. We used cobalt fills to assess the influence of the Octo mutation on the projection

patterns of sensory neurons located on the surface of A 1. Previously, it was shown that there were no segmental differences in the projections of the sensory neurons to the ventral medial neuropil (Levine et al., 1985; Kent and Levine, 1988). However, there were regional differences in the intersegmental projection patterns. Because these studies were carried out primarily on neurons associated with sensilla located on the fourth and fifth abdominal and first thoracic segments, we examined the projection pattern of sensory neurons located on the two most anterior abdominal segments (A1 and A2) and the third thoracic segment (T3) of wild-type larvae. Each of the abdominal segments of the larva consists of a series of eight concentric rings of equal size called annuli. We used these rings to establish the anteroposterior position of a sensory hair along the segment. In the most anterior annuli ( 1 and 2 ) of A2, 96% (22 of 23) of the filled sensory neurons sent anteriorly directed intersegmental projections to A 1 that terminated in the ventral medial region of the A1 neuropil. None of the 29 filled sensory neurons located on the two most posterior annuli of A2 had intersegmental projections. We obtained the opposite result when we examined fills of sensory neurons located on the surface of T3. None of the 16 filled sensory neurons located on the most anterior margin of T 3 had anteriorly directed projections. Of the sensory neurons located on the posterior margin of T3,87% ( l 3 of 15 ) had a posteriorly directed intersegmental projection that terminated in the ventral medial region of the Al neuropil. Our re-

- I 1

"I Figure 4 Intracellular recordings of the responses of the VLE motor neuron to mechanical stimulation of sensory hairs located on the ventral surface of A 1. ( A ) Bar indi- cates the time at which several sensilla located on the ventral medial surface of A 1 were deflected. ( B ) At the arrowhead, a single sensillum located on the coxa of the homeotic leg was deflected. Scale bar = 5 mV. Time scale bar = 5 s.


Table 1 Summary of the Projection Patterns of A1 Sensory Neurons Wild-Type A1 Octopod A I

DorsalILateral Ventral Dorsal/Lateral Ventral Nonleg

Pattern of Projection Anterior Posterior Antenor Posterior Anterior Posterior Anterior Posterior Homeotic Leg

Anterior intersegmental 7/12 017 13/21 0118 18/31 019 11/33 0134 311126 - 0113 - 0118 - 011 I - 2913 I

- 0/18 - O / I I - 3 113 I T3 dorsolateral 017 T3 midline 017 - 0113 A 1 dorsolateral 0112 017 0121 0118 0131 019 0133 0134 111126 Posterior intersegmental 0112 017 0121 0118 0131 019 0133 Of34 11126

sults for A2 and T3 were consistent with those reported earlier for Munduca larvae (Levine et al., 1985; Kent and Levine, 1988) (summarized inFig. 1 ) .

The projection pattern of the sensory neurons located on A1 of wild-type animals differed from those located on the more posterior abdominal segments (Table 1 ). The number of sensory neurons located on the first two annuli of A 1 with anteriorly directed intersegmental projections was intermedi- ate between the patterns observed for anteriorly located sensory neurons of A2 and T3: only 6 1 % (20 of 33) of these anterior sensory neurons had anteriorly directed projections that terminated in T3. The circumferential position (dorsal/lateral versus ventral) of a neuron appeared to play no role in whether an A1 sensory neuron had an intersegmental projection (Table 1 ). The projection patterns of 25 sensory neurons located on the two most posterior annuli of A1 were as described for A2; all lacked intersegmental projections.

An examination of mutant animals revealed that the intersegmental projections of only a subset of the sensory neurons located on the surface of A I in mutant animals were transformed (Table 1 ). Approximately 58% ( 18 of 3 1 ) of the sensory neurons located on the lateral and dorsal surfaces of the two most anterior annuli of mutant larvae possessed anteriorly directed projections that terminated in T3, whereas none of the sensory neurons located on the two most posterior annuli of the lateral and dorsal surface had intersegmental projections, a pattern very similar to that observed in wild-type animals. Of the sensory neurons we filled that were located on the ventral surface of the two most anterior annuli of A 1 in mutant animals, only 33% ( 1 1 of 33) had abdominal-like anteriorly directed projections that terminated in the T3 neuropil.

These results demonstrate that the Octo mutation was capable of transforming the intersegmental projection pattern of a subset of the sensory neu-

rons located on A 1 . The boundary that separated the affected from the unaffected region of the body wall was defined by two features, one external and the other internal. Externally, the boundary coin- cided with the point separating the ventral surface from the lateral surfaces of the larvae. This point on the body surface is easily distinguished because the ventral surface of the segment is flattened rela- tive to the dorsal and lateral surfaces. Internally, the boundary between the affected and unaffected region of the surface of A 1 was correlated with the nerve through which the sensory neuron entered the ganglion. Sensory neurons located on the dorsal and lateral surfaces of A1 enter the ganglion through the more anterior dorsal nerve while the sensory neurons on the ventral surface enter through the more posterior ventral nerve. The effect of the mutation was limited to the sensory neurons that enter the ganglion through the ventral nerve.

It proved difficult to determine the influence of the Octo mutation on the intersegmental projection patterns of sensory neurons located on the homeotic legs. The homeotic legs were often twisted and distorted [Fig. 2 (b ) ] , making it difficult to determine accurately the position of the filled sensory neurons along the anterior to posterior axis of the homeotic leg. For example, 25% (3 I of 126) of the filled sensory neurons associated with the homeotic legs had anteriorly directed projections that terminated in the neuropil of T3, a pattern typical of a sensory neuron located on the anterior surface of A1 (Table 1) . However, many of these anteriorly projecting neurons were located on the posterior surface of the homeotic leg. These results revealed that regardless of their location on the homeotic leg, the sensory neurons could have an intersegmental projection pattern typical of an anteriorly located abdominal sensory neuron. Of the fills of sensory neurons located on the homeotic leg 6% ( 7 of 126) possessed posteriorly directed projections [Fig. 5 (a,d)] ; Table 1 ), a projection pattern that


is characteristic of sensory neurons located on the posterior margin of the thoracic segments. We failed to find sensory neurons located on the two most posterior annuli of A1 in Octo larvae with posteriorly directed intersegmental projections, as would be expected if these annuli were transformed. This could have been a result of our small sample size ( n = 34).

The sensory neurons on the thoracic legs send projections to both the ventral medial region of the neuropil and to a more dorsal and lateral region of the neuropil known as the lateral leg neuropil (Fig. 1 ). We examined the projections of the homeotic leg sensory neurons to determine whether they had projections in the dorsal lateral region of the A1 neuropil (Table I ) . Approximately 9% ( 1 1 of 126) of the filled homeotic leg sensory neurons had such a projection in the dorsal lateral region of the A 1 neuropil [Fig. 5(c-e) and 6(b) ] . The branching observed in the dorsolateral region of A 1 was generally sparse, consisting of one to three short neurites. However, in one case a sensory neuron located on a homeotic leg had eight terminal branches in the lateral region of the A1 neuropil [Fig. 6 (b) ] . We have never observed terminals in the lateral margin of the A 1 neuropil from sensory neurons located on the general body surface of A 1 of wild-type or mutant animals. It appeared that a subset of the sensory neurons located on the homeotic leg were forming projections in a region of the A 1 neuropil that was homologous to the lateral leg neuropil of the thoracic segments. An examination of the shape of the A1 neuropil of mutant animals did not reveal the presence of an outpocketing that would indicate the formation of an extensive homeotic lateral leg neuropil. The occurrence of terminal branches in the dorsal lateral region of the A1 neuropil was independent of the overall segmental identity of the sensory neuron; for example, even sensory neurons with anteriorly directed projections to T3 (suggesting an abdominal segmental identity) had terminal arbors in the lateral margin of the A 1 neuropil.

There were several features of ,the anteriorly directed intersegmental projections of the homeotic leg sensilla that distinguished them from those of sensory neurons located on the general body surface of A1 of either mutant or wild-type animals. Within A l , we observed no difference in the total length of the projections from sensory neurons located on the homeotic legs compared to those on the general body surface (data not shown). How- ever, in T3, the total length of the arbors of the anteriorly directed projections of the sensory neurons

located on the homeotic legs was more than twice that of the sensory neurons located on the general body surface (Figs. 7 and 8 ). The terminal arbors in T3 of the sensory neurons located on the anterior general body surface of A1 of wild-type and mutant larvae typically had only a single primary projection with numerous short secondary branches [Fig. 7 (a)] . All of the anteriorly directed projections to T3 of the sensory neurons located on the homeotic leg had at least two and in most cases three major secondary branches ( n = 31) [Fig. 7 (b ) ] . As a result, the mean total length (k SEM) of the arborization in T3 of the homeotic leg sensory neurons was 753.3 pm & 60 compared to 288.4 pm f 32 and 29 1 pm ? 30.1 for the T3 arbors of sensory neurons located on the general body surface of A 1 of Octo and wild-type animals, respectively. The terminal arbors of the major secondary branches were not randomly distributed through- out the T3 neuropil. More than 90% (29 of 3 1; Ta- ble 1 ) of the homeotic leg sensory neuron projections to T3 had a major secondary branch that terminated in the lateral leg neuropil [Figs. 6 (c ) and 7 (b)] . The terminal arbors of these branches over- lapped with the dorsolateral projections of the sensory neurons located on the thoracic legs. We never observed terminal arbors in the T3 lateral leg neuropil in fills of sensory neurons located on the general body surface of A I of wild-type or mutant animals. The presence of the anomalous projection to the lateral leg neuropil did not account for all of the difference observed in the length of the projections. Excluding the arbors to the lateral leg neuropil, the mean length of the T3 projections of the homeotic leg sensory neurons within the ventral neuropil measured was 66% greater (485.2 k 58.3 pm) than the anteriorly directed projections to T3 of neurons located on the general body surface of A1 . All of the fills from sensory neurons located on the homeotic legs of mutant animals had two major branches in T3, which approached and in many cases crossed the midline of the T3 ganglion in its anterior and posterior ventral commissures, a projection pattern that is characteristic of sensory neurons located on the proximal segments of the thoracic legs.

DISCUSSION

Octopod Mutation Results in the Incomplete Transformation of the A1 Segment The Octo mutation results in the partial transformation of the surface of A1 in the anterior direc-


Figure 5 Camera lucida drawings of cobalt-filled sensory neurons located on the homeotic legs of mutant larvae. A drawing of the larval leg is shown in the center of the figure showing the locations of the filled sensory hairs. In this drawing anterior is to the left. In the ventrome- dial neuropil of the A l ganglion, the projections of all the sensory neurons located on the homeotic leg were indistinguishable from those located on the ventral surface of A l of wild- type animals. However, in other regions of the nervous system the sensory neurons located on the homeotic legs had unique projection patterns. In several instances the sensory neurons located on the homeotic leg had arbors that terminated in the dorsolateral region of the A1 neuropil (C, D, and E, arrowheads). In a few cases the sensory neuron had a posteriorly directed intersegmental projection that terminated in A2 ( A and D). Finally, most of the homeotic leg sensory neurons with an anteriorly directed projection to T3 had an anomalous branch that terminated in the lateral leg neuropil of T3 ( E and F).


Figure 6 The projections of sensory neurons located on the anterior surface of the second segment of the metathoracic leg ( A ) and the projections in A I (B) and T3 (C) of a sensory neuron located on the surface of the homeotic leg of an Octo animal. Below each is a serial reconstruction of the sarne projection from cross-sectioned material. ( A ) Neurons located on the thoracic legs have terminal projections in two regions ofthe ganglion; in the ventral medial region and the more dorsolateral leg neuropil (arrowhead). ( B ) Occasionally, sensory neurons located on the homeotic leg of mutant animals had arbors in a dorsolateral region of A1 (arrowhead), a region that appears equivalent to the lateral leg neuropil ofthe thoracic ganglia. (C) In most cases the anteriorly directed projections of the sensory neurons located on the homeotic leg had a major branch that terminated in the lateral leg neuropil of T3 (arrowhead) projections also crossed the midline of the ganglion through both the anterior and posterior commissures.

tion, leading to the appearance of an extra set of thoracic-like legs on the ventral surface of A 1. In all other respects the A 1 segments o'f mutant animals were normal. The homeotic legs of mutant larvae were often well-formed, including distal leg segments and large bristle mechanosensory hairs, typical of those found on the thoracic legs. The transformation of the external features of these mechanosensory hairs was complete; they were indistinguishable from the sensory hairs found on the thoracic legs. However, none of the homeotic legs possessed the dome-shaped campaniform sensilla normally associated with the thoracic legs. In many respects the phenotype of the Octo mutation is similar to several of the mutations of the E locus of the silkworm Bombyx mori (Tazima, 1964). Many of the E locus mutations alter segmental identity and result in a series of phenotypes similar to mutations within the Bithorax complex (BX-C) of Drosoph- ila (Lewis, 1978 ). The external phenotype of Octo

mutants appears to be most similar to hithoraxoid (bxd), a mutation within the BX-C. Mutations in bxd result in the transformation of the ventral surface of A1 to T3, in some cases resulting in the appearance of an extra set of metathoracic legs. At this point, it has not been determined whether the Octo mutation that lies within Manduca is equivalent to the BX-C. However, it is certain that the key elements found within the BX-C are conserved between flies and moths. This has been confirmed by the isolation of several moth genomic clones that share sequence homology with several of Dro- sophila homeotic genes, including several of those found in the BX-C (Nagy et al, 199 1; Zheng and Booker, 1994).

Octopod Influences the Projection Pattern of the Sensory Neurons The sensory neurons located on the body surface ofManduca exhibit regional differences in their in-

Sensory Projections in u Hovneotic Mutant 291

Figure 7 Drawings of the projections in T3 of two sensory neurons located on the ventral surface of A 1 of an Octopod larva. The projection labeled A was from a neuron located on the anterior ventral body wall of A 1. The projection labeled B was from a sensory neuron located on the homeotic leg. In T3 the projections of the sensory neurons associated with the leg sensilla were much more extensive than those associated with the body wall sensilla.

tersegmental projections (Levine et al., 1985: Kent and Levine, 1988 ). Sensory neurons located on the anterior margin of the abdominal segments have anteriorly directed projections that terminate in the next anterior ganglion. In the thoracic region, it is the sensory neurons located on the posterior margin of the segment that have intersegmental projections that terminate in the next most posterior segment. Like the other abdominal segments, in A1 of wild-type animals only the sensory neurons located on the anterior surface of the segment had intersegmental projections. However, the pro- portion of such neurons was lower in Al, being about 60% of that observed for the more posterior abdominal segments.

Cobalt fills of sensory neurons located on A 1 of mutant animals revealed that the mutation was capable of transforming the intersegmental projection patterns of the sensory neurons. The effect of the mutation was limited to sensory neurons located on the ventral surface. Even on the ventral surface, the influence of the mutation was not uni- form, but exhibited an anterior to posterior gradient of effect along the axis of the segment. In mutant animals the number of sensory neurons located on the anterior ventral surface of A 1 that had the abdominal-like anteriorly directed intersegmental projections to T3 was about half that observed for body wall sensilla of wild-type animals, or the sensilla on the anterior annuli of the lateral and dorsal A 1 body wall of mutant animals (Table

1 ). None of the projections of sensory neurons located on the two most posterior annuli appeared transformed; their projections were typical of sensory neurons located on this part of an abdominal segment. Although the presence of the homeotic legs made it difficult to quantify the effect of the mutation on the intersegmental projections of sensory neurons located on annuli 3 to 6, it was clear that some of the neurons were affected. Fills of the sensory neurons located on the homeotic legs revealed that a small percentage had posteriorly directed intersegmental projections that terminated in A2. We are certain that these sensory neurons were transformed, since in the abdominal segments of wild-type animals there were no sensory neurons associated with the bristle sensilla that had posteriorly directed projections.

The effect of the Octo mutation on the homeotic leg sensory neurons was not limited to their intersegmental projections; they often shared features with sensory neurons located on the thoracic legs. Occasionally, fills of the homeotic leg sensory neurons revealed the presence of terminal arbors in a dorsolateral region of the A 1 neuropil [Fig. 6( B)], an area that appeared homologous to the lateral leg neuropil found in the thoracic ganglia. However, fills ofthe homeotic leg sensory neurons with intersegmental projections were the most revealing. Greater than 90% of the homeotic sensory neurons with a projection to T3 possessed an anomalous branch that terminated in the lateral leg neuropil of T3, a feature that the homeotic leg sensory neurons shared with those located on the thoracic legs. The fact that not all of the anteriorly directed projec-

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Figure 8 Histogram comparing the mean lengths of neurites in T3 of sensory neurons located on the anterior ventral surface of wild-type and mutant moths. The lengths were computed by measuring the arbor from the point it entered the T3 neuropil. n = 6 to 10. Errors rep- resent SEM.


tions of the sensory neurons located on the homeotic leg had terminals in the T3 lateral leg neuropil was not surprising. Most of the sensory neurons we filled were associated with sensilla located on the coxal segment of the homeotic leg. In the thorax, sensory neurons located on the coxal segment occasionally lacked terminals in the lateral leg neuropil (Kent and Levine, 1988 ). A second feature of the sensory neurons located on the homeotic legs also supports the conclusion that they were transformed. Sensory neurons located on the more proximal segments of the thoracic legs have arbors that terminate at the midline of the ganglion (Kent and Levine, 1988), with the projections from the coxal segment actually crossing the midline of the ganglion through one or both of the two ventral commissures. All of the anteriorly directed projections of the sensory neurons located on the homeotic legs had branches that terminated near or past the T3 midline in either one or most often both of the ventral T3 commissures. Thus, once the projections of the homeotic leg sensory neurons reached the T3 neuropil, their projection patterns mimicked those of sensory neurons located on the thoracic legs.

Based on the effect of the Octo mutation on the distribution of postembryonic neuroblasts, we be- lieve that the CNS is only partially transformed (Booker and Truman, 1989). In A1 of mutant animals we often find supernumerary Nbs, which based on a number of criteria, are homologous with those normally restricted to the thoracic ganglia. As a result, the A 1 ganglion of a mutant animal is phenotypically mixed, consisting of both wild-type and mutant tissue. This is similar to what is known about the effect of homeotic mutations on the CNS of Drosophila. The CNS of flies defi- cient for the entire BX-C fail to condense and all of the ganglia share characteristics normally associated with the thoracic ganglia. In flies carrying the bithoruxoid mutation, a supernumerary leg neu- romere often appears in A l , suggesting that the identity of the A1 ganglion is transformed to that ofa thoracic segment (Teugels and Ghysen, 1983). Certain combinations of bithorax mutants can lead to the duplication in T3 of a neural pathway that is normally restricted to T2 (Green, 1981; Schnei- derman et al., 1993). In these BX-C mutants there was also evidence that the morphologies of several of the T3 motoneurons were transformed to resem- ble motoneurons normally found in T2. Given the evidence suggesting that homeotvc mutations can have an effect on the CNS, there is a possibility that the Octo mutation could influence axonaf path-

finding by acting directly on the sensory neurons or by influencing the environment through which the axons grow.

Our analysis suggests that whether the effect of the Octo mutation on the fate of a sensory neuron was direct or indirect depends on which feature of their projection was examined. In the case of intersegmental projections, the evidence suggests that the development of the intersegmental projection is cell autonomous. In mutant animals, all of the sensory neurons located on the anterior surface sent projections to the same region of the ventral neuropil, yet it was only the sensory neurons located on the anterior ventral region of the A1 body wall that were affected by the mutation. It thus seems unlikely that the CNS played a significant role in the establishment of the intersegmental projections. However, since the projections of the sensory neurons located on the lateral and dorsal body wall do not completely overlap with those on the ventral body wall, the possibility that the CNS plays a role in establishing the intersegmental projection cannot be ruled out.

Many of the sensilla located on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons, yet the transformation of the external morphology of the associated sensillum was complete. Thus, the segmental identity of the cuticle secreting cells of the sensillum and the associated sensory neuron did not match. A possible explanation is that the Octo mutation has a differential effect on the segmental identity of the cells that comprise these structures. The cells that secrete the sensillum and the associated sensory neuron are not sisters, but cousins (Lawrence, 1966). It seems reasonable to assume that the different cell types (such as cuticle-secreting cells, sensory neurons) that make up these sensory structures have different thresholds for the cues responsible for establishing their segmental identity. In mutant animals the amount of the signal responsible for transforming segmental identity might be sufficient to transform the cuticle-secreting cells on the homeotic leg of an Octo animal, but still be too low to effect the transformation of the associated sensory neurons.

Although there was a mismatch between the intersegmental projection pattern of many of the homeotic leg sensory neurons and the external morphology of the associated bristle, all of the homeotic leg sensory neurons with an abdominal intersegmental projection pattern established leg- specific projections once they reached the T3 neuropil. If the same cues were responsible for a neu-

Sensory Projections in a Homeofic Mutant 293

ron establishing both intersegmental and leg-specific projections, this result could have been achieved only if the threshold for the elaboration of the leg-specific cues was lower than that of the intersegmental projection. Alternatively, the sensory neurons might use two or more distinct signals in their decision to generate the intersegmental and leg-specific projections. One possible interpreta- tion for this result is that the production of an intersegmental projection is dependent on the segmental identity of the sensory neuron, whereas the development of the leg-specific projections is dependent on positional cues.

In several other insects there is evidence supporting a role for positional information in the establishment of the projection pattern of sensory neurons within the CNS. In Drosophila, the sensory neurons of the thoracic legs generate a somatotopic map within the ventral neuropil (Murphey et al., 1989). Sensory neurons located on the ante- nor surface of the thoracic legs project to the anterior region of the ventral neuropil while the posterior neurons project to a more posterior region. An analysis of the Drosophila mutant Engrailed revealed that compartmental identity did not play a role in determining the projections of leg sensory neurons within ventral neuropil. The results were interpreted as supporting the role of positional information in determining the projection pattern of Drosophila leg sensory neurons. Further evidence supporting a role for positional information in determining the projection pattern of insect sensory neurons has come from a series of heterotopic transplantation experiments (Anderson, 1985; Murphey et al., 1985; Walthall and Murphey, 1986; Kamper and Murphey, 1987). The results of these experiments suggest that during the differen- tiation of the sensilla, positional information pro- vided cues used in determining the projection pattern of the sensory neuron. In the moth, the evidence supporting a role for positional information in the formation of the leg-specific projections is circumstantial. The only neurons in A 1 of mutant animals that developed leg-specific projections were located on the homeotic legs. In wild-type animals, the thoracic legs occupy most of the ventral surface of the segment. In mutant animals the ectopic legs were typically less than half the size of their thoracic homologs. As a result the homeotic legs were often surrounded by untransformed bristle sensilla, which in the thoracic segments would have been part of the thoracic leg. If the presence of the leg-specific projections was only dependent on a sensory neuron’s segmental identity, in mutant

animals we would have expected to find sensory neurons on the general ventral surface of A1 ( nonhomeotic leg) that had established leg-specific projections in T3. In the course of this study more than 200 sensory neurons located on the ventral surface of A1 were filled. The only neurons with leg-specific projections were those located on the homeotic legs. Although this is only indirect evidence, it is consistent with a role for positional cues in the establishment of the leg-specific projections. However, proving this definitively would require a series of transplantation and regeneration experiments.

All of the homeotic leg sensory neurons with intersegmental projections generated leg-specific projections in T3. Unlike the intersegmental projections, the expression of the leg-specific projections was apparently influenced by the segmental identity (abdominal versus thoracic) of the CNS. The probability of finding dorsolateral terminals in the neuropil of T3 was 10 times that of finding such projections in A 1. In addition, more than half of the neurons with terminals in the dorsolateral region of A 1 were found in only two animals. In both cases we had succeeded in filling three of the sensory neurons on the homeotic legs, all of which had at least one small terminal arbor in the dorsolateral region of the A1 neuropil. There are at least two possible explanations for the presence of terminals in the dorsolateral region of the A1 neuropil. The presence of these leglike terminals could reflect particularly strong transformations of the periphery and not a direct effect of the mutation on the A 1 ganglion. If the establishment of the leg-specific projections was primarily influenced by the degree of transformation of the periphery as opposed to the CNS, we would have expected the probability that an individual sensory neuron would establish a dorsolateral projection in both A 1 and T3 would be approximately equal. An alternative explanation is that the presence of the leg-specific projections not only requires that the sensory neuron have a leg identity but also that a signal or signals within the CNS be present to direct the formation of the leg-specific projections. Under normal cir- cumstances, these environmental cues are not found in A1 and only appear under the influence of the Octo mutation. Alternatively, the regional difference in the extent of the leg-specific projections established by the homeotic sensory neurons could result from segmental differences in the abil- ity of the pathfinding axons to respond to the cues that direct the formation of the leg-specific projections. In other words, the lack of terminals in the


dorsolateral region of A I reflects the inability of the homeotic sensory neurons to respond to this cue. Once in T3, the axons became more sensitive to this cue. At this point it is not clear what factors could be used to regulate the responsiveness of the axons to this putative signal.

Sensory Neurons on the Horneotic Leg Form Functional Connections within the CNS

Several homeotic structures in arthropods have been examined with respect to their establishment of appropriate functional connections within the CNS. In the antennapedia mutants of Drosophila all or part of the adult antenna is rleplaced by a thoracic leg (Stocker et al., 1976; Slocker and Law- rence, 198 1 ). The sensory hairs of the homeotic tarsus find their way into the CNS and terminate in a region of the CNS appropriate for antennal fibers, the antennal lobes. These homeotic tarsal sensory neurons form functional connections within the CNS and mediate a reflex response typical of the stimulation of leg tarsal sensory neurons (Deak, 1976). Although this observation is interesting, at this point the pathway that mediates this behavior has not been described. Another method for generating homeotically transformed appendages is through regeneration following amputation. In the stick insect, Carausius moroszis, removal of the distal antennal segments of hatchlings often results in the formation of an antennapedia regenerate (Edwards et al., 1989). The majority of the sensory neurons of the antennapedia tarsus project to the olfactory lobe as in the normal antenna1 nerve. Un- like the normal projection, the antennapedia regenerate does not give rise to the compact olfactory glomeruli typical of the olfactory lobe. There is also evidence that in certain decapod Crustacea, the removal of the eyestalk and the optic lobes results in its replacement by an antennule (Maynard, 1965; Maynard and Cohen, 1965). The antennule, which forms when the eyestalk is severed, makes functional connections within the CNS that appear similar to those elicited by the normal antennules. Using the freshwater crayfish Cherax destructor, Sandeman and Luff ( 1974) found that stimulation of regenerate homeotic antennules elicited behavioral responses that were similar in character to those elicited by stimulation of the normal antennule, not the eyestalk.

Occasionally, we had observed that mutant animals with well-formed homeotic legs were capable of moving them. Thus, it was reasonable to expect

that the homeotic legs might be capable of respond- ing to stimulation of the homeotic sensilla with reflexive movements similar to those of the thoracic legs. We examined this possibility using two different approaches: We used electrophysiological techniques to determine whether the response of the A 1 motor neurons to stimulation of the homeotic sensilla differed from their response to stimulation of the sensilla on the general body wall of A 1. The results for the transformed sensilla on the homeotic leg were essentially the same as those for the sensilla located on the ventral body wall. The motoneurons we tested which innervated ventral intersegmental muscles received excitatory input, and the motoneuron innervating a dorsal lateral muscle received inhibitory inputs from the sensory neurons located on the homeotic legs.

We also analyzed the responses of the homeotic legs to the stimulation of the transformed bristle sensilla. Mechanical stimulation of the transformed sensilla on the homeotic legs of mutant animals occasionally elicited weak reflexive movements of the homeotic legs. In an earlier study (Miles and Booker, 1993), we found that the movements of the homeotic legs were produced by 1 to 4 supernumerary muscles associated with the proximal segments of the homeotic leg. Based on their attachment points, the supernumerary muscles appeared to be homologous to muscles associated with the proximal segments of the thoracic legs. In all the mutants we examined, the VLE motoneuron innervated both its normal target, a ventral body wall muscle, and all of the extra muscles associated with the homeotic legs. Thus, all of the homeotic muscles along with the normal target of the motor neuron formed a motor unit. As a result, the reflexive movements of the homeotic legs par- alleled the movements of the posterior ventral body wall, not the thoracic legs. The VLE motoneuron’s response to stimulation of the homeotic sensilla was indistinguishable from its response to stimulation of ventral body wall sensilla, consisting of a weak inhibition (Fig. 4). This weak inhibitory response to homeotic and ventral body wall sensilla is also shown by at least one other ventrally located external muscle motoneuron, the VEO (C. Miles: personal observation). There was no indication that the response of VLE or any of the other A1 motoneurons to the stimulation of the homeotic sensilla was transformed in any way. Although the VLE motoneuron in mutant animals was capable of innervating both its normal targets and the homeotic muscles, there was no other indication that its developmental fate had been transformed, such

Sensory Projections in u Homeotic Mutunt 295

as the presence of terminal arbors in the dorsolat- era1 region of the A 1 neuropil (Miles and Booker, 1993). Based on both the projection patterns ofthe homeotic sensory neurons and the elicited reflexive movements of the homeotic legs, one could argue that the sensory neurons of the homeotic leg make functional connections within the CNS that are appropriate for a leg. However, in Octo animals, the reflex movements of the homeotic leg appear to be mediated through a unique pathway involving a set of homeotic muscles that are innervated by a single body wall muscle motoneuron. The homeotic muscles appeared to respond to stimulation of the homeotic sensilla as body wall muscles, not leg muscles. This suggests that in the search for func- tionally appropriate connections by homeotic sensory neurons, it is not always sufficient to conduct behavioral and anatomical studies; physiological studies may also be required.

We thank Tatyana Fairietta for technical assistance during phases of this study and Raymond Vazquez for providing the scanning electron micrographs. This study was supported by a grant from the National Science Foundation (BNS-8910275).

REFERENCES

ANDERSON, H. (1985). The development of sensory neurons and connections from transplanted locust sensory neurons. J. Emhrvol. Exp. Morphol. 85207- 224.

BACON, J. P. and ALTMAN, J. S. ( 1977). A silver inten- sification method cobalt-filled neurons in whole mount preparations. Bruin Rex 138:359-363.

BELL, R. A. and JOACHIM F. A. ( 1978). Techniques for rearing laboratory colonies of the tobacco hornworm and pink bollworms. Ann. Ent. Soc. Am. 69:365-373.

BOOKER R. and TRUMAN J. W. (1989). Octopod, a homeotic mutation of the moth Mundzicu sexta, influences the fate of identifiable pattern elements within the CNS. Development 10562 1-628,

DEAK, 1.1. ( 1976). Demonstration ofsensory neurons in the ectopic cuticle of spineless-aristapedia, a homeotic mutant of Drosophilu. Nature 260:252-254.

EDWARDS, J. S., REDDY G. R., and RANI M. U. ( 1989). Central projections of a homeotic regenerate antennapedia in a stick insect curaiisius-morosus. J. Neuro- bid. 2O:lOl-114.

GHYSEN, A., JANSON, R., and SANTAMARIA, P. (1983). Segmental determination of sensory neurons in Dro- sophiiu. Dev. Bio2. 99:7-26.

GREEN, S. H. (1981). Segment-specific organization of leg motorneurons is transformed in bithorax mutants of Drosophilu. Nature 292:152- 154.

KAMPER, G., and MURPHEY R. K. ( 1987). Synapse formation by sensory neurons after cross-species transplantation in crickets: the role of positional information. Dev. Biol. 122:492-502.

KENT, K. S., and LEVINE R. B. ( 1988). Neural control of leg movements in a metamorphic insect: sensory and motor elements ofthe larval thoracic legs in Munducu sextu. J. Comp. Neurol. 271559-576.

LAWRENCE, P. A. ( 1966). Development and determination of hairs and bristles in the milkweed bug, Onco- peltus ,fusciutw (Lygaeidea, Hemiptera). J. Cell Sci.

LEVINE, R. B. and TRUMAN, J. W. (1985). Dendritic reorganization of abdominal motor neurons during metamorphosis of the moth Munducu sextu. J. Neu- rosci. 52424-243 1.

LEVINE, R. B., PAK, C., and LINN, D. ( 1985). The structure function and metamorphic reorganization of so- matotopically projecting sensory neurons in Munducu se,vtu larvae. J. Cornp. Physiol. (A) 157:l-13.

LEWIS E. B. ( 1978). A gene complex controlling segmen- tation in Drosophilu. Nature 276565-570.

MAYNARD, D. M. ( 1965). The occurrence and functional characteristics of heteromorph antennules in an experimental population of the spiny lobsters, Punuli- rus urgus. J . Exp. Biol. 43:79-106.

MAYNARD, D. M. and COHEN, M. J. ( 1965). The function of a heteromorph antennule in a spiny lobster, Punuliviis argzu. J . Exp. Biol. 4355-78.

MILES, C. I. and BOOKER, R. ( 1993). Octopod, a homeotic mutation of the moth, Munduca sexla affects development of both mesodermal and ectodermal structures. Dev. Biol. 155: 147- 160.

MURPHEY, R. K. ( 1986). Competition and the dynam- ics of axon arbor growth in the cricket. J. Comp. Neu- rol. 251:lOO-1 10.

MURPHEY, R. K., BACON, J. P., and JOHNSON, S. E. (1985). Ectopic neurons and the organization of insect sensory systems. J. Comp. Physiol. (A) 156:381- 389.

MURPHEY, R. K., POSSIDENTE, D. R., VANDERVORST, P., and GHYSEN, A. (1989). Compartments and the topography of leg afferents in Drosophila. J. Neurosci.

NAGY, L., BOOKER, R., and L. M. RIDDIFORD ( 199 1 ). The isolation and characterization of a homeotic gene from the moth Munducu sexta. Development 112:

SANDEMAN, D. C. and LUFF, S. E. ( 1974). Regeneration of the antennules in the Australian freshwater crayfish, Cherux destructor. J. Neurobiol. 5475-488.

SCHNEIDERMAN, A. M., TAO, M. L., and WYMAN, R. J. ( 1993). Duplication of the escape-response neural pathway by mutation of the bithorax-complex. Dev. Biol. 157:45 5-473.

STOCKER, R. F. and LAWRENCE, P. A. (1981). Sen- sory projections from normal and homeotically

1~475-483.

9:3209-32 17.

119-129.


transformed antennae in Drosophila. Dev. Biol. 82:

STOCKER, R. F., EDWARDS, J. S., PALKA, J., and SCHUB- IGER, G. ( 1976). Projection of sensory neurons from a homeotic mutant appendage, Antennapedia, in Dro- sophila melunoguster. Dev. Biol. 5 2 ~ 2 10-220.

TAZIMA, Y. ( 1964). The Genetics of the Silkworm. Aca- demic Press, New York.

TEUCELS E. and GHYSEN, A. (1983). Independence of the numbers of legs and leg ganglia in Drosopliila bith- orux mutants. Nature 304:203-207.

TRIMMER, B. A. and WEEKS, J. C. ( 1989). Effects of nic- otinic and muscannic agents on an identified motor-

224-237. neurone and its direct afferent inputs in larval Man- duca sexta. J . Exp. Biol. 144~303-337.

WALTHALL, W. W. and MURPHEY, R. K. (1986). Posi- tional information, compartments and the cercal sensory systems of crickets. DPV. Biol. 113: 182-200.

WEEKS, J. C., and JACOBS, G. A. ( 1987). A reflex behavior mediated by monosynaptic connections between hair afferents motorneurons in the larval tobacco hornworm, Mandzica sexta. J. Comp. Physiol. ( A )

ZHENG, Z. and BOOKER, R. ( 1994). Postembryonic expression of homeobox genes in the moth, Manduca sexta. Soc. Neurosci. Ahstr. 20:46 1.

160:3 15-329.

projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the...

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