catagen in the hairless house mouse

Catagen in the Hairless House Mouse ' D. F. G. ORWIN? H. B. CHASE AND A. F. SILVER Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island

ABSTRACT A morphological and histochemical study was made of some aspects of the catagen phase of the first hair cycle in hairless (hr) mice.

The histochemical tests used included those for sulphydryl and disulfide groups, collagen, and periodic acid-Schiff-reactive materials. Measurements were made of skin thickness, follicle length and for follicles in catagen VI and VII, epithelial column length, length of hair shaft in the skin, the width of glassy membrane and connective tissue sheath, and the width of collagen in the glassy membrane. The results showed that:

1. The mutant epithelial column shortens at a slower than normal rate so that at catagen VII, the total follicle length is greater than normal. The position of the mutant hair shaft in the skin is normal at this stage. 2. The keratinization sequence of the mutant club appears normal. The failure

to form an anchoring brush is associated with a morphological variation in the cell shapes and fibrils of the club. 3. The inner root sheath of the mutant follicle, although apparently keratinizing

in the normal sequence, extends further up the follicle than normal and also further down, enclosing the club.

4. The glassy membrane and connective tissue sheath of the mutant are abnormally narrow due mainly to a marked reduction in collagen, and to a narrower band of PAS-positive material.

Hereditary hairlessness (hr) in the house mouse can be attributed to a single recessive gene (Snell, '31; Chew et al., '31). The first macroscopic manifestation in homozygous hairless animals is hair loss around the eyes and front paws, occurring 10-14 days after birth (David, '31). The loss of hair progresses posteriorly from the head as a wave and is completed for most follicles by 21 days of age (Crew et al., '31; Mann, '61). During the second hair cycle, only vibrissae and some tylotrichs produce hairs and these are usually abnormal (Mann, '61).

Hair loss and the failure of hair to re- grow have been attributed to various events and malformations occurring during the catagen phase (Straile et al., '61) of the hair cycle. According to David ('31), the club ends of the mutant are abnormal and responsible for hair loss. Chase and Mann ('60) stated that hair loss is caused by failure of the anchoring club to keratinize. Both David ('31) and Chase ('54a) re- ported the presence of an abnormal extension of the inner root sheath around the club. Several authors (David, '31; Chase et al., '52; Montagna et al., '52) re- ported that the epithelial column of the

AM. J. ANAT., 221: 489-508.

catagen follicle of the mutant does not undergo shortening but attenuates and eventually breaks, so that the continuity between the follicle and the dermal papilla is destroyed. Chase ('54a, b, c ) found the glassy membrane around the epithelial columns to be unusually thin in the mutant, and attributed this to a deficiency in collagen formation leading to the ultimate failure of the column to shorten.

These varied results suggested the need for a more precise study of events in the late catagen stages of the mutant follicle.

Inbred mice of the Hr/Hech strain of the genotype ccAAbbDDpp Hr/hr were used. The genotype can first be established at 10-14 days when hair loss commences on the front feet of homozygous recessive animals. Comparisons of different char- acteristics were made between mice of the genotype Hr/hr and mice of the genotype

MATERIALS AND METHODS

lThis work was supported by USPHS Research grant CA-00592 and a Training grant GM-00329. The senior author was also assisted by a New Zealand Wool Research Fellowship.

The aid of Miss H. Randelia in histochemical tech- niaues and Dr. James F. &dwell m statistlcal matters is gratefully acknowledged.

New Zealand, Private Bag, Christchurch, New Zealand. 2 Present address: Wool Research Organisation of

489

490 D. F. G. ORWIN, H. B. CHASE AND A. F. SILVER

hr/hr. The paired animals were always littermates of the same sex, and were processed together. Since mutant follicles in substages of catagen beyond catagen VI are abnormal, they were arbitrarily (but not unreasonably) classed in substage VII when the epithelial column was longer than normal for the same relative position of the club in the follicle as in normal catagen VII. In the next stage, catagen VIII, the epithelial column is still longer than normal, and hair loss has usually oc- curred.

Biopsies were taken from the middorsal skin of 42 animals. Six pairs of tissue (animals ages 12-19 days) were treated by the method of Barrnett and Seligman ('52, '54) to demonstrate sulfhydryl and disulfide groups. Five pairs, from animals 15-17 days old, were treated according to the method of McManus ('48) to demonstrate periodic acid-SchifF-reactive (PAS) material. The glycogen test was made according to the procedure of Gomori ('52). Five pairs of tissue were fixed in Bouins' solution at pH 1.8, embedded in paraffin, and stained with V a n Gieson's stain and hematoxylin. An additional five pairs of tissue were processed according to Green ('60) and treated with 2 ml of 0.1% solution of collagenase in 0.9% NaCl for 45 minutes at 37°C.

Sections were cut serially at 7 p, with blocks oriented so as to obtain longitudinal sections of hair follicles.

Measurements were made with an ocular micrometer and an oil immersion lens was used. Measurements of general follicle dimension were made on tissue processed for the collagen test. For each animal, follicles in catagen 11, VI, and VII, which were entirely visible in longitudinal section from dermal papilla to the follicular orifke, were used for measurements. The measurements made were thickness of skin from the epidermis to the top of the panniculus carnosus muscle, the total length of the follicle, the length of the epithelial column from the club to the end of the dermal papilla of catagen VI and VII follicles, and the length of the hair shaft in the skin; i.e. from the club to the surface of the skin, in catagen VI and VII follicles. The same numbers of follicles were measured in both animals of

a pair. Thus, 52 follicles in catagen I1 were measured, 26 follicles per genotype. The breakdown of numbers of follicles measured for the five pairs was four in the Hr/hr animal and four in the hr/hr animal of the first pair and 5,5; 5,5; 6,6 and 6,6 for the other four pairs, respec- tively. Seventy-four follicles in catagen VI were measured, the numbers for the five pairs being 5,5; 5,5; 7,7; 8,8 and 12,12 follicles. Of the follicles in catagen VII, 70 were measured comprising 5,5; 5,5; 6,6; 9,9 and 10,lO follicles for the five pairs of animals. The data were subjected to an analysis of variance developed for nested samples with unequal subsample numbers (Snedecor, '56).

Measurements of epithelial column and related structures were made on PAS- stained material. Follicles used for measurements were those in catagen VI which had been sectioned so that the epithelial column between the club and dermal papilla was clearly definable. The total width of the glassy membrane and connective tissue sheath, and the width of the epithelial column were measured at the same locus of the same section. Random selection of follicles was practiced, the serial sections enabling duplication of measurements to be avoided. Data were obtained from measurements on 20 follicles per animal. Three values were obtained for each follicle at approximately equal intervals along the epithelial column and the results averaged, in order to reduce errors resulting from the plane of section- ing. Both sets of data were subjected to an analysis of variance developed for nested samples (Snedecor, '56). The correlation coefficients for the relationship of width of epithelial column and width of glassy membrane and connective tissue sheath were also determined.

Measurements of the width of the Van Gieson-positive material in the glassy membrane were made in a similar way on the Van Gieson-stained material, The correlation coefficients for the relationship of this parameter to the width of the epithelial columns were also determined.

The formation of the hair club and the internal root sheath (IRS) during catagen were studied in the material demonstra- ing sulfhydryl and disulfide groups.

CATAGEN IN THE HAIRLESS HOUSE MOUSE 49 1

Measurements of the IRS were made along the ectal margin of follicles between the end of the IRS as defined by its form and concentration of sulfhydryl groups, and the dermo-epidermal junction. Data were obtained on 15 follicles per animal. In two pairs, all follicles were in late anagen VI while in the other three pairs the follicles were almost exclusively in catag- ens VI and VII. In all pairs, measurements in follicles at a particular substage of catagen were matched by measurements on an equal number of follicles at the same substage in the other animal. The values obtained were subjected to the analysis of variance developed for nested samples by Snedecor ('56).

OBSERVATIONS

I. Spatial observations: Dimension of skin and follicle components

in catagen The skin of the mutant is thinner than

that of the nonmutant (table 1) . Follicles of both genotypes are approxi-

mately the same overall length at catagen

11, and both types shorten as they pro- gress into catagen VI and VII (table 2). However, the nonmutant follicle becomes considerably shorter than the mutant by catagen VI, and even more so by catagen VII (table 5).

The entire follicle in late catagen may be considered in terms of its two main components, viz. the epithelial column, and the hair shaft within the skin. Both components shorten between the substages of catagen VI and VII (tables 3, 4) . The length of the mutant hair shaft within the skin is deeper than normal in catagen VI (table 4) but at the same level as the nonmutant by catagen VII (table 5, figs. 13, 14). The epithelial column is the same length in catagen VI for both genotypes, but by catagen VII the normal epithelial coIumn is considerably shorter than that of the mutant (table 5).

To sum up, in catagen VI, the mutant follicle is longer than the nonmutant follicle. Although the epithelial column is normal in length, the hair shaft is deeper in the skin. By catagen VII, the follicles of both genotypes have shortened, and the hair shafts lie at similar levels

TABLE 1 Analysis of variance of hairless and normal skin thickness at catagen 11, VI and VII

Source D.F.1 Mean square F

Stage of hair cycle 2 7,081 0.16 Pairs within stages 12 43,123 1.23 Genotypes within pairs within stages 15 35,181 12.42(Po.o1 = 2.16) Error 166 2,832

1 D.F., degrees of freedom. TABLE 2

Analysis of variance of hairless and normal follicle lengths at catagen 11, VI and VII

Source D.F. Mean square F

Stage of hair cycle 2 1,592,278 lO8.6(P0.0t = 6.93) Pairs within stages 12 14,667 0.3 Genotypes within pairs within stages 15 44,054 13.8(Pa.o1 = 2.16) Error 166 3,188

TABLE 3 Analysis of variance of hairless and normal epithelial column lengths measured at

catagen VI and VII


Stage of hair cycle 1 72,865 58.2(P0.01 = 11.3)

Genotypes within pairs within stages 10 18,842 26.1(P0.01= 2.47) Error 124 72 1

Pairs within stages 8 1,564 0.08

492 D. F. G . ORWIN, H. B. CHASE AND A. F. SILVER

TABLE 4

Analysis of variance o f hair shaft lengths remaining in the skin in hairless and normal follicles undergoing catagen VI and catagen VII


Stage of hair cycle 1 884,008 97.3(Po.oi = 11.3)

Genotypes within pairs within stages 10 23,628 5.41(Po.oi = 2.47) Pairs within stages 8 9,088 0.39

Error 124 4,370

TABLE 5 The means and standard deviations o f various follicle dimensions for three stages o f catagen

I1 hr/hr Mean (PI

Hr/hr Mean (a )

VI hr/hr Mean (PI

Hr/hr Mean (PI

VII hr/hr Mean (P I

Hr/hr Mean (PI

St. dev.

St. dev.

St. dev.

St. dev.

St. dev.

St. dev.

297 51

422 121

307 40

38 1 92

329 53

357 77

757 48

736 70

595 74

468 76

480 58

364 61

122 29 96 23

202 35

105 26

472 88

3 74 75

274 62

259 45

in the skin. The mutant follicle, however, shortens in overall length to a lesser ex- tent than does the nonmutant, due solely to the small degree of shortening of the epithelial column.

11. The f m a t i o n of the club The sequence of keratinization of the

normal club is similar to the keratinization sequence of the terminal region of the hair shaft as indicated by the stain for sulfhydryl groups. The hair formed during normal catagen consists of a nonmedul- lated, nonpigmented region of cortex which terminates in the club. The changes in cell shape and in concentration of sulfhydryl groups in this region occur in a characteristic time sequence which is similar to that described for the cortex of hair produced in anagen. During catagen IV to VII, as the cells of the terminal hair shaft move up the follicle, they change from round cells containing weakly staining sulfhydryl (-SH) “fibrils” to elongated cells containing large amounts of intensely staining, -SH-positive, fibrils which run parallel to the longitudinal

axis of the hair shaft (figs. 1, 2). By catagen VII, the cortical cells undergo an abrupt transition from -SH-positive to -SH-negative (fig. 13) and in so doing became positive for disulfide groups.

The sequence of keratinization in mutant follicles is similar to the nonmutant but the fibrils and cells shapes are aber- rant. In catagen IV and V, the cells of most presumptive clubs are round and weakly positive for sulfhydryl groups. In catagen VI, most mutant clubs, like non- mutants, stain maximally for sulfhydryl groups (fig. 4 ) but the cells remain more rounded than normal, and the fibrils are more wavey (fig. 3). In catagen VII, most mutant follicles have completed the sequence with clubs that are unreactive for sulfhydryl groups (fig. 14) and positive for disulfide groups.

In both mutant and nonmutant follicles, the epithelial cells around the club and the epithelial column itself are weakly reactive for sulfhydryl groups. The glassy membrane around the epithelial column is unreactive for sulfhydryl groups while both the fibroblasts of the connective tissue

CATAGEN IN THE HAIR LESS HOUSE MOUSE 493

sheath and the cells of the dermal papilla are weakly reactive.

PAS-stained sections of the same material show differences in nuclear shapes which parallel the differences in cell shapes described above for nonmutant and mutant clubs in catagen VI (figs. 5, 8, 10).

111. The terminal development of the inner root sheath (IRS)

There is a marked difference between the two genotypes in the terminal development of the IRS, but the same sequence of events occurs in the process of keratinization. The term keratinizatim is used here to mean the hardening process undergone by cells which form the IRS. The final hard product of IRS cells is different in several ways from the keratin of the hair cortex, and although IRS cells may contain sulfhydryl groups demonstrable by the Barrnett-Seligman staining method, they do not give a positive disulfide reaction, since, unlike keratinized cortical cells, they contain very little cystine (cf. Mercer et al., ’64).

Of the three layers of the IRS; viz. Henle’s, Huxley’s and the cuticle of the IRS, Henle’s is definable in its final form, first. It does not become strongly -SH- positive, whereas Huxley’s and the cuticu- lar layers attain maximal sulfhydryl- staining at approximately the level of the transition zone from -SH-positive to -SH- negative in the terminal cortex.

The differences in the termination of IRS production between mutant and nonmutant follicles includes both the upper and lower ends of the IRS. At the lower end of the IRS the normal course of events is as follows. Henle’s layer appears in its h a 1 form as early as catagen V. It rarely extends down past the greatest width of the club. As the club keratinizes, Huxley’s layer becomes -SH-positive but only to a region just above the club. This region represents the usual lower terminal point of the IRS (fig. 2). A single and sometimes double line of cells which is apparently continuous with Huxley’s and/or Henle’s layer can usually be defmed extending around the base of the club. These cells represent the capsule and they show only a weak reaction for -SH, similar to the

reaction of the cells of the epithelial column.

In the mutant follicle, the initial development of the IRS during its abnormal club formation is similar to that of the nonmutant IRS (fig. 3) . However, as this club undergoes keratinization, the lower end of the IRS does not terminate above the club. Henle’s layer extends lower down than does the normal (fig. 4), and Hux- ley’s layer, while increasing normally in sulfhydryl group concentration, completely invests the base (fig. 9). Thus, on com- pletion of keratinization the mutant club is completely encased in a hard extension of the IRS.

In both genotypes, the upper terminal of the IRS moves to a more superficial position in the follicle between the stages of anagen VI and catagen VI-VII (tables 6, 7). However, the final upper level of IRS attained, or maintained, by the mutant is higher than in the normal (figs. l l , 12, table 6) .

It was also found, in both genotypes, that the length of the region of the follicle between the top of the IRS, and the dermo- epidermal junction decreases significantly between anagen VI and catagen VI, VII follicles (table 6, 7). This suggests that part of the follicle shortening that takes place during catagen occurs in the region of the outer root sheath between the end of the IRS and the dermo-epidermal junction.

IV. The glassy membrane and connective tissue sheath of follicles in

catagen VI and VII A, Collagen. In both genotypes, the

glassy membrane gives a positive reaction for Van Gieson’s stain which is abolished by collagenase (figs. 15-18). In this way collagen may be demonstrated all along the length of the epithelial column from the base of the club down to the dermal papilla. However, there is considerably less collagen in the glassy membrane of the mutant.

B. Periodic acid - Schiff-positive material. In both genotypes the inner edge of the collagen-containing stratum of the thickened glassy membrane gives a positive PAS reaction which is not abolished by diastase and thus not indicative of glyco-


gen (figs. 19, 20). This region is less well defined in the case of the mutant.

C. Width of glassy membrane and connective tissue sheath. The total width of the glassy membrane and connective tissue sheath is significantly less in the mutant than in the non-mutant follicle (table 8). Considerable variation also occurs in the total width of the glassy membrane and connective tissue sheath among mice of the same genotype.

The total width of the glassy membrane plus connective tissue sheath is poorly correlated with the width of the epithelial column (r Hr/hr = 0.30; r hr/hr = 0.05), indicating that size of the follicles measured is not the reason for the difference observed between genotypes.

D. Width of the Van Gieson-positive material in the glassy membrane. The region containing Van Gieson-positive material is significantly narrower in the

TABLE 6 Analysis of variance of measurements of the distance from the end o f the inner root sheath

to the dermo-epidermal junction o f follicles in anagen VI and catagen VI and VII. All measurements were made along the ectal margin of the follicles


Genotypes 1 164,030 6.68(Po.os = 5.32) Mice of the same genotype 8 24,566 39.97(Po,oi = 2.63) Error 140 615

The means and standard deviations of measurements between the end of the I R S and the demo- epidermal junction of follicles in anagen VI and catagen VI and VII: Hr/h+, 164.70 p & 42.5; hr/hr, 97.50 p 2 45.0.

TABLE 7 Tukey’s test for the significant difference found among the means of mice of the same

genotype in the analysis of variance presented in table 6

Animal Stage of hair cycle -~ ~ ~~~

Means - 119.1 z - 143.8 x - 154.5 - 202.7

1 AnagenVI 2 AnagenVI 3 Catagen VI and VII 4 Catagen VI and VII 5 Catagen VI and VII

Hr/hr 203.2 84.1 59.4 48.7 0.5 202.7 83.6 58.9 48.2 154.5 35.4 10.7 143.8 24.7 119.1

Animal Stage of hair cycle Means - 62.0 z- 70.0 % - 71.7 x- 129.2

P

ht/ ht 1 AnagenVI 2 AnagenVI 3 Catagen VI and VII 4 Catagen VI and VII 5 Catagen VI and VII

159.8 97.8 89.8 88.1 30.6 129.2 67.2 59.2 57.5

71.7 9.7 1.7 70.0 8.0 62.0

D = 28.61.

TABLE 8 Analysis of variance o f the total width of the glassy membrane and connective

tissue sheath o f follicles in catagen VI


Genotypes 1 86.13 10.12(P0.05 5.32) Mice of the same genotype 8 8.51 11.54(Po.oi = 2.61) Error 190 0.74

Mean width and standard deviation of the glassy membrane and connective tissue sheath. Hr/hr, 4.97 p 1.07; h+/hs, 3.65 p 2 0.98.

CATAGEN IN THE HAIRLESS HOUSE MOUSE 495

TABLE 9

Analysis of variance o f the width o f the Van Gieson-positive material in the glassy membrane of mutant and normal follicles in catagen VI


Genotypes 1 122.38 150.8(Po.oi = 11.26) Mice of the same genotype 8 0.81 1.44 Error 190 0.56

The means and standard deviations of the e d t h Of the Van Gieson positive material in the glassy membrane of mutant and non-mutant folhcles in catagen VI. H?./hr, 4.40 p f 0.88; hr/hr, 2.84 fi 2 0.62.

mutant than in the nonmutant (table 9). Since there is a low correlation between the width of this region and the width of the epithelial column ( r HT/hr = 0.12 r hr/hr = 0.12), it is probable that the difference between genotypes is not due to the size of the follicles measured.

DISCUSSION

The study of catagen follicles has indicated that, in both genotypes studied, the cortex produced during catagen undergoes changes in concentration of sulfhydryl groups during keratinization simiIar to those described for the cortex of anagen follicles by Eisen et al. ('53), and Hardy ('52). Furthermore, the catagen cortex becomes positive for disulfide linkages when fully keratinized, which is another characteristic of keratinized anagen cortex (Giroud et al., '51; Montagna, '62).

The club of the normal catagen follicle appears to undergo the same developmental sequence during keratinization as the cortex, with the exception that the nuclei at the bottom of the club appear to elongate only partially. Apart from this, the club appears to be an extension of the cortex.

The mutant club, however, deviates from the described sequence in two re- spects: (1 ) the failure of a comparatively high proportion of club cells to elongate, and (2) the failure of the fibrillar sub- structure of the keratin to be consistently oriented along the longitudinal axis of the hair shaft. The latter situation appears to be a rare phenomenon in animal hairs of this size (Earland, '62). Although the cause of these abnormalities is undeter- mined, it is probable that they are closely associated with each other. The failure of the cells to elongate normally provides a basis for the abnormal club endings depicted by David ('31).

The evidence indicates that the state- ment by Chase and Mann ('60) to the effect that the mutant club fails to keratinize may need modification as, on the basis of the criteria used in this study, the cells of the mutant club appear to kera tinize .

The extension of the hardened inner root sheath around the mutant club re- ported here is consistent with the finding of inner root sheath remains around the mutant club by David ('31) and Chase ('54a). Contact of the club with nonkeratinized cells seems to be necessary to prevent the hair from falling out. In the mutant during late catagen, the club is enclosed in the hardened IRS, and has lost contact with nonkeratinized cells. Hair loss occurs at this time whereas the hair in the nonmutant is not lost until much later. This concept of hair loss is in general agree- ment with David ('31) and Chase and Mann ('60), although these authors placed more importance on the abnormal club endings.

The finding that the inner root sheath of the mutant follicle extends to a higher region of the follicle than the inner root sheath of nonmutant follicles is impor- tant for two reasons. First, this difference was demonstrated in follicles undergoing anagen VI as well as those undergoing catagen VI and VII. Although the youngest animals in this study were only 12 days old their follicles were in the later stages of anagen VI, so it is probable that this difference is present in follicles at the start of anagen VI; i.e., two or three days after birth (Hardy, '49). This difference represents, then, a probable means of identifying mutant animals at a considerably earlier age than has been possible to date. Second, the difference in the extension of the IRS in the higher regions


of the follicle may be helpful in deter- mining the mechanism by which the IRS is broken down. It has been postulated by Montagna (’62) and Straile (’62) that, in the normal follicle, the IRS is degraded by an enzyme (keratinase). Thus, in the mutant follicle, the difference may be ac- counted for by alterations in the enzyme or proteins of the IRS sheath so that the IRS cannot be degraded at the normal level, neither deep nor superficial.

It is possible that, in the mutant follicle, the extension of the IRS up to the vicinity of the pilary canal, could cause the widened hair canals noted by Fraser (’46) and believed by him to be due to hyper- keratosis of the outer root sheath surround- ing the pilary canal.

The fact that the mutant follicle does not shorten normally (David, ’31; Chase et al., ’52; Montagna et al., ’52) has been confmned in this study. Previous findings have drawn attention to the difference in length of the epithelial columns of mutant and nonmutant follicles at catagen VII (Chase et al., ’52; Montagna et al., ’52). This study presents evidence that the entire mutant follicle has a slower rate of shortening throughout catagen than does the nonmutant follicle. It was also shown for both genotypes that the outer root sheath between the end of the inner root sheath and the dermo-epidermal junction, shortens between anagen VI and catagen VI and VII. This suggests that follicular shortening involves other follicle regions besides the epithelial column formed during catagen V and VI. Hair movement is apparently largely unaffected by the different extents of follicular shortening since the mutant hair rises to about the same level in the skin as does the nonmutant hair, by catagen VII.

The demonstration of collagen in the thickened glassy membrane of catagen VI follicles by the collagenase test has con- firmed Wolbach’s interpretation (’51) of stained mouse follicles. The collagen of the thickened glassy membrane of catagen VI presumably represents an increase in thickness of the layer of collagen observed in an electron microscope study of the glassy membrane of developing mouse follicles (Rogers, ’57).

The failure of the glassy membrane to thicken in the mutant follicle has been suggested by Chase as a reason for the failure of the mutant epithelial column to shorten (Chase, ’54a; Chase, 54b; Chase, ’54c). This suggestion is based on the assumption that the glassy membrane is thickened by the laying down of collagen and that the mutant follicle cannot do this. We have shown that both collagen depo- sition and epithelial column shortening are present in the mutant but that both functions are less than normal. This supports the idea that collagen is necessary for the shortening of the epithelial column.

LITERATURE CITED

Barrnett, R. J., and A. M. Seligman 1952 Histochemical demonstration of protein-bound sulfhydryl groups. Science, 116: 323.

1954 Histochemical demonstration of sulfhydryl and disulfide groups of protein. J. Nat. Canc. Inst., 14: 769.

Chase, H. B., and W. Montagna 1952 The development and consequences of hairlessness in the mouse. Genetics, 37: 573.

Chase, H. B. 1954a Growth of the hair. Physiol. Rev., 34: 113.

195413 The causative defect in the hairless mouse. Caryologia VI, suppl., 639.

1954c Somes examples of genecon- trolled functional disturbances in the mouse. J. Nat. Canc. Inst., 15 (3): 655.

Chase, H. B., and S. J. Mann 1960 Pheno- genetic aspects of some hair and pigment mutants. J. Cell and Comp. Physiol., 56: 103.

Crew, F. A. E., and L. Mirskaia 1931 The character “hairless” in the mouse. J. Genet., 25: 17.

David, L. T. 1931 The external expression and comparative dermal histology of hereditary hairlessness in mammals. Zeitschr. f. Zellforsch. u. mikro. Anat., 14: 616.

1962 Molecular orientation of some keratins. Nature, 196: 1287.

1953 Sulfhydryl groups in the skin of the mouse and guinea-pig. J. Nat. Canc. Inst., 14: 341.

Fraser, F. C. 1946 The expression and inter- action of hereditary factors producing hypo- trichosis in the mouse: Histology and experimental results. Can. J. Res., 24D: 10.

Giroud, A., and C. P. Leblond 1951 The kera- tinizatioa of epidermis and its derivatives, especially the hair, as shown by X-ray dsxac- tion and histochemical studies. Ann. New York Acad. Sci., 53: 613.

Gomori, G. L. 1952 Microscopic Histochemis- try. University of Chicago Press, Chicago.

Earland, C., P. R. Blakey and J. G. P. Stell

Eisen, A. Z., W. Montagna and H. B. Chase

CATAGEN IN THE HAIRLESS HOUSE MOUSE 497

Green, J. A. 1960 Digestion of collagen and reticulin in paraffin sections by collagenase. Stain Technology, 35: 273.

Hardy, M. H. 1949 The development of mouse hair in nitro with some observations on pig- mentation. J. Anat., 83: 364.

1952 The histochemistry of hair follicles in the mouse. Am. J. Anat., 90: 285.

Mann, S. J. 1961 The phenogenetics of hair mutants in the house mouse: hairless, rhino, ragged and oppossum. Ph.D. Thesis, Brown University, Providence, Rhode Island.

McManus, J. F. A. 1948 Histological and histochemical uses of periodic acid. Stain Technology, 23: 99.

Mercer, E. H., B. L. Munger, G. E. Rogers and S. I. Roth 1964 A suggested nomenclature for fine-structural components of keratin and keratin-like products of cells. Nature, 201 ; 367.

Montagna, W., H. B. Chase and H. P. Melaragno 1952 The skin of hairless mice: I. The formation of cysts and the distribution of lipids. J. Inv. Derm., 19: 83.

Montagna, W. 1962 The structure and Func- tion of Skin. 2nd Edition, Academic Press, New York.

Rogers, G. E. 1957 Electron microscoae obser- ;ations on the glassy of the hair follicle. Exp. Cell Res., 13: 521.

Snedecor, G. W. 1956 Statistical Methods. Iowa State College Press, Ames, Iowa.

Snell, G. D. 1931 Inheritance in the house mouse, the linkage relations of short-ear, hairless and naked. Genetics, 16: 42.

Straile, W. E., H. B. Chase and C. Arsenault 1961 Growth and differentiation of hair follicles between periods of activity and quiescence. J. Exp. Zool., 148: 205.

Possible functions of the external 1962 root sheath during growth of the hair follicle. J. Exp. Zool., 150: 207.

Wolbach, S. E. 1951 Hair cycle of the mouse with its importance in the study of sequences of experimental carcinogenesis. Ann. N.Y. Acad. Sci., 53: 517.

PLATE 1

EXPLANATION OF FIGURES

Figures 1 through 4 are longitudinal sections of follicIes cut a t 7 p and stained for sulfhydryl groups. X 680.

Normal follicle in early catagen VI. The SH group-positive fibrils are apparent and their orientation can he seen to be generally parallel to the longitudinal axis of the hair shaft (middle arrow). The lighter staining nuclei (lower arrow) are elongated except around the bottom of the club. Henle’s layer (upper arrow) is apparent.

Normal follicle in catagen VI. The club is staining maximally for sulfhydryl groups. The partially elongated nuclei are visible a t the bottom of the club (lower arrow). The inner root sheath has completed its development and terminates well above the club (upper arrow ) . Hairless folicle in early catagen VI. The fibrillar nature of the staining is apparent. The orientation of the fibrils in the club is, i n general, not parallel to the longitudinal axis of the hair shaft. Several lighter staining round nuclei can be seen in the club (lower arrow). Henle’s layer is visible near the club (upper arrow).

Hairless follicle in catagen VI. The nuclei of the maximally staining club have still not elongated (lower arrow). Henle’s layer can be seen extending almost around the base of the club (upper arrow).

498

CATAGEN IN THE HAIRLESS HOUSE MOUSE D. F. G . Orwin, H. B. Chase and A. F. Silver

PLATE I

499

PLATE 2

EXPLANATION O F FIGURES

Figures 5 through 8 are longitudinal sections of follicles cut a t 7 p and stained for periodic acid-Schiff-reactive material. x 680.

Normal follicle in catagen V. The nuclei of the club are still round.

Normal follicle in catagen VI. The nuclei of the cells of the club are elongated except at the bottom of the keratinizing club (arrow). The transition between club and capsule is gradual.

Hairless follicle in catagen V. The round nuclei of the club are apparent (arrow ).

Hairless follicle in catagen VI. A high Froportion of the cells in the club have not elongated (arrow). There is a clear demarcation between club (including Huxley’s layer) and capsule.

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CATAGEN IN THE HAIRLESS HOUSE MOUSE D. F. G. Orwin, H. B. Chase and A. F. Silver

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PLATE 3


9 Hairless follicle in catagen VI. Henle’s layer extends past the club. Huxley’s layer can be seen enclosing the club (arrow). Stained for sulfhydryl groups. x 680.

10 Hairless follicle in catagen VII. Nuclei are not visible in the fully keratinized club. Note the concave end of the club (arrow). PAS stain. ).; 680.

Normal follicle. The upper arrow indicates the orifice of the sebaceous gland. The upper end of the inner root sheath is indicated by the lower arrow. Stained for sulfhydryl groups. x 440.

Hairless follicle. The upper arrow indicates the orifice of the sebaceous gland. The end of the inner root sheath (lower arrow) extends almost to the orifice of the sebaceous gland. Stained for sulfhydryl groups. x 440.

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12

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CATAGEN IN THE HAIRLESS HOUSE MOUSE D. F. G. Orwin. H. B. Chase and A. F. Silver

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EXPLANATION O F FIGURES

13 Longitudinal section of a normal ( H r / h r ) follicle in catagen VII. The club (upper arrow) is situated in the follicle at the level of the junction of the dermis and hypodermis. The lower arrow points to the shortening epithelial column. Stained for sulfhydryl groups. x 210.

14 Longitudinal section of a hairless ( h r / h r ) follicle in catagen VII. The club (upper arrow) is situated in the follicle a t the level of the junction of the dermis and hypodermis. The longer than normal epithelial column (lower arrow) is apparent. Stained for sulfhydryl groups. x 210.

Longitudinal section of a normal follicle. The Van Gieson staining material of the thickened glass); membrane (arrow) is apparent on both sides of the epithelial column. Van Gieson’s stain. X 680.

Longitudinal section of a normal Pollicle. The inability of the glassy membrane to take up Van Gieson’s stain following treatment with collagenase is apparent (arrow). Van Gieson’s stain. X 680.

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16

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CATAGEN IN THE HAIRLESS HOUSE MOUSE D. F. G. Orwin, H. B. Chase and A. F. Silver

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17 Longitudinal section of a hairless follicle. The less well defined Van Gieson-staining material in the thickened glassy membrane is indicated by the arrow. Van Gieson’s stain. X 680.

18 Longitudiiial section of a hairless follicle. Van Gieson-positive material is no longer found in the thickened glassy membrane following treatment with collagenase (arrow). Van Gieson’s stain. x 680.

19 Longitudinal section of a normal follicle. Periodic acid-Schiff-positive material is apparent along the inner margin of the thickened glassy membrane (arrow). PAS stain. X 680.

20 Longitudinal section of a hairless follicle. Periodic acid-Schiff-positive material is present along the inner edge of the thickened glassy membrane (arrow). PAS stain. X 680.

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CATAGEN IN THE HAIRLESS HOUSE MOUSE D. F. G. Orwin. H. B. Chase and A. F. Silver

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catagen in the hairless house mouse

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