Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis

Abstract

In animals, specification of the primordial germ cells (PGCs), the stem cells of the germline, is required to transmit genetic information from one generation to the next. Bucky ball (Buc) is essential for germ plasm (GP) assembly in oocytes and its overexpression results in excess PGCs in zebrafish embryos. However, the mechanistic basis for the excess PGCs in response to Buc overexpression, and whether endogenous Buc functions during embryogenesis are unknown. Here we show that endogenous Buc, like GP and overexpressed Buc-GFP, accumulates at embryonic cleavage furrows. Furthermore, we show that the maternally expressed zebrafish Kinesin-1 Kif5Ba is a binding partner of Buc and that maternal kif5Ba (Mkif5Ba) plays an essential role in germline specification in vivo. Specifically, Mkif5Ba is required to recruit GP to cleavage furrows and thereby specifies PGCs. Moreover, Mkif5Ba is required to enrich Buc at cleavage furrows and for Buc’s ability to promote excess PGCs, providing mechanistic insight into how Buc functions to assemble embryonic GP. In addition, we show that Mkif5Ba is also essential for dorsoventral (DV) patterning. Specifically, Mkif5Ba promotes formation of the parallel vegetal microtubule array required to asymmetrically position dorsal determinants (DDs) towards the prospective dorsal side. Interestingly, while Syntabulin and wnt8a translocation depend on kif5Ba, grip2a translocation does not, providing evidence for two distinct mechanisms by which DDs may be asymmetrically distributed. These studies identify essential roles for maternal Kif5Ba in PGC specification and DV patterning and provide mechanistic insight into Buc functions during early embryogenesis.

Methods to study maternal regulation of germ cell specification in zebrafish

“…Microtubules and microtubule-associated motor proteins (Kinesins) are essential for germ plasm recruitment to the furrow. This has been demonstrated by studies of zebrafish nebel maternal mutants that have a reduced furrow microtubule array and therefore cannot maintain germ plasm within the furrows (Pelegri, Knaut, Maischein, Schulte-Merker, & Nusslein-Volhard, 1999; Theusch et al, 2006), as well as maternal kinesin Kif5Ba mutants that cannot localize germ plasm to the furrows despite intact microtubules (Campbell, Heim, Smith, & Marlow, 2015). …”

“…This technique has higher signal specificity and lower background than conventional in situ techniques and may even be able to detect single RNA molecules. RNAscope has been adapted for use in zebrafish embryos (Gross-Thebing, Paksa, & Raz, 2014) and has also been used along with dual color FISH to examine the intracellular distribution of germ plasm RNAs within PGCs and oocytes (Campbell, Chao, Singer, & Marlow, 2015; Campbell, Heim, et al, 2015; Gross-Thebing et al, 2014; Welch & Pelegri, 2014). …”

“…Whenever conducting a forward genetic screen, one must consider some of the potential limitations of the screening capabilities. For example, some maternal-effect phenotypes may be missed due to earlier zygotic requirements for the gene, or due to later roles during germ cell development that are only detectable with marker analysis (Campbell, Heim, et al, 2015). Finally, a considerable number of mutations may go undetected due to lack of mutant phenotypes owing to the widespread gene duplication present in the zebrafish genome (reviewed in Meyer & Schartl, 1999).…”

“…CRISPR-Cas9 mutagenesis has recently been used to examine the roles of a maternal gene involved in germ cell development. In zebrafish, maternal Kinesin Kif5Ba is required for assembly of germ plasm at the cleavage furrows, and progeny of kif5Ba mutant mothers fail to specify PGCs and are infertile (Campbell, Heim, et al, 2015). Given the ease of use of the CRISPR-Cas9 technology, it is becoming the targeted mutagenesis tool of choice for investigating specific requirements of genes that participate in germ cell specification and maternally regulated embryonic development.…”

Translation repression by maternal RNA binding protein zar1 is essential for early oogenesis in zebrafish

“…These oocytes grow to full-size in females, but degenerate and are replaced by spermatogenesis in males (Uchida et al, 2002). Zebrafish without germ cells develop into male adults, indicating germ cells are essential for female development (Campbell et al, 2015;Draper et al, 2007;Houwing et al, 2007;Siegfried and Nüsslein-Volhard, 2008;Slanchev et al, 2005). When oocytes are depleted in juveniles, zebrafish also develop into males (Dranow et al, 2013;Hartung et al, 2014;Houwing et al, 2008;Rodríguez-Marí et al, 2010Shive et al, 2010;White et al, 2011).…”