APPEARANCE AND LOCALIZATION OF A NERVE GROWTH-PROMOTING PROTEIN DURING DEVELOPMENT

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APPEARANCE

AND

LOCALIZATION

OF

A

NERVE

GROWTH-PROMOTING

PROTEIN

DURING

DEVELOPMENT

Myron Winick, M.D., and Robert E. Greenberg, M.D.

Department of Pediatricc, Stanford University School of Medicine, Palo Alto, Calif.

(Submitted September 28; accepted for publication October 21, 1964.)

These investigations were supported in part by grants from the USPFIS (NB-0513-O1), the John A. Hartford Foundation, and the Lt.

J.

P. Kennedy, Jr., Laboratories for Molecular Medicine.

Dr. Winick is a recipient of fellowships from the Bank of America-Giannini Foundation (1962-1963), the National Institutes of Health (3-F10-Hr-1039-O1S1), and PuS Training Grant (9T1-HD-49), National Institute of Child Health and human Development, Public health Service.

Dr. Greenberg is recipient of a Public Health Service Research Career Program Award (AN15-K3-7263) from the National Institute of Arthritis and Metabolic Diseases.

ADDRESS: (R.E.G.) Department of Pediatrics, Stanford School of Medicine, Palo Alto, California 94304. PRESENT ADDRESS: (MW.) Department of Pediatrics, Cornell University Medical School, New York

hospital.

PEDIATRICS, February 1965

ARTICLES

221

A

NERVE growth-promoting protein

(NGF), capable of stimulating growth

of sympathetic ganglia, has been isolated

and partially purified by Levi-Montalcini

and Cohen, using mouse submaxillaiy

tis-sue as a source.1-4 When antiserum,

pre-pared against this protein, is injected into

young mammals, specific destruction of

cells of sympathetic ganglia ensues.5 Their

studies suggest that growth of sympathetic ganglia, especially during development,

may be subject to regulatory effects

medi-ated by a specific protein. Implicit in these

observations is the possibility that this

fac-tor is a prototype of other chemical

moieties playing a role in the differentia-tion of other structural or functional units of

the nervous system.

The purpose of these investigations is to determine the species distribution of NGF, its tissue localization, the time when it is

first detected during development, and to

correlate these findings with the known sequence of events during differentiation of sympathetic ganglia.

METHOD

Isolation of NGF and Preparation of Antiserum

NGF was purified from adult male mouse salivary glands essentially according to the

method of Cohen.3 Antiserum was

pre-pared by serial injections of purified NGF

with Freunds complete adjuvant into

rab-bits.5

Preparation of Tissues for

Identification of NGF

Tissues were homogenized in four vol-umes of distilled water and then serially

diluted. Chick embryos were removed after

various incubation times and either

ho-mogenized in toto or dissected into three portions before homogenization: head, axial skeleton and rest of body. Frog

em-bryos were staged according to Shumway’s

criteria.6

Criteria for Identification of NGF in Tissues

BIOASSAY AND INHIBITION WITH SPECIFIC

ANTISERUM: The production of a halo of

fibers from ganglia of chick embrves

in-cubated 9-10 days by NGF was used as a

bioassay.7

Tissue homogenates were added to

cul-tures and the response compared with

known NGF and physiologic saline. ho-mogenates showing a positive response

(2)

antiserum and not by normal serum were

considered positive for NGF.

Concentra-tion of NGF could be estimated by

de-termining the highest dilution still giving a

positive bioassay.

COMPLEMENT FIXATION : The ability of

specific antiserum to fix complement in the

presence of a tissue homogenate was used

as a method for detecting NGF in tissues.

A quantitative complement fixation test was

employed. Extraneous complement was

eliminated by incubating antiserum and

control serum at 56#{176}for 30 minutes. All

dilutions were made in Veronal saline

buf-fer 0.147M, pH 7.4. Fresh complement was

obtained by bleeding guinea pigs and

as-sayed by doing a standard titration curve.

A 3% solution of fresh sheep erythrocytes

was sensitized with hemolysin previously

titrated with complement. Test tubes were

prepared containing 0.25 ml of antiserum

or control serum, 0.5 ml of complement,

and 0.2.5 ml of tissue homogenates at

vari-ous dilutions. These were allowed to stand

overnight at 4#{176}C.0.5 ml of sensitized red

cell suspension was then added and the

tubes incubated at 37#{176}and read after 30

minutes.

AGAR DIFFusIoN: The appearance of a

line of identity between purified NGF and

tissue homogenates in a double diffusion

system was accepted as evidence for NGF

in the homogenate. Diffusion plates were

prepared in petri dishes using 1% agar in

0.9% NaCl. Specific antiserum was placed

in the central well and tissue homogenates

or purified NGF in the peripheral wells.8

Readings were made at 24 and 48 hours.

FLUORESCENT ANTIBODIES: NGF was

lo-calized by combining tissue fragments with

antiserum and identifying the

antigen-anti-body complex by staining with a fluorescein

conjugated goat anti-rabbit

gamma-globu-lin (Antibodies, Inc., Davis, California).8

Normal rabbit serum was substituted for

antiserum in the controls. Tissue was

in-cubated with control or antiserum for 30

minutes and then washed five times for 10

minutes each in 0.1M phosphate buffer,

pH

6.8. Fluorescein conjugated goat

anti-rabbit gamma-globulin was then layered

over the tissue for 30 minutes. Tissues were

rewashed in buffer, mounted, and

immedi-ately examined for green fluorescence

un-der the ultraviolet microscope. All tissues

considered to contain NGF showed

posi-tive biologic activity which was neutralized

be specific antiserum plus one or more of

the other criteria described.

RESULTS

Appearance of NGF during Development and Localization within the Embryo, Fetus,

or Newborn Animal

CHICK EMBRYO: In order to determine

the time during development that NGF

first appears, various species were studied:

chick, frog, mouse, and human fetus. In

each specie studied, attempts were made

to localize as far as possible the specific tissue where NGF could be detected. Using

bioassay, antibody neutralization, and

com-plement fixation techniques, NGF can first

be demonstrated in the chick embryo on

the fourth day of incubation, rising sharply

to a sustained maximal value by the fifth

and sixth day (Fig. 1).

When first detected, NGF can be

lo-calized to the axial skeleton region. Figure

2 demonstrates a positive biological

re-sponse of a region of the axial skeleton in

contrast to a negative response seen with

either the head or rest of the body.

FROG EMBRYO: In the frog (Ratio pipiens)

NGF can be first detected at stage 18 (just

prior to hatching). In larger tadpoles it can

be localized to the axial skeleton and in the

adult frog to the sympathetic chain. All

other tissues tested gave no evidence of

either biologic activity or ability to fix

com-plement (Table I).

MOUSE NEWBORN: In the newborn

mouse NGF can be specifically localized

to the sympathetic ganglia. Figure 3

dem-onstrates specific fluorescence of a newborn

mouse sympathetic ganglia. No other tissue

tested including salivary gland exhibited this reaction.

HUMAN FETUS: In the earliest human fetus

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gesta-ARTICLES 223

100 S.

0

75

0,

8)

O -i;h 50

33

m

25

I _Biologic

400

Activity

.

8)

30(

200

c 100

Complement

Fixtion

I 2 3 4 5 6 7 8 9 10 II 12

AGE (days)

Fic. 1. Appearance of NGF in the developing chick embryo. NGF identified by (a) biologic ac-tivity and antibody neutralization and (b)

comple-ment fixation.

tion) NGF could already be detected by

biological assay and antibody

neutraliza-tion techniques. (Fig. 4). In all four fetuses

studied the only tissue in which NGF could

be detected was the sympathetic ganglia.

Natural Distribution of NGF and Immuno-logic Similarity in Various Species

NGF could be detected in bony fish,

amphibia, birds, and mammals and was

not demonstrable in the elasmobranch and

a variety of invertebrates. In all cases

ex-cept in the adult mouse it was localized

specifically to axial regions or sympathetic

ganglia. The NGF found in all species was

immunologically similar to NGF isolated

from mouse salivary gland. Figure 5 shows

lines of identity between purified mouse

NGF and homogenates of fish axial skelton, frog sympathetic chain, chick axial skelton,

and preoperative serum from a child with

tumor of neural crest origin. Biological

ac-tivity of all homogenates was inhibited by

an antiserum to purified mouse salivary

A

Fic. 2. Demonstration of NCF in various tissues of the chick embryo. (a) Axial skeleton showing posi-tive response, (b) inhibition of axial skeleton response with specific antiserum, (c) head showing

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TABLE I

LOCALIZATION OF NGF IN Fiioc Tissuas

Stage Tissue Corn pleinent Fixation Tissue Cu/lure Response

Adult Brain

Heart \riisele

Viscera Skill Liver

Kidney Gallbladder Lung

Spinal Cord Syinp. Chain

Negative

Negative Negative Negative Negative

Negative Negative

Positive (1: 160)

Negative Negative

Negative Negative Negative Negative

Negative Negative Negative Negative Positive (1:80)

Large tadpole Liver

Viscera

Brain

Axial Skeleton

Negative

Positive 1:161))

Negative

Negative Positive (1:80)

Small tadpole

Stage 17 and before \\hole Animal Negative Negative

Stage 18 Whole Animal Positive (1:160) Positive (1:160)

Fic. 3. Localization of NGF to sympathetic ganglia

of newborn mouse. (a) Ganghion incubated with control serum showing no fluorescence, (b) ganghion

incubated with specific antiserum demonstrating characteristic green fluorescence.

Fic. 4. Demonstration of NGF in the sympathetic chain of the human fetus. (a) Positive response of homogenate of sympathetic chain from a 9-week

human fetus, (b) response inhibited by prior

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Tissue Fetus .Vo. 1 Fetus .‘so. 2 Fetus No. 3 Brain Kidney Spiiial iord ‘l’hyinus Iliart

( oiitiet IVU IiSSll(

Liver ‘l’hyroid Lung Skiii Skeletal muscle Smooth muscle Adrenal Salivary glan(l Sympathetic chain Approximmiate age Weight (gin) (‘rovn-runip (cm) Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative PoSITIvE I) Weeks 8.4 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative POSITIVE 3 Months 76 7.0 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative POSITIVE 3 Months 54 6.8 Fetus .\. Negative Negative Negative Negative Negative Negative Negative POSITIVE 4 \1ommtlms 11)3 8.2 ARTICLES

gland material. NGF in axial regions is not

only detectable in vitro but also is sensitive

to the action of antiserum in vivo. Serial

injection of 0.1 cc of specific antiserum for

10 days resulted in a disappearance of

biological activity from the axial skeleton

of bullfrog tadpoles. Three injections of

0.1 cc of the same antiserum into newborn

mice produced a loss of biological activity

from sympathetic ganglia.

COMMENT

225

The demonstration by Levi-Montalcini

and Cohen of a nerve growth-promoting

protein (NGF) which, when injected into

young mammals results in hypertrophy and

hyperplasia of sympathetic ganglia and

hy-perinnervation of the tissues they supply,

has raised the question of the actual

physi-ological significance of this protein.

Sub-sequent studies, showing an increase in the

concentration of catecholamines in tissues

after injection of NGF#{176}and selective

de-struction of sympathetic ganglia following

injection of specific antiserum5 suggest that

NGF participates in the regulation of

growth of sympathetic ganglia. The results

Fic. 5. Immunologic similarity of NGF from

vari-ous sources. Central well contains antiserum pre-pared against NGF purified from mouse sub-maxillary gland. Peripheral wells contain: (a) purified NGF from mouse submaxillary gland, (b)

homogenate of fish axial skeleton, (c) homogenate

of sympathetic chain of the frog, (d) homogenate of axial skeleton of the six-clay chick embryo, (c)

undiluted preoperative serum from a child with a tumor of neural crest origin.

of our studies provide additional evidence

in support of a physiologic role for the

TABLE II

LOCALIZATION OF NGF IN hUMAN FETUS

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‘I’ABLE Ill

J)ISTIIIRUIION OF N(F

Nerre Growth Species Localization Factor A. iIamiimiials 1. IhIiimiami 2. Mouse (mmevbormm)

3. 1OLISe (adult)

It. Bir(ls

1. (‘likk (emmmbryo)

C. Teleosts 1. Goldfish D. Amphibia 1. Frog 2. Tadpole 3. Salamander (Triturus) E. Elasniobrancli

1. Leopard shark F. Invertebrates

1. Cockroach

2. Itound wormmm

‘3.Flatworrmm 4. Snail 5. Clam 6. Starfish Present Present Present Present after day 4 Present Present Present after Stage 18 Present Al)Sent Absent Absent Absent Absent Absent Absent

nerve growth-promoting factor. NGF is

normally present in a wide variety of

ver-tebrate species, always in association with

sympathetic ganglia. Antiserum, when in-jected into newborn mice, can be localized

specifically to sympathetic ganglia and

causes a disappearance of NGF from these

ganglia and from the axial region in

tad-poles.

In addition, NGF is initially detectable

during development at a time when

dif-ferentiation of sympathetic ganglia can first

1)e recognized morphologically. Yntema

and Hammond have proposed a chronolog-ical sequence which occurs during differ-entiation of sympathetic ganglia. This

se-quence proceeds from differentiation of the

neural crest from its ectodermal rudiment

through migration of cells from the neural

crest, differentiation of sympathetic

neuro-blasts, and finally incorporation of

neuro-blasts into ganglia.b0 Although some

in-vestigators have questioned the neural crest

origin of sympathetic ganglia,h112 it is

gen-erally agreed that neuroblasts are

incor-___________

porated into ganglia forming a primary

sympathetic chain during the fourth day of

Symp. chain development in the chick embryo. The

lower thoracic and lumbar portions of this

ymiip. chain chain degenerate on the sixth day and arc

replaced

by

secondary ganglia.11 In other

vertebrates, a single sympathetic chain

er-Axial skeleton sists throughout development. In the frog,

it is clearly recognizable around stage 18.

In the human embryo, the primordia of the

Axial skeleton sympathetic trunk arise at about the 5-mm

Symp. chain stage as a small group of cells lying along

Axial skeleton the dorsolateral aspect of the aorta.

Migra-tion is complete by the 15-mm stage when

a well-marked segmental pattern can be

mp. cmain discerned in the sympathetic trunk.16

Sym-pathetic development in reptilesl7 and fish18

closely approximates that in amphibians.

-

NGF can be initially detected in the

chick and frog embryo approximately at the

time the sympathetic ganglia have

differen-tiated morphologically, and is localized to

the sympathetic chain in the youngest

hu-man fetus examined.

The limited comparative data in these

studies are also consistent with the concept

of a physiological role for NGF. Although

it is not entirely clear what constitutes a

sympathetic ganglion, certain criteria have

been employed. Morphologic criteria have

included cellular location, tissue

innerva-tion, and embryonic derivation.

Physiolog-ically, the nature of response of tissues to

gan glionic stimulation has been studied.

The presence of norepinephrine in

signifi-cant amounts in ganglia represents the

prin-cipal biochemical characteristic unique to

sympathetic tissue.19 Employing suc’i

cri-teria it would appear that there is a

well-defined sympathetic nervous system in all

vertebrates from the elasmobranch through

man. Phylogenetically tile sympathetic

chain is most developed in birds and

rep-tiles, intermediate in mammals and least

developed in amphibians and fish.2#{176}

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ARTICLES 2.27

between bony fish and the elasmobranch,

and both of these species show comparable

values for tissue catecholamines.21

These experiments demonstrate NGF

as-sociated with sympathetic ganglia

through-out the higher vertebrate scale with the

exception of the elasmobranch. Wherever

found, NCF is remarkably similar

immuno-logically suggesting, but certainly not

prov-ing, similarity of molecular structure

throughout

phylogeny.

The

absence of

NFG in the one species of elasmobranch

tested (leopard shark) remains

unex-plained, except to note that during

verte-brate evolution there is a divergence

be-tween tile elasmobranch and other

verte-1)rates included in this study.22

The tissue source of NGF is currently

unidentified. The salivary

gland

was ini-tially considered as the source, since it

con-tains NGF in highest concentration, at least

in the mouse. However, its removal has no

effect on sympathetic ganglia nor does

in-jection of antiserum alter the morphology

of the salivary gland.23 NGF is localized to

the tubular portions of the mouse

submaxil-lary gland and is present in mouse saliva,

suggesting an excretory function.4 Our

studies further exclude salivary tissue as

the primary source: (a) NGF appears in

the sympathetic ganglia of the mouse

be-fore it can be found in the salivary gland;

(b)

it is absent from the salivary gland of

many species, including chicken and

hu-man; (c) it is found in species devoid of

salivary glands, such as frog and fish;

(d) during development, NGF is initially

localized to the area of the sympathetic

chain. These results suggest that the source

and site of action of NGF may well be the

same, namely, sympathetic ganglia.

SUMMARY

A nerve growth-promoting protein (NGF)

has been identified in a number of

verte-brate species, including the human fetus. This protein can be localized to the axial

regions or

the

sympathetic chain directly,

and wherever found is immunologically

similar to NGF from mouse salivary gland.

During development NGF appears

con-comitant with morphologic differentiation

of

the sympathetic chain and always in

association with it. These studies further

implicate the participation of the nerve

growth-promoting

protein in the regulation

of growth of sympathetic ganglia.

REFERENCES

1. Levi-Montalcini, R. : Chemical stimulation of

nerve growth. in The Chemical Basis of

Development, McElroy, W. D., and Glass,

B., Eds. Baltimore: Johns Hopkins Press,

1958, p. 646.

2. Levi-Montalcini, R. : Growth control of nerve

cells by a protein factor and its antiserum.

Science, 143:105, 1964.

3. Cohen, S. : Purification of a nerve growth

r-moting protein from the mouse salivary

gland and its neurocytotoxic anti-serum.

Proc. Nat. Acad. Sci., 46:302, 1960.

4. Levi-Montalcini, R., and Booker, B.: Excessive growth of the sympathetic ganglia evoked

by a protein isolated from mouse salivary

glands. Proc. Nat. Acad. Sci., 46:373, 1960. 5. Levi-Montalcini, R., and Booker, B.:

Destruc-tion of the sympathetic ganghia in mammals by an antiserum to the nerve growth

pro-moting factor. Proc. Nat. Acad. Sci., 46:384,

1960.

6. Shumwav, W.: Stages in the normal

develop-ment of Rana pipiens. I. External form.

Anat. Rec., 78:139, 1940.

7. Levi-Montalcini, R., Meyer, H., and

11am-burger, V.: in vitro experiments on the

effects of mouse sarcoma 190 and 37 on the spinal and sympathetic ganglia of the chick

embryo. Cancer Res., 14:49, 1954.

8. Kabat, E. A., and Mayer, M. M.: Experimental

Immunochemistry. Springfield: Charles C

Thomas, 1981, p. 810.

9. Cram, S. M., and Wiegand, R. C.: Catechol-amine levels of mouse sympathetic ganglia following hpertrophy produced by salivary nerve growth factor. Proc. Soc. Exp. Biol. Med., 107:663, 1961.

10. Yntema, C. H., and Hammond, W. S.: The development of the autonomic nervous sys-tem. Biol. Rev., 22:344, 1947.

11. Raven, C. P.: Experiments on the origin of the sheath cells and sympathetic neuroblasts in

amphibia. J. Comp. Neurol., 67:221, 1937.

12. Brizzee, K. R., and Kuntz, A.: The histo-genesis of sympathetic ganglion cells. An

experimental study. J. Neuropath. Exp.

Neurol., 9:164, 1950.

13. Jones, D. S.: The origin of the sympathetic

trunk in the chick embryo. Anat. Rec.,

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14. Tello,

J.

F.: Sur Ic formation des chaines

primarie et secondarie du grand sympathique

dans l’embryon poulet. Tray. du hab. de Rec.

Biol. de l’Universit#{233}Madrid, 23:1, 1925.

15. Kuntz, A.: Development of the sympathetic

nervous system in amphibia.

J.

Comp. Neurol., 21:397, 1911.

16. Kuntz, A.: The development of the

sympa-thetic nervous system in man.

J.

Comp.

Neurol., 32:173, 1920.

17. Kuntz, A.: The development of the syinpa-thetic nervous system in turtles. Amer.

J.

Anat., 11:279, 1911.

18. Kuntz, A.: The development of the

sympa-thetic nervous system in certain fish.

J.

Comp. Neurol., 21:177, 1911.

19. Von Euler, U. S.: Autonomic neuroeffector transmission. Handbook of

Physiology-Neu-rophysiology, I, Chapter 7. Baltimore:

Wil-hams and Wilkins, 1959, p. 215.

20. Kuntz, A.: The evolution of the sympathetic

nervous system in vertebrates. J. Comp.

Neurol., 21:215, 1911.

21. Ostlund, E.: The distribution of

catechol-amines in lower animals and their effect

on the heart. Acta Phys. Scand., 21, Supp.

112, 1954.

22. Gregory, William King: Evolution Emerging. New York: Macmillan, 1951, p. 86. 23. Levi-Montalcini, R., and Angeletti, P.: Growth

control of the sympathetic nervous system

by a specific protein factor. Quart. Rev. Biol., 36:99, 1961.

24. Levi-Montalcini, R., and Angeletti, P.: Biologi-cal properties of a nerve growth-promoting protein and its antiserum. In Regional

Neu-rochemistry, Kety, S. S., and EWes,

J.,

Eds.,

New York: Pergamon Press, 1961, p. 362.

JUVENILE DLABE1ts: ADJUSTMENT AND

EMO-TIONAL PROBLEMS. Proceedings of a

Work-shop

held

at Princeton, April 22-23, 1963,

Edited by T. S. Danowski, Arthur

Kros-nick, and Harvey C. Knowles, Jr., 151 pp.

Perhaps on no other single subject does such an enormous and complex literature exist as

for Diabetes Mellitus. Yet in the past year the

well-known and often highly distinguished

members of a Workshop, sponsored by the

United States Public Health Service and other bodies, frankly admitted their ignorance both

of the etiology of this common illness and of

the prevention and treatment in childhood of

1965;35;221

Pediatrics

Myron Winick and Robert E. Greenberg

PROTEIN DURING DEVELOPMENT