Systematic studies of all PIH proteins in zebrafish reveal their distinct roles in axonemal dynein assembly

Abstract

Construction of motile cilia/flagella requires cytoplasmic preassembly of axonemal dyneins before transport into cilia. Axonemal dyneins have various subtypes, but the roles of each dynein subtype and their assembly processes remain elusive in vertebrates. The PIH protein family, consisting of four members, has been implicated in the assembly of different dynein subtypes, although evidence for this idea is sparse. Here, we established zebrafish mutants of all four PIH-protein genes: pih1d1, pih1d2, ktu, and twister, and analyzed the structures of axonemal dyneins in mutant spermatozoa by cryo-electron tomography. Mutations caused the loss of specific dynein subtypes, which was correlated with abnormal sperm motility. We also found organ-specific compositions of dynein subtypes, which could explain the severe motility defects of mutant Kupffer’s vesicle cilia. Our data demonstrate that all vertebrate PIH proteins are differently required for cilia/flagella motions and the assembly of axonemal dyneins, assigning specific dynein subtypes to each PIH protein.

Electron cryotomography of intact motile cilia defines the basal body to axoneme transition

“…We next focused on the post-TZ region to determine where axonemal identity arose along the ciliary axis. Axonemes have been extensively studied by cryoEM, resulting in averages from different species (Ichikawa et al, 2017; Lin et al, 2014; Pigino et al, 2011; Yamaguchi et al, 2018). All axoneme structures thus far determined are composed of a 96-nm repeat that propagates along the length of the cilium.…”

Cilia in the developing zebrafish ear

“…The tissue specificity of the dnah expression patterns appears to correlate with the type of ciliary movement present (oscillatory or rotational): notably, the subset of dnah genes expressed in the ear largely seems to overlap with that expressed in the floorplate and Kupffer's vesicle, again highlighting tissues with rotational ciliary movement (Yamaguchi et al, 2018). All four zebrafish PIH (Protein interacting with Hsp90) genes are also expressed in the otic placode or vesicle, some very strongly (Yamaguchi et al, 2018). PIH gene products are DNAAFs implicated in the pre-assembly of dynein arms prior to their transport into cilia.…”

“…As expected for tissues expressing foxj1b, the zebrafish otic placode and vesicle are prominent sites of expression for many genes known to be required for ciliogenesis and ciliary motility (see (Stooke-Vaughan et al, 2012) and references within). These include genes coding for dyneins and dynein axonemal assembly factors (DNAAFs) (Hjeij et al, 2014;Jaffe et al, 2016;Jerber et al, 2014;Sullivan-Brown et al, 2008;van Rooijen et al, 2008;Yamaguchi et al, 2018;Yu et al, 2008). A comprehensive expression analysis of dynein axonemal heavy chain (dnah) genes in the zebrafish revealed several expressed in the ear, including one (dnah9l) that was ear-specific (Yamaguchi et al, 2018).…”

“…These include genes coding for dyneins and dynein axonemal assembly factors (DNAAFs) (Hjeij et al, 2014;Jaffe et al, 2016;Jerber et al, 2014;Sullivan-Brown et al, 2008;van Rooijen et al, 2008;Yamaguchi et al, 2018;Yu et al, 2008). A comprehensive expression analysis of dynein axonemal heavy chain (dnah) genes in the zebrafish revealed several expressed in the ear, including one (dnah9l) that was ear-specific (Yamaguchi et al, 2018). The tissue specificity of the dnah expression patterns appears to correlate with the type of ciliary movement present (oscillatory or rotational): notably, the subset of dnah genes expressed in the ear largely seems to overlap with that expressed in the floorplate and Kupffer's vesicle, again highlighting tissues with rotational ciliary movement (Yamaguchi et al, 2018).…”

“…A comprehensive expression analysis of dynein axonemal heavy chain (dnah) genes in the zebrafish revealed several expressed in the ear, including one (dnah9l) that was ear-specific (Yamaguchi et al, 2018). The tissue specificity of the dnah expression patterns appears to correlate with the type of ciliary movement present (oscillatory or rotational): notably, the subset of dnah genes expressed in the ear largely seems to overlap with that expressed in the floorplate and Kupffer's vesicle, again highlighting tissues with rotational ciliary movement (Yamaguchi et al, 2018). All four zebrafish PIH (Protein interacting with Hsp90) genes are also expressed in the otic placode or vesicle, some very strongly (Yamaguchi et al, 2018).…”