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

Abstract

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.

aura/mid1ip1Lregulates the cytoskeleton at the zebrafish egg-to-embryo transition

“…During oogenesis, yolk granule and CG distribution ( Fig. S2F-I), as well as enrichment of F-actin and formation of the mitochondrial cloud (Marlow and Mullins, 2008;Gupta et al, 2010), appear normal ( Fig. S3A-D).…”

“…Embryos were fixed with paraformaldehyde-glutaraldehyde after dechorionation (Urven et al, 2006) and oocyte labeling was carried out as previously described (Gupta et al, 2010). Primary antibodies: mouse anti-α-Tubulin (1:2500; Sigma, T5168), rabbit anti-β-Catenin (1:1000; Sigma, C2206), rabbit anti-Vamp2 (1:200; Abcam, ab70222), rabbit anti-Rab11b (1:200; GeneTex, GTX127328), rabbit anti-Dynamin 2 (1:100; GeneTex, GTX127330), rabbit anti-Clathrin (1:200; Abcam, ab59710) and rabbit antiVamp4 (1:400; SySy, 136 002).…”

Spectraplakin family proteins – cytoskeletal crosslinkers with versatile roles

“…SxIP motifs exist in multiple MT plus-end-tracking proteins (+TIPs), including the spectraplakins MACF1 and BPAG1 (Akhmanova and Steinmetz, 2008;Gupta et al, 2010;Honnappa et al, 2009). MACF1 directly binds to EB1 (also known as MAPRE1) through its SxIP motif and exhibits MT plus-end tracking (Slep et al, 2005).…”

“…MACF1a (also named Magellan), the spectraplakin found in zebrafish (Dosch et al, 2004), plays a critical role in forming the animal vegetal axis (Gupta et al, 2010). MACF1 regulates the size and localization of the Balbiani body -an aggregate found in developing oocytes, which consists of organelles, including the mitochondria, ER and Golgi.…”

Functional and Genetic Analysis of Spectraplakins in Drosophila

“…p0015 Surprisingly, no human diseases or conditions have been associated with MACF1/ACF7, but mouse and zebrafish models suggest its involvement in a wide range of developmental processes, ranging from early embryogenesis (Chen et al, 2006;Gupta et al, 2010), cell migration of fibroblasts, or skin stem cells (Wu, Kodama, & Fuchs, 2008;Wu et al, 2011), as well as heart and brain development (Fassett et al, 2013;Goryunov, He, Lin, Leung, & Liem, 2010;Jorgensen et al, 2014;Sánchez-Soriano et al, 2009 First, in vivo work is relatively fast in flies, and it is not unusual that new experimental ideas yield experimental data within a few weeks. Notably, there are no legal requirements, meaning that experiments can be performed straight off the drawing board with little or no need for an extensive ethical approval process.…”