,Veurru~~~nce Vol 36.No. I.pp.217-237,1990 Printed inGreatBritain

0306.4522:YO $3.00+ 0.00 PergamonPressplc , lY901BRO

ONTOGENY OF THE FILAMENT PROTEIN,

NEURONAL PERIPHERIN, EMBRYO

INTERMEDIATE IN THE MOUSE

C. M. TROY,K. BROWN, L. A. GREENE and M. L. SHELANSKI Department

of Pathology and Center Surgeons, Columbia

for Neurobiology and Behavior, College University, New York, NY 10032, U.S.A.

of Physicians

and

Abstract--The expression of peripherin, a type III neuron-specific intermediate filament protein, and the middle neurofilament subunit were studied in the mouse embryo using immunofluorescence staining. The earliest staining for both proteins is seen at embryonic day 9 in the myelencephalon, initially as fiber staining followed by cell body staining in the developing facial and acoustic nuclei. As the embryo develops, there is rostra1 as well as cdudal extension of peripherin and staining is seen in the trigeminal ganglia, nerve fibers and in the enteric nervous system. As the spinal cord forms there is anti-peripherin staining in developing motoneurons of the anterior horns while little cell body staining is seen for the middle neurofilament subunit. Both antibodies stain the developing dorsal root and its entry zone, but peripherin is found in the secondary sensory and commissural fibers while the middle neurofilament subunit is not. While both proteins are found in the neurons of the dorsal root ganglia, their distribution varies. The larger peripheral cells of the ganglia contain both proteins while the smaller more central cells. constituting over 60% of the cells in the ganglia, contain only peripherin. A similar picture is found in the sympathetic ganglia where there are cells which contain peripherin, middle neurofilament subunit or both. but where the majority of the neurons have only peripherin in their cell bodies. Peripherin is not found in the developing retina or in the adrenal medulla. Peripherin is also completely absent from cell bodies in the cerebral and cerebellar cortices. These results indicate that peripherin is found in development only in regions in which it is found m the adult. It can either co-exist with neurofilaments in the same neuron or the two may be independently expressed.

Intermediate filament proteins are a group of closelyrelated molecules which form 8-10 nm filaments in the cytoplasm of a variety of cell types.14,‘* These proteins have been divided into subtypes based on their sequences and genomic organization.” One type, the neurofilament proteins, is found exclusively in neurons, and is comprised of a triplet of proteins, designated as NF-L (- 68,000 mol. wt), NF-M (- 150,000 mol. wt) NF-H and (- 210,000 mol. wt).“~” The distribution of NF in the nervous system is widespread, but does not include every neuronal cell type. Such filaments are thought to play a role in the growth and maintenance of neuritic structure. Several lines of evidence have recently suggested that there is another intermediate filament protein specific to neurons (for review see Greene’). The initial evidence was presented by Portier et ~1.” who detected a novel protein of apparent A4, = 57,000 in cultured neuronal cells and differentiating neuroblastoma cells. Subsequently, the protein was shown

to be present in the peripheral nervous system (PNS), but not in non-neural tissues. Based on its presence in the PNS and apparent absence in the CNS, this protein was called “peripherin”.‘5,‘7 The properties of peripherin suggested that it was a member of the intermediate filament family. In 1987 Parysek and Goldman” described a nerve growth factor (NGF)-inducible 57,000 mol. wt protein in PC12 cells that was cross-reactive with. but not identical to, vimentin, and that stained as a filamentous network. This protein was found in the PNS and limited regions of the CNS of the rat.” An antiserum raised against this protein stained nerve bundles and fibers in the tongue, small intestine and adrenal gland. The adrenal medulla was also reported to show staining of chromaffin cells and ganglion cells. In addition, there was staining of small caliber fibers in the sciatic nerve and spinal cord dorsal roots, as well as of dorsal white columns and of neurons in the dorsal root ganglia. In the brain, there was labeling of components of cranial nerves and of fibers in the granular layer of the cerebellum and the corticospinal tract in the brainstem. While characterizing NGF-inducible mRNAs in PC12 cells, Leonard rt a/.‘s.‘h found one (clone 73). that encoded a protein with strong homology to all intermediate filament proteins, but that was distinct in sequence from those of known intermediate

Ahhrc,riurions: DRG, dorsal root ganglion; EX, embryonic day X; FITC. fluorescein isothiocyanate; GFAP, glial fihrillary acidic protein; Ig, immunoglobulin; MAPS, mlcrotubule-associated proteins; NF-(L, M, or H), (low. middle or high) neurofilament protein; NGF, nerve growth factor; PBS, phosphate-buffered saline; PNS, peripheral nervous system; PO. postnatal day 0. 217

filament protein sequences. The sequence of the protein’” and the organization of its gene” indicate that it is a member of the type III intermediate filament protein family, which includes vimentin, glial hbrillary acidic protein (GFAP) and desmin and that it is not of the type IV family which is comprised of the NF triplet proteins. Using in situ hybridization in adult rat tissue. Leonard e/ a/.Ih showed that the clone 73 mRNA is expressed in sympathetic. sensory and ciliary ganglion neurons. in ventral horn motoneurons. in the neurons of the motor nuclei of cranial nerves 111, IV, V. VI, VII. X and XII. and in the dentate nucleus. the red nucleus. the tegmental nucleus of Gudden and scattered reticular neurons, all regions which appear early on the evolutionary scale. Leonard et al.” noted that most of the neurons expressing this message had axons which were partially located outside the CNS, and that most of these axons were long. This distribution was quite distinct from that of the NFs, which are present in a broader spectrum of neurons. Aletta e/ rrl.’ identified and characterized the protein encoded by the clone 73 mRNA and concluded that this intermediate filament protein is the same as peripherin and the intermediate filament protein studied by Parysek et rd.” and Parysek and Goldman.‘2’3 This has been confirmed by recent cloning studies.“,” The development of neuronal intermediate filaments in the mammalian nervous system has been studied by several groups.4.5.7 Cochard and Paulin’ examined the expression of neurofilament proteins in the mouse embryo. They found that the NF triplet proteins first appeared at embryonic days 9 and IO (E9-EIO) and was preceded by the appearance of vimentin. Both vimentin and the NFs co-existed within the same neurons for a short period of time followed by the loss of vimentin. By birth the NF proteins were expressed by the same population 01 neurons that possess NF in the adult. The discovery of peripherin as a biochemically distinct, neuron-specific intermediate filament protein raises a number of issues that can be addressed in part by studying its ontogeny. For instance, when does peripherin first become expressed during neurogenesis? Does it correlate with defined developmental processes? Is there transient expression of this protein? How is peripherin regionally distributed within neurons as they mature? What is the sequence of expression of peripherin in different neuronal populations? How does the development and distribution of peripherin compare with that of the NF intermediate filaments? A preliminary report by Escurat et d.’ has indicated that in the rat embryo

peripherin appears soon after neurons become po\tmitotic and that its expression is limited II) the populations that express it in the adult. In the present study, we have used double-labeling immunotluorcscence to compare the ontogeny and expression al’ peripherin and NF-M in the mouse. Our findings extend prior work to the cellular leve1.7 “” EXPERIMENTAL Preparation

PROCEDURES

of embryos

Embryos from C57 Bl/6 mice were used. The day of appearance of the vaginal plug was designated EO. Birth was on day E19. Embryos were removed from anesthesized pregnant mice at various times between E6 and E17. Early embryos (E66E9) were not removed from the uterus prior to fixing; older embryos (El&El7) were fixed after removing the head. Embryos were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h and then rinsed in phosphate buffer for 2 h and placed in 30% sucrose in 0.1 M phosphate buffer overnight. Prepuration

oj adult

niice

Adult female C57 Bli6 mice were used. Cerebellum, spinal cord, adrenal glands, sciatic nerves, and pieces of small intestine were removed immediately after being killed and either fixed as described above for the embryos of immediately frozen in isopentane on dry ice. In~n~unahistoch~micul

techniques

Fixed embryos were placed on a tissue holder and frozen in a cryostat, and then serially sectioned onto silanized slides.14 For immunofluorescence. sections were first incubated with 3% normal goat serum for 30 min. Sections were then incubated with the various antisera for 1 h at room temperature. Working dilutions were: anti-peripherin and preimmune serum l/200 in phosphate-buffered saline (PBS), anti-NF-M l/4 after reconstituting, as per manufacturer’s instructions. After three rinses in PBS, sections were incubated for 30min at room temperature with fluorescein isothiocyanate (FITC)-conjugated antibodies (goat antirabbit immunoglobulin (Ig), diluted l/l00 in PBS, or goat antimouse Ig, diluted 1140 in PBS). Sections were rinsed three times in PBS and coverslipped with Aqua-mount

(Lerner Laboratories). Sections were incubated with normal goat serum as above, then incubated with both anti-NF and anti-peripherin for 1h at room temperature. After rinsing, the sections were incubated with a mixture of tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit Ig and FITC-conjugated goat anti-mouse IgG for 30min at room temperature. Sections were then rinsed and coverslipped as above.

Preparations were observed with a Nikon fluorescence microscope, equipped with a Nikon AFx camera. Photographs were taken on Kodak Ektachrome 400 ASA color lilm and Kodak TMax 400 ASA black and white film. MaferidY Anti/w&~.

sponding

A synthetic 19 amino acid peptide correto the unique C-terminus of rat peripherin was

Fig. I. Fluorescent photomicrographs of the E9 neural tube stained with (A) preimmune serum, (B) anti-peripherin, (C) anti-NF-M. Straight arrows in B and C indicate examples of fiber staining; curved arrow in C indicates fibers outlining cell body. Bright cells outside neural tube are non-specifically stained blood cells. (A) x 90. (B and C) x 160.

210

(‘. M. .I’KOYc/ t/l

used to generate an antiserum in rabbit (see Ref. I for characterization). A monoclonal antibody to NF-M was purchased from Boehringer-Mannheim (anti-neurofilament 160.000 mol. wt). Fluoresceinand rhodamine-conjugated goat anti-rabbit and anti-mouse were from BoehringerMannheim RESULTS

Centrul nertw4s system Brainstem. Prior to closure of the neural tube (E6 and E8), embryos show no immunoreactivity with either anti-peripherin or anti-NF-M. The first immunostaining is seen at E9 (Fig. 1). This is the stage when the neural tube closes. except for the posterior neuropore and the roof of the myelencephalon. Both antibodies stain fibers along the ventrolateral edge of the neural tube, appearing first at the level of the otocyst. This is the region which will eventually form the myelencephalon. Preimmune serum does not stain any of the fibers in

this and subsequent experiments (Fig. IA). kr~n the onset of the appearance of immunoreactivlt). staining with anti-NF-M is more extensive in the ventral dorsal axis than that seen with anti-peripherln. The pattern of staining with peripherin is restricted to a small portion of the ventrolateral edge of the tube (Fig. IB), while anti-NF-M staining extends over a greater portion of the edge (Fig. IC). Thcrc is no staining near the neurocele, dorsally or ventromedially. All staining is restricted to nerve fibers. By examining various planes of focus at high power we determined that there is no cellular staining, although fibers are seen to surround cell bodies in some cases. As the embryo develops, there is rostra1 as well as caudal extension of peripherin and NF-M that, as described below, extends beyond the myelencephalon. The myelencephalon itself continues to have only fiber staining until E12. The first cell body staining in the myelencephalon appears at E 12. when peripherin stains a line of cells and fibers which are

Fig. 2. Fluorescent photomicrographs showing staining of cell bodies and fibers in the myelencephalon at E13 in the presumptive developing cranial nuclei VII and VIII. (A) Line drawing of entire section. Curved arrows indicate area where staining is located. v, fourth ventricle. (B and C) Double-labeling of same section with anti-peripherin (B) and with anti-NF-M (C). Arrow with asterisk, facial motor nerve; thin arrows, cell bodies which stain only with anti-peripherin. (B and C) x 650.

Ontogeny of peripherin in the mouse embryo located in the parenchyma adjacent to the fourth ventricle. This pattern persists largely unchanged until El3 (Fig. 2A-C). Many of these cell bodies do not appear to contain NF-M, which is largely rcstrictcd to nerve fibers (Fig. 2C). As the embryo develops, between El4 and El7, this area of perikaryal staining curves around the fourth ventricle in the area which will develop into the facial and acoustic cranial nuclei. In the myclencephalon at El? there is heavy peripherin staining in the cell bodies and fibers of the developing Facial and acoustic nuclei. Some of the more dorsal cell bodies show anti-NE‘-M staining at this stage (Fig. 3A-C). Double immunofluoresccnce studies demonstrate that some cells contain immunoreactivity for both antigens. as do some fibers. Other cells contain only peripherin or only NF-M. Through this period no mesencephalic staining is seen with anti-peripherin although there is extensive fiber staining with anti-NF-M. By birth [PO) the facial and acoustic nuclei are coalesced and maintain the high level of peripherin staining in cell bodies and fibers which is seen at E17. Periphcrin is also observed for the first time in the mesenccphalon. At PO both peripherin and NF-M are now detected in the same cell bodies of the oculomotor nuclei complex (III, IV, VI). This pattern persists in the adult (data not shown). S/GUI~ c,ord. At E9 it is difficult to delineate the border between the portion of the neural tube which will become brainstem and that which will become cervical cord. However. by E9.5. there are fibers in the area of the cervical ventral roots which stain with both anti-NF-M and anti-peripherin (Fig. 4A.B). There arc no stained fibers apparent yet in the area of the dorsal root. The neural tube is completely closed in E IO embryos and the ventral horn motoneurons arc not yet fully mature.’ The time of origin of the large ventral horn motoneurons has been variably placed hctween El0 and El I “) and Es.8 and El 1.5.” There arc also rostral-caudal and lateral--medial gradients for the time of origin of the motoneurons.” In general. the lateral neurons innervate limb muscles and the medial innervate axial musculature. At EIO, in addition to fiber staining, anti-periphcrin reacts with a few cell bodies in the ventrolateral portion of the rostra1 spinal cord. These are presumably larpc immature lnotoneurons (Fig. 4C). These results suggest that the cells which stain earliest for periphcrin are destined to innervate limb muscle. Anti-NF-M stains fibers in the ventral cord around the cell bodies and stains few somata (Fig. 4D). Caudal spinal cord does not stain with either antibody. By El I, the spinal cord begins to display its adult organization, with ventral horns and ependyma, but without sharp demarcation between the white matter and gray matter. The motoneurons in the ventral horn have a more mature morphology, with a rounded nucleus and multipolar appearance.’ Antipcriphcrin staining persists in a small number of cell

221

bodies in the lateral-ventral horns, while the network of anti-NF-M positive fibers around these presumptive motoneuron cell bodies is much denser (data not shown). Anti-NF-M stains far fewer cell bodies of these neurons than anti-peripherin (Fig. 4E). The majority of the cells in the ventral horn are unstained by either antisera. The dorsal and ventral roots are well-formed at this point and staining of the dorsal root is observed for the first time with both antibodies. The demarcation of white and gray matter in the spinal cord is more obvious at gestational days 12.-14. in the ventral horn, anti-peripherin stains a larger number of cell bodies which are now clearly located in the peripheral gray motor area. The fibers in this area which stain with anti-NF-M are even dense] than seen at El I. Staining has now progressed to the most caudal portions of the cord. The rostra1 sections show heavier staining than the caudal sections. These observations are in accord with the difference of approximately one day in the rostrocaudal development of the cord. At El2-E14. in the dorsal cord. both antibodies stain the dorsal root and the dorsal root entry zone (Fig. 5A,B). These fibers are seen in cross-section as they began to ascend the dorsal columns. In addition. there was staining with anti-peripherin of the commissural fibers adjacent to the dorsal columns. Double-labeling showed the absence of such staining with anti-NF-M (Fig. SA,B). The spinal cord at El7 shows adult organization with clearly delineated gray and white matter. Staining is equalized over the rostrocaudal axis. The dorsal columns at El7 are clearly seen and are labeled with both antibodies. However, in the entry zone and large diameter fibers in the ascending tracts, staining with anti-peripherin is stronger than with anti-NF-M (Fig. 5C.D). Penetrating primary sensory fibers arc also seen which contain peripherin and NF-M. However. the small diameter fibers contain only pcripherin (Fig. 5C,D). In double-label studies there is cytoplasmic staining of the large ventrolateral motoneurons by anti-peripherin and anti-NF-M at El7. NF-M staining of fibers at the periphery of the cord is far denser than that seen with peripherin. This pattern is not significantly different from that seen at El4 except that the cells are now in their mature positions (Fig. 5E,F). It should be stressed that other motoneurons in the medial areas of the ventral horn do not stain with either anti-peripherin or anti-NF-M. At El7, spinal roots are labeled with both antibodies, with most. if not all. of the fibers containing both peripherin and NF-M (Fig. K,D). Sections taken from cervical. thoracic and lumbar levels of the adult spinal cord show little or no anti-periphcrin staining of ventral motoneuron cell bodies. However, dorsal columns are positive for peripherin as are the dorsal and ventral roots (data not shown).

721 ___

Fig. 3. Fluorescent photomicrographs showing staining of cell bodies and fibers in developing cranial nuclei VII and VIII of the El7 mvelencenhalon. (A) Line drawing of entire section. v, fourth ventricle; curved arrows indicate area where is located. (B and C) Double-labeling of the same section with anti-peripherin (B) and with anti-NF-M (C). Arrows indicate area which stains only with anti-peripherin. (B and C) x440.

Wstaining

Ontogeny of perlpherin m the mouse embryo

223

Fig. 4. Fluorescent photomicrographs showing periphcrin and NF-M localization within developing ventral horns and roots, (A) E9.5: anti-peripherm staining of ventral neural tube and root. (B) Same rection as in (A) double-s~~lned with anti-NF-M. For A and B ventral portion of embryo is at top of tigure, dorsal at bottom. Fluorescence at vemra! edge and middle of neural tube and edge of embryo is non-specific. (C) El& higher power view showing anti-pcripherin staining of immature ventral horn neurons. Note that staimng is eccentric. (D) EIO: higher power view showing anti-NF-M staining of ventral horn fibers. Note fiber staining around the cell bodies. Arrows indicate representative cell bodicr in C and D. (E) Staining of ventral horn at El I with arm-NF-M. Curbed arrow indicates dorsal root. DRG. dorsal root ganglion: WI. ventral horn. (A and B) x 165, (C) x 450. (DJ x 320. (E) x 100.

Upric IIYWCUSC/W&CL Fibers in the optic stalk become morphoio~ically apparent by El 3 and there is no reactivity to either antibody at this age. Ganglion cells are evident m the retina, and these do not stain zither. By El4. the optic fibers have begun to

connect with the brain. At this point. the optic nerve and some retinal ganglion ceils stain with anti-W-M but neither stains with anti-peripherin. In the retina at El7, there is only background staining with antiperipherin (Fig. 6A), but anti-YF-M stains a few

Fig. 5. Fluorescent photomlcrographs showing ventral and dorsdi horns stained with anti-perlphrrin dni; ant]-NF-M. Dorsal horn at El3 double-labeled with (A) anti-peripherin and (B) anti-W-M. Straigftr arrows indicate dorsal roots; curved arrows rndicate dorsal root entry zone; angled arrows point !o ~on~miss~~lfibers, Dorsal horn a~ Eli doubly-addled with (C) anti-~ripherin and (D) anti-NF-M Straight arrows point to dorsal columns; curved arrows point to secondary sensory fibers. Ventral horn at El7 double-labeled with (E) anti-perlpherin and (F) anti-NF-M. Arrows Indicate representative cell bodies. (A and B) x370. (C and D) x75. (E) x 150.

ganglion cells. The optic nerve stains exclusively with anti-NF-M at this age (Fig. 6B). At birth (PO) the same contrast between the distribution of NF-M and ~~ph~~n persists. However, in the adult mouse peripherin, as well as NF-M, is found in the optic

nerve. Double-labeling of cross-sections show that most, if not all, fibers are stained with both antibodies (Fig. 6C,D). Cerebraf and cere~llar cortim. At El4, NF-M staining in the cerebral and cerebellar cortices is

Ontogeny of periphcrin in the mmw

embryo

7?< _-.

limited to occasional fiber bundles. By El 7, anti-NFM staining is widespread in the CNS, with a large number of positive fibers in the cerebral and cerebellar cortices (data not shown). Anti-peripherin staining is minimal in the cortex, with only a few fibers staining. These are presumably ascending fibers from the brainstem (data not shown). There is no staining with anti-peripherin of fibers or cell bodies in the cerebellum. At E17, PO and in the adult there is no peripherin staining of cerebral cortex or cerebellum (data not shown). Cruniul gangliu and the peripheral nerwus

swtem

Crunial gungliu. Prior to E9 there is no staining in the region of the presumptive cranial ganglia. None of the migrating cells that will form these ganglia arc seen with either of the antibodies. At E9, both peripherin and NF-M are seen in nerve fibers and in rare cell bodies in the developing acoustic ganglia (Fig. 7A,B). Cell bodies and fibers around cells in the developing facial and trigeminal ganglia also show immunoreactivity by E9.5. In none of these cranial ganglia are more than 20% of the total cells stained with either antibody (data not shown). At ElO, the trigeminal, facial and acoustic cranial ganglia are distinct blastemal condensations and the ganglia are more clearly developed than at E9. As compared with E9, there is a two-fold increase in the number of cell bodies in the facial, trigeminal and acoustic ganglia that react with anti-peripherin. AntiNF-M stains some cell bodies in these cranial ganglia, but about one-third fewer than stain with anti-peripherin. The staining in the cell bodies of these cranial ganglia by both antibodies is cytoplasmic and eccentric, with a cap towards one end of the cells. This pattern of staining was similar to that seen with anti-peripherin in developing cultured cells prior to neurite outgrowth. ” Within these cranial ganglia. more fibers react with anti-NF-M than with antiperipherin. Double-labeling at El4 (Fig. 7C,D) reveals that there are at least two types of cell staining in the trigeminal ganglia. Cell bodies labeled with both antibodies constitute the majority of the population; cell bodies which stain only with anti-peripherin constitute the remainder. There may also be a few cells staining only with anti-NF-M. These groups contain cells of varying sizes, with no clear association between filament type and neuronal size. By El7, cell bodies in trigeminal, facial and nodose ganglia are heavily stained with anti-peripherin. AntiNF-M also stains these structures but stains fewer cell bodies and more fibers than does anti-peripherin (data not shown). New-d

crest derioatices

Neural crest cells, precursors of most peripheral ganglia, do not stain with either antiserum during their migration (E8-E9). By the 10th day of gestation (E 10) the spinal ganglia are differentiated from neural

crest. At this age, about 10% of the cell bodies of the cervical dorsal root ganglia (DRG) show the prescncc of both peripherin and NF-M, with staining present in a perinuclear distribution. Caudal DRGs (thoracic and lumbar) do not stain with either antibody at this age. The DRGs are well-formed by El 1 and both antibodies stain greater numbers of neurons than at EIO. The appearance of immunoreactivity m DRGs proceeds in a rostral-caudal direction from El0 to El2. By El2 there is staining of DRGs at all levels. However, rostra1 El2 DRGs have a larger proportion of cell staining than do caudal DRGs at this stage. By El7 DRGs at all levels are well stained. In the DRG at El4 and E17, double-labeling (Fig. 8) shows that approximately one-third of the neurons. mostly larger ones near the periphery of the ganglion. contain both NF-M and peripherin. Two-thirds of the neurons present, mostly smaller cells, contain only peripherin. Occasional small neurons appear positive for both proteins. Few if any neurons are positive for NF-M alone (Fig. 8). These data are consistent with the staining of larger and smaller fibers described above in the dorsal regions of the spinal cord. In the adult, the pattern of staining in the DRG changes. The small neurons preferentially stain with antiperipherin while the large neurons are stained with anti-NF-M. At EIO, sympathetic ganglia are forming, and both antibodies stain an evenly distributed ring of cytoplasm around the nucleus in many, but not all, neurons in these ganglia. This pattern of staining is different from that found in the DRGs in which staining is strongest in an eccentric perinuclear “cap”. By E14, sympathetic ganglia arc very distinct, and most cells stain with anti-peripherin. Fewer stain with anti-NF-M. Progression of staining in the sympathetic ganglia proceeds in a rostral-caudal direction. At El7, in the caudal portion of the embryo, the somatic staining is still mainly in a thin peripheral ring, with anti-peripherin staining about four times the number of cells that anti-NF-M does (Fig. 9A,B). There are three groups of cells apparent, peripherinpositive, NF-M-positive and peripherin-NF-Mpositive. However, in the rostra1 portion of the El7 embryo (Fig. 9C,D). the ganglion cells show a fuller, more uniform staining with anti-peripherin in a pattern similar to the staining seen in DRGs at this age. As in the more caudal sections, NF-M stains only about a quarter as many cells as peripherin (Fig. 9C,D). In contrast to the DRG, the peripherin and peripherin/NF-M-positive cells are not readily separable into size classes. In the El2 embryos, the adrenals are distinct and composed of cellular cords representing the cortex. Neither antibody stains the adrenal gland at this age. By El7, the adrenal glands have developed a more continuous medullary region of small cells and they still do not stain with either antibody. In adult adrenal glands. there is non-specific staining of

Ontogeny

of peripherin

in the mouse embryo

Fig. 8. Fluorescent photomicrographs of dorsal root ganglia double-labeled with anti-peripherin (A. C’. E) and anti NF-M (B, D, F). (A and B) E14. Curved arrows indicate representative cell which stains only with anti-~~phe~n, Straight arrows indicate rep~sen~tive cell which stains with both ~ti-~riph~in and anti-NF-M. (C and D) Low power view of El7 DRG, ventral and dorsal roots and spinal cord. Arrows indicate ventral and dorsal roots. (E and F) High power viewof same section:(Ef anti-~~p~rln and (F) anti-NF-M. White arrows indicate representative cell which stains only with anti-peripherin. Black arrows indicate representative cell which stains with both anti-pe~pherin and anti-NF-M. (A and B) x 27t?,tC and D) x 75. (E and F? x 290.

chroma~n granules, but there is no cellular staining with anti-peripherin. Only fibers are stained in the adrenal medulla with anti-NF-Ivl ~~tr~i~testi~l @art. At E9.5 the gut is a simple tube; a few fibers in the wall are stained with both antibodies. As the gut develops, so does immunoreactivity. The gut appears more as it will in the adult by E13. At this age both antibodies stain fibers in the gut wah. and cehs in the parasympathelic ganglia

adjacent to the gut. At El441 7 there was no significant change in the staining of the gut. Using doublelabeling, it is apparent that anti-peripherin and antj-~F-~ stain the same structures (Fig. IO). In the adult, sections ofsmall intestine show staining of the myenteric plexus by both antibodies. P~yip~er~f mwes. Mixed spinal nerves are apparent by Eli and stain with both antibodies. At this age. there is also staining of the braehial plexus by both

Fig, 9. Fluorescent photomicrographs of El7 sympathetic ganglia double-stained with anti-peripherin (A and C) and anti-NF-M (B and D). (A and B) Caudaf ganglia. (C and D) Rostra1 ganglia. Curved arrows (A and B) indicate representative cell which stains only with anti-NF-M. Straight arrows indicate representative cell which stains with only anti-peripherin. Asterisks tC and D) indicate representative ceil Mhich stains with both anti-peripherill and anti-NF-M. (A and B) x 225, (C and D) x 200.

Fig. 10. Fluorescent

photomicrographs

of the El4 gut double-labeled

with (A) anti-peripherin

and (B) anti-NF-M.

Anti-peripherin

diluted

1:200; anti-NF-M

diluted

1:4.

x 100.

Ontogeny of peripherin in the mouse embryo antibodies. As development proceeds, all peripheral ncrvcs show both peripherin and NF-M immunoreactivity (Fig. 11). Double-labeling shows fibers that stain with both antibodies or that stain with one or the other of the antibodies. Both antibodies stain adult sciatic nerve. On double-labeling of the adult sections, there are three groups of fibers seen: those that are stained with only either one of the antibodies and those that stain with both. More than 80% of the fibers fall into the latter category. Olfktory system. At El3, fibers in the lamina propria beneath the nasal mucosa are apparent and stain only with anti-peripherin. This pattern of staining continues to El7 (Fig. 12). These fibers may be axons from the olfactory epithelium or trigeminal fibers. In the adult, there is no staining of the mucosa with either antibody, while both stain a few fibers in the lamina propria. These are presumably trigeminal fibers. Neither antibody stains the olfactory nerve as it enters the olfactory bulb. The results of staining with both antibodies in the CNS and PNS are summarized in Tables 1 and 2, respectively. DECLSSION

Although the functions of intermediate filaments are incompletely understood, it appears that they serve an important role in the spatial organization of the cytoplasm.‘4 In the neuron the number of neurofilaments correlates with and perhaps regulate axonal diamctcr.” The neurofilaments are characteristic of the post-mitotic, post-migratory neuron.’ Earlier in neuronal development many neuroblasts express the mom “generic” intermediate filament protein, vimcntin. Thus neuronal maturation appears to involve a switch from a less specific cell-type to a more specific cell-type intermediate filament type. A similar pattern is seen in astrocytes where a shift from vimentin to GFAP occurs with maturation. In addition to the neurofilament triplet and peripherin, yet another neuron-specific intermediate filament protein, x-internexin, has been recently describcd.4h~7”.‘2~‘.“‘~’ This protein appears to have a different distribution from peripherin. The discovery of peripherin. another neuronspecific intermediate filament protein, adds new complexity to analyses of the role of intermediate filaments in neuronal development. Several questions arise about the respective roles and functions of ncurofilaments and peripherin filaments in neurons. Arc they always present in the same cells? Do they follow each other sequentially in development? Is periphcrin transiently expressed in higher brain regions during development? Do the two types of tilaments serve clearly different functions in specialized ncuronal groups? Is peripherin an evolutionary remnant without a unique role to play in the nervous system’? Does the presence of peripherin correlate with asonal length or diameter? The studies reported

231

here provide some insight into these questions. In this work we have chosen to follow neurofilament development with an antibody to the middle neurofilament subunit, NF-M. This particular antibody was chosen because its reactivity with NF-M is phosphorylation-independent?’ and because it does not recognize peripherin. It is likely that the results obtained apply equally well to the core protein of the neurofilament, NF-L, since the expression of NF-L and NF-M has not been observed to bc dissociated in development4”~‘“.‘” The antibody against peripherin was raised against a I9 amino acid synthetic peptide’ corresponding to the C-terminus of a rat peripherin cDNA.‘~ This antibody is specific for peripherin’ and recognizes its various phosphorylatcd isoforms.’ Both peripherin and NF-M were detected for the first time in the mouse embryo at gestational day 9 (E9). At this point both proteins appeared in the anlage of the acoustic ganglia, in the neural tube and in the myelencephalon, with NF-M more widely distributed at the outset. Both appeared at the outer edge of the neural tube, but peripherin was found in fewer fibers. As the embryo developed, staining with both antibodies increased. However, although many elements were stained by both antibodies. the overall pattern of staining often differed. Early in dcvclopment, E9-El I, both proteins were found in the same parts of the nervous system. At no time during development was either peripherin or NF-M expressed in non-neuronal tissues. Nor were either 01 these intermediate filaments detected in mitotic cells or in cells in the course of migration. In both cases the appearance of immunoreactivity was coincident with or slightly after the initiation of axon outgrowth. Since axon outgrowth can occur in the absence 01 intermediate filaments’4 it would appear that the intermediate filaments are likely to play an important stabilizing role in the axon rather than initiating its formation. In the developing motoneurons of the ventral spinal cord at El 1 there was more prominent staining for NF-M in the nerve fibers which appear to skirt around nerve cell bodies as they course to the developing anterior root. In doing so they often outlined cell bodies but only infrequently could the cell body of origin be clearly identified. When this was the case, filaments appeared to be tightly bundled and to course at the periphery of the perikaryal cytoplasm and then to enter the process. In contrast, many more El I motoneuron cell bodies can be said with confidence to contain peripherin. In these cases. staining was in the form of a rather diffuse perinuclear cap which often extended into the developing process. At El4 a rather similar picture was obtained. By El7 the majority of large motoneurons of the anterior horn were stained by both anti-peripherin and anti-NF-M. The lilaments were similar in distribution in each case, but were more intensely stained by anti-pcripherin. On the other hand. many more nerve fibers at

Fig. 11. Fluorescent

photomicrograph

of an El7 peripheral

nene

double-labeled

with (A) anti-peripherin

and (B) anti-NF-M.

x 180

c‘. M -1KCIY

234 Table

1. Summary

Embryonic age

of ontogeny

of’ peripherin

and middle

Cerebral and cerebellar cortices

E6, E8 E9 El0

(‘i

neurofilament

Brainstem

P in cranial nuclei VII, VIII P and NF-M in cranial nuclei VII. VIII fibers

El2

El3

NF-M in fibers; P absent NF-M in more fibers; P absent

El4

NF-M

protein

expression

m the central

Neural tube/spinal cord

NF-M and P in fibers Same as E9

El1

I//

in more fibers

Same as El2

NF-M and P in dorsal columns; rostrakaudal expression equalized

NF-M in most fibers; P absent

P in many cell bodies in above ganglia, NF-M in fewer

PO

Same as El7

P in most cells in coalescing nuclei VII,

Optic system

NF-M and P in fibers P in ventral horn neural somata; NF-M in many ventral fibers P and NF-M in dorsal horns P in more motoneuron somata and in commissural fibers; NF-M in more fibers Same as El2

Same as El3

El7

ncrvcIu> yst~‘m

NF-M in optic nerve and in retinal ganglion cells; P absent Same as El4

Same as El4

VIII; NF-M in fewer; P and NF-M in nuclei III, IV, VI Adult

-1

not detected;

Same as El7

Motoneuron somata negative for P

P and NF-M nerve

in optic

P, peripherin.

the periphery of the cord were more heavily stained by anti-NF-M than by peripherin. The findings on anti-NF-M staining were in general agreement with earlier work by Cochard and Paulin.s The differences in distribution of the two intermediate filaments were more pronounced in the dorsal root ganglia. Staining was found with both antibodies as early as ElO, although at that stage only 10% of the cells were labeled. As development progressed, it was clear that the large cells at the periphery of the ganglion contained both peripherin and NF-M. However, these account for only onethird of the neurons in the ganglion. The remaining two-thirds of the neuronal cell bodies present, mostly smaller cells at the center of the ganglion contained only peripherin. In the adult, the larger neuronal perikarya lost their reactivity to anti-peripherin, while the small retained their selective staining for peripherin.” These data are consistent with the presence of peripherin, but not NF-M, in the small sensory fibers of the dorsal cord. Similar differences in neuronal cell body staining were found in sympathetic and cranial ganglia. While in some cases the differences in cell body staining could simply reflect the more rapid transport of one or the other filament out of the cell body into the process, in other cases such as the DRG, it is clear that both ceil body and process contained only peripherin and not NF-M. This pattern is also clear in peripheral nerves and spinal roots where three classes of fibers are found:

(1) those that reacted only with anti-peripherin, (2) those that reacted only with anti-NF-M, and (3) those that reacted with both intermediate filament antibodies. In the peripheral nerve it is unclear whether these differences can be correlated with fiber diameter. The one consistent feature found in peripherincontaining cells is that they produce long axons which either exit or enter the CNS. Thus it appears that peripherin and neurotilament proteins are not always present in the same cells, even within a given neuronal population. Although there are cases such as the large peripheral cells of the DRG where peripherin was present during development and then was diminished or absent in the adult, there are many cases in which peripherin, but not NF-M, appears and remains into maturity. Thus it is unlikely that peripherin is a transient step in development which will be replaced by the neurofilament proteins. With a few exceptions, peripherin is found in the embryo where it is found in the adult. It is not found at any stage of development in the higher brain regions where it is absent in the adult. Therefore, in spite of the fact that peripherin is found only in phylogenetically more ancient regions of the brain, it is neither a transiently expressed molecule nor an evolutionary remnant without current function. Our results agree in general with those of Escurat et ~1.’who compared the expression of peripherin and NF-M at low power (8 x ) in sagittal sections of the rat embryo. In addition to the staining which we have

Ontogeny of peripherin in the mouse embryo

3 P

reported above, they have observed peripherin immunoreactivity in the rat retina at El 5. in the optic nerve at El6 and in the nasal mucosa. In the mouse, peripherin immunoreactivity fags behind NF-M in the retina and is not seen during development, although it is present in the adult retina. In the nasal mucosa we observed staining only in the lamina propria in fibers of presumptive trigeminaf origin. Primary olfactory fibers lacked both NF-M and peripherin staining. Other differences, presumably related to species. included staining of the trigeminaf nerve in advance of the cranial nerve ganglia in the rat and the converse in the mouse. While there are no other developmental studies of peripherin. Parysek ef ~1.‘~ have studied the distribution of this inte~ediate filament protein in the adult rat and Leonard rt al.“.” have studied the distribution of the messenger RNA for peripherin in adult rat using in situ hybridization methods. in general our findings agree with these groups. However, in agreement with Leonard, we failed to find peripherin immunoreactivity in the adrenal while this was reported by Parysek cf al.” In contrast to the fatter, we also failed to see fiber staining in the cerebellum. These differences could be due to species differences or to differing specificities of the antibodies used. The present data suggest that peripherin is not a remnant and not merely transiently expressed during development. It is a functional intermediate filament protein which can either co-exist with the neurofilament triplet proteins in the same neuron or function in the absence of the neurolilament subunits. It wouid appear reasonable to assume that it differs in some way in its function from the latter. One possible difference between the two intermediate filament systems could be in the type of cross-bridges they

form in the cytoplasm. In the simplest madei. the NF-derived filament forms two principal types ot cross-bridges-those which link filament to lilament and those which link filaments to microtubulcs. In the case of the neurofilament, the cross-bridges to other filaments are formed by the arms on the filament. composed of the highest molecular weight subunit NF-II.‘” NF-derived filaments are also capable of cross-bridge formation with microtubules mediated by microtubule-associated proteins (MAPS). “I !? The MAP-mediated bridging is directly with the NF-I, core of the neurofilament.” Neurotilaments are apparently unique among the intermediate filaments in being capable of homologous cross-bridging with themselves without need for molecules extrinsic to the filament. Other intermediate filaments appear to be able to form MAP-mediated cross-bridges with their core proteins and are likely IO depend on extrinsic proteins to mediate filament-filament interactions. The ability to form stable filament-filament bridges might underlie the ability of NF-derived filaments to regulate the circular cross-section of the axon over long distances. In contrast. those filaments which depend solely on finks to mi~rotubules or on proteins extrinsic to the filament to form filament-filament interactions might be more labile and subject to rapid rearrangements as a consequence of cellular function. Thus a type III intermediate filament protein such as peripherin might be best suited to axons of‘ smaller diameter or to those in which more rapid remodeling of the cytoskeleton might be desirable. A~iifzon,l~dge~n~nt.~--This work was supported by grants NSl.5076. NSi6036, Basic Research Grant from the March of Dimes; CMT was supported by NRS fellowship I F32 NS08564-01 BNS-I AND CIA I KOX AG00430-OIAI. We wish to thank Carol Mason, Ron Liem. Steve Chin, Jane Dodd and Mike Gershon for helpful advice.

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