Morphogenesis of an identified leech neuron: Segmental specification of axonal outgrowth

DEVELOPMENTAL

BIOLOGY

115,256-260

(1986)

BRIEF NOTE Morphogenesis of an Identified Leech Neuron: Segmental Specification of Axonal Outgrowth C.

JOEL Department

of Biology, Received

GLOVER’

University

September

AND

of California,

9, 1985; accepted

ADRIAN

MASONS

San Diego, in revised

form

La Jolla,

Cal@rnia

December

92093

3, 1985

We have investigated the development of segmental diversity in an identified leech neuron, the Retzius cell. Retzius cells in the genital segments differ from those in other segments in lacking central axons and contacting different peripheral targets: the genitalia. These differences are not apparent during initial axon outgrowth, when all Retzius cells follow the same morphogenetic pattern. Rather, they first appear about the time the peripheral axons of the genital segment Retzius cells contact the genital primordia. This suggests that the pattern of central and peripheral o 1986 Academic press, IX. axonal outgrowth may be modified by an interaction with peripheral targets.

becomes apparent as Retzius cell axons contact segmentspecific peripheral targets.

INTRODUCTION

Many invertebrate and some vertebrate neurons are individually identifiable on the basis of morphology, biochemical characteristics, and the pattern of connections made with other cells. An important objective of developmental neurobiology is to determine how these identities are acquired. Are they expressed autonomously or are they specified by factors in the neuron’s environment? The segmentally homologous neurons of metameric invertebrates provide a particularly favorable opportunity for studying this question because, although generated by outwardly identical precursor cells (Weisblat et aL, 1980; Goodman and Spitzer, 1979), they can exhibit striking segmental differences in morphology and synaptic connectivity (Gillon and Wallace, 1984; Larimer et aZ., 1971; Mittenthal and Wine, 1978; Wilson, 1979; Shafer and Calabrese, 1981; Wine and Krasne, 1972), at least some of which arise during embryonic development (Bate et ok, 1981; Wallace, 1984; Pearson et aa, 1985). We have investigated the development of segment-specific morphology in the Retzius cell, a bilaterally paired serotonergic neuron found in every segmental ganglion of the leech nervous system. This neuron can be identified by position, size, morphology, action potential waveform, and histochemical stains for serotonin (Lent, 1977). Our observations show that, initially, the Retzius cells in all segments follow the same morphogenetic pattern. Later, however, this pattern becomes modified in two segments, resulting in central and peripheral differences in morphology. The modification

MATERIALS

Copyright All rights

$3.00

0 1986 by Academic Press. Inc. of reproduction in any form reserved.

METHODS

The collection and care of Hirudo medicinalis embryos are described in Fernandez and Stent (1982). All embryos were staged by days of development (at 23°C) from the time of laying. Horseradish peroxidase (HRP) was injected into cells and reacted according to the methods of Muller and Carbonetto (1979), modified to produce a cobalt-intensified reaction product (Gillon and Wallace, 1984). Lucifer Yellow was injected according to the methods of Kuwada and Kramer (1983). For serotonin antiserum staining, live embryos were first soaked in 10m4Mserotonin to increase intracellular stores of serotonin in the Retzius cells. They were minimally dissected to expose the nerve cord, then fixed overnight with 4% formaldehyde in phosphate-buffered saline (PBS) at pH 7.4 and 4°C. After several PBS rinses the tissue was incubated for 9 to 12 hr in rabbit antiserotonin antiserum (Immunotech, 1:500 in PBS with 1% bovine serum albumin, 1% goat serum, and 2% Triton X-100 detergent) at room temperature. After further PBS rinses, the tissue was incubated for 3 to 4 hr in fluorescein-isothiocyanate (FITC)-conjugated goat antirabbit IgG (TAGO, 1:lOO) at room temperature. After another PBS rinse (15 min to 3 hr), the tissue was mounted in 80% glycerol and viewed with epifluorescence through FITC filters. RESULTS

’ Present address: Institute of Physiology, University of Oslo, Karl Johans gate 47,0162 Oslo 1, Norway. ‘Laboratory of Physiology, University of Oxford, Parks Road, Oxford, OX1 3PT, UK.

OolZ-1606/86

AND

AND

DISCUSSION

We used HRP to reveal the morphologies of individual Retzius cells in each of the 21 segmental ganglia of adult

256

BRIEF NOTE

H. medicinalis. In 19 of the 21 ganglia,

the Retzius cells were the largest neurons in the ganglion and had axons in each of the four ipsilateral nerve tracts: namely the anterior and posterior interganglionic connectives and the anterior and posterior segmental nerve roots (Fig. 1A). In the genital segments 5 and 6, however, Retzius cell morphology was strikingly different. Retzius (5, 6) (that is, the Retzius cells in segments 5 and 6) had smaller somata and lacked axons in the interganglionic connectives (Fig. 1B). The absence of Retzius (5, 6) connective axons could arise in two ways: either the axons are never produced, or they initially extend as in other segments but are later lost. To distinguish between these alternatives, we studied Retzius cell morphogenesis in H. medicinalis embryos. We examined the earliest development of Retzius connective axons with serotonin antiserum staining, which stains all the serotonin-containing neurons in the nerve cord. At later stages, when it became feasible to impale embryonic neurons with microelectrodes, we injected the fluorescent dye Lucifer Yellow into individual Retzius cells. Figure 2 compares the development of Retzius cell connective axons in segments 4,5,6, and 7 (segments 4 and 7 represent the typical morphogenetic pattern). During their early development (beginning on Day 8) the Retzius cells in these four segments were very similar in structure. In particular, they all extended neurites toward the interganglionic connectives (Fig. 2A), that

A

257

later grew into the connectives and established the connective axons (Fig. 2B). Although initially the lengths of Retzius (4,5,6,7) connective axons were similar, between embryonic Days 9 and 10 the growth of Retzius (5,6) connective axons began to lag behind that of Retzius (4, 7) axons (Fig. 2C). It was difficult to determine precisely when this occurred because of the intrinsic variability of axon length; on Day 10 the difference was clear on average (Fig. 3). By the time Retzius (4,7) connective axons had grown into and beyond adjacent ganglia, and sent branches into the periphery of the adjacent segments, Retzius (5, 6) connective axons had, at most, just reached the proximal margins of adjacent ganglia (Fig. 2D). Subsequently, before most of the Retzius (5, 6) connective axons reached adjacent ganglia, there occurred a decrease in their length relative to the length of the connective (Fig. 3). Eventually the Retzius (5, 6) “connective axons” no longer extended out of their parent ganglia, and if they persisted in the adult we were unable to distinguish them from dendritic branches in the neuropil. Segments 5 and 6 contain the gonopores and reproductive organs (Mann, 1962). Injections of Lucifer Yellow in late embryos showed that Retzius (5,6) innervate the gonopores and associated genital tissues (not shown). This suggested that an interaction with segment-specific peripheral target tissue might be involved in the modification of Retzius cell morphogenesis in the central nervous system. We therefore used serotonin antiserum

B

segmenta nwves FIG. 1. Central morphology of Retzius cells in the adult leech as revealed by HRP injections. Whole-mounts of segmental ganglia. A. Typical morphology. Both Retzius cells (R) have been stained in segment 11. Numerous dendritic branches are present in the neuropil. Axons extend into the ipsilateral segmental nerves and ipsilateral interganglionic connectives (arrowed). B. Morphology specific to segments 5 and 6. Both Retzius cells (R) have been stained in segment 5. No axons are present in the connectives (arrowed). Of 31 Retzius cells stained in ganglia 5 and 6, all lacked the connective axons. By contrast, the incidence of Retzius cells missing one or more axons in other segmental ganglia was roughly 4% (3 of 81).

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FIG. 2. Development of Retzius cell connective axons in segments 4 to 7. Panels A to D present the structure of Retzius cells at 9, 9.5, 10, and 10.5 days of development, respectively. Each segment is identified by number at the bottom of the panel. Scale bars represent 50 pm. In A, Retzius cells were stained in whole mount with serotonin antiserum. In B-D, Lucifer Yellow was injected into individual Retzius cells. This was necessary because the connective axons of Retzius cells in adjacent segments overlap at these stages and become difficult to distinguish when all are stained. To avoid overlap of Lucifer Yellow-filled axons injections were made on alternate sides of adjacent ganglia. In all panels Retzius (5, 6) neurites in or directed toward the connectives are indicated by arrowheads. A. Retzius cells in segments 5-7. Each cell has acquired a stereotyped branching structure that includes neurites directed longitudinally toward both anterior and posterior connectives. B. Retzius cells in segments 4-6. Longitudinally directed neurites have entered, or are entering, the connectives. Variability in axon length is illustrated by Retzius (4), whose posterior axon is much longer than its anterior axon. C. Retzius cells in segments 4-7. Connective axons of Retzius (4, 7) have entered adjacent ganglia and are beginning to send branches to the periphery of adjacent segments via segmental nerve roots. The connective axons of Retzius (5,6) have not reached adjacent ganglia. D. Retzius cells in segments 4-7. Retzius (4,7) connective axons and their peripheral branches continue to elongate, whereas Retzius (5, 6) connective axons have not reached adjacent ganglia. The large autofluorescent spots in the periphery of C and D are the segmental nephridia.

staining in a second group of embryos to examine the growth of Retzius (5, 6) peripheral axons in relation to the developing genital tissue. The anterior and posterior peripheral axons of Retzius (5, 6) emerged from their ganglia on Day 9, and grew laterally or posterolaterally into the periphery, as is typical for the homologous axons in other segments. Between Days 9 and 10, this lateral trajectory brought the posterior axons into contact with the genital tissue developing in their respective segments (Fig. 4). In all preparations examined early on Day 10, the posterior axon of Retzius (5) had encountered the primordium of the ejaculatory duct, grown through the crook in the duct primordium and extended a small branch from this point toward the future location of the male gonopore. The posterior axon of Retzius (6) had encountered the

ovisac primordium, grown along its ventral aspect, and from there extended a small branch toward the future location of the female gonopore. By Day 11, several branches extended from the posterior axons in both segments toward their respective gonopores, along the axes of the elongating genital primordia. The anterior axons of both Retzius (5, 6) had meanwhile extended several branches toward the male gonopore. During Day 12 all of the gonopore-directed peripheral axon branches reached the gonopores (Fig. 4) and contacted them with a profusion of broad lamellipodia and spike-like processes. By Day 14 we observed terminal branches twining about the distal ends of the ovisacs and ejaculatory ducts. Laterally directed branches of the Retzius (5,6) peripheral axons extended only slightly into the body wall, whereas by this time the peripheral axons of other Ret-

BRIEF

vironment. Our observations suggest that the morphogenetic pattern a Retzius cell follows, both in the periphery and in the central nervous system, could be specified by the peripheral targets it contacts. Such “peripheral specification” has also been implicated in the development of the central synaptic patterns made by vertebrate sensory neurons (Frank and Westerfield,

RELATIVE AXON LENGTH (%I

RELATIVE AXON = LENGTH

10

259

NOTE

11

12

13

14

15

Dk2

x 100%

ejaculatory primordium

16-19

FIG. 3. Length of Retzius (5, 6) connective axons (relative to connective length) versus embryo age. Bars represent standard errors. Relative axon length was calculated (inset) by dividing the length of the connective axon within the connective (Di) by the length of the connective itself (Dz). This measure was chosen because of variability among specimens in the absolute lengths of connectives and connective axons, due at least in part to differential stretching of the preparations. We observed no statistically significant decrease in absolute axon length so the decline in relative axon length results primarily from continued growth of the connectives without corresponding elongation of the Retzius cell axons. The average relative axon length of Retzius (4,7) connective axons reached 100% on Day 10 and exceeded this level thereafter.

zius cells had grown the full lateral extent of their respective body wall hemisegments. Retzius (5, 6) are therefore initially identical to the Retzius cells in other segments. Central and peripheral modifications of the typical morphogenetic pattern appear between Days 9 and 10, at about the same time peripheral axons grow into the body wall and contact the genital primordia. The peripheral axons are redirected towards the gonopores, and the growth of central axons is arrested. We expect the resultant morphological specialization of Retzius (5, 6) to be related to the functional specialization of their parent segments. In accordance with a specialized functional role, differences in the intraganglionic synaptic connectivity of Retzius (5, 6) have also been discovered (W. B. Kristan, J. Glover, A. Mason, et ah, manuscript in preparation). Why are Retzius (5, 6) different from other Retzius cells? One possibility is that factors inherited through cell lineage commit Retzius (5,6) to a different morphogenetic pattern. Alternatively, Retzius (5, 6) might be equivalent in morphogenetic potential to the Retzius cells in other segments, with differences arising through interactions with the local cellular and biochemical en-

DAY

duct

primordium /

12

A ganglion

A 5

ganglion

6

FIG. 4. The morphology of Retzius (5,6) peripheral axons as revealed by serotonin antiserum staining at early Day 10 (upper) and Day 12 (lower). The drawings were traced from photographs and represent views through the ventral body wall. The outline of the nerve cord is drawn in thin line, genital tissue is stippled, and the Retzius cells are drawn in artificially thick line. Day 10: relationship of Retzius (5, 6) posterior axons with the ejaculatory duct primordia and the ovisac primordia, respectively. Arrows indicate (on one side of the embryo) the short branches extended toward the future locations of the gonopores, which are not yet evident. Day 12: the two ejaculatory duct primordia have fused at the midline in segment 5 and the two ovisac primordia have fused at the midline in segment 6. Retzius (5) anterior and posterior axon branches and Retzius (6) anterior axon branches have reached the male gonopore (8). Retzius (6) posterior axon branches have reached the female gonopore (P). We are uncertain of the lateral extent of the Retzius (5,6) peripheral axons on Day 12 (note the broken line). Serotonin antiserum staining was performed as described in Materials and Methods, except that HRP-conjugated goat anti-rabbit IgG was used (Cappel 1:250, 7- to 12-hr incubation). The HRP was reacted without the cobalt intensification procedure. Although the serotonin antiserum stains all Retzius cells in the nerve cord, for clarity we have shown only Retzius (5,6). C onnective axon lengths have been estimated from Lucifer Yellow injections in other preparations. At Day 10 the connective axons between ganglia 5 and 6 overlap. We could not use Lucifer Yellow preparations to accurately illustrate the relationship of the growing peripheral axons to the genital primordia because at early stages the dissection used to visualize cells for intracellular injection of Lucifer Yellow damaged or distorted the ventral body wall where the genital tissue lies.

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1982). In the insect embryo, on the other hand, a particular peripheral target, the limb, has been ruled out as a factor in the establishment of segment-specific patterns of neuronal death and differentiation (Whitington et al, 1982). Because we have examined connective axon length and contact of the genital primordia in different embryos and with different techniques, the temporal correlation we have observed between the two is limited in resolution. Support for the hypothesis that peripheral target contact modifies Retzius cell morphogenesis comes from preliminary experiments in which pieces of body wall, that include the presumptive reproductive tissues, are removed from embryos early on Day 10 (J. Jellies, C. Loer, and W. B. Kristan, personal communication). The connective axons of Retzius (5, 6) in these manipulated embryos persist at later developmental stages when normally they would have disappeared. This suggests that at least one morphological characteristic of Retzius (5, 6) can be affected by removal of peripheral targets. We thank W. B. Kristan, in whose laboratory this study was carried out, for advice and encouragement. The work was supported by NIH Research Grant NS.20746 and March of Dimes Grant GRMOD-1-864 to W. B. Kristan and NIH Training Grant GM07048 to Joel C. Glover. REFERENCES BATE, M., GOODMAN, C. S., and SPITZER, N. C. (1981). Embryonic development of identified neurons: Segment-specific differences in the H cell homologues. J. Neurosci. 1,103-106. FERNANDEZ, J. H., and STENT, G. S. (1982). Embryonic development of the hirudinid leech Hirudo medicinalis: Structure, development, and segmentation of the germinal plate. J. Embrgol. Exp. Morphol. 72, 71-96. FRANK, E., and WESTERFIELD, M. (1982). The formation of appropriate central and peripheral connexions by foreign sensory neurones of the bullfrog. J. Physiol. (lb&m) 324,495-505. GILLON, J. W., and WALLACE, B. G. (1984). Segmental variation in the

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arborization of identified neurons in the leech central nervous system. J. Camp. Neural. 223,142-148. GOODMAN, C. S., and SPITZER, N. S. (1979). Embryonic development of identified neurones: differentiation from neuroblast to neurone. Nature (London) 280.208-214. KUWADA, J. Y., and KRAMER, A. P. (1983). Embryonic development of the leech nervous system: Primary axon outgrowth of identified neurons. J. Neurosci. 3,2098-2111. LARIMER, J. L., EGGLESTON, A. C., MASUKAWA, L. M., and KENNEDY, D. (1971). The different connections and motor outputs of lateral and medial giant fibers in the crayfish. J. Exp. Bid 54.391-402. LENT, C. M. (1977). The Retzius cells within the central nervous system of leeches. Prog. Neuro&ol. 8,81-117. MANN, K. H. (1962). “Leeches (Hirudinea): Their Structure, Physiology, Ecology, and Embryology.” Pergamon, New York. MITTENTHAL, J. E., and WINE, J. J. (1978). Segmental homology and variation in flexor motoneurons of the crayfish abdomen. J. Comp. Neural. 177,311-334. MULLER, K. J., and CARBONETTO, S. (1979). The morphological and physiological properties of a regenerating synapse in the C.N.S. of the leech. J: Camp. Neurd 185,485-516. PEARSON,K. G., BOYAN, G. S., BASTIANI, M., and GOODMAN, C. S. (1985). Heterogeneous properties of segmentally homologous interneurons in the ventral nerve cord of locusts. J. Camp. Neural. 233,133-145. SHAFER, M. R., and CALABRESE,R. L. (1981). Similarities and differences in the structure of segmentally homologous neurons that control the hearts in the leech, Hirudo medicinalis. Cell Tissue Res. 214,137153. WALLACE, B. G. (1984). Selective loss of neurites during differentiation of cells in the leech central nervous system. J. Camp. Neural. 228, 149-153. WEISBLAT, D. A. (1981). Development of the nervous system. In “Neurobiology of the Leech” (K. J. Muller, J. G. Nicholls, and G. S. Stent, eds.), pp. 173-196. Cold Spring Harbor Laboratory, New York. WEISBLAT, D. A., HARPER, G., STENT, G. S., and SAWYER, R. T. (1980). Embryonic cell lineages in the nervous system of the glossiphoniid leech Helotdella triserialis. Dew. Biol. 76, 58-78. WHITINGTON, P., BATE, M., SEIFERT, E., RIDGE, K., and GOODMAN, C. S. (1982). Survival and differentiation of identified embryonic neurons in the absence of their target muscles. Science (Washington, D. C) 215,973-975. WILSON, J. A. (1979). The structure and function of serially homologous leg motor neurons in the locust. I. Anatomy. J. NeurobioL 10,41-65. WINE, J. J., and KRASNE, F. B. (1972). The organization of escape behavior in the crayfish. J. Exp. Biol 56,1-18.