RESEARCH ARTICLE Intrinsic curvature in wool fibres is determined by the relative length of orthocortical and paracortical cells Duane P. Harland 1, *, James A. Vernon 1 , Joy L. Woods 1 , Shinobu Nagase 2 , Takashi Itou 2 , Kenzo Koike 2, *, David A. Scobie 3 , Anita J. Grosvenor 1 , Jolon M. Dyer 1 and Stefan Clerens 1 ABSTRACT Hair curvature underpins structural diversity and function in mammalian coats, but what causes curl in keratin hair fibres? To obtain structural data to determine one aspect of this question, we used confocal microscopy to provide in situ measurements of the two cell types that make up the cortex of merino wool fibres, which was chosen as a well-characterised model system representative of narrow diameter hairs, such as underhairs. We measured orthocortical and paracortical cross-sectional areas, and cortical cell lengths, within individual fibre snippets of defined uniplanar curvature. This allowed a direct test of two long-standing theories of the mechanism of curvature in hairs. We found evidence contradicting the theory that curvature results from there being more cells on the side of the fibre closest to the outside, or convex edge, of curvature. In all cases, the orthocortical cells close to the outside of curvature were longer than paracortical cells close to the inside of the curvature, which supports the theory that curvature is underpinned by differences in cell type length. However, the latter theory also implies that, for all fibres, curvature should correlate with the proportions of orthocortical and paracortical cells, and we found no evidence for this. In merino wool, it appears that the absolute length of cells of each type and proportion of cells varies from fibre to fibre, and only the difference between the length of the two cell types is important. Implications for curvature in higher diameter hairs, such as guard hairs and those on the human scalp, are discussed. KEY WORDS: Hair, Wool, Single-fibre curvature, Orthocortex, Paracortex, Cortical cells INTRODUCTION Hair, along with milk production, is a definitive phenotypic trait for mammals. Hair appears to have evolved from scales of synapsid reptiles around 200-million years ago (Alibardi, 2006; Maderson, 2003), and hair was already a well-established trait at the very dawn of mammalian evolution because most aspects of hair biology are highly conserved across all existing mammals, including general fibre morphology. Mammalian coats are typically composed of mixtures of differently functional hair types that are arranged in a three- dimensional (3D) structure, or pelage. The emergent properties of the pelage, and its modification over time (typically seasonal), forms the functional phenotype upon which evolutionary selection occurs, and the adaptability of which has enabled mammals to colonise a wide range of habitats, including climatic extremes (Ryder, 1973). In a typical pelage, for example that of a deer, straight high-diameter guard hairs scaffold a mass of narrow diameter curly underhairs (Brunner and Coman, 1974; Woods et al., 2011), with the combined effect of thermoregulation and mechanical protection. Single-fibre properties, in particular length, diameter and curvature, are important in defining the emergent properties of the pelage. The underpinning mechanism that connects structures at the protein and microstructure level to single-fibre curvature is not well resolved. Here, we use merino wool as a model for understanding the basis of mammalian hair curvature. Whereas wild sheep have a typical pelage of high diameter guard hairs and low diameter underhairs, in that of merino domestic sheep, both guard and underhairs have been selected to be of low diameter and high curvature (Harland et al., 2015; Ryder, 1964). It has long been known that, in wool fibres, there is an approximate correlation between the amounts and distributions of different wool cortical cell types and fleece crimp [the primary parameter of the sheep pelage structure, observed as a wave within a clipped staple (tress/tuft) of wool typically containing thousands of fibres]. Crimp is an emergent effect of single-fibre curvature. Fibre curvature is of two sorts: intrinsic curvature and imposed curvature. Intrinsic curvature is built into a fibre during its development in the follicle, and it is this curvature, sometimes called inherent curvature, to which an undamaged fibre returns when relaxed in water and dried without mechanical constraint (Fish et al., 1999). Imposed curvature is any chemically or physically induced change to intrinsic curvature; for example, from drying a fibre in a constrained state. Here, we investigate intrinsic curvature (Fig. S1). The observation that high-crimp wools tend to have a cortex in which the cell types are bilaterally arranged, with the paracortical cells always on the inside (or concave) half of the fibre curvature, led to the theory that it is the relative proportion and distribution of the orthocortex and paracortex patches that underpins intrinsic single- fibre curvature (Fraser and Rogers, 1954; Horio and Kondo, 1953). The theory has some experimental support from light- and electron- microscopy studies measuring average cell type distribution against average single-fibre curvature (Orwin et al., 1984; Snyman, 1963). The theory explains that, during keratinisation in the wool follicle, a fibre goes from a wet to a dry state, and that this affects the macrofibrils making up the cells of each cell type differently. Due to a greater number of intermediate filaments (IFs) in orthocortical macrofibrils that are initially tilted away from the fibre axis, longitudinal extension occurs during drying, and this extension in the orthocortex is believed to exceed that of paracortex, which is composed of macrofibrils in which the IFs are aligned along the fibre axis. The theory predicts that, in order to relieve internal strain Received 15 October 2017; Accepted 16 January 2018 1 Food and Bio-based Products Group, AgResearch, Lincoln 7608, New Zealand. 2 Hair Beauty Research, Kao Corporation, Tokyo 131-8501, Japan. 3 Farm Systems and Environment Group, AgResearch, Lincoln 7608, New Zealand. *Authors for correspondence (duane.harland@agresearch.co.nz; Koike.kenzo@kao.co.jp) D.P.H., 0000-0002-1204-054X; K.K., 0000-0002-6796-5785 1 © 2018. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2018) 221, jeb172312. doi:10.1242/jeb.172312 Journal of Experimental Biology