Changing Patterns of Gap J unctional Intercellular
Communication and Connexin Distribution
in Mouse E pidermis and Hair F ollicles
During E mbryonic Development
R . CHOUDHRY,1J .D. PIT T S,2and M.B. HODGINS1*
1Department of Dermatology, University of Glasgow, Glasgow, Scotland
2Beatson Institutefor Cancer Research, GarscubeEstate, Bearsden, Glasgow, Scotland
ABST R ACT
embryonic days 12 (E 12) and 16, regular arrays of
epidermal placodes on the mystacial pad develop
into whisker follicles. T his system was chosen for
analysis of gap junctional intercellular communi-
cation during differentiation. T he patterns of
communication were studied by microinjection
of the tracers L ucifer yellow-CH (LY-CH) and
neurobiotin (NB), while immunofluorescent stain-
ing was used to study distribution of connexins
26 and 43. E xtensive communication was seen
between keratinocytes in developing hair pegs
or, in later-stage hair follicles, in the germinative
matrix. Coupling between adjacent hair pegs via
interfollicular epidermis was not observed. Cou-
pling also became restricted as follicular cells
differentiated to form outer root sheath, inner
root sheath, and hair shaft. E xtensive gap junc-
tional coupling is characteristic of keratinocytes
that are rapidly proliferating (as in hair pegs and
germinative matrix). F ollicular keratinocytes
commence differentiation shortly before restric-
tion of gap junctional coupling becomes evident.
Dermal mesenchymal cells undergoing different
modes of differentiation also exhibit differences
in gap junctional coupling, as evidenced by poor
transfer of LY-CH between cells in dermal conden-
sations of hair follicles compared with extensive
transfer elsewhere in the dermis. LY-CH and NB
were not transferred between epidermal or fol-
licular epithelium and mesenchyme, arguing
against a direct role for gap junctions permeable
to known second messenger molecules or nucleo-
tides in epithelial-mesenchymal interactions in
this system. T he distribution of connexins 26 and
43 in epidermis and hair follicles changed during
differentiation but there was no correlation with
changing patterns of dye transfer, indicating an
unexpected degree of complexity in the relation-
ship between gap junctional intercellular commu-
nication and connexin protein distribution dur-
ing development. Dev. Dyn. 1997;210:417–430.
?1997 Wiley-L iss, Inc.
In the mouse embryo betweenK ey words: gap-junctions; connexins; skin; hair
follicle; epidermis; embryo; develop-
INT R ODUCT ION
Development of vertebrate skin appendages involves
complex cellular interactions within the epithelium
and between the epithelium and mesenchyme (Sengel,
1976; Kratochwil et al., 1996). These interactions in-
volve cell adhesion molecules (Chuong and Edelman,
1985; Hirai et al., 1989; Zhang et al., 1996), extracellu-
lar matrix components (Goetinck and Carlone, 1988;
J iang and Chuong, 1992; Zhang et al., 1996), and
secreted growth factors (Hogan et al., 1994; Finch et al.,
1995; Guo et al,. 1996; TingBerreth and Chuong
1996a,b). Gap junctions provide opportunities for fur-
ther forms of control in tissue differentiation (Pitts et
al., 1988a,b; Katz, 1995; Paul et al., 1995; Bruzzone et
al., 1996; Kumar and Gilula, 1996) and generation of
pattern (Wolpert, 1989).
Gap junctional communication, which is widespread
in animal tissues, exposes cells to a particular form of
environmental influence arising from the fluctuating
concentrations of small ions and molecules within the
cytoplasms of surrounding cells (Lawrence et al., 1978;
Lowenstein, 1979). The rapid movement of the second
messengers and other small ions and molecules down
concentration gradients between cells, generates a form
of homeostasis within groups of cells joined by gap
junctions. In cells in culture, this leads to coordination
and uniformity of phenotype and tends to inhibit the
expression of cellular differences (Pitts et al., 1988a,b).
It has therefore been suggested that the formation of
communication compartments (groups of cells joined to
each other by gap junctions but not to cells in different
compartments) is likely to be associated with the
expression of new phenotypes during morphogenesis
and patterning (Pitts et al., 1988a,b; Kam and Hodgins,
Grant sponsor: bbsrc; Grant number: 17/S03206.
*Correspondence to: M.B. Hodgins, Department of Dermatology,
University of Glasgow, Glasgow G12 8QQ, Scotland.
Received 24 March 1997;Accepted 30August 1997
DE VE L OPME NTAL DYNAMICS 210:417–430 (1997)
?1997 WILEY-LISS, INC.
1992; Serras et al., 1993). Although studies in several
systems have provided general support for these ideas,
thereis very littledetailedinformation relating changes
in gap junctional intercellular communication to spe-
cific stages in tissue morphogenesis. Much recent work
has concentrated on documenting the expression pat-
terns of connexins, a family of gap junction proteins
containing at least 13 distinct types that are expressed
during embryogenesis in complex tissue-specific pat-
terns (Risek et al., 1992, 1994; Goliger and Paul, 1994;
Dahl et al., 1996). Much less is known about the
relationship between theobserved patterns of connexin
staining and the compartmentation of gap junctional
communication. It is often assumed that connexin
staining is sufficient evidence of functional coupling,
but as gapjunctions may adopt open or closedconforma-
tions, this assumption may not bejustified.
The mouse embryo whisker follicle is a particularly
suitable mammalian system for analysis of cellular
interactions involved in pattern formation and differen-
tiation. Interactions between mesenchyme and epithe-
lium and interactions within the epithelium are all
important during organogenesis (Sengel, 1976). Indi-
vidual whisker follicles develop from epidermal plac-
odes established in a simple pattern of rows in the
mystacial pad (Davidson and Hardy, 1952). Epidermal
cells giverisetotheprogenitor cells of thetwomain cell
lineages of the follicle, the outer root sheath and the
germinative matrix. The outer root sheath is appar-
ently continuouswith theepidermis, whereasthegermi-
native matrix in the hair bulb is a population of rapidly
proliferating keratinocytes that gives rise to the six
morphologically distinct cell types of the hair shaft and
inner root sheath during anagen (Hardy, 1992). A
condensation of mesenchymal cells, which interacts
with the epidermal placode to establish the follicle,
eventually forms the dermal sheath of the follicle and
dermal papilla. The dermal papilla, which is enveloped
by the hair bulb, plays an instructive role in the growth
of the whisker (Oliver, 1970; Reynolds and J ahoda,
We have compared patterns of gap junctional cou-
pling, using the fluorescent tracer Lucifer yellow-CH
(LY) and neurobiotin (NB), with the patterns of con-
nexin staining during development of whisker follicles
in the mouse embryo. We show that morphogenesis is
associated with separation of differentiating cells into
new communication compartments and with changes
in expression of connexins 43 and 26. However, func-
tional compartmentation is not explained by the differ-
ential expression of thesetwoconnexins. Our studies do
not support a role for gap junctions in epithelial-
R E SULT S
Development of Hair F ollicles in the Mouse
At embryonic days 12 (E12) to 13, the whisker pad
epidermis is stratified into three cell layers (including
the outer periderm layer) with a pattern of hair pegs at
different stages of differentiation according to position
within the rows (Hardy, 1992). The pegs range from
simple epidermal thickenings associated with underly-
ing condensations of dermal cells (Hardy’s stage 1) to
protrusions of circumferentially arranged epithelial
cell surrounded by densely packed dermal cells (stages
2/3a). By E14, the outer columnar cells of the upper
follicle have the appearance of outer root sheath, while
a well-formed bulb with germinative matrix surrounds
the dermal papilla in some follicles (stages 3b/4). By
E16, the squamous layers of the interfollicular epider-
mis are cornified and several rows of fully formed
whisker follicles are present, some with hair shafts
reaching theskin surface(stages 5/6). However, even at
this stage, some late-developing follicles at the early
stages of differentiation are alsoobserved. Perhaps the
most striking contrast between embryonic and adult
follicles is the higher density of mesenchymal cells in
theembryonic dermal sheath and papilla.
Analysis of J unctional Communication
by Dye Injection
LY-CH (or NB, see below) was injected into cells at
different sites as described in Experimental Proce-
dures. After fixation (and fluorescein-avidin staining
for NB), all dye spreads were analysed either after
embedding and cutting complete sets of serial sections
or by complete sets of optical sections imaged by
confocal microscopy.By following thestructuresthrough
serial sets of sections, it was possible to establish an
accurate morphological description of each section. To
facilitate interpretation, morphological features have
been marked on the selected sections presented in the
J unctional Communication in the
Interfollicular E pidermis
When injected into a basal epidermal cell at E12,
LY-CH spread only to neighbouring basal and su-
prabasal cells (Fig. 1A,B). Dye did not spread into the
periderm (outermost) layer. At E14, after cornification
of the outermost epidermal cells, LY-CH injected intoa
basal or suprabasal cell spread vertically through all
living layers, leaving sharp boundaries at the dermo-
epidermal junction and cornified layer (Fig. 1C,D).
Lateral spread of dye was not restricted by such
morphologically identifiable boundaries but was no
greater, and often less in extent, than the vertical
spread. This is consistent with the previous report of
Kam et al. (1986), which suggested that the epidermis
is divided into many small communication compart-
ments. However, the spreads seen at this stage of skin
development do not suggest specific boundaries, just
limitedlateral coupling.Therestriction in lateral spread
of LY-CH was more pronounced within the basal layer
(Fig. 1C,D). When LY-CH was injected into an epider-
mal cell close to the edge of a developing hair peg
(Hardy stage 2), there was limited transfer with some
CHOUDHRY ET AL.
transfer in threedimensions (krypton/argon laser, exci-
tation 488 nm, emission filter 522 nm). In some cases
this analysis provided sufficient resolution, but to ob-
tain more precise information about the relationship
between zones of dye transfer and tissue architecture,
sections of LY-CH embedded tissue were prepared as
previously described (Kam and Hodgins, 1992). Speci-
mens were dehydrated, embedded in LR-White resin,
and serially sectioned at 3 µm. Sections were photo-
graphed using phase contrast and epifluorescence on
Kodak Ektachrome 400 film (Rochester, NY) with an
exposure time of 2.0 min. Colour photographic slides
were then scanned into Photoshop, composite images
were assembled with Micrografx Windows Draw, and
printed on a Kodak Coloreaseprinter.
Mouse embryo upper lip skin was embedded in OCT
compound and frozen in liquid nitrogen. Sections 5- to
7-?mthick werecut in a cryostat andfixedin acetoneat
room temperature for 10 min and blocked (20 min) in
20% normal horse serum or normal goat serum diluted
in PBS. Sections were incubated with one of the follow-
ing antibodies for 2 hr at room temperature: a 1/1,000
dilution of rabbit polyclonal antipeptide-antiserum
against amino acids 101–119 of rat connexin 26 (S.
J amieson, personal communication, 1995), a 1/200 dilu-
tion of an affinity-purified rabbit polyclonal antipeptide
antiserum against amino acids 108–122 of mouse con-
nexin 26 (Goliger and Paul, 1994), a 1/1,000 dilution of
a monoclonal mouse antipeptide-antiserum against
amino acids 252–271 of rat connexin 43 (Beyer et al.,
1989; ZymedLaboratories, San Francisco, CA), or 1/200
dilution of an affinity-purified rabbit antipeptide-
antiserum against aminoacids 252–271 of rat connexin
43 (Beyer et al., 1987). In negative controls primary
antibody was omitted. After washing in PBS, the sec-
tions were incubated with fluorescein-conjugated anti-
rabbit and anti-mouse immunoglobulins (Vector Labo-
ratories, Peterborough, UK) for 30–45 min, washed in
PBS, mounted in Vectashield (Vector), and examined by
epifluorescence and with a BioRad MRC 600 confocal
microscope. The confocal microscope was used to scan
immunofluorescent slides in both green (excitation 488
nm, emission filter 522 nm for fluorescein) and red
(excitation 568 nm, emission filter 585 nm for back-
ground). Scanning of tissue autofluorescence at 585 nm
in frozen sections provided the equivalent of a counter-
stain to visualise tissue architecture and illustrate
localisation of fluorescein-labelled immunoglobulins.
Dual false-colour images were transferred into Micro-
grafx PhotoMagic, and colour intensities were in-
creased to give a sufficiently strong red tissue back-
ground without obscuring the fluorescein (this results
in fluorescein appearing yellow in the colour plates).
Images were than assembled in Micrografx Windows
Draw and printed on a Kodak Coloreaseprinter.
ACK NOWL E DGME NT S
We thank Rita MacKay for technical assistance, Dr.
S. J amieson and Professor D. Paul for the gift of
connexin antibodies, and Drs. M. Finbow and C.A.B.
TABL E 2. Summary of Connexin Antibody Staining R esults
Time in development
Connexin 43 staining
E12–E13 (stage 2–3a)
Epidermis Hair pegsHair follicles
E14–E15 (stage 3c–6)Suprabasal?/?
E16–E17 (stage 3c–6) Suprabasal?
Connexin 26 staining
E14–E15 (stage 3c–6)—Henley’s, IRS??
E16–E17 (stage 3c–6)Suprabasal??
ORS, outer root sheath; IRS, inner root sheath; GM, germinativematrix.
GAP J UNCTIONS AND CONNEXINS
J ahoda for helpful discussions. We also thank Dr. D.
Neil for theloan of an oscilloscope.
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