The hair follicle contains stem/progenitor cells that supply progeny for skin development and the hair cycle. Several signaling molecules belonging to the Wnt, BMP, shh, and transforming growth factor β (TGF-β) signaling cascades are involved in the normal hair follicle cycle. However, the systemic mechanism of how these humoral factors are controlled remains largely unknown. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions extracellularly as a key coordinator of multiple signaling networks. Here, we show that TSK is expressed at the restricted areas of hair follicle during the morphogenesis and the hair cycle. Targeted disruption of the TSK gene causes the hair cycle to be delayed with low levels of TGF-β1 and phosphorylated Smad2/3 (pSmad2/3) expression. Biochemical analysis indicates that TSK directly binds to TGF-β1. Our data suggest that TSK controls the hair cycle by regulating TGF-β1 signaling.

Lens growth involves the proliferation of epithelial cells, followed by their migration to the equator region and differentiation into secondary fiber cells. It is widely accepted that fibroblast growth factor (FGF) signaling is required for the differentiation of lens epithelial cells into crystallin-rich fibers, but this signaling is insufficient to induce full differentiation. To better understand lens development, investigatory and functional analyses of novel molecules are required. Here, we demonstrate that Equarin, which is a novel secreted molecule, was expressed exclusively in the lens equator region during chick lens development. Equarin upregulated the expression of fiber markers, as demonstrated using in ovo electroporation. In a primary lens cell culture, Equarin promoted the biochemical and morphological changes associated with the differentiation of lens epithelial cells to fibers. A loss-of-function analysis was performed using zinc-finger nucleases targeting the Equarin gene. Lens cell differentiation was markedly inhibited when endogenous Equarin was blocked, indicating that Equarin was essential for normal chick lens differentiation. Furthermore, biochemical analysis showed that Equarin directly bound to FGFs and heparan sulfate proteoglycan and thereby upregulated the expression of phospho-ERK1/2 (ERK-P) proteins, the downstream of the FGF signaling pathway, in vivo and in vitro. Conversely, the absence of endogenous Equarin clearly diminished FGF-induced fiber differentiation. Taken together, our results suggest that Equarin is involved as an FGF modulator in chick lens differentiation.

We have shown that draxin is a repulsive axon guidance molecule for a variety of neuron classes and that genetic deletion of draxin in mice results in the absence of all forebrain commissures. Moreover, we also identified a secreted molecule, Tsukushi (TSK), that belongs to the small leucine-rich proteoglycan family (SLRP) and inhibits signaling molecules, such as BMP and Wnt. TSK knockout mice show malformation of the corpus callosum (CC) and agenesis of the anterior commissure (AC), suggesting the importance of TSK function in forebrain commissure formation. There is a possibility that the combined function of these two proteins is essential for the formation of these commissures. In this study, we investigate this possibility by generating draxin/TSK doubly heterozygous mice and comparing their forebrain commissure phenotypes with those of singly heterozygous mice. We found that, although draxin and TSK did not interact directly, their genetic interaction was evident from the significantly higher prevalence of CC malformation and agenesis of the AC in the draxin/TSK doubly heterozygous mice. Importantly, in this study, we demonstrated a new function of TSK in guiding anterior olfactory neuronal (AON) and cortical axons. TSK bound to and provided growth inhibitory signals dose-dependently to AON and cortical axons in outgrowth assay. TSK also induced growth cone collapse when applied acutely to these cultured neurons. Furthermore, TSK and draxin had additive effects in inhibiting cortical and AON neurite outgrowth. Thus, based on a combination of genetic analyses and in vitro experiments, we propose that the combined guidance activities of draxin and TSK regulate forebrain commissure formation.

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self‐renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.