Developmental Brain Research, 58 (1991) 147-150 147 Elsevier

BRESD 60387

The immunolocalisation of the neuroendocrine specific protein PGP9.5 during neurogenesis in the rat

Christine Kent and Pamela J. Clarke

Department of Human Morphology, Medical School, Queen's Medical Centre, Nottingham NG7 2 UH (U. K.)

(Accepted 2 October 1990)

Key words: Neurogenesis; Neuronal marker; Protein gene product 9.5; Ubiquitin

We have examined the immunolocalisation of the protein gene product (PGP) 9.5 during neurogenesis in the rat embryo. PGP9.5 was first present at 11.5 days gestation (Ell.5): all morphologically recognisable nerve cell bodies and fibres were immunoreactive. In routinely processed, wax-embedded tissues, using a standard immunocytochemical technique, PGP9.5 polyclonal antibody specifically demonstrated the developing nervous system and primitive adrenal chromaffin cells.

A relatively recent addition to the well established list

of neuronal cell markers is protein gene product 9.5 (PGP9.5), a soluble protein isolated from human brain y.

It is a major protein component of neuronal cytoplasm present in a wide range of species 8. PGP9.5 has a more

extensive distribution amongst neuronal subtypes than

neurofilament protein (NF) and neuron-specific enolase

(NSE) and occurs throughout the cell from perikaryon to fine terminals 6. However, its function was unknown until

recently. Evidence has now been reported that identifies PGP9.5 as a carboxyl-terminal hydrolase for ubiquitin, a

molecule involved in the posttranslational targeting of cell proteins for further processing H. The observations

that the ubiquitin COOH-terminal hydrolase isozyme is tissue-specific and the role of ubiquitin varies according

to tissue type could be the basis for the neuronal

specificity of PGP9.5. It is also possible that variations occur during differentiation. We have therefore investi-

gated the appearance of the molecule in the developing

nervous system of the rat from the day of neural tube

closure (El0). Using a polycional antibody to PGP9.5 raised in rabbit 4 we have demonstrated, with a specificity

and clarity comparable to that described in adult tissue 12,

the presence of the marker in embryonic nervous tissue as early as E l l .5. This is the first report we are aware of

on the use of PGP9.5 as a marker during neurogenesis; some of the work has already been presented 3.

Wistar rats kept under standard conditions of heating

(20 °C) and lighting (12h light-12h dark) with free access to food (Heygate rat breeding diet) and water were allowed to mate and the morning of the appearance of a vaginal copulatory plug taken as day 0 (E0) of gestation.

Embryos were obtained from dams deeply anaesthetised

by an intraperitoneal injection of sodium pentobarbitone

(60 mg/kg b.wt.) on embryonic days El0, E l l , El l .5 , E12, E12.5, E13 and E15, dissected free from surround-

ing membranes and immersed in Bouin's fixative for 6 h

then embedded in paraffin wax. Serial 5-6 ktm sections in either transverse or longitudinal planes were mounted on

polylysine-coated slides. Immunohistochemistry. Antigen was visualised by the

avidin-biotin method using the Vectastain ABC perox- idase kit (Vector Laboratories, Peterborough) with DAB

(diaminobenzidine tetrahydrochloride). Primary anti-

body (rabbit polyclonal anti-PGP9.5; Ultraclone Ltd., Wellow, Isle of Wight, U.K.) diluted between 1:800 and

1:1600 was applied at 4 °C overnight. Every 10th section from each of 3 embryos at each time interval was

processed. Adjacent sections were stained with haematoxy- lin to facilitate the identification of the tissue architecture.

Control sections in which non-immune rabbit serum

was substituted for primary antibody showed no specific immunostaining.

Progenitor cells. The dividing cells of the ventricular zone and neuroblasts in the mantle layer of the neural tube were not immunostained with PGP9.5 antibody.

Neither were migrating neural crest cells. The only evidence of cells expressing the PGP9.5 antigen before

reaching their normal destination came from one El3 embryo (Fig. 1), in which cells anterior to the para-

vertebral sympathetic ganglia and lateral to the aorta on the predicted pathway of neural crest cells destined for the pre-aortic ganglia or the adrenal medulla were immunoreactive.

Correspondence: C. Kent, Department of Human Morphology, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, U.K.

0165-3806/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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Nerve cell bodies. PGP9.5 was first de tec ted in the

neuronal cell bodies of cranial nerve nuclei (Fig. 2) and

in dorsal root ganglia ( D R G ) and sympathet ic ganglia

(SR) in a more rostral region of the spinal cord in E l l . 5

embryos. The cell bodies of the pr imary motor neurons

in the ventral horn region also stained at E l l . 5 . In

general , cells were PGP9.5-posi t ive as soon as they were

recognisable as being neuronal e i ther by their posit ion or

by their morphology.

Nerve fibres. In the spinal cord the per ipheral ly

d i rec ted processes of early unipolar pr imary motor

neurons s tained at E l l . 5 and at later stages were seen to

consti tute the axons that formed the ventral root.

Immunos ta ined sensory axons of dorsal root ganglion

cells had reached the spinal cord by E l 2 (Fig. 3) and in

t ransverse sections could be seen to enter it at a densely

s tained area, the oval bundle of His. This region, where

the first afferent fibres to reach the spinal cord begin to

bifurcate , was part icular ly densely stained up to day E l 5

(Fig. 4). Mixed spinal nerves were strongly immunoposi -

tive at E l 3 (Fig. 5).

Dorsal root ganglia and the 'boundary cap'. The dorsal

root ganglia from stages E l l . 5 to E l 3 were divided into

two regions, a ventrola tera l region in which the cyto-

plasm of the major i ty of the cells was immunosta ined and

a dorsomedia l region where few cells were positive and

which corresponds to the ' boundary cap ' (Fig. 6).

Enteric neurons. The earliest evidence of PGP9.5-1ike

immunosta ining in the enteric nervous system was in the

oesophagea l plexus of an E l l . 5 embryo. Neurons with

posit ively s tained cytoplasm and fine processes were seen

in the wall of the s tomach at day E12.5 and by E l 5 had

formed a more clearly defined myenter ic plexus. In no

stages were there any signs of immunostaining of neu-

roendocr ine cells in the gut epi thel ium (Fig. 7).

Adrenal medullary cells. Islands of PGP9.5-posi t ive

cells were seen in the medul lary region and at the medial

pole of the adrenal gland at E l5 .

Structures outside the nervous system. The only struc-

tures o ther than those of nervous origin observed to stain

with PGP9.5 ant ibody were groups of cells in the

developing gonad at E l 3 (Fig. 2).

The results show that polyclonal PGP9.5 ant ibody

specifically s tained neuronal derivat ives in the embryo

and that the PGP9.5 antigen is expressed early in the life

of the neuron. In the dorsal root ganglion at E l 2 the

major i ty of neurons expressed PGP9.5 at a stage,

according to Al tman and Bayer 1 and Lawson, Caddy and

Briscoe t°, before the peak day for the genera t ion of these

neurons. It is possible, therefore , that some of the

PGP9.5-posi t ive cells were premitot ic . The ' boundary

cap ' region of the dorsal root ganglion is thought by

Al tman and Bayer I to consist of non-neuronal , possibly

presumpt ive glial e lements or it may represent the

immature B neurons descr ibed by Lawson et al. ~°. Ei ther

explanat ion would be consistent with the absence of

PGP9.5 expression. Pr imary moto r neurons were immu-

nostained as soon as they were dist inguishable from

neuroblasts by their slightly e longated shape, and neu-

ronal processes seemed to be PGP9.5-posi t ive as soon as

they sprouted from nerve cell bodies. The cells dest ined

to form the enteric nerve plexuses conta ined PGP9.5 at

a primit ive stage and the first neuroendocr ine cells to

appear in the presumpt ive adrenal medul la were a l ready

immunoposi t ive , as were p robab le phaeochromoblas t s at

the medial pole of the adrenal .

There were no convincing cases of migrat ing neural

crest cells staining with PGP9.5. The neural crest is a

fleeting structure that appea red throughout E l l embryos

and in the caudal region of E l l . 5 embryos , and it can be

difficult to distinguish whether isolated cells are stained

in tissue sections if there is any degree of background

staining. Since cells in division in the ventr icular zone and

neuroblasts in the mantle zone of the spinal cord were

also negat ive, it is unlikely that precursor cells express

PGP9.5 until they have progressed some way along the

neuronal l ineage, al though there is always a possibil i ty

that antigen is present in amounts below the sensitivity of

the technique.

PGP9.5 is a soluble cytoplasmic prote in which does not

appear to be associated with any cytoarchi tectural feature

of the neuron 6. Wilkinson et al. 1~ have shown the

neuronal prote in PGP9.5 to be ubiquit in CO O H - te rmina l

hydrolase isozyme, which they speculate may have a role

Fig. 1. Abdominal region of an El3 embryo showing immunostaining of ventral horn (VH), sympathetic ganglion cells (S), presumptive pre-aortic ganglion cells (arrowed) and a positive reaction in the gonad (G). Bar = l id ktm. Fig. 2. PGP9.5-positive cranial nerve nucleus at Ell.5. Bar = 10/~m. Fig. 3. Longitudinal section through the spinal cord and dorsal root ganglia (DRG) of an El2 embryo. Sensory axons (arrowed) have reached the spinal cord and are more heavily immunostained than the cell bodies. Ventral horn motor neurons (VH) are immunostained with PGP9.5 but precursor cells of the ependymal and mantle layers are unreactive. Bar = lid/~m. Fig. 4. Dorsal root ganglion at El3 showing bipolar neurons with intensely immunostained processes. Oval bundle arrowed. Bar = 20/~m. Fig. 5. Longitudinal section of an El3 embryo showing immunostaining of DRG and mixed spinal nerves. Bar = l id ktm. Fig. 6. A: sagittal section of DRG at El3 showing division into a region of bipolar neurons, the majority positive for PGP9.5, and a dorsal region (BC) in which few cells are immunostained. B: the adjacent section stained with haematoxylin and eosin illustrating the presence of smaller non-immunostained cells in the dorsal region, the boundary cap (BC). Bar = 20/~m. Fig. 7. Enteric neurons in the wall of the stomach immunostained for PGP9.5 at E12.5. Bar ~ 20/~m.

15(1

in the disassembling of ub iqu i t in -pro te in complexes

formed during normal metabol ism. The varying role of

ubiquit in in different tissues means that the expression of

the isozyme is tissue-specific. The results of the present

study suggest that it is a cytoplasmic component which

becomes tissue-specific at an early stage in differentia-

tion. The dis tr ibut ion of immunostaining for PGP9.5 was

consistent with that descr ibed in the adult of several

mammals , including the human. As well as consistent

immunosta ining of all nervous and most neuroendocr ine

systems, Wilson et ai. ~2, using the same polyclonal

PGP9.5 ant ibody and two monoclonal ant ibodies, de-

scribe localisation in germ cells and Leydig cells of the

testis and in ova and the theca externa of the ovary. We

have shown that undifferent ia ted gonadal cells are also

immunoposi t ive with the polyclonal ant ibody. This may

be due to cross-react ion with similar epi topes in gonadal

prote ins or the neuronal ubiquitin isozyme may have a

function in gonadal tissue. The lack of immunostaining

with the PGP9.5 polyclonal ant ibody in the neuroendo-

crine cells of the a l imentary tract is also in keeping with

the findings of Wilson et a1.12, although they found that

the cells did stain with the monoclonal PGP9.5 ant ibody

(13C4) in mater ia l fixed in acetic acid/alcohol. It is

possible that PGP9.5 could be detected in fetal gut

neuroendocr ine cells with the use of the appropr ia te

an t ibody and fixation method.

In an investigation of cardiovascular innervat ion of the

guinea-pig, Gulbenkian et al. 6 found that PGP9.5 was

more widely dis t r ibuted amongst neuronal subtypes than

ei ther NSE or neurof i lament tr iplet protein and demon-

s t ra ted that it occurred in both cholinergic and adrenergic

nerves. We have now demons t ra ted the widespread

occurrence of PGP9.5 in the developing nervous system.

Surface binding sites for tetanus toxin (TT) have been

1 Altmann, J. and Bayer, S.A., The development of the rat spinal cord. In F. Beck, W. Hild, W. Kriz, R. Ortmann, J.E. Pauly and T.H, Schiebler (Eds.), Advances in Anatomy, Embryology and Cell Biology, Vol. 85, Springer, Berlin, 1984, pp. 53-95.

2 Bennett, G.S., Hollander, B.A. and Laskowska, D., Expression and phosphorylation of the mid-sized neurofilament protein NF-M during chick spinal cord neurogenesis, J. Neurosci. Res., 21 (1988) 376-390.

3 Clarke, P.J. and Kent, C., The ontogenesis of the neuronal marker PGP 9.5 in some neural crest derivatives of the rat, J. Anat., 167 (1989) 270.

4 Doran, J.E, Jackson, P.J., Kynoch, P.A.M. and Thompson, R.J., Isolation of PGP 9.5, a new human neurone specific protein detected by high resolution two-dimensional electropho- resis, J. Neurochem., 40 (1983) 1542-1547.

5 Grothe, C. and Unsicker, K., Reciprocal age-dependent pattern of two neuronal markers, tetanus toxin and neuron-specific enolase, in postnatal rat sensory and sympathetic neurons, Dev. Brain Res., 39 (1988) 1-8.

6 Gulbenkian, S., Wharton, J. and Polak, J.M., The visualisation of cardiovascular innervation in the guinea pig using an antiserum to protein gene product 9.5 (PGP 9.5), J. Auton. Nerv. Syst., 18 (1987) 235-247.

7 Jackson, P. and Thompson, R.J., The demonstration of new

suggested as one of the earliest indicators of neuronal

differentiat ion 9, but Gro the and Unsicker 5 repor t that TI"

may only serve as a neuronal marker up to the neonatal

stage in the rat. Af te r that there is a gradual transit ion

from the major i ty of the neurons being TT-posit ive and

NSE-negat ive at birth to the adult si tuation in which

neurons are TT-negat ive and NSE-posi t ive. They recont-

mend TT as a marker for the newborn, NSE for the adult

and a combinat ion of the two during adolescence. In

contrast , PGP9.5 immunoreact ivi ty is detected from the

bir thdate of neurons (e.g. in the D R G ) and increases in

intensity into adul thood. The o ther marker that can be

detected in the fetus is neurof i lament protein, and the

pat tern of deve lopment of NF immunoreact ivi ty in the

chick spinal cord 2 is remarkably similar to that of PGP9.5

repor ted here, part icular ly the intense reaction of the

oval bundle; however , N F is not usually found in

neuroendocr ine cells. Al though PGP9.5 does not appear

to be associated with structural proteins such as those

constituting neurofi laments , the observat ion that it arises

so early in the life of the neuron underl ines the suggestion

by Wilkinson et ai. ~ that it plays an impor tant role in

neuronal metabol ism.

In the opinion of Wilson et al. 12, PGP9.5 is ' the best

immunohis tochemical marker for nerves current ly avai-

lable ' . We have shown that it has the addit ional advan-

tage that it can be used in convent ional ly processed tissue

to label neurons and neuroendocr ine cells at very early

stages in differentiat ion, as early as E l i . 5 in the rat, and

believe it will prove a useful tool in studying the

developing nervous system.

The authors wish to thank Alison Bexon for the secretarial work and Alan Pyper and Ivan Harriman for the photography.

brain-specific proteins by high-resolution two-dimensional poly- acrylamide gel electrophoresis, J. Neurol. ScL, 49 (1981) 429-438.

8 Jackson, P., Thompson, V.M. and Thompson, R.L, A compar- ison of the evolutionary distribution of the two neuroendocrine markers, neurone-specific enolase and protein gene product 9.5, J. Neurochem., 45 (1985) 185-190.

9 Koulakoff, A., Bizzini, B. and Berwald-Netter, Y., A correla- tion between the appearance and the evolution of tetanus toxin binding cells and neurogenesis, Dev. Brain Res., 5 (1982) 139-147.

10 Lawson, S.N., Caddy, K.W.T. and Biscoe, T.J., Development of rat dorsal root ganglion neurones. Studies of cell birthdays and changes in mean cell diameter, Cell Tissue Res., 153 (1974) 399-413.

11 Wilkinson, K.D., Lee, K., Deshpande, S., Duerksen-Hughes, P., Boss, J.M. and Pohl, J., The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase, Science, 246 (1989) 670-673.

12 Wilson, P.O.G., Barber, P.C., Hamid, Q.A., Power, B.E, Dhillon, A.P., Rode, J., Day, I.N.M., Thompson, R.J. and Polak, J.M., The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies, Br. J. Exp. Pathol., 69 (1988) 91-104.