Methods to study maternal regulation of germ cell specification in zebrafish

Abstract

The process by which the germ line is specified in the zebrafish embryo is under the control of maternal gene products that were produced during oogenesis. Zebrafish are highly amenable to microscopic observation of the processes governing maternal germ cell specification because early embryos are transparent, and the germ line is specified rapidly (within 4–5 h post fertilization). Advantages of zebrafish over other models used to study vertebrate germ cell formation include their genetic tractability, the large numbers of progeny, and the easily manipulable genome, all of which make zebrafish an ideal system for studying the genetic regulators and cellular basis of germ cell formation and maintenance. Classical molecular biology techniques, including expression analysis through in situ hybridization and forward genetic screens, have laid the foundation for our understanding of germ cell development in zebrafish. In this chapter, we discuss some of these classic techniques, as well as recent cutting-edge methodologies that have improved our ability to visualize the process of germ cell specification and differentiation, and the tracking of specific molecules involved in these processes. Additionally, we discuss traditional and novel technologies for manipulating the zebrafish genome to identify new components through loss-of-function studies of putative germ cell regulators. Together with the numerous aforementioned advantages of zebrafish as a genetic model for studying development, we believe these new techniques will continue to advance zebrafish to the forefront for investigation of the molecular regulators of germ cell specification and germ line biology.

Primordial Germ Cell Development in the Poeciliid, Gambusia holbrooki, Reveals Shared Features Between Lecithotrophs and Matrotrophs

“…The relatively high expression of PGC markers tested (dazl, dndα, piwi II, and vasa but not nanos1) in unfertilised eggs of G. holbrooki implies their maternal inheritance and suggests PGC specification is determined by preformation mode. This pattern is similar to those observed in oviparous species such as zebrafish (Kaufman and Marlow, 2016) and medaka (Herpin et al, 2007) as well as those of Drosophila (Rongo and Lehmann, 1996), and Xenopus (Chan et al, 2007). However, in mice, mechanical ablation of vegetal or animal pole of the fertilised egg did not arrest the development of viable and fertile offspring (Zernicka-Goetz, 1998).…”

rbpms2 functions in Balbiani body architecture and ovary fate

“…To test if Rbpms2 proteins can localize to granules in the zebrafish germline, we used a commonly employed assay in which in vitro transcribed RNA encoding GFP-rbpms2a/2b was injected into fertilized zebrafish eggs. For many germ plasm RNAs and interacting proteins, the translated product of the exogenous RNA will localize to germ granules of primordial germ cells (PGCs) in 24-30hpf embryos (reviewed in [ 1 , 30 ]). PGCs were marked by cytoplasmic expression of RFP- nanos3’UTR .…”

Mechanism of Germ Cell Specification in Early Zebrafish Embryogenesis

“…Later during oogenesis in stage III oocytes, the Balbiani body is dispersed at the vegetal pole (Dosch, 2015;Kloc et al, 2014;Bontems et al, 2009;Heim et al, 2014; O.H. Kaufman and F.L. Marlow, 2017).…”

“…Marlow, 2017). The germ plasm comprises of maternally supplied RNAs, proteins, cell organelles like endoplasmic reticulum (ER), Golgi and mitochondria (Raz, 2003;Kloc et al, 2004;O.H. Kaufman and F.L.…”