Identification of recessive maternal‐effect mutations in the zebrafish using a gynogenesis‐based method

Abstract

In animal species, early developmental processes are driven by maternally derived factors. Here, we describe a forward genetics approach to identify recessive mutations in genes encoding such maternal factors in the zebrafish. We used a gynogenesis-based approach to identify 14 recessive maternal-effect mutations. Homozygosity for these mutations in adult females leads to the inviability of their offspring. Confocal microscopy of embryos labeled with a DNA dye and a membrane marker allowed us to further analyze mutant embryos for defects in nuclear and cellular divisions. The mutations result in a range of defects in early developmental processes, including egg activation, early nuclear events, mitosis, cytokinesis, axial patterning, and gastrulation. Our effort constitutes a systematic attempt to identify maternal-effect genes in a vertebrate species. The sample of mutations that we have identified reflects the diversity of maternally driven functions in early development and underscores the importance of maternal factors in this process. Developmental Dynamics 231:324 -335, 2004.

Analysis of axis induction mutant embryos reveals morphogenetic events associated with zebrafish yolk extension formation

“…Even though morphogenetic movements begin after the initiation of zygotic transcription, perduring maternal gene products can affect post-MBT processes, including cell movements (Pelegri et al, 2004;Wagner et al, 2004). Thus, mutant phenotypes for maternal factors can lend insight into the complexities of development and demonstrate how later processes build upon early cell fate decisions.…”

“…Thus, mutant phenotypes for maternal factors can lend insight into the complexities of development and demonstrate how later processes build upon early cell fate decisions. The recessive maternal-effect mutation in the gene hecate (hec) was identified in a gynogenesisbased genetic screen for maternal-effect mutants (Pelegri et al, 2004). Embryos from homozygous females lack dorsoanterior structures at 24 hpf due to a lack of activation of the Wnt/␤-catenin signaling pathway thought to specify the zebrafish dorsal organizer (Lyman Gingerich et al, 2005).…”

Transient window of resilience during early development minimizes teratogenic effects of heat in zebrafish embryos

“…Despite this, transient heat shocks have been shown to be an effective genome doubling strategy in zebrafish (Streisinger et al, 1981). The practical utility of experimentally doubling ploidy in a genetically tractable vertebrate such as zebrafish lies in the expedited identification of zygotic and maternal recessive mutations (Streisinger et al, 1986, 1981; Beattie et al, 1999; Pelegri and Schulte‐Merker, 1999; Pelegri et al, 2004). In zebrafish, a typical F3 screen for zygotic mutations takes ∼6 months and F4 screen for maternal‐effect mutations takes ∼9 months (Driever et al, 1996; van Eeden et al, 1998; Amsterdam et al, 1999; Pelegri and Schulte‐Merker, 1999; Patton and Zon, 2001; Pelegri et al, 2004; Pelegri and Mullins, 2016).…”

“…The practical utility of experimentally doubling ploidy in a genetically tractable vertebrate such as zebrafish lies in the expedited identification of zygotic and maternal recessive mutations (Streisinger et al, 1986, 1981; Beattie et al, 1999; Pelegri and Schulte‐Merker, 1999; Pelegri et al, 2004). In zebrafish, a typical F3 screen for zygotic mutations takes ∼6 months and F4 screen for maternal‐effect mutations takes ∼9 months (Driever et al, 1996; van Eeden et al, 1998; Amsterdam et al, 1999; Pelegri and Schulte‐Merker, 1999; Patton and Zon, 2001; Pelegri et al, 2004; Pelegri and Mullins, 2016). However, genome doubling strategies can reduce the mutation screen time by at least one generation wherein heterozygous F2 haploid progeny can be diploidized and the resultant gynogenic diploids in which the mutation is now homozygous, can be directly screened for recessive zygotic mutations, or raised to adults and the F3 embryos can be screened for maternal‐effect mutations (Streisinger et al, 1986, 1981; Beattie et al, 1999; Pelegri and Schulte‐Merker, 1999; Pelegri et al, 2004; Trede et al, 2008).…”

“…In terms of ease of experimental technique, the transient pressure paradigm requires the use of a pressure chamber, whereas heat shocks can be done using a simple water bath immersion method. However, despite the simplicity of experimental procedure and predictable homozygosity efficiency, genome doubling strategies in zebrafish and Xenopus use pressure as the experimental paradigm of choice (Pelegri et al, 2004; Noramly et al, 2005; Goda et al, 2006; Trede et al, 2008). …”