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Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo
Published on: September 20, 2017
Microtubule-associated protein 9 (Map9/Asap) is required for the early steps of zebrafish development
Laura Fontenille1, Sylvie Rouquier2, Georges Lutfalla3
1Institute of Human Genetics; UPR 1142; CNRS; Montpellier, France; Université de Montpellier 1; Montpellier, France.
Abstract:
Microtubules are structural components of the cell cytoskeleton and key factors for mitosis and ciliogenesis in eukaryotes. The regulation of MT dynamics requires non-motor MAPs. We previously showed that, in human cells in culture, MAP9 (also named ASAP) is involved in MT dynamics and is essential for mitotic spindle formation and mitosis progression. Indeed, misexpression of MAP9 leads to severe mitotic defects and cell death. Here, we investigated the in vivo role of map9 during zebrafish development. Map9 is expressed mainly as a maternal gene. Within cells, Map9 is associated with the MT network of the mitotic spindle and with centrosomes. Morpholino-mediated depletion of map9 leads to early development arrest before completion of epiboly. Map9 localizes to the MT array of the YSL. This MT network is destroyed in Map9-depleted embryos, and injection of anti-map9 morpholinos directly in the nascent YSL leads to arrest of epiboly/gastrulation. Finally, map9 knockdown deregulates the expression of genes involved in endodermal differentiation, dorso-ventral and left-right patterning, and other MT-based functions. At low morpholino doses, the surviving embryos show dramatic developmental defects, spindle and mitotic defects, and increased apoptosis. Our findings suggest that map9 is a crucial factor in early zebrafish development by regulating different MT-based processes.
Insights
Microtubule-associated protein 9 (MAP9) is essential for early zebrafish development. Depleting MAP9 disrupts microtubule networks, causing developmental arrest and mitotic defects.
Area of Science:
- Cell Biology
- Developmental Biology
- Genetics
Background:
- Microtubules (MTs) are vital for eukaryotic cell structure and function, including mitosis and ciliogenesis.
- Non-motor Microtubule-Associated Proteins (MAPs) regulate MT dynamics.
- MAP9 (ASAP) was previously implicated in human cell MT dynamics and mitosis.
Purpose of the Study:
- To investigate the in vivo role of map9 in early zebrafish development.
- To understand MAP9's function in microtubule-based processes during embryogenesis.
Main Methods:
- Morpholino-mediated knockdown of map9 in zebrafish embryos.
- Analysis of embryonic development, cell division, and gene expression.
- Localization studies of Map9 within embryonic cells.
Main Results:
- Map9 is maternally expressed and associates with mitotic spindles and centrosomes.
- Map9 depletion caused early developmental arrest before epiboly completion.
- Disruption of the Yolk Syncytial (YS) MT network and gastrulation arrest were observed.
- Knockdown deregulated genes involved in endodermal differentiation and patterning.
- Surviving embryos exhibited developmental, spindle, and mitotic defects, with increased apoptosis.
Conclusions:
- Map9 is crucial for early zebrafish development.
- It regulates essential microtubule-based processes, including spindle formation and gastrulation.
- MAP9 plays a significant role in embryonic patterning and differentiation.
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