DEVELOPMENTAL BRAIN

RESEARCH

ELSEVIER Developmental Brain Research 85 (1995) 140-145

Short communication

Cyclic remodelling of growth cone lamellae and the effect of target tissue

Gianluca Gallo, Emanuel D. Pollack * Department of Biological Sciences and Committee on Neuroscience, The University of Illinois at Chicago, 1640 West Roosevelt Road,

Chicago, IL 60608, USA

Accepted 16 November 1994

Abstract

We report the existence of cyclical fluctuations in the total size of growth cone lamellae, represented by membrane protrusions and retractions, and show that aspects of this behavior can be regulated by the target tissue for the nerve fibers. The transition of the growth cone from a high to a less motile state, which occurs in the presence of the target tissue, has implications for the mechanisms that underlie nerve fiber elongation during development.

Keywords: Growth cone; Lamella; Cyclic remodelling; Target tissue; Axon guidance; Motility

A number of studies has shown that the target tissue for extending nerve fibers plays an important role in establishing directional growth cues [19], and that the growth cone detects and responds to environmental signals in bringing about oriented growth [33,341. In vitro, the presence of lamella at the growth cone corre- lates with elongation of the nerve fiber [2,3,15,28]. Nerve fiber extension has been described in detail [l,lO] and is considered to result from a sequence of three processes identified as (i) protrusion of lamellar membrane at the growth cone followed by (ii) engorge- ment of the lamellae by cytoplasmic flow and (iii) consolidation of the lamellar membrane into the nascent nerve fiber. Elongation of the nerve fiber tends to be discontinuous with alternating phases of active extension and inactivity [1,3,6,10,13]. For example, spinal motor axons demonstrate cycles of accelerated growth followed by deceleration and intermittent qui- escence [23,26]. The relationship between growth cone type, nerve fiber elongation, and the periodicity of elongation, suggested that periodic phenomena may also be manifested in growth cone behavior. We are able to demonstrate the existence of cyclical fluctua- tions in growth cone lamellae that can be regulated by

* Corresponding author. Fax: (1) (312) 413-1543. e-mail Emanuel.D.Pollack@uic.edu

0165-3806/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94)00201-O

the presence of the target tissue for the extending nerve fiber.

Using cultured spinal cord explants and premuscle limb tissues from larval frog, it has been possible to delineate the changes that the growth cone lamellae undergo as a function of time and cocultured target tissue. Cross-sectional explants, typically about 300 ,um of stage V larval Rana pipiem [32] lumbosacral spinal cords, and epidermis-free hind limb buds, were cul- tured on poly-DL-lysine substratum (1.0 mg/ml) in Sykes-Moore chambers using a defined medium of Eagle’s minimum essential medium in Earle’s salts as previously reported [22]. Limb buds were obtained by amputation at the most proximal end. Following re- moval of the epidermis, the mesenchymal limb buds were placed facing the ventral half of the spinal cord explant at a distance of 0.7 to 1.0 mm. Under cocultur- ing conditions, one limb bud was cocultured with each spinal cord section. A total of 13 cultures from the same number of tadpoles was used for data collection, 7 of which were cord-limb bud cocultures. Growth cones were videotaped for periods of 1 h through an inverted microscope with phase contrast optics and an attached NC-15 CCD color camera (NEC) that fed into a SONY VCR connected in series with an IBM PS/2 computer. For each growth cone studied, framegrabbed video images separated by 1-min intervals were ana- lyzed morphometrically using image analysis software

G. Gallo, E.D. Pollack /Decelopmental Brain Research 85 (1995) 140-145 141

(JAVA, Jandel, Inc.). The area of the lamellae was determined by outlining the perimeter of the lamella, which usually included the central domain of the growth cone. For the growth cones of this study, the central domain was very small relative to the lamellae. This method precludes the possibility of including the en- gorged lamellar area in the measurement of retracted area, thereby providing a more accurate measure of lamellar retractive behavior. Repeated measurements on the same growth cone provided a measurement error of approximately l-2%.

All analyzed growth cones (n = 35) exhibited cyclic changes in the area of the growth cone occupied by lamellae as a function of time (Fig. 1A). In the pres- ence of target tissue, 16 of 20 growth cones examined underwent a steady decline in the average lamellar area, a process we term ‘lamellar rundown’ (Fig. 1B). Rundown is phenomenologically different from contact mediated growth cone collapse (e.g. with heterotypic neurites). Rundown involves a steady decrease in lamellar area over 20-60 min, while contact mediated collapse in our system occurs in 2-5 min (unpublished observations). All of the growth cones (n = 15) exam- ined in the absence of target tissue maintained a con- stant average lamellar area over the l-h observation period without any indication of lamellar rundown.

A B _ 2501

0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (ain)

The lamellar cycle peak-to-peak periods ranged from 2-12 min for all growth cones whether or not limb tissue was present. There was no significant difference between the mean period of the cycle for growth cones in the presence and absence of target tissue (t-test, P = 0.75), the mean period of a lamellar cycle being approximately 4.5 min, nor between the lamellar cycle periods of individual growth cones (Kruskal-Wallis nonparametric ANOVA, corrected for ties, P > 0.05). Since the average lamellar area of individual growth cones ranged from 10 to 387 pm’, the period is not related to the lamellar area of a growth cone. The amplitudes of the lamellar cycle are represented by an ascending (A + 1 and a descending (A - ) phase (Fig. 1A). The average amplitudes of A + and A - for the cycles of growth cones not engaged in lamellar run- down were highly correlated (r* = 0.95, slope = 0.92). This was expected if the growth cone was to maintain a constant average lamellar area over time. The ampli- tudes of both phases of the cycle were positively re- lated to the average lamellar area of the growth cone (Fig. 1C).

Difference images (Fig. 2) produced by overlaying video images demonstrated that changes in the relative proportion of expansion and retraction of lamellae are related to the amplitudes of the ascending and de-

Fig. 1. Characteristics of growth cone lamellar cycling behavior. A: typical fluctuations in lamellar area occurring at a single growth cone over a l-h period in the absence of target tissue. The amplitude of the descending phase of the cycle (A - ) was determined by subtracting the lamellar area at y from the lamellar area at x. The amplitude for the ascending phase of the cycle (A + ) was determined by subtracting the lamellar area at y from the lamellar area at z. B: a representative graph of a growth cone undergoing rundown in the presence of target tissue. Note that the periodicity in lamellar fluctuations is not abolished during rundown. C: the mean amplitude of the phases of lamellar cycles within growth cones is positively correlated to the mean lamellar area of the growth cones (r* = 0.71). Data from growth cones growing in the absence of target tissue is shown. Since A + and A - were found to be highly correlated (see text), the relationship between mean growth cone size and cycle amplitude holds for both A + and A - .

142 G. Gallo, E. D. PoNack / Developmental Brain Research 85 (1995) 140-l 45

Fig. 2. Example of the difference image method used in this study to quantify aspects of growth cone morphological remodelling. Super- imposing of images of a growth cone at intervals of 1-min results in a difference image that shows changed areas of lamellar protrusion (black areas) and retraction (stippled areas). Portions of the lamellar perimeter not undergoing retraction or protrusion can also be visual- ized in this manner (lower right hand side of the difference image). For further description of difference images see [14].

scending phases of the cycle. Overall remodelling of the growth cone was quantified using a motility index [14] defined as M.I. = (area protruded + area re- tracted)/(area of earlier image + area of later image). MI. equals 1.0 if the growth cone lamellae are fully displaced from one minute to the other, and equals 0 if the growth cone lamellae are fully quiescent. Measure- ments of M.I. showed that the motility of the growth cone was similar during both phases of the cycle (Fig. 3A), although the relative amounts of retraction and protrusion differed (Fig. 3B). During A - , the magni- tude of lamellar retraction was greater than that of protrusion while during A + the magnitude of protru- sion was greater than that of retraction.

Although the data suggest that the episodic nature of nerve fiber elongation can be correlated with events at the growth cone, the complexity of the interrelation- ships requires additional consideration. The ratio, R,

A

of the number of l-min intervals during which growth cones spent in A + to the number of 1-min intervals which growth cones spent in A - provides some insight in this regard. R = 1 if the time spent in A + equals that spent in A - . Since the value of R for intervals during which elongation was occurring is less (0.55) than for intervals during which there was no elongation (0.871, elongation is then related to the descending phase (A - ) of the lamellar cycles, a relationship which is not statistically significant (Fisher’s Exact Test, two- tailed, P = 0.33). Observations of elongating nerve fibers showed that the lamellae at the sides of the engorged portion of the lamellae are retracted in a process analogous to consolidation [l,lOl. Hence, the occurrence of nerve fiber elongation in conjunction with a reduction of lamellar area most likely is due to the loss of lamellae through consolidation. In the pre- sent case, consolidation is estimated to take from one to three min and may be initiated during the engorge- ment phase. This implies that the retraction of the lamellae observed during consolidation is not necessar- ily a part of the retraction process that occurs at the growth cone during lamellar cycles; rather it may be a separate, independent event. This analysis similarly explains why R has a value of only 0.55 during elonga- tion instead of zero as expected if nerve fiber elonga- tion were causally related to the decrease in lamellar size during the cycle. The question remains as to what signal causes lamellar retraction during the consolida- tion period following cytoplasmic engorgement.

In both cord only and cord + target tissue conditions there was no relationship between the amount of growth cone lamellae and the growth rate (pm/h) of the nerve fiber (r* = 0.0). Under most in vitro condi- tions growth cones require lamellar presence in order to elongate, but this requirement may be relaxed on highly adhesive substrata [20]. Lamellar production and

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Fig. 3. Comparison of the phases of the lamellar cycle. A: the motility of the growth cone was similar during both the ascending (A + ) and descending (A - ) phases of lamellar cycles (t-test, P = 0.77). Only motility index measurements from growth cones in the absence of target tissue were used in this analysis. However, the motility of growth cones in the presence of target tissue did not vary between cycle phases (t-test, P = 0.84). B: the relative magnitudes of protrusion and retraction of lamellae vary as a function of cycle phase (Pro = protrusion, Ret = retraction). During A + the relative magnitude of protrusion was greater than that of retraction (Welch’s approximate t-test, P = 0.004). During A - the relative magnitude of retraction was greater than that of protrusion (Welch’s approximate t-test, P < 0.0001). Bars are f standard error of the mean.

G. Gallo, E.D. Pollack /Deuelopmenral Brain Research 85 (1995) 140-145

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Fig. 4. Effects of target tissue on lamellar cycles. A: the phase amplitudes of growth cones not undergoing rundown in the presence of target tissue were diminished (A + , bar IV; Welch’s approximate r-test, P = 0.008; A - , bar V; Welch’s approximate t-test, P = 0.004) with respect to those of similarly sized growth cones in the absence of target tissue (A + , bar I; since A + and A - for individual growth cones are strongly correlated this bar is also representative of A - 1. The A + of growth cones in the presence of target tissue undergoing rundown (bar II) was also diminished with respect to the A + of growth cones in the absence of target tissue (bar I; Welch’s approximate f-test, P = 0.004). However, the A - of growth cones in the presence of target tissue undergoing rundown (bar III) did not differ from the A - of growth cones not in the presence of target tissue (indirectly represented by bar I; t-test, P = 0.986). B: the motility of growth cones in the presence of the target tissue not undergoing rundown was decreased during both A + (crosshatch bar; Welch’s approximate f-test, P = 0.0108) and A - (diagonal line bar; Welch’s approximate r-test, P = 0.0022) with respect to the motility of growth cones not in the presence of target tissue (A + , open bar; A - , vertical line bar). C: the % of lamellar perimeter remodelling was found to be diminished in growth cones in the presence of target tissue not undergoing rundown (crosshatch bar) as compared to the % of lamellar perimeter remodelling in growth cones in the absence of target tissue (open bar) (Welch’s approximate f-test, P < 0.0001). Bars are f standard error of the mean.

dynamics seem to be regulated differently than the process underlying the elongation of the nerve fiber [6,12]. While protrusion may be required for elongation to occur, in particular for directed elongation [5], these and other data suggest that it is not coupled to en- gorgement [9,30].

The mesenchymal limb bud, the developmentally appropriate target tissue of the nerve fibers studied here, has been shown to affect both nerve fiber elonga- tion and the morphology of the growth cone [24,25,31]. In the presence of the limb bud, 80% of the observed growth cones underwent lamellar rundown accompa- nied by a transition from the lamellar to the filopodial growth cone form. Both the A + and A - of growth cones not undergoing rundown in the presence of the target tissue were significantly less than those of simi- larly sized growth cones in the absence of target tissue (Fig. 4A). Thus, the limb bud caused a decrease in the amplitudes of both phases of the lamellar cycle. The motility index of these growth cones also was signifi- cantly diminished in comparison to that of spinal cord only cultures during each phase of the cycle (Fig. 4B). Motility decrease was due mainly to a reduction in the

extent of the lamellar perimeter undergoing remod- elling (Fig. 40 Therefore, the effect of the limb bud on the cycle results from a decrease in growth cone motility. The A + of growth cones with target tissue, and undergoing rundown, is not different from that of growth cones with target tissue not undergoing lamel- lar rundown (Fig. 4A). However, the A - of the growth cones undergoing rundown in the presence of the target tissue is indistinguishable from the A - of the growth cones grown without target tissue. As a result, growth cones undergoing rundown have a greater mean magnitude for the descending slope than for the as- cending slope of the cycle, the outcome being a loss of lamellar area with each cycle. The sequential loss of lamellae with each cycle results in rundown. It is note- worthy that as rundown progressed, lamellar cycles remained evident, although with smaller amplitudes. Limb bud tissue had two effects on the lamellar cycles: (i> a diminishing of the cycle amplitudes due to an inhibition of growth cone motility, and (ii) an increase in the frequency of transition between the lamellar and the filopodial morphologic types.

In accord with previous reports [2,3,15,28], we have

144 G. Gallo, E.D. Pollack /Developmental Brain Research 85 (1995) 140-145

found that the presence of lamellae correlates with the capacity of the nerve fiber for growth. However, the amount of growth bears no relationship to the amount of lamellae present at the growth cone, suggesting that growth cone morphology is not directly related to nerve fiber elongation (i.e. engorgement and consolidation). The lamellar cycles at the growth cone arise from an interplay between retraction and protrusion, the rela- tive differences in magnitude possibly being due to mechanisms at the growth cone as suggested by the activities of isolated growth cones [29], or related to the provision of cellular materials via axoplasmic flow in an intermittent manner [11,16,21,27]. Interestingly, the transition from an active (lamellar) to a quiescent (filopodial/blunt ended) growth cone type seems to require a signal from the cell body [8]. It is thus possible that the signal which initiates rundown may not be intrinsic to the growth cone. We have also observed lamellar cycles in growth cones of dorsal root ganglia of larval Rana pipiens (n = 7) and spinal cord explants of larval Xenopus lueuis (n = 3) indicating that lamellar cycling is a property restricted to neither Rana pipiens nor spinal neurites.

Recent studies on the dynamics of membrane recy- cling at the growth cone have demonstrated the pres- ence of membranous organelles in the central region of the growth cone with life spans greater than 5 min, and similar organelles with life spans less than 5 min at the periphery of the growth cone [7]. Whether these cellu- lar components are related to a domain of retraction along the growth cone perimeter remains to be deter- mined. Engorgement occurs in regions of the lamellae that presage a trajectory change by the nerve fiber [17,18,4]. The decrease in growth cone motility, due to a reduction in the extent of the lamellar perimeter undergoing remodelling, may provide the growth cone with sites of reduced activity at which engorgement preferentially can occur, thereby facilitating the di- rected growth of the nerve fiber. Thus, the decrease in growth cone motility may be related to the processes responsible for the production of a tropic response by the nerve fibers to the target tissue. The discovery of growth cone cyclic remodelling provides additional in- sight into the levels of control that bring about directed nerve fiber growth.

We thank R. Dang, F. Demavivas, and D. Phan for invaluable assistance. This study was supported in part by the Campus Research Board of the University of Illinois at Chicago.

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