Hair follicle (HF) miniaturization is a degenerative process that proportionally reduces the dimensions of the epithelial and mesenchymal compartments, and leads to the conversion of thick, terminal hair to fine, vellus hair4
. HF miniaturization is most commonly observed in androgenetic alopecia, but is also characteristic of a rare, autosomal dominant form of hair loss, known as hereditary hypotrichosis simplex1
(HHS; OMIM 146520) (Supplementary Note
). To gain insight into the molecular underpinnings of HF miniaturization and identify a gene underlying HHS, we performed a linkage study in two Pakistani families (HHS1 and HHS2) ( and S1a-l
). After excluding the CDSN locus on chromosome 65
, we used Affymetrix 10K SNP arrays for genotyping, and linkage analysis using a dominant model yielded a maximum LOD score of Z=4.6 on chromosome 18p11.22 (Fig. S1m
). We narrowed the candidate interval to a 1.8 Mb region () containing 8 genes, 4 pseudogenes and 3 predicted transcripts (Fig. S1n
). Direct sequencing identified a heterozygous mutation 26T>G (L9R) in the signal peptide of the A
) gene (Fig. S1o
. The mutation L9R cosegregated with the disease phenotype in both families, and was absent in 200 unrelated, unaffected controls and in the SNP databases (Fig. S1p
; data not shown). Unexpectedly, we identified the identical APCDD1
mutation in an Italian family with autosomal dominant HHS that had previously been mapped to the same region of chromosome 18p11.22 (Fig. S2
, providing independent genetic evidence in support of this finding.
The HHS phenotype maps on chromosome 18p11.2 in a point mutation in APCDD1 gene
APCDD1 was abundantly expressed in both the epidermal and dermal compartments of the human HF, consistent with a role in HF miniaturization. APCDD1
mRNA and protein was present in human scalp skin by RT-PCR (Fig. S3a
), and a western blot using an APCDD1 antibody (). APCDD1 mRNA and protein were also highly expressed in the HF dermal papilla (DP), the matrix, and the hair shaft (). Apcdd1 orthologs are conserved throughout vertebrate evolution (Fig. S4a,b
), suggesting that a role in mouse3
and human HF growth emerged recently in mammalian species.
Several lines of evidence led us to postulate that APCDD1 may function as a negative regulator of Wnt signaling, including the observation that it is a direct target gene of Wnt/β-catenin 6
; its similarity in expression pattern with another Wnt inhibitor, Wise8
; the abundance of Wnt inhibitors in the HF9
; and the conservation of 12 cysteine residues (Fig. S4a
), a structural motif important for interaction between Wnt ligands and their receptors10,11
To test if APCDD1 is an inhibitor of Wnt signaling, we first determined if APCDD1 interacts with ligands and receptors of the canonical Wnt pathway. No interaction was found with Fzd2, Fzd8, and Dkk4 (data not shown). In contrast, the extracellular domain of APCDD1 (APCDD1ΔTM) coprecipitated with recombinant tagged forms of Wnt3A and LRP5, two proteins important for HF induction 12
(, S3b and S5
), suggesting that APCDD1 can modulate the Wnt pathway via potential interactions with WNT3A and LRP5 at the cell surface. To determine the effect of APCDD1 on Wnt signaling, we performed TOP/FOP Flash Wnt reporter assays in HEK293T cells. Reporter activity induced by WNT3A alone, or in combination with LRP5/Fzd2, was downregulated ~2-fold by APCDD1 in a dose-dependent manner (), indicating that APCDD1 inhibits the Wnt/β-catenin pathway.
Wild-type, but not L9R mutant APCDD1, inhibits canonical Wnt signaling
To determine if APCDD1 can function as a Wnt inhibitor in vivo
, we selected two systems in which the role of Wnt/β-catenin pathway has been well-defined: neuronal specification in the developing spinal cord13–15
, and axis determination in the frog16,17
. In the chick spinal cord, a Wnt/β-catenin gradient promotes proliferation of neural progenitors and generation of some neuronal classes13–15
. Transfection of the Wnt reporter TOP
::eGFP in the chick neural tube revealed strong activation of the pathway in the dorsal and intermediate progenitors, as previously shown15
. However, overexpression of APCDD1 strongly reduced eGFP expression levels (Fig. S6a-d
), decreased by ~20–30% the number of Sox3+
neural progenitors, as well as various neuronal subtypes of dorsal and ventral origin ( and S7a-d
). This effect was stronger with mouse Apcdd1, a closer ortholog of the chick protein (Figs. S8a-i and S9a-e
). These findings are consistent with the hypothesis that APCDD1 functions as a Wnt inhibitor.
Overexpression of Wt-APCDD1, but not L9R mutant, inhibits progenitor proliferation and neuronal specification in the chick spinal cord
The maternal Wnt pathway is required for the formation of dorsal and anterior structures in early Xenopus
. Overexpression of APCDD1 in dorsal blastomeres (n=35) reduced the anterior structures, such as the eyes and cement gland, at the tadpole stage (), consistent with maternal Wnt inhibition. APCDD1 also inhibited transcription of the Siamois
) reporter gene (), activated by the maternal Wnt pathway20
. A zygotic Wnt pathway is subsequently activated on the ventral side of the embryo21
, and its inhibition produces secondary axes with incomplete heads16,17
. Ventral overexpression of APCDD1 induced secondary axes (n=43, 28% duplicated axes, ), consistent with an inhibitory effect on zygotic Wnt signaling. The inhibition of Wnt activity by APCDD1 was also seen in transcription assays with Wnt8
RNA, but not β-catenin (), indicating that it acts upstream of β-catenin.
APCDD1 inhibits the Wnt pathway in Xenopus embryos
We next investigated which domain of APCDD1 mediates its activity and in which cell APCDD1 exerts its function. First, western blot of APCDD1 expressed in HEK293T cells revealed that the protein is glycosylated and forms a dimer ( and S10a-c
). Misexpression of mApcdd1ΔTM (lacking the transmembrane domain) in the chick neural tube mimicked the effects observed with mApcdd1 (Figs. S8j-r and S9f-j
), suggesting that the Wnt inhibitory activity resides within the extracellular domain. Secondly, APCDD1 could affect either the signaling cell, by regulating Wnt secretion 22
, or the receiving cell. In Xenopus
transcription assays, Wnt8
RNA injected in one cell activated the Sia
reporter in an adjacent cell. APCDD1
RNA inhibited transcription when coinjected with the Sia
reporter, but not with Wnt8
(), suggesting that APCDD1 inhibits Wnt signaling cell-autonomously in the receiving cell. Finally, since Wt-APCDD1 contains a transmembrane domain (), and was localized to the plasma membrane ( and Fig. S11a,c,f,i
), we tested whether APCDD1 undergoes cleavage to generate a diffusible inhibitor (APCDD1ΔTM), however, it was undetectable in the medium of transfected cells (Fig. S10d
). Collectively, these data reveal that APCDD1 is likely a membrane-tethered Wnt inhibitor that acts as a dimer at the surface of the Wnt-receiving cell.
The L9R mutation disrupts the hydrophobic core of the signal peptide critical for co-translational processing (Fig. S4b,c
. We analyzed protein stability and localization by western blotting and immunofluorescence in two cell lines (HEK293T or Bend3.0) transfected with either wild-type (Wt) APCDD1 or two different mutant forms (pathogenic mutation, L9R, and conservative substitution, L9V). Two fragments (68 KDa and 130 KDa) were detected in lysates of the Wt- and the L9V-APCDD1-transfected cells, whereas only a faint 68 KDa fragment was detected in the L9R mutant (). In addition, Wt- or L9V-APCDD1 protein was localized to the cell membrane, while L9R-APCDD1 was retained within the ER ( and S11a-j
). Furthermore, unlike the Wt isoform, N-terminally GFP-tagged L9R-APCDD1 could not be cleaved to localize at the membrane (Fig. S11l-n
). Finally, when the Wt- and L9R-APCDD1 were co-transfected either in cells or injected into Xenopus
embryos, some Wt protein was degraded (), and the rest sequestered in the ER along with the L9R isoform ( and S11k,o-q
). Therefore, the L9R mutation likely functions in a dominant-negative manner, to destabilize the Wt protein and prevent it from reaching the plasma membrane.
We next tested if the L9R mutation affects APCDD1 protein function in vivo
. In the chick neural tube, expression of L9R-APCDD1 only weakly inhibited eGFP transcription from the Wnt reporter (Fig. S6e,f
), and had no effect on Sox3+
neural progenitors and neuronal subtypes ( and S7e-h
), in contrast to Wt- or L9V-APCDD1 (Fig. S7m-u
). Moreover, L9R-APCDD1 was able to block Wt protein function in vivo
when they were co-transfected ( and S7i-l
), indicative of a dominant-negative effect. The same results were observed in Xenopus
, where the inhibitory effect of Wt APCDD1 on Wnt8-induced transcription was blocked by coexpression of the L9R mutant ().
We then determined the consequences of Xenopus
APCDD1 (Xapcdd1) protein depletion on axis formation in Xenopus
mRNA is expressed maternally throughout development, with the highest levels in animal (future ectoderm) and marginal (future mesoderm) cells of stage 10 embryos (Fig. S12
). Depletion of Xapcdd1 protein with a specific translation-blocking MO oligonucleotide (Fig. S13
) resulted in loss of anterior and dorsal structures ( and Table S1
). This phenotype was rescued by either injection of MO-resistant 5’ mutant Xapcdd1
RNA, or by DNWnt8
RNAs ( and Table S1
), which inhibit zygotic Wnt signaling24
. Therefore, the loss-of-function phenotype is consistent with ectopic activation on the dorsal side of zygotic Wnt activity, and supports the notion that endogenous APCDD1 is a Wnt inhibitor.
In conclusion, we suggest that APCDD1 may prevent formation of the Wnt receptor complex () since it interacts in vitro with LRP5 and WNT3A. The L9R mutant is unable to repress Wnt-responsive genes, by trapping the Wt protein in the ER where it may undergo degradation ().
Our findings underscore the requirement for exquisitely controlled regulation of the Wnt signaling pathway in HF morphogenesis and cycling 25
. It is known that forced activation of Wnt signaling exclusively in the epidermis leads to increased hair follicle density and tumors26,27
. We postulate that in HHS, Wnt signaling is indirectly increased through loss of the inhibitory function of APCDD1 in both the epidermal and dermal compartments of the HF, although the lack of HHS scalp samples precluded us from verifying this assumption. This notion is supported by mice with targeted ablation of another Wnt inhibitor, Klotho
, which exhibit a reduction in HF density due to indirect upregulation of Wnt signaling and a depletion of HF bulge stem cells28
. Since APCDD1 is expressed in both epidermal HF cells as well as the dermal papilla, we postulate that the simultaneous deregulation of Wnt signaling in both compartments may lead to the proportional reduction in organ size of the HF, resulting in miniaturization.
Our study provides the first genetic evidence that mutations in a Wnt inhibitor result in hair loss in humans. APCDD1 may be implicated in polygenic HF disorders as well, since it resides within linkage intervals on chromosome 18 in families with androgenetic alopecia29
as well as alopecia areata30
. Furthermore, since APCDD1 is expressed in a broad range of cell types3
, our findings raise the possibility that APCDD1 is involved in other Wnt-regulated processes, such as morphogenesis, stem cell renewal, neural development and cancer.