differences that lead to distinct programs of differ- 
entiation in the epidermis and hair foUicie. The em- 
bryonic basal layer expresses low levels of the basal 
epidermal keratins K5 and K14. At this early stage, 
aggregates of specialized mesenchymal cells, or der- 
mal papillae, form beneath this layer. Where embry- 
onic basal cells come into contact with dermal pa- 
pillae, they suppress expression of K5 and K14, 
grow downward, and appear to become committed 
^^to form hair follicles. In the absence of this stimu- 
lus, embryonic basal cells stratify to give rise to epi- 
dermis. K5 and K14 expression is increased in the 
basal layer and downregulated during terminal dif- 
ferentiation. 
As the hair follicle develops, mitotically active 
matrix cells, which remain in direct contact with 
dermal papillae and which remain relatively undif- 
ferentiated, do not seem to contain keratin fila- 
ments, nor do they express Kl4 and K5. These cells 
can choose among six different programs of differ- 
entiation to give rise to specialized cells of the hair 
shaft and the inner root sheath. Only upon com- 
mitment to one of these programs is expression of 
hair-specific keratin mRNAs induced. In contrast, 
the mitotically active cells of the developing outer 
root sheath do not remain in contact with dermal 
papillae; concomitant with this loss of interaction, 
the cells induce expression of K5 and K14, which is 
maintained in the adult. Further investigation is 
needed to determine whether there is a specific 
hair follicle-stimulating factor produced by dermal 
papillae, whether this factor plays a role in control- 
ling K5/K14 expression, and whether there is a nar- 
row window during development whereby the epi- 
thelial component can respond to these cells. 
III. Function and Structure of Keratin Filaments. 
To form a single 8 nm keratin filament, —10,000 
subunits each of type I and type II keratins interact 
in a complex self-assembly process. To elucidate the 
mechanisms underlying this process and to exam- 
ine the dynamics of the keratin filament network 
and its interactions with other cellular organelles, 
the laboratory has focused on deletion and site-di- 
rected mutagenesis of the coding portions of the 
cDNAs encoding human K14. To distinguish the ex- 
pression of the mutant keratins in epidermal cells, 
the group replaced the carboxyl-terminal sequence 
encoding the antigenic portion of K14 with a small 
sequence encoding the antigenic portion of neu- 
ropeptide substance P An antiserum, anti-Kl4, rec- 
ognized only the endogenous K14, while an anti- 
body, anti-P, recognized only the foreign (mutant) 
K14-P. An SV40 promoter/enhancer was used to 
drive the expression of these mutant K14-P cDNAs 
in a variety of cells in vitro. Even when sequences 
encoding the entire nonhelical end domains of 
K14-P were removed (—150 amino acids), the mu- 
tant protein, composed largely of a 310-amino acid 
residue central helical domain, was able to inte- 
grate into the existing keratin filament network. 
However, when a single point mutation was intro- 
duced into a highly conserved region at the end of 
the a-helical domain, the mutant protein caused 
complete collapse of the existing keratin filament 
network. Other mutations showed various pheno- 
types, and in many cases the phenotype was domi- 
nant. The behavior of these mutant keratins in cul- 
tured epidermal cells suggested that 1) the keratin 
filament network is far more dynamic than pre- 
viously recognized, 2) the network appears to initi- 
ate from the nuclear envelope and grow toward the 
cell periphery, and 3) alterations of the intracellular 
keratin network in one epidermal cell can influence 
keratin networks in adjacent cells, presumably indi- 
rectly, through desmosomal contacts. In vitro fila- 
ment assembly studies with these mutants should 
help to unravel the precise nature of keratin sub- 
unit interactions. Transgenic mice expressing these 
mutants should test the possibility that some keratin 
mutations might lead to various genetic skin diseases. 
lY Regulation of Keratin Gene Expression. 
As possible agents for targeting the expression of 
foreign genes in the skin and for altering the ex- 
pression patterns of genes normally involved in epi- 
dermal differentiation, the promoters/enhancers of 
the epidermal keratin genes should be invaluable. 
The laboratory has focused on the human genes 
encoding K5 and K14, the keratins expressed in the 
mitotically active cells of the epidermis, and has iso- 
lated and characterized the functional genes encod- 
ing these keratins. The transcriptional rates of 
these genes in cultured human basal epidermal 
cells appear to be the highest of all genes ex- 
pressed in these cells. The group has begun to elu- 
cidate the sequences important in directing the 
proper tissue-specific and diflferentiation-specific 
expression of these genes. Preliminary studies have 
revealed that sequences encompassing 2,500 bp of 
a 5' upstream regulatory sequence of the human 
K14 gene are sufficient to drive the expression of 
the tagged K14-P cDNA (see section III) in the basal 
layer of the epidermis of transgenic mice. Addi- 
tional studies are in progress to delineate the regu- 
latory elements responsible for this pattern of ex- 
Continued 
62 
