Proc. Nat. Acad. Sci. USAVol. 70, No. 2, pp. 433-437, February 1973

Structure and Development of Neuronal Connections in Isogenic Organisms:Cellular Interactions in the Development of the Optic Lamina of Daphnia*

(neuronal specificity/serial section reconstruction/crustacea)

V. LOPRESTI, E. R. MACAGNO, AND C. LEVINTHAL

Department of Biological Sciences, Columbia University, New York, N.Y. 10027

Contributed by C. Levinthal, December 4, 1972

ABSTRACT Some details of the growth and initialcellular interactions of optic nerve axons were examinedin a parthenogenetic clone of Daphnia magna. Resultsare summarized as follows: (i) the final structure of theoptic lamina is dependent upon interactions betweengrowing optic nerve fibers and optic lamina neuroblastsclosest to the midplane of the animal, which trigger themorphological differentiation of the neuroblasts; thespecificity of connections is achieved by well-defined se-quences of cell migration in the ganglion; (ii) only one ofthe eight optic nerve axons growing back from each omma-tidium in the eye possesses a structure similar to thegrowth cones seen on termini of nerve fibers growing invitro; and (iii) undifferentiated neuroblasts in the ganglionreact to surface contact by this "lead axon" by envelopingthe axon in a glial-like relationship.

The formation of specific neuronal connections has been stud-ied by various methods in several different biological systems(1, 2). Very little is known, however, about either the morpho-logical or biochemical phenomena that take place at thecellular level and are responsible for the resulting adultneuronal patterns. A straightforward procedure for studyingthe anatomy of embryological events is to examine serialelectron micrographs of identified growing nerve fibers atvarious well-defined stages, especially at the time when thetarget cells are contacted and functional interactions estab-lished. Studies of this type have been made with the computermethod of three-dimensional reconstruction from serialsections (3, 4). We have examined embryos of the smallcrustacean, Daphnia magna, grown under conditions in whichthe organisms reproduce as a parthenogenetic clone. It is pos-sible to obtain embryos that are accurately staged withrespect to time of development and to identify individual opticnerve axons as they grow from the eye to the optic ganglionwhere primary synaptic contacts take place in the adultorganism. The comparison of three-dimensional reconstruc-tions at various stages allows a determination of the sequenceof events from which overall patterns of development can bededuced.In several instances, the nature of the phenomena only

became apparent after the three-dimensional reconstructionswere seen on the computer. But in virtually every case it wasthen possible to confirm the findings by examination of theelectron micrographs themselves. For example, one of theearly interactions between an undifferentiated target cell andthe first optic nerve axon that touches its surface involved awrapping of the target cell around the optic axon. Once thisprocess was recognized, it was possible to examine the detailsin a high-magnification electron micrograph.

There are three general aspects of the interactions betweenoptic axons and ganglion cells that have been clarified in theinitial studies. First, a well-defined temporal sequence ofgrowth and migration can account for most, if not all, of thespatial specificity of the nerves that are establishing their con-nections between the eye and the optic ganglion. Second,only a small fraction of the fibers that grow from the eye to theganglion have the flattened expansions with filopodial-likeprojections at their termini that have been called growth conesfor fibers growing in vitro (5). Third, neuroblasts in theganglion with which the growing axons first make contactrespond with rather elaborate and characteristic changes,which are associated with the onset of morphological dif-ferentiation and seemingly with establishment of functionalcontacts. These results raise a series of questions concerningthe nature of the information transfer between optic fibers andganglion neuroblasts that can, at least in principle, beanswered by further experiments.

MATERIALS AND METHODS

Embryos are staged by isolation of large females with darkovaries and observation of the time at which the eggs arepassed from the ovaries into the dorsal brood pouch. This timeis taken as time zero of development; the total gestation timein vivo is about 55 hr. In almost all animals, no more than 5min elapse between entrance of the first and last eggs into thebrood pouch, the brood size ranging between 10 and 30 forthese large adults. Broods that did not enter the brood pouchwithin a 5-min interval were not used in these studies. Theembryos are allowed to develop in vivo at 240 until the desiredstage is reached (between 26 and 38 hr for these studies), atwhich time they are removed and placed into fixative.Alternatively, only part of the brood is immediately fixed;the remainder are allowed to develop further in vitro at 24°and subsequently fixed. We have found that in order for de-velopment in vitro to occur, embryos must remain in themother for the initial 7 hr of gestation. They may then betransferred to plastic trays containing standard medium at240; development then proceeds with a gestation timeequivalent to that in vivo.

All other techniques are identical to those described in thefirst paper of this series (3) with the following exceptions: thefixative and buffer contain 0.40% NaCl rather than 0.85%(this reduction in osmolarity is necessary to prevent cellshrinkage in embryos); the fixation time is 45 min rather than1 hr, any longer times resulting in extensive leaching of mem-branes; finally, the uranyl acetate staining solution used was3% in 50% ethanol rather than 0.5%.

433

* This is paper no. II in a series. The previous paper is ref. 3.

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434 Cell Biology: LoPresti et al.

FIG. 1. Computer reconstruction of the outline of the em-bryonic eye (arrow) at 35 hr of gestation, with the two lobes ofthe optic ganglion displayed in their proper position relative tothe eye. The approximate areas of eye pigmentation at this stagehave been added to the reconstruction. Bar = 20 Am.

RESULTSThe following results were obtained from studies of fiveembryonic stages, namely 29, 32, 34, 35, and 37.5 hr ofgestation. The uniqueness and reproducibility of each stagehave been checked by looking at various embryos of a singlebrood, fixed at the same time. We find that the relevantfeatures are the same for all specimens at any one stage.In adult Daphnia, each bundle of eight optic nerve axons

from the receptor cells of one ommatidium is surrounded, asthe bundle enters the ganglion, by the cell bodies of five opticlamina neurons, forming a structure similar to an opticcartridge, as seen in other crustaceans and insects (6, 7).The axonst of these five lamina cells receive primary afferentinput from the optic axons (3). There are 22 such structures,11 in each lobe of the optic lamina. In discussing their forma-tion, the term "proximal" refers to locations close to the eye,and hence anterior.

Early in the development of a cartridge, all five lamina cellbodies are located at different anteroposterior levels; progres-sion toward adult morphology involves proximal migration ofthe cells as one of its aspects until all five cell bodies come tobe located in about the same plane. The five lamina cells ofany cartridge can be numbered sequentially 1 through 5, alower number indicating both an earlier association withoptic axons and a more proximal initial location.At these embryonic stages, the ganglion consists of two

symmetric and separate lobes, which develop simultaneously

t The term "axon" refers to a process containing the normallyobserved ultrastructural aspects of axonal processes. For mono-polar invertebratene urons the term is used independently ofwhether the process is pre- or post-synaptic. "Neurite," on theother hand, refers to a process extending from the cell body whosecytoplasmic morphology resembles that of the cell body and thatdoes not yet show characteristic axonal elements such as orga-nized microtubular arrays.

(Fig. 1). In each lobe the 11 sets of optic nerve axons growinto the lamina over a period of about 10 hr, from gestationtimes of 28 hr to about 38 hr. Table 1 shows the numbers andrelative positions of developing optic cartridges present at thefive stages examined within this period (see Fig. 2). Twofeatures of the system are evident in this table. First, thenumber of cartridges formed increases with time in a regularfashion; and second, the older cartridges have migratedlaterally away from the midplane, at any stage the youngestcartridges being closest to the midplane. This observation isillustrated in Figs. 2a and 2b. At 35 hr (Fig. 2a), optic axonslIIa, b, and c occupy the midplane position and interact withthe neuroblasts present there. At 37.5 hr (Fig. 2b), cartridgesIlla, b, and c have moved laterally with the arrival of newneuroblasts in the midplane; these neuroblasts are contactedby optic axons IVa, b, and c, which come from the last om-matidia to differentiate.As a consequence of the sequential growth of optic axons

into the midplane of the lamina, one finds cartridges at dif-ferent stages of development within one embryo. The mostlateral cartridges are always closest to the adult morphology,the most medial are the youngest. We can, therefore, followthe time course of cartridge development both by examiningthe same cartridge at different developmental stages and bycomparing the morphology of cartridges at different medio-lateral levels in one embryo.

In 35-hr embryos, three general stages of cartridge de-velopment can be found. In cartridge IJIb, located adjacent to

TABLE 1. Numbers and relative positions of developing opticcartridges present in both lobes of the optic lamina atfive different

embryonic stages

Hr ofgestation

V

L

D

For reference to Fig. 2: 1 and 2 in this table correspond to laand lb, respectively; 3, 4, and 6 to Ila, Ib, and MIc; 6, 7, and 8to lIla, Illb, and Mlic; and 9, 10, and 11 to IVa, IVb, and IVc.D = dorsal; V -= ventral; L = lateral. The double line representsthe midplane of the animal.

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Development of Neuronal Connections 435

- -

FIG. 2. Tracings made from electron micrographs of sections at equivalent anteroposterior levels of the optic lamina in 35-hr (a) and37.5-hr (b) embryos. Clear areas represent lamina cell cytoplasm; stippled area, glial cell cytoplasm; and slanting parallel-lined areas, thebundles of optic axons. Note that the relative positions of the cartridges are the same in each embryo (e.g., Ia with respect to lIb and lic),but that lateral movement of those present at 35 hr has occurred at 37.5 hr with the arrival of new undifferentiated cells and lead opticaxons (IVa, b, c) in the midplane. In (b) there is seemingly a shift in the positions of Ab and Ha with respect to their positions relativeto the other cartridges in (a). This is solely a function of a difference in sectioning plane between the two animals; the three-dimensionalreconstructions confirm that these two cartridges do maintain the same position relative to the others at 37.5 hr. D = dorsal; V = ventral.Bar = 1Oum.

the midplane at this stage, the central of the eight optic axonshas grown much further than the rest, and is the only one topossess a terminal dilation which resembles the growth conesdescribed in vitro. Filopodia are short and few in number, afeature consistently observed for all growth cones in thesestudies. The seven "follower fibers" in this cartridge growalong, and in contact with, the "lead fiber," but do not possessany terminal dilations (Fig. 3a and b). The lead axon in thiscartridge has contacted only three midplane neuroblasts outof the final number of five; the growth cone makes surfacecontact with the two more distal cells (cells 2 and 3). None ofthe three cells shows any signs of axon proliferation. Incartridge Ilb, located more laterally at the 35-hr stage, thelead and follower relationship is evident, although the fol-

TABLE 2. General structure of cartridge Ia at fivedifferent stages

Hr of gestation

29 32 34 35 37.5

Optic axons+GC 1 1 6 8 2-GC 7 7 2 0 0+S 0 0 0 0 6

Lamina neurons1 U* N A A Ab2 U U N A Ab3 U U N N A4 U U N A5 U U U A

For the eight optic axons, +GC indicates the number of axonswith growth cones, -GC the number without growth cones, and+S the number with nascent synapses. For lamina neurons, U =morphologically undifferentiated; N = distally directed neurite;A = axon; Ab = branched axon. U* for cell 1 at 29 hr indicatesthat the cell is wrapping around the lead axon as shown in Fig. 5.

lowers are further advanced in growth with respect to the leadthan in IJIb. Of the five associated lamina cells, cells 1 and 2have short neurites along the surface of the optic bundle and,like the follower optic fibers, do not show terminal growthcones; cells 3, 4, and 5 are devoid of distal processes. In a mostlateral optic cartridge, Ia, in the same embryos, all eight opticaxons have grown to more or less the same level and allpossess terminal dilations. Lamina cells 1 and 2 have well-defined axons without growth cones, cells 3 and 4 have shortneurites, and cell 5 is morphologically undifferentiated (devoidof processes). The lamina cell axons lie along the bundle ofeight optic axons and are in contact with its surface, thusforming what is finally a fascicle of thirteen nerve fibers. Inaddition, the five lamina cell bodies in the cartridge are

b

FIG. 3. (a) Illustration made from computer reconstructionof a lead axon (L) with two of its follower axons (Fi, F2). (b) Com-puter reconstruction of two different lead axons showing theirgrowth cones (arrows) and one follower axon displayed separately.The receptor cell bodies would be at the top of the figure. Thefollower is not shown in its correct relative position with respectto either lead. Bar = 2 ,m.

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436 Cell Biology: LoPresti et al,

1 1~~~3 t.

a b 4 '

FIG. 4. Computer reconstruction of cells 1 and 3 of cartridgela displayed with only one of the eight optic axons (arrow) at 35hr (a) and 37.5 hr (b). Note that at 35 hr, cell 1 has a well-definedaxon, cell 3 has a short neurite in the eventual direction of theaxon, and the terminal dilation of the optic axon appears to be agrowth cone. At 37.5 hr, the axon of cell 1 is longer with significantbranching, while cell 3 now has a well-defined axon. The terminusof the optic axon at this stage contains forming synapses. Bar =2Mm.

significantly closer to their adult coplanar positioning than arethe five lamina cells of cartridge IIb at this stage.The same and additional stages in cartridge development

have been investigated by tracing cartridge Ia at varioustimes. These results are summarized in Table 2. Computerreconstructions of part of this cartridge at 35 and 37.5 hrare shown in Figs. 4a and 4b. In Fig. 4b, the terminal portionof the optic axon shown contains forming synapses. It followsfrom the data in this table and from data obtained for othercartridges that axon proliferation by lamina cells in a car-tridge occurs in the order 1 to 5, which corresponds to theorder in which the cells are contacted by the lead axon in themidplane.The results for the 35-hr embryo show that the optic nerve

axons from the eight receptor cells in an individual om-matidium are not proliferated at the same time. It was con-sistently found in all newly differentiated ommatidia thatthere is a lead axon and that it is the only one to possess agrowth cone. It was also found that it is always the lead axonfrom each ommatidium that makes the initial cellular contactsin the midplane. Although they are clearly growing, thefollowers lag behind without growth cones, until the stagewhen the lamina cells of the cartridge have begun to pro-liferate axons. The followers then catch up to the lead, and theoptic fibers form synaptic expansions onto the lamina cellaxons.

In both cartridge Ia at 29 hr and cartridges lIIa and b at35 hr, the lead axon has contacted only three midplane cellsof the final five, the growth cone touching the cell surface ofthe two distal neuroblasts (cells 2 and 3). The most proximaland, therefore, the first contacted neuroblast wraps itselfaround the axon in a glial-like relationship (Figs. 5a and 5b).The relationship is clearly transient, as at 37.5 hr the cellbody is adjacent to the entire bundle of eight optic axons(Fig. 5c), rather than surrounding any one of the eight.Three-dimensional reconstructions not shown here demon-

strate that at 34 hr, IIIb is in a configuration where thegrowth cone of the lead axon is touching the surface of twomidplane neuroblasts, confirming, by comparison with 35 hr,that the lead axon first contacts a cell with its growth coneand the cell then wraps around the axon as the growth conemoves distally to contact additional midplane cells. In IVaand b, midplane cartridges at 37.5 hr, the lead axon has con-tacted four neuroblasts, the growth cone touching the sur-face of the distal two cells (cells 3 and 4). Cell 2 is wrappedaround the lead in the same fashion as above, while cell 1 isonly partially wrapped around the bundle of eight optic axons,the followers having grown to this level.

DISCUSSION

Three-dimensional reconstructions of growing optic nerveaxons and of the neurons they contact in the optic laminamake it possible to identify and visualize various stages in thedevelopment of these structures. From a comparison of thesestages and the variations in morphology of the cells involved,several generalizations appear to be justified:

(i) Growth of new axons from receptor cells toward theoptic lamina occurs along the midplane. When the lead axonin the group of eight from one ommatidium has contacted five

a b

C

FIG. 5. (a) Electron micrograph showing the glial-like rela-tionship between a lead axon (L) and one of its recently contactedmidplane neuroblasts (C1) Bar = 0.5,m. (b) Illustration madefrom computer reconstruction of the anterior half of the cellshown in (a). Bar = 1 ,um. (c) Electron micrograph of the samecell (C1) shown in (a), but 2.5 hr later in development. Sevenfollowers now surround the lead axon (L) and C1 is only partiallysurrounding the whole bundle of eight. The second cell originallytouched by the lead fiber in the midplane (C2) now lies almost onthe same anteroposterior level as C1. Bar = 1 ,m. The lead axonin this figure was traced from its receptor cell body in the eye toits cellular contacts in the lamina in a low magnification series ofelectron micrographs. It was the examination of the three-dimensional reconstruction that then led to the higher-magnifi-cation electron micrograph shown in (a).

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Development of Neuronal Connections 437

undifferentiated neuroblasts in one lobe and formed an opticcartridge, the whole structure migrates laterally, even whilemany of its fibers are still growing and making connections.The process is then repeated by the next arriving group ofaxons and by the neuroblasts that have moved into the mid-plane position. Previous results on adults (3) have shown thatthe arrangement of cartridges and the ommatidia that map

on to them is the same in all genetically identical animalsstudied, reflecting the constancy of the developmentalsequence just described.Within any one cartridge, axon proliferation by the five

neuroblasts occurs in the same temporal order as the order inwhich the cells were originally contacted by the lead axon fromthe eye. When surface contact is first made, the neuroblastsare spread out in the anteroposterior direction, and, therefore,the first one contacted is closest to the eye and the last one isfurthest away. As the cartridge develops, the lamina cellaxons grow distally along the surface of the bundle of eightoptic nerve axons to form a bundle of 13 fibers. Concomitantwith axon formation is the proximal migration of the laminacell bodies in order to achieve their final adult positioningwithin a cartridge.

(ii) The overall pattern of cell and fiber migration sum-

marized above allowed us to study in detail the structure ofthe end of the growing axons. In all cases, the initial cellularcontacts are made solely by one axon among the eight fromone ommatidium. This lead axon is the only one to possess a

growth cone; the seven follower axons clearly grow along thesurface of the lead, but end in a rounded tip with no terminalenlargements. Except for the close proximity between the leadand its followers, we have not yet observed any specializedinteractions between them.

(iii) At least for the first two lamina cells to be touched bylead fibers, there is a characteristic response of the neuroblaststhat results in their being wrapped around the lead axon in a

glial-like relationship. This reaction has not yet been observedfor cells 3, 4, or 5 in any of the cartridges studied, but thismay well be because our methods give us only brief glimpsesof the ongoing developmental processes. In any case, thewrapping around lasts for only a short time, and the type ofclose interaction shown in Fig. 5a has not been observed ex-

cept for the lead fiber. By the time the followers have grown

beyond the positions of neuroblasts 1 and 2, these cells haveloosened their grasp on the lead axon and have taken a posi-

tion adjacent to the bundle of eight nerve fibers without theindividual wrapping that is evident earlier.There are several interpretations that seem to us to be sug-

gested by the above results. First, although the adult Daphniaoptic system is connected with a well-defined three-dimen-sional geometry, it is possible to design a model for the eventsthat occur during establishment of neural connections thatonly requires cells or fibers to receive very limited signals todetermine their positions or appropriate targets for con-

nections. Wolpert (8) discussed the need for developing sys-

tems to establish "positional information" to which individualcells could respond, depending on their genes and their physio-logical states. In the present case, the only "positional in-formation" that the growing axons seem to require is an

indication as to whether or not they are near the midplane,and which positions are anterior and posterior. If we assume

that only morphologically undifferentiated neuroblasts areable to act as targets for growing axons, then the relativelycomplex three-dimensional relationships could be determinedif cell behavior were simply controlled by (i) contact with alandmark, such as the midplane column of glial cells of theembryonic eye, and (ii) a more or less stereotypic response ofa neuroblast when it is touched by the growth cone of a leadaxon. A model of this type would imply that the connectionsbetween eye cells and optic ganglion cells are made with what-ever undifferentiated neuroblast happens to be touched bythe growing lead axon. After a cartridge has been completedand moved laterally, the next optic axons that grow back fromthe eye are limited in their available contact to those cellsthat are now undifferentiated midplane cells. All lateral cellsfacing the base of the eye are already occupied and in theprocess of further differentiation.The type of model outlined here suggests that the particular

cell that is contacted by a lead axon is not necessarily identi-fied before being touched. The wrapping around process couldbe thought of as the equivalent of the neuroblasts respondingto a particular lead fiber in the manner, "You touched me,I am yours." A different type of model, more similar to theone proposed by Sperry (9), would imply that the appropriatephrase for then neuroblasts to express before contact is madewould be, "I am yours, come and get me."The first type of model discussed above is functionally

equivalent to that proposed by Jacobson (1), in which neuro-blasts in the optic system are assumed to be "labeled" by thefirst optic nerve fiber to grow back from the eye and make con-tact with them. The transient wrapping around reaction of theneuroblasts could then be thought of as a morphological repre-sentation of Jacobson's proposed labeling.

Finally, it should be emphasized that only one of the eightmembers of a bundle of axons from a single ommatidium hasa growth cone at its terminus. This terminal flattened enlarge-ment resembles those described in vitro, except that wherefilopodia are present they are rather short. The other sevenoptic axons grow without such a terminal specialization, asdo the five axons from the lamina neuroblasts. It is clear thatgrowth per se does not require the so-called "growth cone."It seems, therefore, likely that the growth cone is in factfunctionally associated with the process of "recognizing"cell surfaces or otherwise detecting position in space, and thatthis information can be then shared with other growingprocesses in order to create a spatial arrangement.We thank Yu-Chih Jao for technical assistance and Dorothea

Goldys for illustrations. This work was supported by NIH GrantsRR00442(04) and 5R01-A1-08902(04), and a gift from the RGKFoundation.

1. Jacobson, M. (1970) in Developmental Neurobiology (Holt,Rinehart and Winston, Inc.), pp. 252-260 and 305-330.

2. Gaze, R. M. (1970) in The Formation of Nerve Connections(Academic Press, London), pp. 178-215.

3. Macagno, E. R., LoPresti, V. &, Levinthal, C. (1973) Proc.Nat. Acad. Sci. USA 70, 57-61.

4. Levinthal, C. & Ware, R. (1972) Nature 236, 207-210.5. Yamada, K. M., Spooner, B. S. & Wessells, N. K. (1970)

Proc. Nat. Acad. Sci. USA 66, 1206-1212.6. Hamori, J. & Horridge, G. A. (1966) J. Cell Sci. I, 257-270.7. Trujillo-Cen6z, 0. (1963) J. Ultrastruc. Res. 13, 1-33.8. Wolpert, L. (1969) J. Theor. Biol. 25, 1-47.9. Sperry, R. W. (1963) Proc. Nat. Acad. Sci. USA 50, 703-709.

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