The Wnt-inhibitor Dkk4 is required for hair follicle initiation and patterning

Summary: Abstract Body: Hair follicles (HF) are skin appendages covering the surface of the mammalian body. They arise during embryonic development in regularly spaced patterns through a continuous crosstalk between the epithelium and underlying mesenchyme. Several secreted signaling factors are intricately involved in the determination of HF formation sites. Notably, inhibition of Wnt signaling completely prevents the formation of HF epithelial precursors called placodes (Pc), suggesting that Wnt resides at the top of the hierarchy of Pc initiation. We hypothesized that the interaction of Wnt activators and inhibitors underlies the pattern of HF formation. We focused on the Wnt inhibitor and direct target Dickkopf 4 (Dkk4), which is specifically expressed in Pcs, and generated Dkk4 knockout (Dkk4-/-) mice using CRISPR/Cas9. Dkk4-/- embryos show profound disruptions in HF development and patterning, suggesting that Dkk4 plays a crucial role in these processes, likely through its negative feedback interaction with Wnt signaling. To genetically test whether DKK4 acts as an inhibitor of the Wnt pathway during HF formation, we crossed Dkk4-/- mice with the BAT-GAL line, a canonical WNT reporter mouse. Dkk4-/-; BAT-GAL embryos showed abnormal Wnt activity pattern, suggesting that the loss of DKK4 disrupts the balance of Wnt inhibitors and activators. To further characterize the role of Dkk4 in HF formation and patterning, we generated and used Dkk4-Cre knockin mice and performed lineage tracing analysis to test whether entire HFs are derived from Dkk4-expressing cells. Our data showed that not all Pc cells originate from Dkk4-Cre expressing cells, and instead mature HFs show a broad and patchy distribution of Dkk4-derived cells. Overall, we propose that Dkk4 is initially expressed in the skin to mark the position of HFs and modulate Wnt activity for a refined regular pattern. Hirofumi Teshima¹, Houda Khatif², Hisham Bazzi^{1, 2} 1. Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, United States. 2. Cell Biology of the Skin, Universitat zu Koln, Cologne, NRW, Germany. Stem Cell Biology, Tissue Regeneration and Wound Healing