Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
PLoS Genetics (Impact Factor: 7.53). 08/2010; 6(8):e1001073. DOI: 10.1371/journal.pgen.1001073
Source: PubMed


Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.

Download full-text


Available from: Tripti Gupta,

Click to see the full-text of:

Article: Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

5.15 MB

See full-text
  • Source
    • "Forward genetic screens have isolated more key regulators of maternally controlled processes than any other approach. Nonetheless, a key limitation of all genetic screens is the identification of the underlying molecular lesions, since in maternal-effect mutations require an additional generation of breeding (Bontems et al., 2009, 2011; Fukazawa et al., 2010; Gupta et al., 2010; Holloway et al., 2009; Kanagaraj et al., 2014; Mei et al., 2009; Yabe et al., 2007, 2009). From this and other screens, many mutants with exciting phenotypes remain to be molecularly identified showing the potential of forward genetic screens as a cornucopia to discover novel hitherto uncharacterized regulators of processes such as egg activation, fertilization, early nuclear events (Abrams et al., 2012), zygotic genome activation (Lee et al., 2013; Leichsenring et al., 2013) and cell migration (Fukazawa et al., 2010; Holloway et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In many animals, factors deposited by the mother into the egg control the earliest events in development of the zygote. These maternal RNAs and proteins play critical roles in oocyte development and the earliest steps of embryogenesis such as fertilization, cell division and embryonic patterning. Here, this article summarizes recent discoveries made on the maternal control of germline specification in zebrafish. Moreover, this review will discuss the major gaps remaining in our understanding of this process and highlight recent technical innovations in zebrafish, which allow tackling some of these questions in the near future.
    Critical Reviews in Biochemistry and Molecular Biology 11/2014; 50(1). DOI:10.3109/10409238.2014.985816 · 7.71 Impact Factor
  • Source
    • "Consistent with an important function in mammalian cells, the role of MACF1 in maintaining microtubule stability is conserved in non-mammalian systems too. For example, mutations in the MACF1 gene cause a loss of stable microtubule localization to the periphery of the zebrafish oocyte (Gupta et al., 2010). A model of how centrosomes move in the absence or presence of MACF1 is presented in Fig. 9A. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Neuronal migration and subsequent differentiation play critical roles for establishing functional neural circuitry in the developing brain. However, the molecular mechanisms that regulate these processes are poorly understood. Here, we show that microtubule actin crosslinking factor 1 (MACF1) determines neuronal positioning by regulating microtubule dynamics and mediating GSK-3 signaling during brain development. First, using MACF1 floxed allele mice and in utero gene manipulation, we find that MACF1 deletion suppresses migration of cortical pyramidal neurons and results in aberrant neuronal positioning in the developing brain. The cell autonomous deficit in migration is associated with abnormal dynamics of leading processes and centrosomes. Furthermore, microtubule stability is severely damaged in neurons lacking MACF1, resulting in abnormal microtubule dynamics. Finally, MACF1 interacts with and mediates GSK-3 signaling in developing neurons. Our findings establish a cellular mechanism underlying neuronal migration and provide insights into the regulation of cytoskeleton dynamics in developing neurons.
    Developmental Biology 11/2014; 395(1). DOI:10.1016/j.ydbio.2014.09.009 · 3.55 Impact Factor
  • Source
    • "In zebrafish, bucky ball has been shown to be essential for the assembly of germ plasm mRNAs and aggregate of organelles leading to BB formation (Marlow and Mullins, 2008; Xu et al., 2001). Similarly, the zebrafish macf1, a member of the highly conserved spectraplakin family of cytoskeletal linker proteins is required for BB formation and hence in establishment of oocyte polarity (Gupta et al., 2010). In medaka it has been shown that dazl RNA is localized in the BB but boule is not (Xu et al., 2009), and the same is seen in trout (Li et al., 2011b). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fertility genes boule and dazl constitute the evolutionarily conserved DAZ (Deleted in AZoospermia) family of RNA binding proteins essential for germline development across animal phyla. Here we report the cloning and expression analysis of boule and dazl from the Asian seabass (Lates calcarifer), a marine fish that undergoes sequential male-to-female sex reversal. Molecular cloning and sequence comparison led to the identification of boule and dazl cDNAs. RT-PCR analysis showed that both boule and dazl RNAs were restricted to the gonads among adult organs examined. Chromogenic in situ hybridization revealed germ cell-specific expression for both boule and dazl in female and male adults. Importantly, distinct differences were found between boule and dazl in terms of temporospatial expression and subcellular distribution. The boule RNA was abundant in late gametogenic cells except sperm. Interestingly, dazl expression increases in early oocytes and concentrates in a perinuclear speckle that appears to develop ultimately into the Balbiani body in advanced oocytes. The dazl RNA was found to be abundant in spermatocytes but hardly detectable in sperm. These data demonstrate that boule and dazl are germ cell markers in the adult Asian seabass, and that bisexual germline-specific expression has been conserved for boule and dazl in fish.
    Gene 10/2014; 549(2). DOI:10.1016/j.gene.2014.07.068 · 2.14 Impact Factor
Show more