Segmental determination of sensory neurons in Drosophila

DEVELOPMENTAL

BIOLOGY

99,7-26

Segmental

(1983)

Determination ALAIN

Laborattire

de Gkitique, *Centre

of Sensory Neurons in Drosophila

GHYSEN,RENAUDJANSON,ANDPEDRO University libre de Gbnnitique

Received

October

de Bruxelks, Molkulaire,

SANTAMARIA*

67, rue des Chevaux, CNRS, 91190 Gif-sur-

26, 1982; accepted

in revised

fwm

Rhode St Gem&se, Belgium; Yvette, France

1640

March

21,

and

1985

The analysis of flies where one segment is transformed into another by mutation or by experimental treatment shows that the central projection of a sensory neuron depends on its segmental determination. The genetic functions that control the segmental determination of neurons and of epidermal cells appear to be distinct, but they have common regulatory features.

anterior segment. Such mutations have been called homoeotic (Bateson, 1894). These results all derive from observations on the cutitular morphology of mutant or experimental flies, and therefore they bear only on the development of the epidermis. Nevertheless it might be that similar principles are at work for the development of other tissues as well, and in particular for the development of the nervous system. It has been reported that the central projections of segmentally homologous neurons are substantially similar yet show clear-cut differences (Ghysen, 1978), indicating that some sort of segmental determination might be involved in the establishment of appropriate projections. Since sensory neurons and epidermal cells share a common lineage during most of the larval life (Wigglesworth, 1953; Garcia-Bellido and Merriam, 1968), it might have been expected that genetic manipulations known to cause segmental transformations of the epidermis would induce similar transformations in the sensory neurons. This, however, proved not to be the case in the one system analyzed thus far (Palka and Schubiger, 1980). In the present paper, we analyze the effect of genetic or experimental interference with the normal process of segmental determination in three different systems of homologous neurons. We conclude that the central projection of a sensory neuron depends on its segmental determination, and that the elements involved in the control of this segmental determination are similar, but not identical, to those involved in the segmental determination of epidermal cells.

INTRODUCTION

Segmentation is a common feature of all higher organisms. The segmental organization is particularly conspicuous in the epidermis and nervous system of arthropods. Its developmental significance, however, remained much less obvious until recently. Thanks largely to the pioneering work of Lewis and Garcia-Bellido, a coherent picture of the mechanism and function of segmentation is now emerging in the case of the epidermis of Drosophila (Morata and Lawrence, 1977). It appears that a set of instructions is available in the genome of the fruitfly that leads to the development of a “basic” (or “archetypic”) body segment (Garcia-Bellido, 1977). This set of instructions is fully expressed in the mesothoracic region. Another set of genes is responsible for various modifications of this basic program, leading to the morphological differences between the different thoracic and abdominal segments posterior to the mesothorax (Lewis, 1963). The genes of this second set are clustered in a single region of the genome, the bithorax complex (BX-C) (Lewis, 1978). The bithorax complex itself appears to be regulated by other genes, some of which apparently act as repressors (Lewis, 1978; Struhl, 1981) and others as activators (Garcia-Bellido and Capdevila, 1979; Ingham and Whittle, 1980). As a result of the interaction of these regulatory genes, early during embryogenesis, the different genes of the BX-C would be sequentially activated in the segments posterior to the mesothorax, and would lead to the successive modifications of the basic program into a metathoracic variant (reduction of the wings to halteres and near suppression of the notum), to a first abdominal variant (suppression of wings and legs) and so on. When one of those genes is inactivated by mutation, the corresponding modification does not take place and the relevant segment is morphologically “transformed” into a more

MATERIAL

AND METHODS

Fly strains. Canton S was used as wild type. All mutant strains were obtained from E. B. Lewis except for trx, obtained from P. Ingham. Ant#, Ns, and wg were obtained from I. Deak. The mutations abx, ti, and ti 7 0012-1606183 Capyriyht Al1 rlyhts

$3.00 R: 1983 by Academic Press. Inc. of wproduciinn in any form rtwxwd.

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DEVELOPMENTALBIOLOGY VOLUME99,1983

pbx were used as homozygous or were made hemizygous with the deficiencies Df(3)P9, Df(3)P2, or Df (3)bxd’? The mutation trx was used as homozygous or as hemizygous with the deficiency Df(3)redPs2. Methods. Peroxidase backfill (Ghysen, 1978) and ether phenocopying (Santamaria, 1979) were done as described previously. Misroutings. Misroutings of haltere fibers through the wing nerve were obtained either surgically (Ghysen and Deak, 1978) or by using the mutation wg. This mutation replaces the wing and haltere with a duplicated mesoand metanotum, respectively (Sharma and Chopra, 1976). The penetrance of this phenotype is about 50%; in those cases where the haltere develops normally, it is found that the haltere nerve is frequently misrouted and joins a dorsal mesothoracic nerve (Palka and Shubiger, 1980; Ghysen and Janson, 1980b). The same effect is found when the haltere is transformed to a homoeotic wing by the ti pbx mutation. Figures. Photographs have been used whenever possible. However, the three-dimensional complexity of many projections made it necessary to resort to camera lucida drawings in a number of cases. Furthermore in mutant flies the transformed appendage is often filled with hemolymph. This usually results in a substantial background staining in the ganglion, low enough to allow the tracing of backfilled axons but too high for photographic purposes. Photographs were made with a 16X Zeiss objective and camera lucida drawings were made with 50X or 100X Wild objectives. Nomenclature. Since many of the experiments involve nerve misroutings or segmental transformations, it was necessary to devise a set of abbreviations to deal separately with the different aspects of segmentation. We call the three thoracic segments Tl, T2, and T3, irrespective of how they differentiate: thus these abbreviations refer to the position of the segments along the body axis of 14 segments. Likewise the corresponding segmental nerves will be called NTl, NT2, and NT3, and the roots of these nerves, i.e., the sites where they enter the ganglion, RTl, RT2, and RT3. On the other hand we call mesothoracic (MS) and metathoracic (MT) the pattern of differentiation that is normally observed in T2 and T3, respectively. Thus in normal adult flies T3 differentiates as MT, and the T3 sensory axons follow NT3 to enter the ganglion at RT3. Misroutings can cause T3 axons to enter the ganglion at RT2, while homoeotic mutations can cause T3 cells to differentiate as MS. RESULTS 1. Thwack

Projections

(a) Sensory projections in wild type. We have analyzed the projections from four sets of sensory neurons present

on the wing and the haltere. The first two sets innervate campaniform sensilla, a family of dome-shaped sensory structures which are believed to act as strain receptors. About 40 sensilla of slightly varying shapes are found on the base of the wing, while about 150 others are tightly clustered in rows on the stalk of the haltere. All sensilla on the base of the wing seem to share the same projection, in spite of the fact that they can be divided into subgroups on the basis of their pattern and detailed morphology, and therefore we will refer to them collectively as wing proximal campaniform sensilla (pcs). The same is true for the sensilla on the stalk of the haltere, which we will call haltere PCS. The base of the wing and the stalk of the haltere are developmentally homologous (Morata, 1975) and therefore wing and haltere pcs are usually considered homologous even though they differ in number, size, and pattern (see Discussion). The central projections from wing and haltere pcs have been described previously (Ghysen, 1978) (Figs. la and b). Interestingly, the wing and haltere axons follow the same longitudinal pathway in the dorsal layer of the fused thoracic-abdominal ganglion. Besides this striking similarity, two clear-cut differences can be observed between wing and haltere projections: the former extends further into the subesophageal ganglia (Figs. la, b and 2a, b) and presents a medial branch in the thoracic ganglion (arrows in Fig. la). Other differences may be noted (Palka et al, 1979) but are less reproducible or less easily scored than the above-mentioned two. The longitudinal and the median bundles do not correspond to different subsets of wing sensilla, since backfilling of single sensilla showed axons branching to follow both paths (Ghysen, 1980). Furthermore, the size of the median and longitudinal bundles appears very similar, indicating that most if not all wing pcs share a common pattern of projection. The other two sets of sensory structures that we analyzed are setae, or sensory hairs, which act as tactile receptors. About 500 setae of varying shapes are arranged in rows along the anterior margin of the wing, while only about 15 tiny setae are found on the haltere knob. The wing setae can be subdivided according to their shape and pattern, yet we will treat them collectively as they share a common pattern of projection. The developmental homology between wing blade and haltere knob indicates that wing and haltere setae may be considered homologous in spite of their differences in number, size, and pattern (see Discussion). The projections from wing and haltere setae clearly resemble each other, in that both are local: they extend ventrally and remain confined to a small area limited by the adjacent leg neuromeres (Ghysen, 1978; Figs. 3a and 4a, b). However, there is one marked difference between wing and haltere projections: a few of the hal-

GHYSEN,

a

JANSON,

b

AND SANTAMARIA

C

Segmental

Determination

d

of Neurons

9

e

FIG. 1. Projections in the thoracic and suboesophageal ganglia from the proximal campaniform sensilla in a wild type wing (a) and haltere (b), in a haltere partly transformed to wing in tm homozygous (c) and hemizygous (d) flies, in a haltere completely transformed to wing in a W $rx homozygous fly (e) and in a zug; W &x homozygous fly where the nerve from the transformed haltere was misrouted and joined the ganglion at the place appropriate for the normal wing nerve (f). The projection of misrouted haltere pcs in a t%r+&r+ fly is indistinguishable from that shown in (f), whether the misrouting is obtained by surgery or by using the mutation wg. The projections were marked by peroxidase backfill; the variably wavy aspect of the bundle is due in part to distortion of the ganglion and in part to some effect of the peroxidase itself (Strausfeld and Ghysen, unpublished). The suboesophageal ganglion (at the top of the drawings) and the fused thoracio-abdominal ganglia are outlined in each case. Arrows indicate the features of the normal wing projection that differentiate it from the normal haltere projection.

tere axons follow a very ventral longitudinal course, which wing axons never do. The observation that not all haltere fibers follow this longitudinal path may indicate that there are subgroups among the haltere setae; alternatively it may be due to incomplete staining. These axons are difficult to stain properly (Ghysen, 1978) and their projection was completely overlooked in early cobalt backfills (Palka et aZ., 1979). This projection is consistently observed after cobalt intensification (Strausfeld and Singh, 1980) or using a high concentration of peroxidase, but even so it remains possible that some very fine fibers escape detection. The haltere fibers that follow the longitudinal pathway anteriorly ramify in the region between pro- and mesothoracic leg neuromeres where the wing setae project (Figs. 4a, b); furthermore these fibers often send branches along another more medial and ventral path (arrow in Fig. 4b). We tentatively identify both the local and the longitudinal components as parts of the haltere setae projection because they are observed when peroxidase is applied to a cut in the haltere capitellum, and the setae are the only sensory structures known to be present on that part of the haltere. Proof of this identification, as well as evidence as to whether the longi-

tudinal projection corresponds to a specific subgroup of haltere setae, would require single setae backfills, which has not been achieved yet. (b) Sensory projections after nerve misrouting. It is conceivable that the differences between the projections of homologous wing and haltere neurons are a simple consequence of the fact that wing and haltere axons travel along different segmental nerves and hence enter the ganglion at different sites (Figs. la, b). For example, in the case of the PCS, the haltere axons might simply miss the branching point to the medial branch, which is slightly lateral to their main pathway. We have examined this possibility by misrouting the haltere nerve so that it now joins the ganglion at the same site as the wing nerve. To clarify the description of the following experiments, we will call the second and third thoracic nerves NT2 and NT3, and their root or site of entry into the ganglion RT2 and RT3, respectively (see Nomenclature under Material and Methods). Thus in normal flies, wing axons follow NT2 and enter the ganglion at RT2 while haltere axons follow NT3 and enter at RT3. Two techniques can be used to misroute haltere fibers so that they now enter the ganglion at RT2 rather than at RT3. The first is to

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FIG. 2. Projections in the suboesophageal ganglion of the proximal campaniform sensilla of a normal wing (a) and haltere (b), of a partly transformed haltere in a tra: homozygous fly (c), and of the slightly enlarged haltere in U!m’“/+ (d), Df(S)bxd’w/+ (e) and Df(S)PS/+ (f) flies. In all cases the suboesophageal ganglion and cervical connective are outlined by a dashed line, the oesophageal canal is marked by an asterisk. Arrows show abnormal (wing-like?) fibers.

perform surgical operations aimed at breaking the segmental nerve NT3 connecting the haltere disc to the ganglion in mature larvae (Ghysen and Deak, 1978). The second is to use the mutation, wingless, which by itself has no effect on the central projections analyzed here. In homozygous wg flies, the nerve from the haltere is occasionally misrouted to RT2 (Ghysen and Janson, 1980b). The two methods gave identical results. In the case of pcs neurons, the projection established by misrouted haltere axons still lacks a medial branch and stops shortly after entering the subesophageal ganglion

(Fig. If), i.e., they retain their haltere properties in spite of the fact that they now enter the ganglion at the place appropriate for wing axons. The projection from haltere setae is shown in Fig. 4c in the case of a fly where the misrouting was induced by larval surgery. Again the misrouted fibers retain a typical haltere behavior in that in addition to the local component, now extending ventral to RT2, one or two fibers extend longitudinally along the meso- and metathoracic leg neuromeres. Furthermore another fiber, or branch, extends more medially and ventrally (arrow in Fig. 4~). Both longitudinal

a

FIG. 3. Projections from wing setae: normal wing setae in a wild type fly (a), wing-like setae on the transformed haltere in &.z/Df(s)P.2 (b), in b3c9/&zs (c) and in b2/Dj(8)PZ (d) flies. In all preparations only the setae axons were backfilled. The arrow indicates the ventralmost of the two anterior tracts typical of haltere setae projections (see text). The ganglia are outlined by a dashed line; MS, MT, P: meso-, metaand prothoracic leg neuromeres. 11

DEVELOPMENTALBIOLOGY VOLUME 99, 1983

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,a

b

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FIG.4. Projections from normal haltere setae: normal projection (a, b) and projection in a fly where the haltere fibers were misrouted and entered the ganglion at the place appropriate for normal wing fibers (c). Arrow and abbreviations as in Fig. 3.

paths appear identical to those followed by normal haltere fibers (Figs. 4a, b). These experiments indicate that the differences between wing and haltere projections do not depend on the site of entry of the fibers into the ganglion. Furthermore they allow us to analyze the specificity of these projections. In the case of the PCS, it appears that the axons recognize and follow a unique central pathway irrespective of where they reach it, a behavior typical of fibers that extend over long distances within the central nervous system (Ghysen, 1978). The same is true for the few fibers from the haltere setae that extend longitudinally. On the other hand, the local component of the haltere setae projection may form indifferently between the pro- and mesothoracic leg neuromeres when the axons enter the ganglion through RT2, or between the meso- and metathoracic leg neuromeres when they enter through RT3. This suggests that these fibers extend ventrally from wherever they enter the ganglion, rather than seeking and following a unique pathway. A similar behavior has been reported for other locally projecting fibers, the axons from leg chemoreceptor neurons (Teugels and Ghysen, 1983). The wing setae projection resembles very much the local component of the haltere projection, and we expect it also to extend ventrally from wherever the fibers enter the ganglion. Unfortunately, this could not be demonstrated because it has not been possible so far to misroute wing fibers through RT3. This may well be because two larval nerves make contact with the wing disc (Reinhardt et al, 1977) so

that it is very unlikely that both connections can be broken simultaneously either by mutation or by surgery. (c) Projections in flies with altered segmental determination. Since the specific features of the haltere projections remain unaffected by nerve misroutings, it may be that they depend on the segmental determination of haltere neurons. If this is so, one should observe transformations of the projections when the process of segmental determination is altered. Before describing the experiments designed to test this hypothesis, we will define the abbreviations we will use in the following section (see Nomenclature under Materials and Methods). Any structure on the second or third thoracic segments will be referred to as T2 or T3, respectively. This expresses the origin of the structure along the axis of the 14 body segments, irrespective of how they differentiate. On the other hand, we will call mesothoracic (MS) and metathoracic (MT) the programs of differentiation normally observed in T2 and T3. Thus the wing is a MS structure which normally develops on T2; the haltere is the homologous MT structure that develops on T3. The segmental determination is the process that leads T3 cells to differentiate as MT, this process depends on the expression of the bithorax complex (BX-C). Altering this process may lead T3 cells to differentiate as MS rather than as MT, for example, as wing rather than haltere. We used two methods to interfere with the expression of the BX-C, and hence with the process of segmental

GHYSEN, JANSON, AND SANTAMARIA

determination. The gene “Regulator of bithwrax” (Rg!z), located outside the BX-C, is apparently required for the expression of the BX-C functions (Capdevila and Garcia-Bellido, 1981). Rg-bx mutations are homozygous lethals; however, a leaky allele named ttithoraz (trx) has been described (Ingham and Whittle, 1980). Flies homozygous for trx show patches of MS tissue in Tl and T3. Another means to alter the segmental determination is to treat early embryos with ether (Gloor, 1947). Such treatment also results in patches of T3 cells differentiating as MS. A detailed analysis of this phenomenon (Capdevila and Garcia-Bellido, 1974,1978) has led to the conclusion that ether acts by interfering with the action of Rg-bx+ (Capdevila and Garcia-Bellido, 1981), so that the BX-C locus is not properly expressed in some T3 cells. We have examined the projection of the pcs and setae in ether-treated flies where variable parts of the T3 appendage differentiate as MS, i.e., where variable parts of the haltere were transformed to wing. Two extreme results are presented in Fig. 5. In one case (Fig. 5a) all sensilla distal to the cut belong to the transformed patch and are arranged in a pattern typical of wing PCS; the analysis of the corresponding projection (Fig. 5~) shows that most or all pcs fibers display a behavior typical of normal wing fibers. In the other case (Fig. 5b) more than 100 sensilla belong to the normal haltere tissue and only 5 are found within the wing patch. In the resulting projection (Fig. 5e), many fibers follow the normal haltere pattern and only one shows the features typical of normal wing axons. In only three ether-treated flies was the projection from the T3 setae clearly resolved; in all three flies the projection was wing like in that it remained local, with no fiber following the longitudinal path typically observed in normal haltere setae projections (Figs. 5d and f; the two longitudinal fibers in f most likely come from normal haltere setae). We have also analyzed the projection of the pcs and setae in trx flies. Out of 40 homozygous or hemizygous mutants that showed a patch of wing-like tissue in the haltere (Fig. 6), 16 gave a projection consisting of a mixed population of wing-like and haltere-like fibers, as shown in Figs. lc and d, and 2~. For technical reasons, it was not possible to determine whether the presence and number of wing-like fibers were correlated to the number of pcs in the patch of wing-like tissue. This correlation, however, could be examined in the case of the setae. This is because patches of wing-like setae are occasionally found at the tip of long stripes of winglike tissue, so that their axons can be specifically backfilled. We examined the projection from the wing-like setae in 10 such appendages. In three of the cases, the projection was wing like (i.e., purely local) while in the other seven cases the projection showed a prominent

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longitudinal component, very similar to but much larger than the normal haltere component. These results further support the conclusion that the difference between wing and haltere projections is unrelated to the site of entry of the fibers in the ganglion. They also show that treatments known to alter the segmental determination of T3 cells may transform the projections of T3 sensory neurons, so that they show features typical of normal wing neurons (the fact that in trx flies, the neurons of transformed halteres may either establish a wing-like projection or retain a haltere-like one will be discussed later). Thus it would seem that the establishment of a unique projection is part of the program of differentiation of a neuron, and that it depends on the segmental determination of that neuron. We will call MS and MT the projection normally observed for T2 (wing) and T3 (haltere) sensory neurons. Misrouting T3 fibers through RT2 does not change their pattern of projection, which remains MT; on the other hand interfering with the expression of the BX-C may cause T3 neurons to differentiate as MS (appearance of wing-like projections) much as it causes T3 epidermal cells to differentiate as MS (appearance of wing-like epidermis). (d) Projections in mutants of the BX-C: ti. We have analyzed two mutations of the BX-C that make anterior T3 cells differentiate as MS rather than as MT. These two mutations, bithorati (ti) and anterobithorax (abx), have a very similar phenotype: both extensively transform the anterior haltere into an anterior wing (Figs. 7b, c). In hemizygous W/Df(3)PZ and abx/Df (3)PZ flies, the transformation appears complete as far as the setae are concerned, and most or all pcs are also clearly transformed as judged by their pattern and shape (Figs. 8b, c). A detailed examination of the morphology of T3 pcs in bti and abx flies revealed that all but a small subset of them differentiate as MS in ti, while the transformation is more variable in abx flies (Cole and Palka, 1980). Unfortunately this analysis was carried out on homozygous rather than on hemizygous mutants. The projection from the T3 pcs was analyzed in homoand hemizygous ti mutants, or in the double mutant bti pbx where the entire T3 segment differentiates as MS. A typical result is presented in Fig. le: the projection has remained MT, in that it clearly lacks all MS features characteristic of normal wing fibers. In none of 90 projections could MS features be observed, except for a slight departure from the MT pattern in the brain projection of hemizygous mutants (see below). The projection from the T3 setae is shown in Figs. 3c, d: it too has remained typically MT in that it shows a large bundle of fibers along the longitudinal pathway characteristic of the normal haltere setae projection. More specifically two anterior bundles are observed, one more medial and

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FIG. 5. Projections from partly transformed halteres in ether-treated flies. (a) and (b) represent the cut appendages. Each appendage gave three projections: a dorsal one (c and e) comprising the fibers from the proximal sensilla, a middle one (not shown) comprising the fibers from the distal sensilla, and a ventral one (d and f) comprising the fibers from the setae. The projection from the distal sensilla (not shown) mimicks that of normal wing distal sensilla, as expected (see Discussion and Ghysen, 1978). Appendage (a) bears many wing-like proximal sensilla and few wing-like setae. The projection from the former (c) is essentially wing-like while the projection from the latter (d) is very reduced. On the contrary, appendage (b) bears mostly haltere-like proximal sensilla and many wing-like setae; the projection from the former (e) is mostly haltere-like while the projection from the latter (f) is massively local, i.e. wing-like. The 1-2 haltere-like fibers in (f) probably arose from setae in the haltere territory, compare with Fig. 4a. In (a) and (b), the boundary between wing and haltere (labeled H) territories is marked by a dashed line. The thin arrows show clusters of wing-like proximal sensilla, the thick arrows show haltere-like proximal sensilla. MS, MT, P: meso-, meta- and prothoracic leg neuromeres.

ventral than the other (arrows in Figs. 3c, d); both ramify in the region where normal wing fibers project. These two paths match exactly those observed in the normal haltere projection (Figs. 4a, b). In addition one bundle travels posteriorly, again matching the normal haltere projection. These results indicate that in ti flies, T3 neurons behave as MT even though the surrounding epidermis and the sensory structures themselves are clearly transformed to MS.

(e) Projections of mtirouted axons in bx? The contrasting behavior of T3 epidermis and sensory structures, which become MS in ba?, and of the sensory neurons, which apparently remain MT in the same flies, prompted us to examine the behavior of misrouted T3 axons. Misroutings were obtained by using homozygous wg flies, where fibers from T3 sensory neurons are occasionally misrauted through RT2. The same effect is observed when the haltere is transformed to wing in ti

GHYSEN,

JANSON,

AND SANTAMARIA

or b$ p&x homozygous or hemizygous flies. Eleven hemizygous ti flies (wg/wg; bxs/Df(3)P2) that showed a misrouting of T3 fibers through RT2 were analyzed. In all of them, the T3 pcs axons still behave as MT (Fig. If) in spite of the fact that they come from morphologically MS sensilla and enter the ganglion at the site appropriate for MS axons. Likewise in all cases the projection from the T3 setae was clearly different from the MS pattern observed in the case of normal T2 setae, and very reminiscent of the projection obtained when normal T3 setae are misrouted through RT2 (compare Fig. 9 with Fig. 4~). These experiments demonstrate that in ti flies, where most or all T3 sensory structures are clearly MS (wing like), the sensory projections nevertheless remain MT (typical of haltere neurons). In the case of T3 PCS, a similar conclusion had been reached on the basis of other differences between MS and MT projections (Palka and Schubiger, 1980). The projection of T3 setae, on the other hand, was believed to indicate that some T3 neurons are transformed to MS and therefore seek and find their normal target in the ganglion, thereby establishing the anterior branches observed in Figs. 3c, d. (Palka et al, 1979). This explanation is ruled out by our misrouting experiments. Indeed if the T3 axons become MS, one would definitely expect them to establish a MS projection when they enter the ganglion through RT2. This expectation is clearly not fulfilled (compare Figs. 9a-c with Fig. 3a). From these results we conclude that in ti flies the T3 neurons behave as MT, whether they enter the ganglion through RT2 or RT3, and in spite of the fact that the sensory structures they innervate are clearly MS in shape and pattern. (f) Projections in a&$ies. As mentioned previously, anterior T3 epidermis and sensory structures differentiate as MS in abx flies. The projection of T3 sensory neurons has been followed in the thoracic ganglion only. In homozygous flies, the projection of the pcs was usually MT, i.e., typical of normal haltere neurons. However, in some flies (4 out of 14) the medial branch characteristic of the MS (wing) projection was followed by one or two fibers (Figs. lOa, b). The same result was obtained in hemizygous abx/Df(.?)P2 flies. In addition, the few homozygous and hemizygous flies that showed fibers following the medial branch also had supernumerary medial branches more posteriorly; in one case where only three neurons were filled and the three axons could be individually traced, it appeared that the medial branch characteristic of the normal wing projection (Fig. 10~) and the supernumerary medial branch belong to the same fiber (Fig. lob). This suggests that the mutation abx might affect the central nervous system as well. A similar observation has been made in the case of the

Segmental

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of Neuron.~

FIG. 6. Partly transformed haltere in a trz fly. (a) X250, bright field; (b) X1000, Normarski. The size and shapes of haltere and wing proximal sensilla (Hpcs, Wpcs) and of haltere and wing setae (Hse, Wse) are identical to those in normal halteres and wings.

giant fibers, which normally extend from the brain down to a level dorsal to the mesothoracic leg neuromeres, where they bend laterally. In hemizygous abx bx pbx flies, these fibers extend further and show a supernumerary lateral bent at a level dorsal to the metathoracic leg neuromeres (J. Thomas and R. Wyman, personal communication). The projection from T3 setae in homo- and hemizygous abx flies also appeared partly MS, in that most fibers remained confined to the local component (Fig. 3b). However, the interpretation of this observation is complicated by the fact that the central nervous system might be affected: for example, it is conceivable that the ventral longitudinal pathway normally recognized by haltere fibers is altered in this mutant. This question will have to be settled by using mosaic flies.

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FIG. ‘7. Normal wing (a) and anterior haltere transformed to wing in ti/Dj($)P~ (b) and abx/Dj(~)P~ (c) flies, at the same magnification. Note the similarity of the transformation in (b) and (c). The size and pattern of the setae along the anterior margin, and in particular the various types of setae that make up the triple row, are identical in wild type wings and in the mutant appendages.

These results indicate that, contrary to ZPJ?,the abx mutation makes at least some T3 sensory neurons behave as MS, and probably has an effect on central neurons as well. (g) Projecticms in j-lies hemixygws for the BX-C. Deficiencies for the entire BX-C (Df(3JP9) or for its left half (Df (3)bxiF’) are homozygous lethals and have a dominant effect on the development of T3 which results in a slight enlargement of the halteres in Df(3)P9/+ and Df/(3)bxdlw/+ flies. We have analyzed the projection of the T3 pcs in these hemizygous flies. While the projection is completely MT in the thoracic ganglion, there is a slight but reproducible departure from the normal haltere projection in the subesophageal ganglion (Figs. 2d, e). We have also examined the mutation UIX”~ which

behaves genetically as a deficiency for the functions bx+, p&r+, and bxd+, and which has the same effect on the size of the haltere as the true deficiencies. Figure 2f shows that Ubx also has a slight dominant effect on the subesophageal projection of the T3 PCS,very similar to that observed with the deficiencies. The projections in heterozygous ti pbx/Df (3)P9, ti pbx/Df (3)bxdlw, and ln? pbx/lJh~~~~ flies do not differ from those of +/ Df (3)P9, +/Of (3)bxd’O”, and +/U!U’~~.

2. The Abdominal

Setae

The abdominal segments of Drosophila provide an excellent system to investigate the effect of segmental determination on sensory projections. Indeed they pos-

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of Neurons

17

sess mechanosensory setae which are obviously homologous from one segment to the next. Each segment comprises a dorsal plate (tergite) and a much smaller ventral plate (sternite). Except for the first abdominal segment, the arrangement and appearance of setae on all tergites closely resembles each other, and the same is true for the sternal setae. There is, however, a clear difference in size and pattern between the tergal and the sternal setae. The first abdominal segment differs from all others in that it lacks a sternite, and the setae on its tergite are much smaller than those in the following segments (Fig. 11). The neurons underlying these setae were filled singly or in groups. In all cases the axons enter the central nervous system through the corresponding segmental nerve. The first and second abdominal nerves enter the ganglion at slightly different places (solid and open arrows in Fig. 12), while all the segmental nerves from the more posterior segments merge to form the main abdominal nerve and enter the ganglion at its posterior tip. Within each segment, the projections of dorsal setae differ markedly from those of the ventral setae; for technical reasons we limited cur analysis to the former. The projections of the dorsal setae on consecutive segments are similar to each other in that all of them extend anteriorly along the abdominal neuromere, turn medially to enter this neuromere, and ramify when they come close to the midline (Fig. 12). There are, however, discrete differences between the projections of the different segments. The axons from the first abdominal segment differ from all others in that they do not remain confined to the abdominal neuromere, but extend anteriorly between the metathoracic leg neuromeres (Figs. 12a and c). The axons from the next segments differ from each other in the place where they turn medially to enter the abdominal neuromere, this place being more posterior for more posterior segments (illustrated in Fig. 12b for the second and third segments). In addition to the thoracic transformations mentioned above, the mutation ttithoraz (try) gives patchy abdominal transformations toward more anterior abdominal segments. Thus patches of dorsal setae typical of the first abdominal segment may be seen on the second and less frequently on the third or following abdominal segments (Ingham and Whittle, 1980). Given the difference in size between first abdominal and second abdominal FIG. 8. Proximal companiform sensilla on the base of a normal wing (a) and of the transformed haltere in td/Df(S)PZ (b) and abx/Df(.?)P2 (c) flies, at the same magnification. Two clusters of sensilla typical of the wing in pattern and shape are marked by the arrows. Note also the similarity of the setae.

18

DEVELOPMENTALBIOLOGY VOLUME99,1983

FIG. 9. The projection of wing-like setae on the transformed haltere of three wg; bx’/Df(S)PZ flies where the fibers from the transformed haltere are misrouted and enter the ganglion at the place appropriate for wing fibers. The presence of longitudinal fibers makes these projections different from the normal wing projection (see Fig. 3a) and more like the projection of misrouted haltere fibers (see Fig. 4~). Arrow and abbreviations as in Fig. 4.

setae, such patches are easily recognized (Fig. 11). Backfilling of the neurons underlying these transformed setae gave the following results: (i) in 29 out of 30 cases, all

a

transformed setae of a given patch project in the same manner; (ii) transformed setae on the second segment may project either as first abdominal (10 cases, Fig. 12f)

b

FIG. 10. Projection from the proximal sensilla in the transformed haltere of an abx/abx fly. In (a), the total projection is shown; since only three fibers were backfilled, it was possible to trace them individually and to observe that one of them showed both the medial branch typical of a normal wing projection, and a supernumerary medial branch (b). A backfill of a single sensillum in a normal wing is shown in (c) for comparison. The arrows point to the medial branch typically found in the normal wing projection (see Fig. la, b) and to a supernumerary (serially homologous?) medial branch.

GHYSEN,

JANSON,

AND

SANTAMARIA

Segmental Determination

of Neurms

or as second abdominal (5 cases); (iii) transformed setae on the third abdominal segment may project as first abdominal (4 cases, Fig. 12g) or as third abdominal (10 cases) or, in one case, as second abdominal. The mutation Ultra-abdominal (Uab), which is located within the BX-C, produces a transformation of the first and second abdominal segments to third abdominal (Lewis, 1978). The transformation of the first segment appears complete as judged by the size and pattern of setae and by the pattern of pigmentation. We backfilled the neurons of the transformed first or second segments on one side, and of the normal third segment on the other side of 23 Uab5 flies. In all cases (15 for the first segment and 8 for the second) the projection remained totally unaffected by the mutation (Figs. 12d and e). 3. Head and Leg Bristles Besides the bithorax complex, which affects the thoracic and abdominal segments, another major homoeotic locus is known in Drosophila: the Antennapedia complex (Lewis et al., 1980), which affects the prothoracic and cephalic segments. Some mutations in this locus result in the transformation of the antenna into a mesothoracic leg. The range of mutant phenotypes extends all the way from a slight disorganization of the anteriormost vibrissae (in some Ns/+ individuals) to an essentially complete transformation of the antenna and the surrounding regions of the head capsule into a complete mesothoracic leg and sternopleural plate (in some Ns/Ant# flies, Fig. 13). An examination of many intermediate transformations shows that the large sternopleural setae which are clearly identifiable in many N/An@ individuals are homologous to the large anteriormost vibrissae in normal flies. We filled these vibrissae in 15 wild-type flies and the largest homoeotic sternopleural in 10 Ns/Ant$ that showed a transformation at least as extreme as that shown in Fig. 13, left. The results are illustrated Figs. 14a and b: in all cases the projection was the same in wild-type and mutant flies. The projection from the normal sternopleural setae is shown for comparison in Fig. 14~. DISCUSSION

In Drosophila, different sensory neurons have been shown to establish normal projections even if they enter the central nervous system at an inappropriate place (Ghysen, 1978). A similar result was observed in the locust (Anderson and Bacon, 1979). The misrouting experiments reported here extend these observations to the cases of the proximal campaniform sensilla of the haltere, and to the case of the haltere trichoid sensilla. Thus all cases studied thus far support the proposal

FIG. 11. Abdominal transformation in a trdtm mutant. The dashed line delineates a patch of transformation of second abdominal to first abdominal setae. A smaller patch can also be seen on the third abdominal segment. Abl, Ab2, Ab3: first, second and third abdominal segments.

that axons which travel long distances in the central nervous system recognize and follow preexisting pathways (Ghysen, 1978). In this paper we analyze the projections of homologous sensory neurons in consecutive segments of Drosophila. There is no simple definition by which two neurons can be unequivocally recognized as homologous. Thus our criteria for homology should be discussed first. We considered as homologous those neurons that underly the same type of sensory structure and that are located in corresponding regions of consecutive segments. Neither of these two criteria is absolutely straightforward. While it is clear that two identical sensory structures may be

20

DEVELOPMENTAL

BIOLOGY

VOLUME

99, 1983

e

FIG. 12. Projections from abdominal setae in wild type (a, b, c), in the mutant Uab* where the first and second segments are transformed to third (d, e) and in trse/tm: flies where patches of setae on the second and third segments are transformed to first (f, g). a: left, first segment; right, second segment. b: left, second segment; right, third segment. c: left, first segment; right, third segment. d: left, transformed second segment; right, third segment. e: left, transformed first segment; right, third segment. f: left, first segment; right, transformed setae on second segment. g: left, first segment; right, transformed setae on third segment. The abdominal neuromere is outlined by a dotted line, the eagittal plane is marked by a dashed line. Solid and open arrows indicate the roots of the first and second abdominal nerves respectively.

considered to be of the same type, the problem arises for sensory structures that are not identical. In this paper, we considered the proximal campaniform sensilla of the wing and haltere to be of the same type because they subserve the same sensory modality (strain receptors), yet they differ in the detail of their external morphology. Likewise we assumed the mechanosensory bristles on the different abdominal segments to be homologous in spite of the fact that the bristles on the

first segment can easily be distinguished from those on the following segments on the base of their smaller size. The size difference is even greater in the case of the bristles on the haltere capitellum and on the wing margin, so great that the former are referred to as trichoid sensilla and not as bristles. A classification of sensory structures based on their sensory modality appeared to us more meaningful than one strictly based on the external morphology; yet it is not as clear cut as one might

GHYSEN,

JANSON,

AND

SANTAMARIA

Segmental

Determination

21

of Neurom

FIG. 13. Homology of sternopleural setae and vibrissae. From the left, heads of three NdAntpflies with good, moderate and poor transformation of the antenna to leg, and head of a wild type fly. The seta that is clearly identified as a large sternopleural in the most extreme transformation appears homologous to the anteriormost vibrissa in a normal head (arrows).

hope. Indeed sensory structures whose external appearance is intermediate between bristles and campaniform sensilla may be observed in mutants (Lees, 1942). While it would certainly be of interest to analyze the projections of neurons underlying such intermediate

a

structures, their very existence raises some doubt about the ground of a classification based on sensory modality. The second criterion, that neurons should be considered homologous when they are located in corresponding (homologous) regions of different segments, also lacks

I

\

C FIG. 14. Projections from the largest sternopleural seta on the head of a NdAntp fly (a), from the anteriormost vibrissae on the head of a wild type (b), and from the two large sternopleural setae on a wild type thorax (c). The homoeotic sternopleural axon (a) was always observed to enter the brain through the antenna1 nerve, cross it posteriorwards and ventralwards, and ramify close to or within the suboesophageal ganglion. The axon from the normal anteriormost vibrissae (b) usually (but not always) join the antenna1 nerve close to the brain. They then follow the same path as the homoeotic axons and ramify in the same region. The axons from normal sternopleural setae join the mesothoracic leg nerve close to the ganglion, travel anteriorwards under the mesothoracic leg neuromere, and then follow a specific pathway along the prothoracic and mesothoracic leg neuromeres with profuse ramifications in the region between these two leg neuromeres. This pathway appears specifically recognized in the sense that it is followed by neurons innervating sternopleural setae that develop on the pro- and metathoracic segments in try flies, even though their axons enter the ganglion far away from the normal site (Ghysen, unpublished). AN: antenna1 nerve; CC: cervical connective; LN: mesothoracic leg nerve; MS, MT, P: meso-, meta- and prothoracic leg neuromeres. In (a) and (b), the saggital plane is marked by the dashed line.

22

DEVELOPMENTAL BIOLOGY

a precise operational value. While the homology between the various legs seems obvious, it is less so in the case of the dorsal appendages (wing and haltere) or other ventral appendages (legs, antenna, and proboscis). The difficulty increases when we consider regions within such appendages: thus the homology we assume in this paper to exist between proximal regions or distal regions of the wing and haltere may be consistent with all we know about the development, phylogeny, and genetics of these appendages; yet this homology is not a fact but a reasonable hypothesis. We must acknowledge, therefore, that our criteria to assume homology are rather precarious; nevertheless, it seemed to us that the clarifying value of these assumptions validates their use. The major result of our investigations is that sensory neurons are segmentally determined, and that this determination is involved in the choice of their pattern of projection; nevertheless, mutations that alter the segmental determination of epidermal cells may have no effect on neurons. These two conclusions will be discussed separately. The most salient results are summarized in a simplified form in Table 1. Sensory Neurons are Segmental& Determined We have extensively analyzed three different systems of homologous neurons. The systems differ in the type of sensory structure (campaniform sensilla or bristles), in the region (body or appendage), in the segments (thoracic or abdominal), and in the pattern of projection (these neurons follow three completely different central pathways). In all three cases, clear differences can be observed between the projections of homologous neurons on consecutive segments. In two of the three cases, it has been shown that these differences do not depend on the point of entry of the axons in the ganglion. (This is probably true in the third case as well, for we have observed one exceptional wild-type fly where a spontaneous misrouting of the first abdominal axons through the second abdominal nerve had occurred; the central projection of the misrouted axons was nevertheless typical of first abdominal neurons.) On the other hand, we observed in all three cases that the projection can be readily transformed from one segmental type to another by means which are known to alter the process of segmental determination, namely, the mutation trx and ether phenocopying. Thus we conclude that the behavior of sensory axons depends on the segmental determination of the neuron. It should be noted that the transformations are patchy, so that all individuals are in effect mosaics. We may then examine whether or not the establishment of a given projection is a cell-autonomous process in the case of the abdominal bristles, where the transformed bristles may be individually

VOLUME 99. 1983 TABLE 1 PATTERN OF PROJECTIONOF SENSORYNEURONS: SUMMARY OF THE RESULTS

Sensory structure

Wild type

Ether treated

T2 pcs T3 pcs T2 setae T3 setae AbI setae Ab2 setae Ab3 setae

W H’ W H* AI* A2 A3

W W W W

tm W W, H W W,H AI AI, A2 AI, A2,

bx

atm

W w

W W,H w

H’

W,H

H*

uob

AI A2 AS

A3

Note.The projections are labeled as W (wing-like), H (haltere-like), Al to A3 (typical of the first to third abdominal segments). An asterisk means that the projection retains the same properties when the axons are misrouted. Italicized letters indicate that the projection does not correspond to the external morphology of the sensory structure. For other explanations, see text.

identified. In all cases where a patch of transformed bristles gave a transformed projection, the control bristles on the contralateral side showed a normal projection. It seems very unlikely that in all those cases the central nervous system was also transformed on the same side as the patch of bristles, and therefore we conclude that the type of projection depends exclusively on the segmental identity of the projecting neurons, and not on some other effect on the central nervous system. Furthermore in all but one case, all the neurons of a patch gave the same pattern of projection, which indicates that the transformed neurons are not influenced by the normal surrounding neurons. Thus it appears that the choice of a pathway is truly a cell-autonomous process. The effect of ether occurs at about the blastula stage (Capdevila and Garcia-Bellido, 1974; Santamaria, 1979), early during embryogenesis, is then clonally inherited (Capdevila and Garcia-Bellido, 1978), and is finally expressed in the adult epidermis. The transformation of the neural projections also depends on an early effect of ether, since the treatment is limited to a brief exposure of early embryos. In the case of trx, an early time of action is indicated by the existence of a maternal effect, by the synergy of trx and ether, and by a very early thermosensitive period (Ingham and Whittle, 1980). However, in trx flies the early decision seems not to be irreversible since early clones are occasionally found to contain both transformed and nontransformed epidermis (Ingham, 1980a,b). Our observations are consistent with an early effect of trx, since with one exception the neurons innervating a patch of transformed bristles were either all normal or all transformed. Thus it appears that the segmental determination of sensory neurons depends on a decision taken very early, long before these neurons were generated.

GHYSEN,

Tissue Speci&ity

of Hmoeotic

JANSON,

AND

SANTAMARIA

Mutations

We now turn to the lack of effect on sensory neurons of mutations within the bithorax complex (BX-C). It might be that the epidermal specificity of these mutations is fortuitous. Indeed various alleles of bx (and of many other genes) show some level of regional specificity (Morata and Kerridge, 1980), and one may imagine that some alleles are more effective in one cell type than in another. However, the fact that the same discrimination between epidermis and neurons is found in bx’, pbx, Uab’, and Antp suggests that this is not a mere allele-specific eccentricity. The lack of effect of ti and pbx on the neurons innervating the proximal campaniform sensilla of the haltere was already reported, and was proposed to result from some leakiness of these two mutations (Palka and Schubiger, 1980). However, we feel that our results offer some support for an alternative explanation, namely, that the control of segmental determination is distinct for adult neurons and for adult epidermis. It is certainly conceivable that the mutations ti and pbx do not completely abolish the activity of these genes, and that the residual amount of gene function is responsible for the nontransformed behavior of the sensory neurons within transformed regions of the epidermis. Interfering with the control of these genes by ether treatment or trx might, on the other hand, lead to a complete lack of expression, and therefore to a transformation of the neuronal projection as well as of the epidermis. If this explanation were correct, we would expect that only genetic combinations showing the most extreme epidermal phenotype should show a transformed neuronal projection. This, however, does not seem to be the case: indeed the amount of bx and pbx products is certainly much higher in Df(3)P9/+ than in homozygous Mpbx flies, yet the projection is slightly transformed in the former but not at all in the latter. Furthermore there is no difference between Df(3)P9/+ and Df(3)P9/td pbx flies. This is the first argument to support the idea that a gene distinct from bx’ and pbx’ is responsible for the metathoracic variant of the proximal sensilla projection. If this is so, the results also indicate that either this gene belongs to the BX-C, or it maps elsewhere but is stoechiometrically controlled by an element of BX-C (the fact that the mutation abx, which is localized within BX-C, induces an occasional transformation of the metathoracic proximal sensilla projection certainly supports the idea that elements of BX-C are involved in the control of segmental determination in neurons as well as in epidermis). However, this first argument is admittedly tenuous, because we do not understand why the supposedly transformed fi-

Segmental Determination

of Neurcms

23

bers in Df@)P9/+ flies never show the contralateral branch typical of wing fibers. Thus the argument relies only on the presence of wing-like extensions in the subesophageal ganglion: such extensions may result from some other effect, e.g., a direct effect of the deficiency on the brain, rather than from a true transformation of some metathoracic neurons. The second argument pleading against a differential sensitivity of neurons and epidermis to residual amounts of BX-C products is provided by the analysis of the Uab5 mutation. This mutation results in the transformation of the first and second abdominal segments into more posterior segments. It is believed that this effect is due to the expression, in these segments, of BX-C functions that are normally expressed only in the more posterior segments. If neurons were much more sensitive than epidermal cells to the BX-C products, then one would expect that the abnormal expression of BX-C in Uab5, being sufficient to transform the epidermal cells, should certainly also be sufficient to affect the sensory neurons. Yet we observed that this is not the case: Uab’ has no effect on the sensory neurons, even though the epidermal transformation appears complete. The third argument supporting the idea of separate controls for neurons and for epidermis comes from the analysis of the effect of trx. We have observed, both for the abdominal and for the wing margin bristles, that in some cases a patch of transformed bristles gives rise to a normal (nontransformed) projection. In one revealing case, we observed a simultaneous transformation of third abdominal bristles to first abdominal, and of their neurons to second abdominal. Thus there is no doubt that the cell that makes up the bristle shaft, and the cell that becomes the underlying neuron, may express different segmental determinations even though they are siblings or, at most, cousins (Wigglesworth, 1953). These observations certainly support the idea that separate controls are involved in the segmental determination of neurons and of epidermis, so that in a given clone the two controls might be set independently to two different segmental determinations. It is clear, however, that the two events are not completely independent because a high proportion of the epidermally transformed clones also show a neural transformation. If we take into account the early effect of ether and trx, we come to the conclusion that early during embryogenesis the defect in trx function may result in both controls being erroneously set in a given cell (and in the resulting clone), and that at differentiation one is relied upon in epidermal cells and the other in neurons. This would result in some of the epidermally transformed clones having transformed neurons, others having normal neurons, and others where third abdominal

24

DEVELOPMENTALBIOLOGY VOLUME99,1983

epidermal cells and neurons are transformed independently to first and second abdominal. According to this view, one would expect to find cases in trx flies where the neurons are transformed while the epidermal cells are not. In our crosses, the size of the transformed patches is about l/10 of a tergite, the frequency of patches is about 0.5 per tergite, and we can backfill about 10 contiguous bristles in each fly. If the frequency of neuronal transformation were the same as that of epidermal transformation, we should then detect about 1 instance of neuronal transformation in 10 flies. We analyzed normal second abdominal bristles in 20 trx flies and found no case of transformed projection. This negative result is no evidence against our explanation, however, since we have no independent estimate of the penetrance of the mutation for sensory neurons. In view of the arguments presented above, we favor the hypothesis that the lack of effect of ti, pbx, and Uub5 on neurons is likely to reflect their involvement in the control of epidermal, as opposed to neuronal, segmental determination. Segmental

Determination

in Central Neurons

The lack of effect of b2 on sensory neurons may seem surprising in view of the claim, based mostly on mosaic experiments, that this mutation has some direct effect on the central nervous system (Palka et aL, 1979). However, the data on mosaics are difficult to interpret, in part because some of the abnormal projections reported to be found only in mosaics have also since been observed in nonmosaic mutant flies (Strausfeld and Singh, 1980) and in part because the cell lethality and genetic background associated with the generation of somatic recombination may have affected the results. A recent reexamination of this question (Strausfeld and Singh, 1980) led to the conclusion that no autonomous modification of the central nervous system could be detected in mutant flies, and that the minor differences between wild-type and mutant ganglia are most likely accounted for by the increased sensory input present in the ti pbx flies. Thus it would seem that ti has no effect on either sensory or central neurons. More recently it has been suggested that the triple mutant abx tipbx has some effect on the central nervous system (Green, 1981). In view of our results, this may indicate that a&r has some effect on the central as well as on the sensory neurons. Unfortunately, only the triple mutant was analyzed; furthermore, the claim for segmental transformation relies only on the position of the cell bodies of the leg motoneurons. The only clear evidence so far that the BX-C is involved in the development of the central nervous system, and therefore that seg-

mental determination is involved in this development, comes from the analysis of embryos deficient for the whole complex. Such embryos show a failure of condensation of the central nervous system (Lewis, 1978); furthermore, all the segmental ganglia display a staining pattern normally observed only in the thoracic ganglia (Jimenez and Campos-Ortega, 1981). Apparent Variety of Behaviour Homoeotic Neurons

among

In a previous study we examined the central projections of homoeotic sensory structures that have no homologs on the normal (nontransformed) segment (Ghysen, 1978). Such is the case of the bristles on the notum and the distal campaniform sensilla on the wing, which are only found in the mesothorax. It was found that in the double mutant, ti pbx, the bristles on the homoeotic notum and the distal sensilla on the homoeotic wing give rise to the normal mesothoracic projections. Thus the complex projection from a homoeotic wing contains both mesothoracic-like and metathoracic-like components (corresponding, respectively, to the distal and the proximal campaniform sensilla), as well as an apparently new component (corresponding to the enormous increase of the minute trichoid projection of the haltere). This led to the conclusion that different classes of sensory neurons obey different rules once they enter the central nervous system (Palka et al, 1979). This apparent disparity of behaviors can now be explained as follows. The homoeotic neurons that establish a mesothoraciclike projection innervate sensory structures that have no homologs in a normal metathorax. Since these structures do not normally develop on the metathorax, there is no metathoracic variant of the basic program of projection. Thus in mutant flies these homoeotic neurons will establish the unmodified basic projection exactly as if they had developed on the mesothorax. On the other hand, the homoeotic neurons of the second group innervate sensory structures which are also present on the normal haltere. The projection of the metathoracic sensory structures is modified relative to the projection of their mesothoracic homologs. If the genetic function that controls this segmental modification is somehow unexpressed, then the homoeotic neurons will project as mesothoracic. This seems to be the case in ether phenocopies, in the trx mutant, and to some extent in abx flies, but not in the In? pbx mutant. Antenna

to Leg Tranzfnmation

The conclusion that the segmental determination of epidermis and neurons is under distinct control may not be limited to the case of the BX-C. Indeed it has

GHYSEN,

JANSON,

AND SANTAMARIA

been shown that mutations which transform the distal part of the antenna into a distal part of a leg leave most of the antenna1 projection unchanged and have no detectable effect on the central nervous system (Stocker and Lawrence, 1981). The one clear difference that was observed between the projections from normal and mutant antennae is the presence of a new component in the latter. This new component may well be due to the development of a class of sensory structures which is present on the distal leg but not on the normal antenna, such as sugar receptors. This would be identical to the situation found in Lzr pbx mutants, where the projection from the transformed metathorax contains both an unmodified haltere component (from the proximal campaniform sensilla) and components not found in the normal metathoracic projection (from the distal sensilla on the homoeotic wing and the bristles on the homoeotic notum). In the case of the antenna to leg transformation, the sensory structures from which the various components arise have not been identified. However, the one experiment reported here on the Antp mutant certainly supports this explanation, as it shows that an identified bristle neuron gives rise to exactly the same projection whether it is found on a normal head or on a typical sternopleural plate as a result of the homoeotic transformation. CONCLUSION

We propose that the establishment of sensory projections in Drosophila follows the same general principles that underly the development of the epidermis (Garcia-Bellido, 1977; Lewis, 1978). The analysis of wildtype flies shows that projections from nonhomologous neurons are completely different from each other, while projections from homologous neurons on consecutive segments show marked similarities. This suggests that homologous neurons use the same program of projection, on which segment-specific features are superimposed. Thus the genome of a fly would contain the instructions, mostly in terms of pathway recognition (Ghysen and Janson, 1980a), allowing the establishment of a basic set of projections in the central nervous system (archetype). In addition to these instructions, there would be another set of genetic functions responsible for the various segmental modifications of these basic projections. This second set of functions appears very similar to the known BX-C functions: their expression is affected by exposure to ether at the blastoderm stage and requires the activity of the trx+ gene, and they probably map within the BX-C. However, various indirect arguments indicate that they are distinguishable from the BX-C functions involved in the segmental determination

Segmental

Determination

of Neurons

25

of the adult epidermis. (The fact that one mutation, abx, affects both the epidermis and to some extent the sensory neurons may indicate that there is some overlap between epidermal and neuronal elements within the BX-C; alternatively it may be due to a “polar” effect as has been observed for other BX-C mutations (Lewis, 1974).) This does not imply that we postulate yet another battery of genes within the BX-C. There is no reason to assume that these functions are genes in the classical sense; they might as well be controlling elements or sites, in which case the interpretation of mutant phenotypes may take on a rather different aspect. This leads us to believe that a complete understanding of the BX-C complex will require a more extensive analysis of its function in nonepidermal tissues, and in particular in sensory and central neurons. We thank E. Lewis for information on and mutations in the BXC; A. Garcia-Bellido, for discussions; P. Ingham, for providing the trzr mutation; R. Thomas and a referee, for comments on the manuscript. This work was supported by a FNRS research grant to A.G. and by a contract between the Belgian government and the University of Brussels. A.G. is Chercheur Qualifie of the FNRS (Belgium). Note added in proox We have recently isolated a monoclonal antibody that labels the neuromeres and used it to assess the effect of BX-C mutations on the organization of the central nervous system. We have shown that the mutation &rdiw, which transforms the first abdominal epidermis to thoracic, has a dramatic effect on the central nervous system as well (Teugels and Ghysen, Nature (London), in press). Preliminary results suggest that absc also affects the organization of the central nervous system, while t&has little or no effect, in agreement with our results on sensory neurons. REFERENCES ANDERSON, H., and BACON, J. (1979). Developmental determination of neuronal projection patterns from wind-sensitive hairs in the locust, Schistocerca gregaria Da? Biol 93, 549-575. BATESON, W. (1894). “Materials for the Study of Variation.” McMillan, London. CAPDEVILA, M. P., and GARCIA-BELLIDO, A. (1974). Development and genetic analysis of &thorax phenocopies of Drosophila Nature (Lendon) 250, 500-502. CAPDEVILA, M. P., and GARCIA-BELLIDO, A. (1978). Phenocopies of &thorax mutants. Wilhelm Roux’s Arch. 185, 105-126. CAPDEVILA, M. P., and GARCIA-BELLIDO, A. (1981). Genes involved in the activation of the Bithorax Complex of Drosophila. Wilhelm RwuxL Arch. 190. 339-350. COLE, E. S., and PALKA, J. (1980). The pattern of campaniform sensilla on the wing and haltere of Drosophila melanogaster and several of its homoeotic mutants. J. Emtnyol. Exp. Mmphol 71, 41-61. GARCIA-BELLIDO, A., and MERRIAM, J. R. (1968). Genetic analysis of cell heredity in imaginal discs of Drosophila melanogaster. Proc. Nat. Acaa! Sci. USA 68, 2222-2226. GARCIA-BELLIDO, A. (1977). Homoeotic and atavic mutations in insects. Amer. Zool 17, 613-629. GARCIA-BELLIDO, A., and CAPDEVILA, M. P. (1979). Initiation and maintenance of a developmental pathway in Drosophila. In “The Clonal Basis of Development” (S. Sobtenly and I. Susex, eds.), pp. 3-21. Academic Press, New York.

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MORATA, G. (1975). Analysis of gene expression during development in the homeotic mutant Contrabithorax of Drosophila melanogaster. J. Emlnyol. Exp. Morph& 34.19-31. MORATA, G., and KERRIDGE, S. (1980). An analysis of the expressivity of some b-ithorax transformations. In “Development and Neurobiology of Drosophila” (0. Siddiqi, P. Babu, L. Hall, and J. C. Hall, eds.), pp. 141-154. Plenum, New York. MORATA, G., and LAWRENCE, P. A. (1977). Homoeotic genes, compartments and cell determination in Drosophila Nature (London) 265, 211-216. PALKA, J., LAWRENCE, P. A., and HART, H. S. (1979). Neural projection patterns from homoeotic tissue of Drosophila studied in &thorax mutants and mosaics. Den Biol. 69,549-575. PALKA, J., and SCHUBIGER, M. (1980). Formation of central patterns by receptor cell axons in Drosophila In “Development and Neurobiology of Drosophila” (0. Siddiqi, P. Babu, L. Hall, and J. C. Hall, eds.), pp. 223-246. Plenum, New York. REINHARDT, C. A., HODGKIN, N. M., and BRYANT, P. J. (1977). Wound healing in the imaginal discs of Drosophikz. I. Scanning electron microscopy of normal and healing wing discs. Deu. Biol 60, 238257. SANTAMARIA, P. (1979). Heat shock induced phenocopies of dominant mutants of the bithorax complex in Drosophila melanogaster. Mol Gen . Genet. 172, 161-163. SHARMA, R. P., and CHOPRA, V. L. (1976). Effect of wingless (wg) mutation on wing and haltere development in Drosophila melanogaster. Dev. Biol. 48,461-465. STOCKER, R., and LAWRENCE, P. A. (1981). Sensory projections from normal and homoeotically transformed antennae in Drosophila Dev. Biol. 82, 224-237. STRAUSFELD, N. J., and SINGH, R. N. (1980). Peripheral and central nervous system projections in normal and mutant (bithwaz) Dr+ sophila melanogaster. In “Development and Neurobiology of L3ro sophila” (0. Siddiqi, P. Babu, L. Hall, and J. C. Hall, eds.), pp. 267290. Plenum, New York. STRUHL, G. (1981). A gene product required for correct initiation of segmental determination in Drosophila Nature 293, 36-41. TEUGELS, E., and GHYSEN, A. (1983). Two mechanisms for the establishment of sensory projections in Drosophila. Progr. Brain Res., In press. WIGGLESWORTH, V. B. (1953). The origin of sensory neurons in an insect. Quart. J. Microscop. Sci 94, 93-112.