Developmental Brain Research, 61 (1991) 173-182 ~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0165-3806/91/$03.50 A DONIS 0165380691512972

BRESD 51297

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The pattern and timing of cutaneous hair follicle innervation in the rat pup and human fetus

Jonathan Payne, Jacqueta Middleton and Maria Fitzgerald Department of Anatomy and Developmental Biology, University College London, Gower Street, London (U. K.)

(Accepted 9 April 1991)

Key words: Development; Skin; Hair; Somatosensory; Innervation; Human; Rat; Terminal

The postnatal development of hair follicle innervation was studied in the rat hindlimb using a silver stain which detects large and medium calibre cutaneous nerve fibres. The pattern and timing of innervation in relation to postnatal changes in follicle growth were studied providing new data on nerve-target interactions in the developing peripheral nervous system. Sensory axons begin to leave the dermal plexus and grow towards follicles at P (postnatal day) 3 but do not start to innervate them until P7 or achieve an adult appearance until P19. The first terminals are circumferential, followed some days later by the appearance of palisade endings. The number of axons innervating a hair follicle increases steadily with age until P19 and there is no evidence of exuberant innervation of follicles during development. Hair follicle density in the rat is maintained during development due to waves of small, vellus follicle growth later in postnatal life as the skin grows. The percentage of follicles innervated however, decreases from the second postnatal week onwards presumably because late developing veilus hairs do not become innervated. Comparative analysis in human fetal abdominal skin using the same silver stain reveals a similar sequence and pattern of innervation to the rat over the period of 22 to 35 weeks EGA (estimated gestational age). Human skin does not, however, undergo the late waves of follicle growth seen in the rat. Follicular density decreases and the percentage of innervated follicles increases in the third trimester of fetal life.

INTRODUCTION

The sensory innervat ion of hair follicles in the skin of the rat 2°'26'27 and man has been well descr ibed 21'33. There

is still much to be learnt , however , about how this innervat ion develops 3'17As'32. The pos tnata l matura t ion

of sensory afferents in the rat mystacial pad has been

s tudied and shown to occur in successive waves over a

p ro longed pe r iod 3'22'32. Here we concentra te on the

pos tna ta l matura t ion of follicle innervat ion in the less

special ized skin regions of the rat hindlimb.

There are several reasons for wishing to examine this

deve lopment . In the rat , considerable informat ion is

accumulat ing about the outgrowth of cutaneous per iph-

eral and central processes of lumbar dorsal root ganglion cells 1'13'25, the format ion of central cutaneous afferent

terminals in the spinal cord 25'28'30 and of functional

connect ions with dorsal horn cells leading to the transfer

of cutaneous sensory informat ion to the CNS 6'10. In o rde r

to in terpre t these events and unders tand the interact ions

be tween the format ion of per iphera l and central connec-

tions during deve lopment we need to know not only the

t iming of cutaneous innervat ion but also the process by

which it reaches its final pat tern . Hindl imb hair follicles

are a par t icular ly useful target for this s tudy since they

are easily identif ied and are innervated by myel ina ted

fibres in the skin which form morphologica l ly distinctive terminals a round the foUicle 2°'26'27. Fu r the rmore , the

central terminals of these hair follicle afferents form

characteris t ic ' f l ame-shaped ' arbors within the spinal cord

dorsal horn 29'34 and can therefore always be ident if ied

anatomical ly at their central ends.

Hairs form a significant ' sensory surface ' in the human

fetus and little is known about the deve lopmen t of their

innervat ion 21'23. A fur ther aim of this s tudy was to

compare the sequence and pa t t e rn of hair innervat ion in

the rat with that in human skin. Such compara t ive data

is impor tan t if studies on the l abora to ry rat are to

contr ibute to our unders tanding of sensory deve lopment

in man.

MATERIALS AND METHODS

Tissue collection Rat. Sprague-Dawley rat pups aged 0.5 (where P0 is the day of

birth), 3, 5, 7, 10, 12, 15 and 19 days old and 2 adult rats were sacrificed with an overdose of barbiturate anaesthetic. They were perfused intracardiaUy with normal saline followed by 4% parafor- maldehyde in 0.1 M phosphate buffer. Approximately 0.5 cm 2

Correspondence: M. Fitzgerald, Dept. of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, U.K.

174

TABLE 1

Postnatal changes in mean interfollicle distance in rat hindlimb

Age (days) Lateral leg Dorsum o f foot

Distance (l~m) n Distance (Itm) n +_ S.D. + S.D.

0.5 95 + 26 54 70 _+ 26 48 3 76 + 35 82 83 + 27 40 5 58 + 21 75 59 _+ 24 70

10 91 + 36 82 160 + 68 80 19 84 + 44 55 100 + 28 82 Adult 67 + 38 39 130 _+ 65 50

pieces of skin were dissected from both the lateral thigh and dorsum of the foot and placed in Winkelmann post-fix solution; 10% formaldehyde, 15% sucrose, 0.35% ammonium in 0.1 M phosphate buffer for a minimum of 7 days.

Human. Small samples of human fetal skin from the midline abdominal region were obtained from the pefinatal pathology department at University College Hospital. Samples were obtained from fetuses at each of the following ages: 18, 21 22, 24, 29.5 and 35 weeks estimated gestational age (EGA). The 18 and 35 week fetuses were stillborn, the 21, 22, 24 and 29.5 week fetuses were spontaneous abortions, the 29.5 week fetus surviving only 33 h postnatally. Tissue was either removed a few hours after death or after two to three days chilled storage. Upon removal the skin was placed in Winkelmann post-fix solution for at least 7 days and then processed exactly as the rat skin.

Histological processing Tissue was defatted twice for optimum results and then 100-/~m

frozen sections cut on a freezing mierotome. Sections were stained using the Winkelmann silver method described in detail elsewhere 2°. The tissue was then examined and analyzed under the light microscope and the follicle innervation drawn using a camera hicida.

RESULTS

General features o f postnatal hair follicle development in

the rat hindlimb

Hai r follicles are easily ident if iable in the skin of both

the la teral leg and the dorsum of the foot from birth.

They do not begin to produce hairs, however , until P3

and there cont inues to be a large number of quiescent

follicles until P5. Sebaceous glands associated with the

follicles cannot be de tec ted until P7. Ha i r follicle densi ty

on the la teral leg does not change significantly with

pos tna ta l age (Table I). On the dorsum of the foot,

however , densi ty decreases in the second postnata l week

and remains about half that of the lateral leg in the adult

(Table I). Since hair follicle densi ty will depend on the

rate of product ion of new follicles and the change in skin

area , it must be concluded that whereas these two factors

balance out on the lateral leg, pos tnata l hair follicle

product ion is not as great on the dorsum of the foot.

The adul t hair follicle types cannot be proper ly

dis t inguished until P7, when they clearly fall into small

TABLE II

Postnatal changes in the percentage of small follicles in the rat hindlimb

Age (days) % Small foUicles (<300pro)

Lateral leg Dorsum of foot

0.5 67 76 3 58 74 5 70 80 7 9 7

10 0 0 12 3 0 15 14 0 19 21 26 Adult 79 53

and large size categories , cor responding to vellus and

guard (and a small number of very large tylotr ich) hairs.

Table II shows that hair follicle deve lopmen t in the lateral

leg appears to occur in two waves. The propor t ion of

small follicles (less than 3 0 0 / z m length) falls s teadily

be tween P5 and P10 as they grow into larger guard hair

follicles but P15 marks a second increase in the popula-

t ion of small follicles which will p resumably become the

vellus hairs that form a significant percentage o f the total

popula t ion in the adult 2°. A similar second wave of small

follicle deve lopment occurs in the dorsal foot skin but

slightly la ter at P19.

Postnatal development o f hair follicle innervation in the

rat hindlimb

The WLnkelmann silver stain technique was used to

detect the onset of follicle innervat ion by large and

medium calibre cutaneous axons. A t the ear l ies t ages

studied, P0.5 and P3 nerve fibres are seen but are

restr ic ted to the dermis. Very occasional ly, at P3, small

bundles of 1-3 axons leave the dermal ne twork and are

seen to ascend towards the epidermis , approaching hut

TABLE III

Percentage of rat hindlimb hair follicles innervated at different postnatal ages

Age (% of all follicles innervated)

Lateralleg Oorsura of foot

0 . 5 -

3 ~ -

5 58* 52* 7 70 42

10 53 78 12 80 73 15 77 94 19 54 97 Adult 62 52

* Axons associated with follicles, not innervating them.

175

P5

I

P7 P7

Fig. 1, Camera lucida drawings of 100-#m skin sections from the lateral leg of P5 and P7 rat pups. Skin surface is uppermost. At P5 some axons are seen growing towards and becoming associated with hair follicles. At P7 fibres are beginning to grow circumferentiaUy around the follicles. Scale bar: 100/~m.

not reaching the base of developing hair follicles.

A t P5, bundles of fibres can be seen to have reached

the base of follicles and in some cases grow up into the

epidermis alongside and in association with a hair shaft

(Fig. 1). Table I I I shows that over half the follicles have

such a re la t ionship with growing axons. However , there

TABLE IV

Mean number o f axons per rat hindlimb follicle at different postnatal ages

Age Mean number o f n axons + S.D.

0 . 5 w _

3 1.9 + 0.9 11 5 2.5 + 1.1 32 7 2.5 + 1.0 19

10 2.8 + 0.9 22 12 3.3 + 1.2 30 15 3.3 + 1.1 28 19 4.0 + 1.3 63 Adult 4.0 + 0.9 19

was no sign of any morphologica l special izat ion of the

nerve endings at this age or even any growth a round the

follicles. The axons had apparen t ly reached the follicles

and begun to grow in associat ion with them but not yet

te rminate on them.

By P7 follicle innervat ion has begun. Bundles of up to

20 fibres leave the dermal plexus and travel superficially

before dividing in the region of the hair follicles, typically

giving off two branches that fur ther subdivide to supply

nearby or ad jacent follicles (Figs. 1 and 2). Some fibres

grow on up into the epidermis and in t raep idermal fibres

are just visible at this age. The number of axons

associated with each follicle increases to be tween 2 and

6 (Table IV). A t this age the beginning of morphologica l

specialization of terminals is clear in that some fibres are growing circumferent ial ly a round the follicles. These

circumferent ial or presumpt ive Ruffini terminals are the

first clear morphologica l te rminal type to appear in

association with the hair follicles.

A t P10, circumferentiaUy organized terminal fibres can

be seen a round many of the hair follicles. In addi t ion it

~ 0 ~r

"U

"0

177

i"

P12 P19

Fig. 3. Camera lucida drawings of 100-/gm skin sections from the lateral leg of P12 and P19 rat pups. The density of innervation was at its highest at P12 and decreased at P19. The increase in complexity of innervation compared to that in Fig. 1 can be clearly seen. Arrows show part of a circumferential ending at P12 and a palisade ending at P19. Scale bar: 100/~m.

18wks

22wks

21wks Fig. 4. Camera lucida drawings of 100-/~m sections of human fetal abdominal skin at 18, 21 and 22 weeks estimated gestational age. Axons only begin to approach follicles at 22 weeks. Scale bar: 100 gm.

178

29½ wks

/

f 35wks

Fig. 5. Camera lucida drawings of 100-/~m sections of human fetal abdominal skin at 29.5 and 35 weeks estimated gestatioaal age, The follicle innervation at 35 weeks is markedly more mature than that seen at younger ages. Open arrow indicates part of a circumferential ending and closed arrow indicates a palisade ending. Scale bar: 100/zm.

is now possible to detect terminal fibres arranged vertically along the hair follicle shaft beginning to form

'palisades' , arrays of 1-5 lanceolate terminals around the

long axis of the follicle. These are increasingly common at P12 and by P19 were similar in appearance to those

seen in the adult (Fig. 3). Furthermore, at this age the other elements of adult innervation of follicles such as

TABLE V

The development of hair foUicles and their innervation in human skin

Age (weeks) Interfollicle % Follicles distance (l~m) innervated

18 300-500 - 21 200-400 <1" 22 100-300 28* 29.5 300-500 21 35 700-1000 56

* Axons associated with follicles but not yet innervating them.

single axons innervating the mouth of the follicles and entering their connective tissue sheaths were also iden-

tifiable. Tables I I I and IV illustrate two general points about

the development of hair follicle innervation in the rat. Firstly, the number of axons contributing to this inner-

vation or innervation density increases steadily over the first postnatal weeks and is mature by P t9 (Table IV).

Secondly, the percentage of follicles innervated also increases postnatally but then drops substantially in the third postnatal week (or later in the ~ of the dorsum

of the foot) to approach the low value found in the adult z°. This presumably reflects the overall increase in number of hairs in the adult due to late waves of vellus hair development (Table II) which do not become innervated.

Development o f hair follicle innervation in human skin At 18 weeks E G A , follicles are clearly present but

179

%

2 2 w k s ~ ' 3 5 w k s ' '

Fig. 6. Photomicrographs of 100-/~m sections of human fetal abdominal skin at 22 and 35 weeks. At 22 weeks the sensory axons (arrow) approach but do not yet innervate the follicle, whereas at 35 weeks a complex palisade terminal (arrow) has formed around the follicle. Scale bar: 100/~m.

none are innervated. Only the occasional nerve fibre is

seen running within the dermis and hypodermis. By 21

weeks more nerve fibres can be detected and the occasional axon can be seen beginning to grow towards

the hair follicles although they have not yet reached them (Fig. 4). At 22 weeks there is a marked maturation and

many larger bundles of nerves (5-10 axons) can be

detected in the dermis and hypodermis running parallel to the skin surface and fibres branching off from these are

beginning to organize themselves around the follicles but still not actually contact them (Figs. 4 and 6 and Table

V). At 24 and 29.5 weeks many fibres can be detected in the hypodermis and dermis in bundles of 20-30 fibres

running parallel to the skin surface and sending branches up towards the epidermis. The follicles become inner- vated and start to form recognizable morphological endings on the hair follicles (Fig. 5). These are all circumferentially arranged around the hair follicle in a similar pattern to that seen in the rat pup at P7.

At the oldest age studied, 35 weeks, there is a qualitative change in the skin and its innervation (Figs. 5

and 6). Thick nerve bundles following the path of blood

vessels send numerous branches up towards the epider-

mis. Many of these terminate in free nerve endings in the

dermis and others are associated with sweat glands which are now very clearly present. Numerous circumferentially

arranged terminals are seen and in addition clear palisade arrangements of fibres are now present. The majority of

follicles have nerve fibres associated with them at this age

but not all are yet organized into identifiable morpho- logical patterns.

DISCUSSION

While there are several earlier studies on the produc- tion of hair fol l ic les 1921'23'31 and on the development of skin and follicle innervation 3'5'14'28'32, this is the first

attempt to study the two together in the rat hindlimb. The aim was to view the growth of sensory fibres in relation to the growth of their target follicles and build up a picture of the density as well as the morphology of developing sensory innervation.

180

The findings confirm that follicles are formed prena- tally and that their development occurs in waves 19"23. In

the mouse the largest tylotrich follicles develop first, followed by intermediate follicles and finally the small vellus hairs 19 and the findings here in the rat are consistent with this. Waves of hair growth occur in the first and third postnatal week in the rat, and presumably beyond. The earlier waves appear to produce guard hairs (and presumably some tylotrichs) whereas the later waves remain small and become vellus hairs resulting in this hair follicle type making up the majority of the total popu- lation in the adult hindlimb 2°. A wave of hair follicle growth can also be seen in our human material at 22 weeks. This sequence of events is consistent with earlier findings in human fetal skin, showing that the first follicles appear randomly distributed over the skin surface but at relatively fixed intervals and then as the skin grows and the interfollicular distance reaches a certain critical size a new wave of follicular growth is triggered 3~. The clear difference in human and rat skin is seen in the final follicular density. In humans follicle growth is complete by 5 to 6 fetal months and from then on becomes 'diluted' by skin growth 31. This explains the considerable decrease in interfollicular distance observed here at 35 weeks which continues through fetal and neonatal life 31. In the rat, follicle growth continues well into postnatal life at a rate that compensates for skin growth and maintains follicle density. The final density is lower on the foot than on the lateral leg as described elsewhere 2°.

The Winkelmann silver technique used here to stain cutaneous nerves is the same as that used to study the innervation of hair follicles in the adult rat hindlimb and face 20"26"27. The technique only stains large- and medium-

sized dorsal root ganglion cells and hence A beta and A delta fibres in the skin and not C fibres z°. While we have no data here on unmyelinated fibres, the technique is capable of detecting very fine endings of myelinated fibres, which were the subject of the present investiga- tion. Inspection of sections from a different study 25 using GAP-43 immunostaining which labels all growing axons, confirmed that the Winkelmann technique was detecting the earliest innervation of the follicles.

In the adult the pattern of innervation of tylotrichs, guard and vellus hairs has been described in detail and the complexity of innervation shown to increase with follicle size 2°'z6'a7. While less than 10% of vellus hairs in the adult are innervated, 80% of guard hairs and all tylotrichs have nerve terminals associated with the follicles. A further striking feature of adult hair follicles is their polyneuronal innervation whereby up to 15 fibres can innervate a single follicle, depending on its size 2°. In the present study we have been able to describe how this

pattern of innervation emerges. The first important point is the relative delay in onset

of follicle innervation. This is also found in the rat facial region although innervation of vibrissae begins before birth z2. Cutaneous innervation of the hindlimb begins in the upper lateral leg at E14-15 and reaches the tip of the toes by birth 25. There is no evidence for any postnatal increase in the number of peripheral axons, in fact if anything, there is a decrease 17. It is likely, therefore, that axons 'wait' in the dermal plexus for about a week before innervating target follicles. The possibility that earlier innervation by fine axons not detected by the methods used here cannot be entirely excluded. Nevertheless neither GAP-4325 nor CGRP immunostaining, both of which label fine unmyelinated axons in the skin reveal such early innervation. While studies have been made of chemotropic factors responsible for initial cutaneous innervation 17'1s nothing is known of the trigger for axons to leave the plexus and grow to the follicles. Target recognition may be related to the basement membrane- specific proteoglycan molecules that are expressed in developing follicles 4.

The different proportions and complexity of innerva- tion of different follicle types may simply be a reflection of timing. The late development of veUus hairs may mean that they miss the period of active growth and terminal formation of cutaneous axons which occurs in the first 3 postnatal weeks. These hairs are presumably used purely for insulation and protection rather than for sensory detection. While the definition of distinct morphological endings during development is not easy, it was clear from our results in both rat and human that circumferential endings are the first to differentiate and that palisade endings develop somewhat later. This is in agreement with the earlier findings of Bressler and Munger in the primate face 3.

Examination of the number of axons associated with individual follicles provide a picture of the development of their polyneuronal innervation. This is achieved gradually over the first 3 postnatal weeks, as additional axons grow up from the dermal plexus to join those that have already reached the follicles. This is in complete contrast to the situation in the neuromuscular junction where multiple or polyneuronal innervation of end plates occurs initially early in development and subsequently withdraws to leave one axon per end plate 15'24. There is no evidence of such excessive or exuberant innervation of follicles at any stage. This does not mean that there is no exuberant cutaneous innervation earlier in the prenatal period when the initial plexus is formed 25 before dorsal root ganglion cell death takes place 5, although derma- tomal boundaries are specific from the outset 2s.

The innervation of follicles has little qualitative effect

181

on the physiological proper t ies of afferents. Recordings

of the recept ive field and response proper t ies of individ-

ual afferents in the neonata l rat h indl imb show that the

typical rapidly adapt ing low threshold responses associ-

a ted with hair follicle afferents are clearly identif iable

from before bir th in the absence of hairs or follicle

innervat ion 7'9. The deve lopment of innervated follicles

appears to amplify the responses and increase the

f requency discr iminat ion of such rapidly adapt ing affe-

rents rather than produce totally new receptor propert ies 7.

One point that emerges from the postnata l innervat ion

of follicles is that it occurs after the format ion of

character is t ic hair follicle afferent terminals in the spinal

cord dorsal horn. Whi le , ear l ier in deve lopment , the

initial growth of pr imary afferents into the spinal cord

does corre la te with the arrival of those afferents at their

per iphera l target , the e labora t ion of specific central

te rminal pa t te rns appears to be independent of per iph-

eral events. Golgi studies have shown that ' f l ame-shaped '

te rminal arbors , characteris t ic of hair follicle afferents

appea r at E l 8 in the lumbar cord 2 which is well in

advance of follicle innervat ion in the per iphery . This

result implies that it is not the per iphera l target that

de te rmines the characteris t ic growth pa t te rn of the

central terminals of a p r imary afferents but some central

factor. This is suppor ted by the finding t h a t such

terminals can develop a different morpho logy when

induced to grow into inappropr ia te central regions, in

spite of continuing to innervate hair follicles in the

per iphery 12.

Finally, some compara t ive da ta can be gained from our

human material . Follicle growth in the human takes place

in the 4th and 5th fetal month 23 and here we show that

innervat ion takes place soon after. Desp i te differences in

the final hair densi ty of rats and man the pa t te rn of

follicle innervat ion is very similar. The deve lopmenta l

t imetable in the two species is different but hair follicle

innervat ion appears to be one of a number of events in

the developing somatosensory system that mature in the

3rd t r imester of human fetal life and the second postnata l week in the rat 11.

Acknowledgements. This work was supported by the MRC.

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