Complete rescue of the nude mutant phenotype by a wild-type
Vincent T. Cunliffe, Andrew J.W. Furley, Deborah Keenan
Centre for Developmental Genetics, School of Medicine and Biomedical Science, University of Sheffield, Firth Court, Western Bank,
Sheffield, S10 2TN, UK
Received: 14 November 2001 / Accepted: 18 January 2002
Abstract. In this paper we describe the production and analysis of
mice carrying a 110-kb transgene that encompasses the wild-type
Foxn1 genomic locus. Mutations in Foxn1 cause the nude pheno-
type. We show that in the hair follicles, transgenic mice with
increased Foxn1 gene dosage exhibited increased Foxn1 expres-
sion that was restricted correctly to the nascent, post-mitotic cells
of the differentiating hair cortex and hair cuticle lineages. We also
demonstrate for the first time that a Foxn1 transgene rescues com-
pletely both the hair follicle and the thymus defects in animals that
are also homozygous for the nude mutation at the endogenous
Foxn1 locus, causing the development of a full coat of hair and a
normal population of peripheral blood T lymphocytes. We con-
clude that sufficient cis-acting regulatory information resides
within this 110-kb transgene to direct reliable and appropriate
tissue-specific expression of the Foxn1 gene.
The Foxn1 gene is required for normal development of the hair
follicles and thymus in mice, since mutations in this gene cause the
nude mutant phenotype (Flanagan 1966; Pantelouris 1968; Nehls
et al. 1994). Homozygous nude mice lack both a coat of hair and
a functional thymus. Hair follicles are specified in nude mutants,
and their development is initiated correctly, but the developing hair
rudiments subsequently exhibit impaired keratinization and mor-
phological defects in cuticle formation (Flanagan 1966; Kopf-
Maier et al. 1990; Meier et al. 1999). As a result of defective hair
differentiation, fragments of broken hair shaft accumulate in the
upper portion of each hair follicle, giving this region of the follicle
an abnormally distended and mis-shapen appearance. In the nude
mutant thymus, epithelial cells fail to differentiate normally, which
prevents normal T lymphocyte development in this organ and so
causes severe immunodeficiency (Nehls et al. 1996).
The Foxn1 protein is a member of the forkhead family of
transcription factors and possesses both a winged-helix DNA-
binding domain and a potent transcription activating domain
(Nehls et al. 1994; Schlake et al. 1997; Schuddekopf et al. 1996).
In the hair follicle, the Foxn1 gene is transcribed predominantly in
post-mitotic precursors of the hair cuticle and cortex (Lee et al.
1999), a pattern of expression that requires Bone Morphogenetic
Protein (Bmp) signaling (Kulessa et al. 2000); however, it is not
yet known if Foxn1 is a direct target of this signaling pathway.
Taken together, the mutant phenotype and the Foxn1 expression
pattern imply that Foxn1 may function at an early stage in the
differentiation of hair cortex and cuticle cells.
A cosmid-derived transgene containing 26 kb of mouse geno-
mic DNA that encompassed the coding exons of Foxn1 plus 8.5 kb
of 5Ј-flanking sequence and 3 kb of 3Ј-flanking sequence was able
only to rescue partially the phenotypic effects of homozygosity for
the nude mutation at the endogenous Foxn1 locus (Kurooka et al.
1996). Homozygous nude mutant animals that also carried this
transgene exhibited an incomplete coat of hair, suggesting that
expression of the transgene was variable in the hair follicles, pos-
sibly owing to the absence of critical cis-regulatory elements de-
termining the transcriptional stability or efficiency of the Foxn1
transgene in hair follicle cells. Moreover, peripheral T lympho-
cytes were completely absent from these transgenic animals, and
thymus development was arrested as in non-transgenic nude ani-
mals, indicating that critical regulatory elements required for thy-
mus-specific Foxn1 expression were absent in the transgene em-
ployed. In order to begin to dissect the developmental mechanisms
regulating Foxn1 expression in the hair follicle and thymus, we
sought to define a region of genomic DNA encompassing the
murine Foxn1 locus that caused the complete transgenic rescue of
both hair follicle and thymus phenotypes in animals homozygous
for the nude mutation at the endogenous Foxn1 genomic locus. In
this paper we describe transgenic mice carrying a 110-kb Foxn1
transgene that fulfills these requirements.
Materials and methods
The nude mutation was maintained on a Balb/c background from a
stock originally supplied by Harlan, UK. Homozygous nude mutants could
be distinguished from littermates shortly after birth by the absence of
whiskers. The identity of nude homozygotes was confirmed by PCR-based
sequence analysis of exon 3 of Foxn1 in genomic DNA samples.
Histology, histochemistry, and immunocytochemistry.
was dissected from the anterior back region of sex- and age-matched ani-
mals and fixed in 4% paraformaldehyde in Phosphate-Buffered Saline
(PBS) overnight at 4°C. Samples were dehydrated through an ethanol
series, cleared in xylene, and embedded in paraffin wax. Histological sec-
tions of 8 m thickness were taken onto Vectabond-subbed slides, dehy-
drated, and cleared, then stained with Hematoxylin and Eosin and mounted
in DPX. Cell types were identified by reference to histological texts under
a compound microscope.
For immunostaining wax-embedded skin sections, tissues were
mounted on Vectabond-subbed slides, then cleared in xylene, dehydrated
through an ethanol series, and rinsed sequentially in distilled water fol-
lowed by PBS.
To prepare sections for immunostaining with the AE13 monoclonal
antibody (Lynch et al. 1986), the anti-Foxn1 (Lee et al. 1999) or anti-
phospho-histone H3 polyclonal rabbit antisera (Santa Cruz Biotechnol-
ogy), slides were boiled for 15 min in citrate buffer (Vector Laboratories,
Burlingame, Calif.), then cooled to below 60°C over a period of 20 min.
After rinsing all slides in PBS, we blocked tissues with 3% heat-inactivated
goat serum (HIGS) in PBS/0.1% Triton X-100, for 30 min at room tem-
perature in a humidified chamber. Slides were then drained, and primary
antibody was added after dilution in 3% HIGS/PBS/0.1% Triton X-100
(1:50 for anti-Foxn1 and AE13; 1:1000 for anti-phospho-H3). Slides were
Correspondence to: V.T. Cunliffe; E-mail: email@example.com
Present address of D.K.: Affymetrix UK, Ltd, Voyager, Mercury Park,
Wycombe Lane, Wooburn Green, High Wycombe HP10 0HH, UK
Mammalian Genome 13, 245–252 (2002).
© Springer-Verlag New York Inc. 2002
Incorporating Mouse Genome