Roles of basal keratinocytes in actinotrichia formation

Abstract

The embryonic fins and the tip of adult fins of teleost fish are supported by rows of straight, unmineralized fibrils called actinotrichia. The proximal ends of the actinotrichia are bundled and the mineralized bones called lepidotrichia are made along them. Since malformation in actinotrichia causes wavy fin bones, the correct configuration of actinotrichia is essential for the correct construction of the fin shape. Past studies suggested that two types of cells, basal keratinocytes, and mesenchymal cells involve in the formation of actinotrichia. However, the mechanism how these cells contribute is unknown. In this study, we elucidated the role of basal keratinocytes in actinotrichia formation. First, we developed the imaging tool that specifically visualizes the basal keratinocytes and actinotrichia. Then, we established the in vitro culture method of the basal keratinocytes and found that the keratinocytes developed fine needle-like structures in it. The TEM image of them showed the specific shadow pattern of actinotrichia, indicating that the fine needle-like structures are the newly made actinotrichia. Finally, we cultured the basal keratinocytes with mature actinotrichia and observed that the basal keratinocytes actively holded actinotrichia with their membrane, and often generated a linear array of cells holding a single actinotrichium. This behavior suggested a mechanism with which long actinotrichia are made by relatively small cells. Our results clarified the role of basal keratinocyte and provided a novel insight into understanding the mechanism of actinotrichia formation.

The Physical Role of Mesenchymal Cells Driven by the Actin Cytoskeleton Is Essential for the Orientation of Collagen Fibrils in Zebrafish Fins

“…Prior to conducting the in vitro study, we established a new actinotrichia-visualized zebrafish line [5x and1 (2k): And1 full -GFP] to allow more detailed observation of the cell and actinotrichia dynamics ( Supplementary Figure 1 ). The fins of this new actinotrichia-visualized zebrafish showed even greater fluorescence intensity of the actinotrichia compared to the previously reported visualization line ( Kuroda et al, 2018 ) ( Supplementary Figures 1 , 2 ). Using previously reported methods ( Kuroda et al, 2018 ), we first isolated the actinotrichia and mesenchymal cells from the larval fins of our new transgenic F1 line [5x and1 (2k): And1 full -GFP/5x and1 (MC): Lifeact-mCherry] and cultured them in culture dishes coated with various kinds of substrates.…”

“…Previous studies have suggested that actinotrichia are distributed between basal keratinocytes and mesenchymal cells in fins ( Wood and Thorogood, 1987 ; Smith et al, 2008 ; König et al, 2018 ; Kuroda et al, 2018 ). Basal keratinocytes are in contact with the outer part of the two actinotrichia rows whereas mesenchymal cells are in contact with the inner part.…”

“…First, we visualized each cells by expressing mCherry under the control of a cell-specific promoter ( Lalonde et al, 2016 ; Kuroda et al, 2018 ; Phan et al, 2019 ). Actinotrichia were also simultaneously visualized by expressing And1-GFP under the control of and1 promoter, as we reported previously ( Kuroda et al, 2018 ). Figures 1A,C show the larva used for the observation and Figures 1B,D show the fin fold.…”

“…All larvae were at the stage of 3 dpf. MC, mesenchymal cell specific promoter designated as 2PΔepi in the previous paper ( Lalonde et al, 2016 ); BK, basal keratinocyte specific promoter designated as 1.4 k and1 pro in the previous paper ( Kuroda et al, 2018 ). Scale bars: 500 μm in (A,C) , 50 μm in (B,D) .…”

“…If the interaction between purified cells and actinotrichia could be observed in vitro , it would complement the weaknesses of in vivo experiments. As a first example of such an experiment, in vitro culture of basal keratinocytes purified from fins has shown that actinotrichia formation begins intracellularly ( Kuroda et al, 2018 ).…”