developmental increases in expression of neurofilament mrna selectively in projection neurons of the...

THE JOURNAL OF COMPARATIVE NEUROLOGY 364:383401 (1996)

Developmental Increases in Expression of Neurofilament mRNA Selectively in

Projection Neurons of the Lamprey CNS

ALAN J . JACOBS, GARY P. SWAIN, AND MICHAEL E. SELZER Department of Neurology and David Mahoney Institute for Neurological Sciences,

University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19 104-4283

ABSTRACT Neurofilaments of the sea lamprey are unique in being homopolymers of a single subunit

(NF-180). Digoxigenin-labeled RNA probes complementary to NF-180 were used to determine the distribution and timing of expression of neurofilament message in the brain and spinal cord of the lamprey. In the brainstem, detection of NF-180 mRNA was restricted to neurons with axons projecting to the spinal cord or the periphery. The majority of brainstem neurons, whose axons project locally, did not express NF- 180 within the detection limits of this technique. NF-180-positive neurons included cells with a wide range of axon diameters, suggesting neurofilament mRNA expression was linked to axon length rather than caliber. To further evaluate this hypothesis, expression was studied in animals of different developmental stages between larvae and adults. In younger (shorter) larvae, the large Mauthner and rhombencephalic Muller cells did not express NF-180 mRNA, even though their axons are among the largest caliber in the animal and extend the entire length of the spinal cord. In contrast, many other reticulospinal neurons, whose axons are smaller in diameter than those of the Muller and Mauthner cells, expressed NF- 180 message throughout larval development. Furthermore, neurons of the cranial motor nuclei did not express NF-180 until later developmental stages and the extraocular motor neurons did not label until metamorphosis. Therefore, while detectable neurofilament mRNA expression in the lamprey is restricted to neurons with long axons, its expression in this population of neurons appears to be developmentally regulated by factors still not determined. I t is postulated that need for NF message is determined by a balance between the volume of axon to be filled and the rate of turnover of NF in that axon. , 1996 Wiley-Lm, Inc

Indexing terms: metamorphosis, reticulospinal, larvae, intermediate filament, digoxigenin

Neurofilaments (NFs) are members of the intermediate filament family and constitute the largest component of the neuronal cytoskeleton, predominantly extending longitudi- nally within axons. They have been detected immunohisto- chemically in many mammalian neurons of the central nervous system (CNS) but are strikingly absent from small neurons with scant cytoplasm (Trojanowski et al., 1986). The reason for this heterogeneity of NF expression still unknown. During neurogenesis the three mammalian NF subunits (NF-L, NF-M, and NF-H) are differentially expressed under the control of separate genes. NF-L and NF-M are coexpressed with vimentin early in embryonic development, when postmitotic neurons bean to grow processes (Shaw and Weber, 1982; Cochard and Paulin, 1984; Carden et al., 1987). NF-H expression replaces that of vimentin during a period that overlaps with synaptogenesis (Shaw and Weber, 1982; Cochard and Paulin, 1984; Pachter and Liem, 1984; Julien et al., 1986; Carden et al., 1987).

Studies such as these, correlating the time of acquisition of particular phenotypic features with the appearance of NFs during development, are complicated in mammals due to the heteropolymeric nature of their NFs. The staggered appearance of these subunits makes it difficult to study the relationship between the presence of NFs and particular neuronal features.

NFs are thought to be involved in the regulation of axon diameter, which is a determinant of conduction velocity and other vital neuronal characteristics. The mechanism for this is not known but in some circumstances, it may be mediated by the phosphorylation of the carboxy terminus sidearm of higher molecular weight NF subunits. I t has

Accepted July 3, 1995. Address reprint requests to Michael E. Sclzer, M.D., Ph.D., Department of

Neurology, University of Pennsylvania Medical Center, 452 Stemmler Hall, 3600 Hamilton Walk, Philadelphia, PA 19104-4283.

L 1996 WILEY-LISS, INC.

584 A.J. JACOBS ET AL.

been suggested that during their transport down the axon (Nixon et al., 1987), phosphorylated NFs constitute a stable, slowly transported pool of cytoskeletal elements (Nixon and Logvinenko, 1986), which therefore accumulate along the length of the axon and cause it to increase in diameter (Nixon, 1993; Nixon et al., 1994). In large myelin- ated fibers, the number of NFs increases proportionally to axon cross-sectional area (Friede and Samorajski, 1970). During development, a progressive increase in NF synthesis and decrease in NF transport rate accompany the radial growth of axons (Willard and Simon, 1983; Hoffman et al., 1984, 1987). This is correlated with the progressive phosphorylation of NFs (Carden et al., 1987; de Waegh et al., 1992). Heavily phosphorylated NFs have generally been found in large caliber axons (Sternberger and Sternberger, 1983; Cohen et al., 1987; Lee et al., 1987; Pleasure et al., 1989; de Waegh et al., 1992). Further evidence that NFs control axon diameter was provided by observations during regeneration of peripheral axons, in which reduced NF message and NF transport correlated with somatofugal reductions in axon caliber following distal axotomy (Hoff- man et al., 1985, 1987; Cleveland et al., 1991). Axon caliber is similarly reduced in the NF-deficient quail expressing a nonsense mutation of NF-L (Osamu et al., 1993). However, overexpression of NF-L to twice baseline levels did not produce overt pathology and had no effect on axon caliber in transgenic mice (Monteiro et al., 1990). Further increases in NF-L expression by mating of two independent, highly expressing transgenic lines did induce aberrant perikaryal and axonal accumulations of NFs and muscle atrophy resembling human motor neuron diseases (Xu et al., 1993). The effect of this magnitude of NF overexpression on axon caliber remains to be determined. Interference with the normal NF composition of neurons by expressing an NF-H fused to p-galactosidase protein in transgenic mice had the striking effect of inducing perikaryal aggregation of all three NF subunits and axons virtually devoid of NFs (Eyer and Peterson, 1994). These NF-deficient axons were of reduced caliber.

Thus, while interfering with the synthesis of NF subunits and their transport into the axon appears to produce axons of decreased caliber, overexpression of NFs has not resulted in larger diameter axons as expected. Rather, excess production of either NF-L or NF-H in transgenic mice induced perikaryal aggregates and proximal axon swellings containing densely packed NFs accompanied by reduced distal axon caliber (Cote et al., 1993; Xu et al., 1993). These findings suggest that appropriate functions of NFs require proportional changes in all three subunits, such that the normal 6:2:1 ratios of the low, middle and high MW (molecular weight) subunits, respectively, are maintained (Shecket and Lasek, 1980).

Studies of the relationship between NF expression and neuronal structure are simplified in the most primitive living vertebrate, the sea lamprey, in which the 10 nm NFs are composed of a single 180 kDa subunit (NF-180; Lasek et al., 1985; Pleasure et al., 1989; Jacobs et a]., 1995), with features found in each of the mammalian NF subunits. In addition, the architecture of the lamprey brain provides an opportunity to study in wholemount the structure of neurons and neuronal nuclear groups concomitant with measurements of gene expression at different stages of development (Swain et al., 1993; Jacobs et al., 1995). The large Muller and Mauthner reticulospinal neurons of the lamprey have been extensively described (Rovainen, 1967;

Nieuwenhuys, 1972; Swain et al., 1993) and permit intera- nimal comparison of gene expression in individually identifiable neurons. We have previously cloned and character- ized the complete NF-180 cDNA and demonstrated mRNA expression in the lamprey brain and spinal cord by in situ hybridization (Jacobs et al., 1995).

In the present report, we show that NF-180 mRNA is expressed selectively in neurons with long axons projecting both within and out of the CNS. These findings suggest that NFs might serve to provide longitudinal stability to axons experiencing stretching and shearing forces during normal body movement. Moreover, NF expression was delayed in many neurons. NF-180 message was not expressed in ocular motor neurons until metamorphosis, when maturation of extraocular muscles and initiation of eye movements occur.

MATERIALS AND METHODS Tissue preparation

Wild-type larval lampreys (4.5-16 cm), and recently transformed adult specimens, were obtained from the Connecticut River in Massachusetts and from streams feeding Lake Champlain in Vermont. Animals were kept in fresh water tanks at 16°C until the day of dissection. They were anesthetized by immersion in 0.1% tricaine methane sulfonate. For wholemount preparations brains were removed, stripped of overlying meninxprirnatiua and choroid plexus, incised along the dorsal midline through the posterior and cerebrotectal commissures and pinned flat on Sylgard strips.

In situ hybridization of digoxigenin-labeled riboprobes

Hybridization of digoxigenin-labeled riboprobes to sectioned and wholemounted lamprey brainstem was performed using the techniques similar to Swain et al. (1994). Tissue was fixed in 2% paraformaldehyde overnight, washed three times in phosphate-buffered saline (PBS) and stored in 70% EtOH at 4°C. Tissue for sectioning was cut into 1-2 cm segments, fixed in 2% paraformaldehyde, washed in PBS, dehydrated in serial ethanols, cleared in toluene and infiltrated with Paraplast.

Digoxigenin-labeled cRNA riboprobes were constructed from subclones of NF-180 cDNA encompassing the long carboxy-terminal sidearm (i.e., LIF13). This resulted in greater specificity of neuronal labeling than was obtained by probes that represent substantial portions of the rod domain (Swain et al., 1994), which contains sequences that may be conserved across all intermediate filament proteins. Two other probes have been used to detect NF-180 mRNA in situ but neither was more sensitive than LIF13. In vitro transcription was performed with an RNA transcription kit (Stratagene, La Jolla, CA) as recommended by the manufac- turer except for the addition of digoxigenin- 11-UTP (Boeh- ringer Mannheim, Indianapolis, IN) at a 35:65 ratio with unlabeled UTP. Transcripts were fragmented into approximately 100 nucleotide polymers through incubation with sodium carbonate (0.1 M Na2COt3, 65"C, 85 minutes) and ethanol precipitated.

Prior to hybridization, tissue sections were stripped of paraffin in toluene and rehydrated through serial ethanols. Both whole mounted and sectioned tissue was hybridized to digoxigenin-labeled riboprobes with the following method: tissue was washed in PTw (0.1% Tween-20 in PBS),

EXPRESSION OF LAMPREY NEUROFILAMENT (NF-180) 385

prehybridized at 55°C in hybridization solution (50% deion- ized formamide; 5 x SSC; 100 pgiml Torula yeast RNA; 100 pgiml wheat germ tRNA; 50 pgiml heparin; 0.1% Tween- 20) followed by hybridization overnight at 55°C in the same solution plus 400 ngiml digoxigenin-labeled cRNA. The next day, specimens were washed in hybridization solution a t 60°C followed by room temperature washes in PTw and PBT (0.1% bovine serum albumin; 0.2% Triton X-100 in PBS). Alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (0.75 Uiml, Boehringer Mannheim, India- napolis, IN) were diluted 1:1,000 and applied to tissue overnight at 4°C. Tissue was washed sequentially in PBT and SMT (100 mM NaCl, 50 mM MgClz, 100 mM Tris pH 9.5,0.1% Tween-20). The chromogenic reaction was carried out in ice-cold SMT containing 175 pg/ml5-Bromo-4-chloro- 3-indolyl-phosphate and 350 pgiml 4-Nitro blue tetra- zolium chloride for 30 minutes on ice in the dark. Finally, specimens were washed in PBS, dehydrated in serial ethanols, cleared in methyl salycilate and mounted under DPX (Fluka, Switzerland).

Retrograde labeling of reticulospinal neurons Reticulospinal neurons were retrogradely labeled by

horseradish peroxidase (HRP) applied to a transection of the spinal cord of 2 small larvae 7.9 and 8.0 cm long as previously described (Swain et al., 1993). The reticulospinal system of larger larvae and adults had been described previously but that of the smaller larvae had not. Briefly, animals were anesthetized by immersion in 0.1% tricaine methane sulfonate and a pledget of Gelfoam soaked in 40% HRP placed at the site of a complete spinal transection performed at 25% of body length. The incision was closed with 2-3 sutures and the animal returned to fresh water and maintained at 4°C for 7-21 days. Animals were then reanesthetized and the CNS removed as described for in situ hybridization. Retrograde transport of HRP into brainstem neurons was detected by incubation in Hanker-Yates reagent, followed by dehydration in serial ethanols, clearing in methyl salicylate and wholemounting under DPX.

Semiquantitative evaluation of message level The level of expression of NF-180 message was estimated

in brainstem wholemounts following hybridization to digoxigenin-labeled NF- 180 ribonucleotide probes. Labeling of certain giant reticulospinal neurons in small larvae appeared to be weaker than in the same neurons of large larvae and adults. To determine whether this difference was statistically significant, a semiquantitative scale from 0 to 4 was used to grade the intensity of labeling for NF-180 message in individual giant reticulospinal neurons of young and old larvae. The criteria for each score were as follows: 0, no label observed in intact neurons identified by light and differential interference contrast microscopy at 40 x magn- fication; 1, faint staining limited to the perimeter of the perikaryon; 2, faint staining throughout the cytoplasm; 3, intense staining throughout the cytoplasm but not suffi- ciently dark to obscure the nucleus; 4, dark staining with completely obscured nucleus. To confirm the reproducibil- ity of this scale, scoring was performed independently by two observers, one of whom was blind to the stage of development of the animals. There was a 95% concordance between the two raters. Eighty-nine percent of the discrep- ancies were a difference of one grade level and none were more than two levels. The distribution of scores for each identified reticulospinal neuron was compared between

small larvae (6-8 cm long; 2-3 years old) large larvae (12-14 cm long; 4-5 years old) and adults. Contingency table analysis of the labeling scores of each identified giant reticulospinal neuron was performed to determine chi- square and P values.

Immunohistochemistry Labeling for NFs was performed by a metal-enhanced

immunoperoxidase technique on paraffin sections cut at 6 pm. Most of the methods were those previously described (Pleasure et a]., 1989), except as indicated below. The antibody used was LCM3, a core-specific mAb raised against lamprey NF-180. Since this mAb binds to a phosphorylation- independent epitope, it labels all axons. LCM3 was diluted 1:1,000 in 0.1 M Tris and 2% fetal calf serum, applied to sections and incubated overnight at 4°C. Sections were then reacted by the avidin biotin complex method as described in the ABC kit (Biomedia, Foster City, CAI, using metal- enhanced DAB substrate (Pierce, Rockford, IL).

Electron microscopy Miiller axons were imaged by electron microscopy in

transverse 60-70 nm sections of Epon-embedded spinal cord, using methods as previously described (Lurie et al., 1994). Sections were photographed with a Zeiss 10 EM at 1 2 . 5 0 0 ~ .

RESULTS Wholemount in situ hybridization for NF-180 The pattern of cellular expression of NF-180 mRNA was

analyzed by in situ hybridization of digoxigenin-labeled cRNA probes specific for the carboxy-terminus sidearm of NF-180 (Fig. 1). Sidearm rather than core sequences were selected for construction of riboprobes because the long carboxy-terminus distinguishes NFs from all other intermediate filament proteins. The chromogenic reaction cata- lyzed by alkaline phosphatase linked to the anti-digoxigenin Fab fragments yielded a blue precipitate, which accumu- lated within intact neurons of the wholemounted brainstem. Intensity of labeling for NF-180 message was variable among neurons in the same brainstem preparation, ranging from no label in intact neurons visible by light and differential interference contrast microscopy to intense staining which completely obscured the normally visible nucleus of giant reticulospinal neurons. This differential labeling of neurons exposed to identical hybridization and development conditions presumably reflects different concentrations of NF-180 message within the cell. The intensity of cell labeling was also dependent on the concentration of ribonucleotide probe in the hybridization mix, the hybridization temperature and the incubation time with chromogen. Labeling visible to the naked eye typically appeared after 10-15 minutes and attained maximal levels by 30 minutes. Longer incubation times in chromogen increased nonspecific background, but staining of weakly labeled or unlabeled cells did not increase further with incubation for up to 60 minutes. Absence of labeling did not necessarily imply total absence of message, but only that the levels were below the detection limits of the technique. Many cells that were unlabeled stained for NFs by immunohistochemistry (Swain et al., 19941, had axons that contained NFs (see below) and therefore presumably expressed some low level of NF-180 mRNA. The distribution of NF-180 expression in

386

Head lOkD

Core 36.5 kD

A.J. JACOBS ET AL.

Sidearm 76 kD

Proline Glutamate K P E K P Rich Rich Rich Rich Rich

_ - -

9 1027 1110 1 1 103 414 479

c t t t t r t r t t e r E E E E E E E E E E E E E E E E E E E E E E E E .AAAAA

77 1 908 929 1027 1110

I LIF13 I

602

Fig. 1. Diagram of the cDNA subclone (LIF-13) of the single lamprey neurofilament subunit, NF-180, used to generate cRNA probes for in situ hybridization. This subclone encodes the carboxy terminus

the brains of larval and adult lampreys is illustrated in Figure 2.

NF-180 mRNA expression in mature larval lamprey

Figure 3 illustrates diagramatically the major anatomical features of the lamprey brainstem and the locations of identified neurons and neuron groups that strongly expressed NF-180 mRNA. In the mature larval brainstem, NF message was also detected in several cranial nerve nuclei and in scattered cells of the alar plate. Expression was absent in cells of the telencephalon, olfactory lobes and optic tectum. Message was similarly not detectable in ependymal cells, areas rich in glial cell bodies such as the fiber tracts of the posterior commissure and habenulopeduncular tract, and in the small interneurons located throughout the CNS. No cells were labeled by NF-180 sense RNA probes. NF-180 mRNA was abundant in all of the identifiable giant reticulospinal neurons (Muller and Mauthner cells) and in the majority of other spinal-projecting neurons (Figs. 4 and 5).

Within the diencephalon, typically 20-30 medial piriform- shaped neurons weakly expressed NF-180. Reaction prod- uct was asymmetrically distributed towards one pole of these cells (Fig. 4A). NF-180 mRNA was detected in approximately 60 small, spindle-shaped and rounded multi- polar neurons within the nucleus reticularis mesencephali (Fig. 4B; Nieuwenhuys, 1972). These cells correspond in shape and location to mesencephalic reticulospinal neurons (Swain et al., 1993). At the anterior end of the rhombencephalic basal plate (Fig. 4C), expression was detected in a cluster of 10-20 bipolar and spindle-shaped neurons similar in location, shape and number to isthmic reticulospinal neurons (Swain et al., 1993). Within the rhombencephalic basal plate, two rostrocaudal columns of NF- 180-expressing neurons were observed that corresponded in location to the somatomotor and visceromotor columns (Nieuwen- huys, 1972). Abundant NF-180 mRNA was detected in neurons of the nucleus reticularis rhombencephali (Fig. 5B). Labeled cells represented approximately 80% of the neurons in the middle and medial inferior reticulospinal groups that were previously labeled retrogradely by HRP injected into the spinal cord (Swain et al., 1993). In the alar

sidearm of NF-180 subunit, encompassing amino acids 602 to 1110 and the 3' untranslated region including the polyA tract. Numbers correspond to amino acid residues of NF-180.

plate, octavomotor neurons (Fig. 5C) and medullary dorsal cells (presumed primary sensory neurons; Fig. 5D; Nieuwen- huys, 1972) also expressed NF-180. The large octavomotor neurons send axons into the ventral funiculi of the spinal cord and synapse directly on spinal motoneurons and lateral spinal interneurons (Rovainen, 1979; Ronan, 1989).

NF-180 mRNA was readily detected within neurons of cranial nerve nuclei V, VII, IX and X (Figs. 4C, 5A,B), but was conspicuously absent in neurons of the larval oculomotor, trochlear and abducens nuclei. NF message was addi- tionally detected in neurons of the trigeminal sensory ganglion, anterior lateral line ganglion, ganglion of nerve VIII, and the posterior lateral line ganglion (Fig. 6). Expres- sion was also weakly detected within a subset of cells of the olfactory epithelium and outer layers of the retina (not shown).

The spinal cord of the lamprey exhibits primitive struc- tural features, most notably an absence of myelin and lack of penetrating blood vessels. There are approximately 105 nerve cells in the lamprey spinal cord (Rovainen, 1967), many of which are recognizable as to type based on position, morphology and physiology. Several labeled neurons were found in most 8-pm sections sampled over a 3-cm length of spinal cord, spanning the gill region and part of the abdomen. Since there are approximately 1,000 neurons per segment and since each segment is approximately l-mm- long, there should be approximately 8 neurons per 8-pm section. From this we conclude that NF-180 expression was detected in a substantial fraction of spinal neurons, including the dorsal cells and edge cells shown in Figure 7. It is likely that many of the labeled cells included other previously described neurons, such as motoneurons and lateral interneurons, which could not be unambiguously identified in these randomly sampled paraffin sections. We did not identify types of neurons that were systematically unlabeled. Expression was absent from ependymal cells surrounding the central canal and from neuroglia within the central gray.

Developmental regulation of NF-180 mRNA expression during larval growth

Miillar and Mauthner cells. Expression of NF-180 message in reticulospinal neurons and in neurons of the cranial

Fig.

2.

Dev

elop

men

tal

chan

ges

in

the

patt

ern

of N

F-18

0 ex

pres

sion

in th

e la

mpr

ey b

rain

. In

sit

u hy

brid

izat

ion

who

lem

ount

s w

ere

prep

ared

us

ing

brai

ns

of

anim

als

at t

hree

sta

ges.

A

: Sm

all

(65-

mm

-lon

g) la

rva,

app

roxi

- m

atel

y 2

year

s ol

d. B

: Lar

ge (

147-

mm

- lo

ng) l

arva

, app

roxi

mat

ely

5 ye

ars

old.

C: Y

oung

adu

lt (

145-

mm

-lon

g). N

ote

the

prog

ress

ive

over

all

incr

ease

in

NF-

180

mes

sage

and

the

inc

reas

e in

the

num

ber

of n

euro

ns e

xpre

ssin

g N

F-18

0 m

RN

A w

ith d

evel

opm

ent.

Spec

ifica

lly, n

ote

the

incr

ease

in

labe

ling

of m

any

gian

t re

ticul

ospi

nal n

euro

ns (

solid

arr

ows)

bet

wee

n th

e sm

all a

nd l

arge

larv

ae a

nd th

e pr

ogre

ssiv

e in

crea

se in

the

num

ber

and

size

of l

abel

ed n

euro

ns in

the

trig

emin

al (

open

arr

ows)

and

othe

r cr

ania

l m

otor

nuc

lei d

urin

g de

velo

pmen

t. Se

e Fi

gure

3 f

or c

ytoa

rchi

tect

onic

dia

gram

. Sc

ale

bar,

250

km

.

inf.

I

Figure 3

I2 ?

1:

I6

0m.n.

' 0

0 8

8 1

m.d.c. 1 t

Telencephalon

Mesencephalon

Rhombencephalon


nerve nuclei increased during larval growth. In young larva (approximately 2-3 years old, based on length), NF-180 mRNA was undetectable or present at very low levels in many of the identified reticulospinal neurons and cranial nerve nuclei. Mauthner cells, for example, rarely expressed detectable levels of NF in young larva (Fig. 8). Expression in the middle rhombencephalic giant reticulospinal neurons (i.e., Bi, Bs, B4, etc.) was more variable, ranging from no detectable expression within these cells (Fig. 8A) to prominent expression in some of the bulbar neurons. In the anterior rhombencephalic (isthmic) reticulospinal neuron group, approximately half the number of neurons labeled for NF-180 message in the young larva compared to their older counterparts. However, retrograde labeling with HRP demonstrated that the full complement of these neurons, as well as each of the 32 identified reticulospinal neurons, projected to at least 25% body length in even the smallest larvae (Fig. 8B). Thus, the difference between large and small larvae in the number of isthmic reticulospinal and identified reticulospinal neurons expressing NF-180 message did not reflect an increase in their absolute numbers, nor in the number that projected to the spinal cord.

Considering the variability of staining seen in reticulospinal neurons within brainstem preparations of different aged animals, it seemed probable that the labeling intensity reflected different magnitudes of NF-180 mRNA expression in these cells. To test the hypothesis that NF expression increased during larval growth, specimens were grouped by length and evaluated for degree of NF expression. A semiquantitative grading scale was applied to each of the identified reticulospinal neurons as described in Materials and Methods. Whole number scores from 0 to 4 were gven to neurons based on the intensity of labeling. Examples of neurons with each of these scores are shown in Figure 9. The distribution of these scores between small larvae (less than 10 cm in length) and larger larvae (12-14 cm in length) was evaluated by contingency table analysis, with chi-squared and p values calculated for each identified reticulospinal neuron. The average labeling score for these neurons (Fig. 10) demonstrated significantly ( P < 0.05) increased levels of NF-180 message in several of the giant reticulospinal neurons of large larvae compared to smaller larvae, especially B1 and the Mauthner neuron. There was no further increase in intensity of labeling between large larvae and adults, and indeed for some cells, there was a modest but statistically significant reduction (Fig. 10).

The reduced amount of NF-180 message in the Muller and Mauthner perikarya of smaller larvae was not accompanied by an equivalent reduction in NF protein concentra-

Fig. 3. Schematic drawing of mature (5-year-old) larval sea lamprey brainstem showing major anatomical features and the locations of identified neurons and neuron groups that express NF-180 are shown. The view is from the dorsal (ventricular) surface following removal of mesencephalic and rhombencephalic choroid plexus, transection of the cerebrotectal commissure and obex, and lateral reflection of the alar plate. All of the identified spinal-projecting neurons, i.e., MIA, I,-6, B,_6, the Mauthner (Mth) and auxiliary Mauthner (Mth') neurons, strongly expressed NF-180 mRNA, as did neurons of the isthmic reticulospinal (isth. retic. 1, middle reticulospinal (mi-.) and medial inferior reticulospinal (m.i.r.) cell groups. NF-180 message was also detected in some cranial nerve nuclei (Vm, VII, IX and X), neurons of the octavomotor nucleus (0m.n.i and in medullary dorsal cells (m.d.c.i. s.l., sulcus limitans; s.m.i., sulcus medianus inferior; inf., infundibulum; habbped. tr., habenulopeduncular tract. Note: The precise location of isthmic reticulospinal axon decussation is unknown.

tion in their axons. In Figure 11A, C and E, transverse sections of spinal cord from the gill region are immunola- beled with LCM3, a mAb specific for the core region of NF-180. Staining of the giant axons was equivalent in small larvae, large larvae and adults, despite an approximately fourfold increase in axon diameters between the small larvae and adults. Electron micrographs of Muller axons also showed that NF packing densities were similar at all three stages (Fig. 11B, D and F). If anything, NFs appeared to be slightly less densely packed in the adult, consistent with the inverse correlation noted between axon diameter and NF packing density (Pijak et al., 1995). Thus, compared with the same neurons in large larvae and adults, Muller and Mauthner cells of small larvae require lower message levels to maintain comparable concentrations of NF in their axons. Perhaps this is due to the great increase in volume of these axons in older, larger animals (see Discussion).

The number of cells expressing NF-180 within each of the cranial nerve nuclei was also reduced in the smaller larvae compared to large larvae (Fig. 12), although the most dramatic changes occurred with transformation to the adult form (see below).

Cranial nerve nuclei.

Metamorphic initiation and expansion of NF-180 expression in cranial nuclei

Metamorphosis of Petromyzon marinus from larva to young adult spans a period of 4-6 months and typically begins when the animal has achieved a length of 12-13 cm (Potter et al., 1978). Changes during metamorphosis in- clude eruption and maturation of the eye, modification of the prebranchial region into an oral disc specialized for suction, development of teeth and a tongue-like piston, and the conversion of gill structure to allow for tidal respiration. During this period, internal changes to the opisthonephric kidney, endostyle and intestine are occurring along with differentiation of the retina, growth and lamination of the optic tectum (Rubinson, 1990), enlargement of the trigeminal motor nucleus and thickening of the spinal cord (Damas, 1935, 1951). Concomitant with these morphologic changes, we found alterations in brainstem expression of NF-180 mRNA.

Within the trigeminal motor nucleus, there was a dramatic increase in the number of small (10-15 Fm diameter), round cells expressing NF-180 during larval growth and metamorphosis (Fig. 12). The area occupied by these NF-180-expressing cells increased approximately fourfold between the larval and young adult stages 14.0 x lo4 Fm2 (n = 5) and 16.5 x lo4 pm2 (n = 4), respectively]. A less dramatic but prominent increase in NF-180 expression also occurred within the glossopharyngeal nucleus. The terri- tory of the labeled cells enlarged approximately 3.5-fold, along with a dramatic increase in the intensity of labeling of neurons within this nucleus. The size of the facial nucleus expanded to a lesser degree (less than %fold) and cells within this nucleus did not appear to increase their level of NF expression. Prior to transformation, NF-180-expressing cells were distributed in clusters throughout the vagal nucleus (Fig. 5B). In the young adult, gaps between these clusters were filled in with labeled cells as the nucleus increased 2- to %fold in width. An increase in the number of NF-180-expressing cells also occurred in the medial inferior reticulospinal cell group, in which the number of neurons projecting to at least 258 of body length has been shown to expand twofold during transformation (Swain et al., 1995).


Fig. 4. Distribution of NF-180 mRNA in wholemounted larval lamprey diencephalon (A), mesencephalon (B), and anterior rhombencephalon (C). Message was detected by nonradioactive in situ hybridization with digoxigenin-labeled cRNA probes. NF-180 message was primarily found in neurons with long projection axons (Swain et al., 19931, such as the identified giant reticulospinal neurons W 4 , neurons of the isthmic reticulospinal group (isth. retic.), and neurons of

the trigeminal motor nucleus (V,,,). Staining was not observed in neurons of the oculomotor, trochlear or abducens nuclei. Inset shows the locations of photomicrographs. Note that this specimen appears to have duplications of 13 and Is, located just rostra1 to each of these cells; such duplications are not uncommon. Labels for identified neurons are immediately adjacent to the cells. Scale bars, 100 pm.

-4 I I I I I I I I I I I I I I I I I


The most striking increase in NF-180 expression took place within ocular motor neurons. Preceding metamorphosis, expression of NF-180 mRNA was not detected within motoneurons of the oculomotor, trochlear or abducens nuclei. Following transformation into the young adult, neurons within each of these nuclei labeled with NF-180 cRNA. In a location corresponding to the oculomotor nucleus (Fritzsch et al., 19901, a cellular band of small NF-180-expressing neurons (10-15 pm diameter), ventro- medial to identified Miiller cell M3 and extending from the ventricle to the ventral meningeal surface, was seen in postmetamorphic lampreys (Fig. 13A, B). Several large neurons (25 pm) located at the edge of the oculomotor nerve root were also labeled. Caudal to the oculomotor nucleus, in the dorsal mesencephalic tegmentum between the mesencephalon and rhombencephalon, was a cluster of bipolar-shaped NF-180-expressing cells which, by their form and location, corresponded to trochlear motor neurons (Fig. 13C; Fritzsch and Sonntag, 1988; Fritzsch et al., 1990; Rodicio et al., 1992). Streaming from this nucleus ventrad toward the oculomotor nucleus, were numerous spindle-shaped neurons that weakly expressed NF-180. The shape and location of these motoneurons are reminis- cent of nascent postmitotic trochlear motor neurons migrat- ing to a more dorsal position as described by Fritzsch and Northcutt (1993). Postmetamorphic expression of NF-180 was similarly detected within motoneurons of the abducens nucleus (not shown). NF-180 cRNA also labeled an elon- gated group of bipolar-shaped cells occupying a submenin- geal position ventral to the VIIth and IXth nuclei in the young adult hindbrain, but not in that of larva.

DISCUSSION Selective expression of NF-180 mRNA

in projection neurons In situ hybridization indicated that, within the detection

limits of the wholemount technique, NF-180 message in the brainstem was expressed primarily in neurons whose axons projected into the spinal cord or out nerve roots. In large larva and adults, for example, NF-180 expression was detected only in the cranial motor nuclei and in reticulospinal neurons. However, these neurons represent less than 10% of the total neurons in the lamprey brainstem (Rovainen, 1982). Despite the wide range of axon diameters in the reticulospinal neurons, labeling for NF- 180 message was quite homogeneous. NF-180 expression in the large Miiller and Mauthner reticulospinal neurons, whose axons can attain diameters of 50-100 pm, was qualitatively similar to that of smaller reticulospinal and cranial motor neurons, some of whose axons have diameters less than 1 pm. Thus, NF-180 expression appeared to be independent of axon caliber and present at detectable levels only in neurons whose axons extend substantial distances within the CNS or into the peripheral nervous system (PNS), suggesting that neurons require active NF transcription only when their axons grow beyond a certain length or volume.

In adult mammals, immunoreactivity for each of the three NF subunits has been seen in the cerebral and cerebellar cortices, hypothalamus, spinal cord and retina (Trojanowski et al., 1986). Large numbers of neuronal perikarya, axons and dendrites were stained in each of these regions. In axon-rich areas of the subcortical white matter, optic nerve and spinal cord, all identifiable axons

expressed each of the three NF subunits. However, almost a third of neuronal perikarya in any given area did not stain for NF subunits. These were typically smaller neurons with minimal cytoplasm. Neurons of the granular layers of the cerebral cortex and granule cell neurons of the cerebellar cortex were also devoid of NF immunoreactivity (Tro- janowski et al., 1986). The axons of these neurons probably terminate locally rather than projecting out of the brain region of origin. As pointed out by the authors, it cannot be assumed that the absence of perikaryal labeling implies a lack of NF synthesis. The axons of these small neurons were not identified and might contain NF. Except for NF-H, the distribution of mRNA expression for the mammalian NF subunits has not been systematically investigated. In the rat, NF-H mRNA is expressed in thalamic neurons, in the motor neurons of the spinal cord and brainstem and in the corticospinal and reticulospinal neurons (see below). Thus, in mammals as in the lamprey, although axons in general contain NFs, these are detected in perikarya primarily when the neurons have long axonal projections.

The message for an NF-M-like subunit isolated in the elasmobranch Torpedo californica was expressed exclu- sively in the electric lobe and did not appear to be present in the brain, spinal cord, or electric organ (Linial and Scheller, 1990). Other NF subunits have not yet been reported in this species. In the squid, two low molecular weight subunits (NF-60 and NF-70) were expressed in the neuropil of the optic lobe, the large neurons of the stellate ganglion, axoplasm of the giant axon and in a few other locations (Szaro et al., 1991). Messenger RNA for a larger subunit, NF-220, has been similarly detected in neurons of the squid stellate ganglion (Way et al., 1992). I t would seem that in the squid, as in vertebrates, high levels of NF expression are required primarily in neurons with long axons.

Late appearance of NF-180 mRNA during development

The developmental increases in NF- 180 expression detected in reticulospinal neurons were fundamentally different from that reported for the development of higher vertebrates. In the developing mammalian CNS, the onset of NF-L and NF-M immunoreactivity coincides with the time of initial axon extension, while NF-H expression is delayed until after synaptogenesis (Cochard and Paulin, 1984; Carden et al., 1987). In the lamprey, however, NF- 180 message was not detected in numerous reticulospinal neurons or in some cranial nuclei of young animals despite projection of their axons into the spinal cord and peripheral structures. Identified Muller and Mauthner reticulospinal neurons did not express detectable levels of NF-180 message until the larvae were 3-4 years old. Yet, axons of these neurons were demonstrated to project well into the spinal cord. Despite dramatically higher levels of NF-180 expression in older larvae, young larvae were behaviorally indistinguishable from older ones. Further- more, oculomotor neurons, whose axons project to immature extraocular muscles in the larva (Rodicio et al., 1992; Fritzsch and Northcutt, 1993), did not express NF-180 mRNA until metamorphosis. Thus, despite a definite neuronal phenotype and projection of axons to distal targets, many brainstem projection neurons did not express detectable levels of NF message until later stages of development.

The delayed onset of NF expression may reflect anatomical changes such as dendritic sprouting or radial or longitudinal axon growth. During transformation, the eyes of the

Figure 5


Fig. 6. Sections through cranial ganglia hybridized to NF-180 cRNA probes. A Most neuronal perikarya of the anterior lateral line (all) and trigeminal sensory (V,) ganglia stained for NF-180 message. Neurons of

the vestibulocochlear and posterior lateral line ganglia (B and C, respectively) also labeled for NF-180. sr, sensory root. Scale bars, 100 ym.

Fig. 7 NF-180 mRNA in neurons of larval lamprey spinal cord. Labeled neurons were identified in virtually all 8-pm serial sections through the spinal cord. A Section of spinal cord illustrating labeling of a dorsal cell IDC) and a probable motoneuron (MN). Expression is

absent from ependymal cells surrounding the central canal (star) and neuroglia. B: Spinal cord section with labeling in an edge cell (EC) and several unidentified neurons. Scale bars, 100 ym.

Fig. 5. NF-180 mRNA expression in the middle and posterior rhombencephalon. A Labeling was found in identified neurons B,-6, the Mauthner (Mth) and auxiliary Mauthner neurons (Mth’), and neurons of the middle reticulospinal group (Rovainen, 1967, 1982; Swain et al., 1993J, trigeminal motor nucleus (V,) and facial nucleus ( V H . B: NF-180 message in the glossopharyngeal (IX), vagal (X) and medial inferior reticulospinal (mir) nuclei. C: Labeled neurons in the octavomotor nucleus. D: Large labeled neurons in the rhombencephalic alar plate. presumed to be medullary dorsal cells. Scale bars: 100 ym in A and B; 25 Fm in C and D.

lamprey emerge from their subcutaneous location and the head grows considerably in width, even though the length of the animal remains relatively constant. Thus, the extraocular motor axons become substantially longer and this may require the development of a more elaborate cytoskeleton. However, the Muller and Mauthner axons are already several cm long at the earliest stages of development examined in this paper and thus, a length hypothesis alone does not entirely explain the low level of NF message. Moreover, these axons clearly do have plentiful NF protein, as seen in electron micrographs and immunohistochemi-


Fig. 8. Absence of NF-180 message from identified reticulospinal neurons of young lamprey larva. A Message was not detected in identified reticulospinal neurons BI-s or the Mauthner neuron (dashed outlines) of this young animal (2-3 years old, based on length) despite its abundant presence in other nonidentified reticulospinal neurons of the middle reticulospinal group. Weak labeling is seen in the auxiliary

Mauthner (Mth’) and Bs neurons. B: Retrograde labeling of spinal- projecting neurons in a similar age animal by HRP injection into a complete spinal cord transection. All of the identified reticulospinal neurons project axons to 25% or more of body length by 2-3 years of age. Scale bars, 100 IJ-m.

cally. This paradox may be explained by the fact that the NF contained in these giant axons is highly phosphorylated, while that in smaller caliber axons is not (Pleasure et al., 1989; Pijak et al., 1995). Post-translational phosphorylation of the carboxy sidearm of NFs as they migrate down the axon may greatly slow NF transport and turnover (Nixon and Logvinenko, 1986; Nixon et al., 1987, 1994; Nixon, 1993), thus allowing a lower message level to maintain large amounts of NF in the axon. If so, the rates of NF transport and degradation may be lower in the giant axons than in smaller caliber axons. Thus a smaller level of message would be required per axon volume in a large caliber axon than in a fine axon of equal length.

NF immunoreactivity first appeared in mouse embryos at about the 15-somite stage, with expression restricted to a few cells and processes in the midbrain and hindbrain, but not in more rostra1 and caudal portions of the developing CNS (Cochard and Paulin, 1984). NF expression progressed rapidly from this stage and within one day (at E 10.51, NF immunoreactivity appeared in the telencephalon, diencephalon, rhombencephalon and all but the most caudal portion of the spinal cord (Cochard and Paulin, 1984). Similarly in the rat, NF labeling first appeared on E l 2 in neurons of the mesencephalon, in clusters of cells destined to form the trigeminal, facial and vestibulocochlear ganglia and in cells

on the outer edge of the rhombencephalon (Carden et al., 1987). Telencephalon and retina were NF-negative at this stage.

In mammalian CNS, NF expression was generally pre- ceded by that of vimentin and in most cases, expression of the two types of IFs were mutually exclusive (Cochard and Paulin, 1984). Although NF and vimentin can coexist in some immature CNS neurons, vimentin synthesis has presumably ceased by the time neurites are extended, since neuronal processes stained only with anti-NF antibodies (Cochard and Paulin, 1984). In these newly formed neurons, only NF-L and NF-M subunits were detectable and rapidly attained adult levels, while NF-H production was delayed (Shaw and Weber, 1982, 1983; Willard and Simon, 1983; Pachter and Liem, 1984; Harry et al., 1985; Nona et al., 1985; Carden et al., 1987). Moreover, NF-H expression continued to increase during the first few postnatal weeks and was expressed only in neurons with well developed axons. This and the fact that neurons in culture express primarily NF-L and NF-M (Lee, 1985; Shaw et al., 1985) led Carden et al. (1987) to suggest that recruitment of NF-H is triggered by factors related to synaptogenesis. Because lamprey NF is a homopolymer, the possible different functions of NFs are not segregated among the subunits and the late expression of NF-180 might reflect a delay in attaining


Fig. 9. Examples of scoring of NF180 mRNA expression in wholemounted lamprey brainstem. A semiquantitative scale from 0 to 4 was applied to the intensity of staining by digoxigenin-labeled cRNA. The morpholopcal criteria for each score are discussed in Materials and Methods. A Small larva with no labeling in the Mauthner neuron (scored ’0’) despite prominent staining of other reticulospinal neurons including the auxiliary Mauthner (Mth’). B: Variable labeling intensity in bulbar reticulospinal neurons. Faintly stained neurons (‘1’) with

label limited to perikaryal perimeter were scored as one, while neurons with similar staining intensity distributed throughout the cytoplasm (‘2’) were scored two. Arrowheads point to cells identified by adjacent score. C: A score of three was applied to neurons with more intense staining throughout the cytoplasm, but without obscuring the nucleus ( ‘3’ ) . Open arrowhead points to the nucleus of this Mauthner cell. D: The maximum score of four was restricted to darkly stained neurons in which label completely obscures the nucleus (‘4’). Scale bars, 100 km.


Small Larva LargeLarva Adult

4 7 * I *

* p < 0.05

*

B1 B2 B3 B4 B5 B6 Mauthner Aux Mauthner

MI M2 M3 M4 I1 I2

Reticulospinal Neuron

Fig. 10. Developmental changes in NF-180 mRNA expression in the identified large reticulospinal neurons. Average labeling scores are graphed for each identified reticulospinal neuron in 7 small larvae, 5 large larvae and 5 adult specimens. Since the neurons are paired, for each identified neuron, a total of 14, 10 and 10 cells were examined in the three developmental stages, respectively. Occasionally, cells were missing or could not be identified, so that the number of cells included in the calculated means was not always identical. The cell I6 was difficult to identify with certainty in the adult because it was superim- posed on the expanded trigeminal motor nucleus. Therefore, its label-

full NF function in higher vertebrates until NF-H is incorporated into the cytoskeleton. Expression of additional NF subunits in the lamprey or splice variants of NF- 180 is unlikely, as a thorough immunochemical analysis of intermediate filaments in the lamprey CNS detected only three different glial keratins and only one NF subunit (Pleasure et al., 1989; Merrick et al., 1995).

Detailed reports of neuron-specific NF mRNA expression in the developing and adult mammalian CNS are limited. Except perhaps for NF-H message expression in the rat, studies aimed at outlining the specific types of neurons that

I3 I4 I5 I6

ing intensity is not graphed in the adult. Note the generally reduced labeling intensity for NF-180 message in the smaller larvae. Nearly all reticulospinal neurons increased their expression of NF-180 during larval growth. This trend was statistically significant ( P < 0.05, chi- squared) in several of the identified bulbar neurons (B,-B4 and B6), in M? and in the Mauthner neuron. Note also the more modest reduction in labeling of several cells in the adult compared with the large larva. Asterisk indicates statistically significant change from the immediately preceding developmental stage.

make large amounts of NFs have not been performed. Since neurons are heterogeneous in their NF contents, and since the timing of NF expression during development is highly variable, detailed analysis of which populations of neurons produce NFs is essential to a full understanding of NF functions. In the adult rat brain, NF-H mRNA was detected by in situ hybridization in most of the cranial motor nuclei (e.g., mesencephalic oculomotor nucleus, facial nucleus, trigeminal motor nucleus, hypoglossal nucleus), in cortical pyramidal cells, in reticulospinal and thalamic nuclei and in the ventral horn of the spinal cord (Dautigny et al., 1988;


Fig. 11. Neurofilaments are abundant in axons of giant reticulospinal neurons at all three examined stages of development. Panels on the left are 6-pm transverse paraffin sections through the gill region of spinal cords immunostained with LCM3, a mAb that is specific for the core of NF-180 and labels axons of all sizes. A, C, and E are from a small larva (55-mm-long), a large larva (125-mm-long) and a young adult ( l.lO-mm-long), respectively. The large labeled profiles in the ventrome- dial columns are Miiller axons (M); the solitary large labeled profiles in the lateral columns are the Mauthner axons (Mth). Note the progres-

sive increase in diameter of these axons during development, concomitant with the overall increase in spinal cord dimensions. Scale bar, 25 Fm. Panels on the right are 12,500~ electron micrographs of trans- versely sectioned giant axons also in the g l l region of animals size- matched to those on the left. Each electron micrograph is through the most dorsomedial Miiller axon in the section, possibly Mil (Rovainen et al., 1973). Note that the density of NFs is comparable in small and large larvae, and slightly reduced in the adult. Scale bars, 2 pm.


Fig. 12. Increased NF-180 message expression in trigeminal motor nucleus during larval growth and following metamorphosis. Within the trigcminal motor nucleus Wm), a dramatic increase in the number of NF-180-labeled neurons is seen concomitant with a fourfold enlarge- mcnt in nuclear area during metamorphosis. A NF-180 expression in V,, and Isthmic reticulospinal neurons of young a larva 8 cm long. The stars indicate the locations of the I, and Mauthner (Mth) cells, which

are not labeled in this animal. B: Older, larger larva 13 cm in length. Note the increase in the number of neurons staining for NF-180 message and the onset of expression in I2 reticulospinal and Mauthner (Mth) neurons. C: Recently transformed adult lamprey with dramatic increase in area of V,, and in number of NF-180-positive neurons. Arrows point to the cells identified by adjacent labels. Scale bars, 100 pm.

Fig. 13. Emergence of NF-180 expression in ocular motor neurons in adult lamprey. A Oculomotor neurons adjacent and deep to M:i labeled with NF-180 cRNA in this newly transformed adult lamprey (compare with premetamorphic specimen in Fig. 6B). B: Labeled

neurons of the oculomotor nucleus viewed from the meningeal (ventral) surface. C: Trochlear neurons in the dorsal mesencephalic tegmentum. NF-180 message was not detected in the oculomotor and trochlear nuclei of larvae prior to transformation. Scale bars, 100 pm.

Roussel et al., 1991). NF-H mRNA has also been detected in the larger neurons of the hippocampus, cerebellum and thalamus, although the magnitude of expression is much lower than seen in the cranial nerve nuclei (Roussel et al.,

1991). In embryonic rats, NF-H message could be detected in ventral portions of the metencephalon and myelencepha- lon as early as E l 7 (Roussel et al., 1991). Thus, as with expression of NF-180 message in lamprey larvae, the


distribution of NF-H mRNA expression in rat brain is concentrated in brainstem motor and reticulospinal nuclei and expands dramatically during embryonic development.

Among nonmammalian species, developmental expression of NF has been studied previously only in the frog. In Xenopus laeuis, three proteins homologous to the mammalian NF subunits have been identified. An additional type IV intermediate filament protein (XNIF) has been found with a molecular weight smaller than that ofXenopus NF-L (Szaro et al., 1991). XNIF and the middle molecular weight Xenopus NF (XNF-M) are the first NF subunits to appear in the embryo. They appear roughly simultaneously in reticu- lar neurons, Mauthner neurons, cranial motor neurons, ventral longitudinal axon tracts, primary motoneurons and in dorsal Rohon-Beard cells (Szaro et al., 1989; Charnas et al., 1992). Many of these NF-positive neurons are large reticulospinal neurons, like those of the lamprey. In Xeno- pus, some of these neurons (Rohon-Beard cells) degenerate during metamorphosis. Others may persist but are no longer identifiable after the growth of neighboring reticulospinal neurons. Nevertheless, the pattern seems to be similar to that in lamprey. Neurons most strongly expressing NF are those with long axonal projections.

These observations must be interpreted with caution because many factors contribute to the expression of NF protein by a neuron. Since NFs are rapidly transported into the axon, it may well be that the number of neurons whose axons contain NFs exceeds the number whose perikarya stain positively for NF protein. Furthermore, from the present observations, the number of neurons whose axons contain NFs exceeds the number of neurons that label for NF message by in situ hybridization, at least in younger animals. The distribution of NFs within a neuron depends not only on the rate of transcription, but on factors regulating translation and turnover. As indicated above, the phosphorylation of NFs may reduce their transport and turnover (Nixon and Logvinenko, 1986; Nixon et al., 1987), which has been postulated to be a mechanism by which NFs accumulate along the axon and regulate axon diameter (Nixon et al., 1994). In the lamprey, a correlation has been found between the caliber of an axon and the phosphorylation state of its NFs (Pleasure et al., 1989; Pijak et al., 1995). This could lead to the apparent paradox that a neuron with a very large axon volume due to a large diameter may require less NF message than one with a smaller axon volume due to a small diameter. In order to understand the level of message required, it is necessary to factor in both the volume of axon to be filled and the rate of NF message turnover. In Muller and Mauthner neurons, whose axons contain highly phosphorylated NFs, NF message might not be observed until the volume becomes so great that it overwhelms the turnover-retarding effect of NF phosphorylation. Much more information will be needed concerning the regulation of NF synthesis, transport and turnover before the significance of variations in message levels will be fully understood.

Conclusions It has long been speculated that NFs determine the

diameter of axons. This is based on observations in mammalian peripheral nerve that there is a nearly constant density of NFs among axons of different diameter (Friede and Samorajski, 1970; Hoffman et al., 1984) and that following a distal axotomy, axon caliber decreases in a somatofugal manner at a rate coincident with the rate of NF transport

(Hoffman et al., 1985). However, axon diameter might dictate the number of NFs rather than the reverse. In fact, studies of transgenic mice overexpressing either the NF-L or NF-H subunit showed a progressive neuropathy with perikaryal and proximal axon accumulations of NFs and a reduction in overall axon caliber (Monteiro et al., 1990; Cote et al., 1993; Xu et al., 1993). Thus, the true function of NFs remains unclear. The pattern of expression of NF in the lamprey and in the other species where it has been studied could be interpreted to indicate that NFs are required for axons to span regions of the nervous system rather than that they are required to control axon diameter. This might be the case if NFs were involved in the mechanisms of slow axonal transport. Alternatively, NFs might provide axial and longitudinal strength that protect axons from being sheared by stretching or torque forces such as those experienced by peripheral nerves or the spinal cord during limb or body movements. The axons of local interneurons would presumably experience much smaller shearing forces and might therefore not require the additional strength imparted by intermediate filaments. I t is interesting in this context that the extraocular motoneurons of the lamprey did not express NF-180 until metamorphosis to the adult form. While the ocular motoneurons project to the extraocular muscles in larvae, the eyes remain subcutaneous and relatively immobile until transformation. In the adult, these extraocular motor axons are subject to stresses caused by eye movement and therefore might require additional mechanical strength provided by NFs.

ACKNOWLEDGMENTS This research was supported by NIH Grant NS14837.

A.J.J. was supported as an NIH Medical Scientist Training Program Trainee, Grant 5-T32-GM07170. Thanks to D.S. Pijak for help with immunohistochemistry and electron microscopy.

LITERATURE CITED Carden, M.J., J.Q. Trojanowski, W.W. Schlaepfer, and V.M.-Y. Lee (1987)

Two-stage expression of neurofilament peptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J. Neurosci. 7:3489-3504.

Charnas, L.R., B.G. Szaro, and H. Gainer (1992) Identification and developmental expression of a novel low molecular weight neuronal intermediate filament. protein expressed in Xrnopus laeuis. J. Neurosci. 12:3010- 3024.

Cleveland, D.W., M.J. Monteiro, P.C. Wong, S.R. Gill, G.J.D., and P.N. Hoffman (1991) Involvement of neurofilament in the radial growth of axons. J. Cell Sci. Suppl. 1585-95.

Cochard, P., and D. Paulin (1984) Initial expression of neurofilaments and vimentin in the central and peripheral nervous system of the mouse embryo in viva. J . Neurosci. 412080-2094.

Cohen, R.S., H.C. Plant, S. House, and H. Gainer (1987) Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion. J. Neurosci. 7:2056-2074.

Cote, F., J:F. Collard, and J:P. Julien (1993) Progressive neuronopathy in transgenic mice expressing the human neurofilament heavy gene: A mouse model of amyotrophic lateral sclerosis. Cell 7335-46.

Damas, H. (1935) Contribution a l’etude de la metamorphose de la tete de la Lamproie. Arch. Biol. 45171-227.

Damas, H. (1951) Observations sur le developpement des ganglions craniens chez Lampetru flr~viatilis (L.1. Arch. Biol. 62:65-95.

Dautigmy, A. , D.D. Pham. C. Roussel, J.M. Felix, J.L. Nussbaum, and P. Jolles (19881 Thct large neurofilament subunit (NF-H) of the rat: cDNA


cation of neurofilament proteins by phosphate during axoplasmic transport in retinal ganglion cell neurons. J. Neurosci. 7.1145-1158.

Nixon, R.A., P.A. Paskevich, R.K. Sihag, and C.Y. Thayer (1994) Phosphory- lation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: Influences on regional neurofilament accumulation, interneurofilament spacing, and axon caliber. J. Cell Biol. I26:1031-1046.

Nona, S.N., S.C. Trowell, and J.R. Cronley-Dillon (1985) Postnatal developmental profiles of filamentous actin and of 200 kDa neurofilament polypeptide in the visual cortex of light- and dark-reared rats and their relationship to critical period plasticity. FEBS Lett. 186.111-115.

Osamu, O., Y. Gahara, T. Miyake, H. Teraoka, and T. Kitamura (1993) Neurofilament deficiency in quail caused by nonsense mutation in neurofilament-L gene. J . Cell Biol. 121,387-395.

Pachter, J.S., and R.K.H. Liem (19841 The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve. Dev. Biol. 103:200-210.

Pijak, D.S., G.F. Hall, P.J. Tenicki, AS . Boulos, D.I. Lurie, and M.E. Selzer f 1995) Neurofilament spacing, phosphorylation and axon diameter in regenerating and uninjured lamprey axons. J . Comp. Neurol. fin press).

Pleasure, S.J., M.E. Selzer, and VM-Y. Lee 119891 Lamprey neurofilaments combine in one subunit the features of each mammalian NF triplet protein but are highly phosphorylated only in large axons. J . Neurosci. 9:698-709.

Potter, I.C., G.M. Wright, and J.H. Youson (1978) Metamorphosis in the anadromous sea lamprey, Petromyzon marinus. Can. J . Zool. 56:561- 570.

Rodicio, M.D., E. de Miguel, M.A. Pombal, and R. Anadon f 1992) The origin of trochlear motoneurons in the larval sea lamprey, Petromyzon marinus. An HRP study. Neurosci. Lett. 138.19-22.

Ronan, M. (1989) Origins of the descending spinal projections in petromy- zonid and myxinoid agnathans. J . Comp. Neurol. 281,5448.

Roussel, G., J.M. Felix, A. Dautigny, D. Pham-Dihn, C. Hindelang, P . Jolles, and J .L. Nussbaum (1991) In situ localization of NF-H neurofilament subunit mRNAs in rat brain. Dev. Neurosci. I3:98-103.

Rovainen, C.M. (1967) Physiological and anatomical studies on large neurons of the central nervous system of the sea lamprey Petromyzon marinusl. I. Muller and Mauthner cells. J . Neurophysiol. 30.1000-1023.

Rovainen, C.M. ( 1979) Electrophysiology of vestibulospinal and vestibulore- ticulospinal systems in lampreys. J. Comp. Physiol. 42.745-766.

Rovainen, C.M. (1982) Neurophysiology. In M.W. Hardisty and I.C. Potter (ed): The Biology of Lampreys, Vol. 4A. London: Academic Press, pp. 1-136.

Rovainen, C.M., P.A. Johnson, E.A. Roach, and J.A. Mankovsky (1973) Projections of individual axons in lamprey spinal cord determined by tracings through serial sections. J. Comp. Neurol. 149.193-202.

Rubinson, J. (1990) The developing visual system and metamorphosis in the lamprey. J. Neurobiol. 21:1123-1135.

Shaw, G., and K. Weber (1982) Differential expression of neurofilament triplet proteins in brain development. Nature 298.277-279.

Shaw, G., and K. Weber (19831 The structure and development of the rat retina: An immunofluorescence microscopal study using antibodies specific for intermediate filament proteins. Eur. J . Cell Biol. 30.219-232.

Shaw, G., G.A. Banker, and K. Weber (1985) An immunofluorescence study of neurofilament protein expression by developing hippocampal neurons in tissue culture. Eur. J . Cell Biol. 39.205-216.

Shecket, G., and R. Lasek (1980) Preparation of neurofilament protein from guinea pig peripheral nerve and spinal cord. J . Neurochem. 35.1335- 1344.

Sternberger, L.A., and N.H. Sternberger f 1983) Monoclonal antibodies distinguish phosphorylated and non-phosphorylated forms of neurofilaments in situ. Proc. Natl. Acad. Sci. U.S.A. 80:6126-6130.

Swain, G.P., J.A. Snedeker, J. Ayers, and M.E. Selzer f 19931 The cytoarchi- tecture of spinal-projecting neurons in the brain of the larval sea lamprey. J. Comp. Neurol. 336.194-210.

Swain, G.P., A.J. Jacobs, E. Frei, and M.E. Selzer f 1994) A method for in situ hybridization in wholemounted lamprey brain: Neurofilament expression in larvae and adults. Exp. Neurol. 126256-269.

Swain, G.P., J . Ayers, and M.E. Selzer (1995) Metamorphosis of spinal projecting neurons in the brain of the sea lamprey during transformation of the larva to adult: Normal anatomy and response to axotomy. J . Comp.

cloning and in situ detection. Biochem. Biophys. Res. Comm. 154:1099- 1106.

de Waegh, S.M., V.M.-Y. Lee, and S.T. Brady (1992) Local modulation of neurofilament phosphorylation, axon caliber, and slow axonal transport by myelinating Schwann cells. Cell 68.451-463.

Eyer, J., and A. Peterson (19941 Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament- P-galactosidase fusion protein. Neuron 12.389-405.

Friede. R.L., and T. Samorajski (1970) Axon caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice. Anat. Rec. 167.379-387.

Fritzsch, B., and R.G. Northcutt (1993) Origin and migration of trochlear, oculomotor, and abducent motor neurons in Petromyzon marinus. Dev. Brain Res. 74:122-126.

Fritzsch, B., and R. Sonntag (1988) The trochlear motoneurons of lampreys fLarnpetra fluuiatilis): Location, morphology, and numbers as revealed with horseradish peroxidase. Cell Tissue Res. 252223-229.

Fritzsch, B., R. Sonntag, R. Dubuc, Y. Ohta, and S. Grillner (1990) Organization of the six motor nuclei innervating the ocular muscles in lamprey. J . Comp. Neurol. 294.491-506.

Harry, J.G., J .F . Goodrum, and P. Morel1 f 1985) The postnatal development of glial fibrillary acidic protein and neurofilament triplet proteins in rat brain stem. Internat. J. Dev. Neurosci. 3.349-352.

Hoffman, P.N., G.W. Thompson, J.W. Griffin, and D.L. Price (1984) Control of axonal caliber by neurofilament transport. J. Cell Biol. 99.705-714.

Hoffman, P.N., G.W. Thompson, J.W. Griffin, and D.L. Price (1985) Changes in neurofilament transport coincide temporally with alteration in the caliber of mans in regenerating motor fibers. J. Cell Biol. 101.1332- 1340.

Hoffman, P.N., D.W. Cleveland, J.W. Griffin, P.W. Landes, N.J. Cowan, and D.L. Price f 19871 Neurofilament gene expression: A major determinant of axonal caliber. Proc. Natl. Acad. Sci. U.S.A. 84.3472-3476.

Jacobs, A.J., J. Kamholz, and M.E. Selzer (1995) The single lamprey neurofilament subunit (NF-180) lacks multiphosphorylation repeats and is expressed selectively in projection neurons. Molec. Brain Res. 29.43- 52.

Juiien. J .P. , D. Meyer, D. Flavell, J. Hurst, and F. Grosveld (19861 Cloning and developmental expression of the murine neurofilament gene family. Brain Res. 387.243-250.

Lasek, R.J., M.M. Oblinger, and P.F. Drake (1985) Function and evolution of neurofilament proteins. Ann. N.Y. Acad. Sci. 455.462-478.

Lee, V.M.-Y. ( 1985) Neurofilament protein abnormalities in PC12 cells: Comparison with neurofilament proteins of normal cultured rat sympa- thetic neurons. J. Neurosci. 5:3039-3046.

Lee, V.M.-Y., M.J. Carden, W.W. Schlaepfer, and J.Q. Trojanowski (1987) Monoclonal antibodies distinguished several differentially states of the two largest rat neurofilament subunits (NF-H and NF-M) and demon- strate their existence in the normal nervous system of adult rat. J. Neurosci. 7.3474-3488.

Linial, M., and R.H. Scheller (1990) A unique neurofilament from Torpedo electric lobe: Sequence, expression, and localization analysis. J. Neuro- chem. .54:762-770.

Lurie, D.I., D.S. Pijak, and M.E. Selzer (1994) The structure ofreticulospinal axon growth cones and their cellular environment during regeneration in the lamprey spinal cord. J . Comp. Neurol. 344.559-580.

Merrick. S.E., S.J. Pleasure, D.I. Lurie, D.S. Pijak, M.E. Selzer, and V.M.-Y. Lee f 1995) Glial cells of the lamprey central nervous system contain keratin. J . Comp. Neurol. 355.195-210.

Monteiro, M.J., P.N. Hoffman, J.D. Gearhart, and D.W. Cleveland (1990) Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: Increased neurofilament density in axons without affecting caliber. J. Cell Biol. lllr1543-1557.

Nieuwenhuys, R. (19721 Topological analysis of the brainstem of the lamprey Lampetra fluuiatilis. J. Comp. Neurol. 145:165-178.

Nixon, R.A. (19931 The regulation of neurofilament protein dynamics by phosphorylation: Clues to neurofibrillary pathobiology. Brain Pathol. 3-29-38,

Nixon, R.A., and K.B. Logvinenko (1986) Multiple fates of newly sysnthe- sized neurofilament proteins: Evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons. J. Cell Biol. 102:647-659.

Nixon, K.A.. S.E. Lewis, and L.A. Marotta (19871 Post-translational modifi- Neurol. 362.453467.


Szaro. B.G., V.M.-Y. Lee, and H. Gainer (1989) Spatial and temporal expression of phosphorylated and non-phosphorylated forms of neurofilament proteins in the developing nervous system ofXenopus laeuis. Dev. Brain Res. 48:87-103.

Szaro, B.G., H.C. Pant, J. Way, and J. Battey (1991) Squid low molecular weight neurofilament proteins are a novel class of neurofilament protein. J. Biol. Chem. 225:15035-15041.

Trojanowski. J.Q., N. Walkenstein, and V.M.-Y. Lee (1986) Expression of neurofilament subunits in neurons of the central and peripheral nervous system: An imrnunohistochemical study with monoclonal antibodies. J. Neurnsci. 6:650-660.

Way, J., M.R. Hellmich, H. Jaffe, B. Szaro, H.C. Pant, H. Gainer, and J. Battey ( 1992) A high-molecular-weight squid neurofilament protein contains a lamin-like rod domain and a tail domain with Lys-Ser-Pro repeats. Proc. Natl. Acad. Sci. U.S.A. 89:6963-6967.

Willard, M.B., and C. Simon (1983) Modulations of neurofilament axonal transport during the development of rabbit retinal ganglion cells. Cell 35551-559.

Xu, Z., L.C. Cork. J.W. Griffin, and D.W. Cleveland (1993) Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease. Cell 73:23-33.

developmental increases in expression of neurofilament mrna selectively in projection neurons of the...

Documents