Chapter 11 - Imaging and quantitative analysis of cytokinesis in developing brains of Kinesin-6 mutant mice

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Abstract

Cytokinesis in neural progenitors occurs with specialized constraints due to their highly polarized structure and the need for both symmetric and asymmetric divisions. They must produce proper numbers of progenitors, neurons, and glia in a precise order. Yet very few functional studies of cytokinesis have been done in the developing brain. To elucidate mechanisms of cytokinesis during brain development, we designed a novel method to study cytokinesis in whole mount “slabs” of embryonic mouse cerebral cortex. It takes advantage of cytokinesis occurring on the ventricular surface of the cortex and allows examination of cytokinesis across many cells in the context of an intact brain tissue. The cortical slabs can be fixed for immunohistochemistry or used in live imaging experiments. In particular, we investigated mutants of the Kinesin-6, Kif20b, which show defects in cytokinetic abscission and have small brains. Here, we describe how to dissect neocortex from embryonic cerebral hemispheres, immunostain the cortical slabs for cytokinetic midbodies and other structures, and image the apical surface. We show how to quantitatively analyze apical structures including midbody numbers, organization, and morphology. New images and analyses of Kif20bmagoo loss of function mutants are shown. Applying and adapting these types of analyses to other cytoskeletal proteins and mouse mutants will help advance our understanding on how the embryonic neuroepithelium generates neurons and builds the brain.

Introduction

Cytokinesis consists of two sequential events: first, cleavage furrow ingression to split the cell in two, and then the final severing event, abscission. After the segregation of the sister chromatids in anaphase by kinetochore microtubules, the central spindle (also called the midzone) induces furrow ingression at the equatorial plane by forming an actomyosin ring under the plasma membrane. This actomyosin ring cleaves the cell, generating the two sister cells and compressing the microtubules of the central spindle into a microtubule-dense structure, called the midbody. The midbody contains a plethora of proteins needed for control and completion of abscission. The final cut to separate the sister cells may take one or more hours to complete in the next G1 phase. Abscission may take place on one or both sides of the midbody (for reviews see Agromayor and Martin-Serrano, 2013, Green et al., 2012, Mierzwa and Gerlich, 2014).

Cytokinesis must be strictly regulated to repair tissues, achieve proper embryonic development, and prevent cancers. However, it is primarily studied in vitro using isolated single cells. Though this approach has yielded a lot of valuable insight for the events occurring and proteins required in cytokinesis, cell division in tissues adds different constraints. For example, epithelia undergo a specialized form of cytokinesis that appears critical for maintaining epithelial polarity, integrity, and tension, as demonstrated recently in Drosophila and Xenopus (Founounou et al., 2013, Guillot and Lecuit, 2013, Herszterg et al., 2013, Kieserman et al., 2008, Morais-de-Sá and Sunkel, 2013).

Neural epithelia have even more specialized constraints on cell division, particularly during development of the mammalian brain. Cortical neuroepithelial progenitors (also called apical progenitors) undergo interkinetic nuclear migration, with mitosis and cytokinesis taking place at the apical (ventricular) surface (Figure 1(A) and (B)). These cells become very tall and thin over the course of development, so the apical membrane surface is called the apical “endfoot.” In most divisions of cortical neuroepithelial progenitors, the actomyosin ring of the cleavage furrow ingresses from the basal to apical side of the cell, instead of as a contractile ring, and midbody formation occurs on the apical side (Dubreuil, Marzesco, Corbeil, Huttner, & Wilsch-Bräuninger, 2007). In early embryonic cerebral cortex, symmetric cell division ensures the expansion of the progenitor pool (Farkas and Huttner, 2008, Taverna et al., 2014). Later, neurons are generated through asymmetric cell divisions. Symmetric and asymmetric cell divisions add another layer of complexity that is not found in cell lines (Farkas and Huttner, 2008, Taverna et al., 2014). Thus, the complexity of cytokinesis in developing neural tissue highlights the necessity to investigate it within the context of both whole tissue and single cell assays.

In mammals, several kinesins are required for cytokinesis (Breuss & Keays, 2014). Two members of the mammalian Kinesin-6 family have been strongly associated with cytokinesis, Kif23/MKLP1, and Kif20a/MKLP2. Both localize to the midbody during cytokinesis. Knockdown of either in human cell lines leads to cytokinesis failure. Furthermore, Kif20a or Kif23 knockdown results in abscission failure and an increase of polyploidy; additionally there is no midbody formation when Kif23 is depleted (Barr and Gruneberg, 2007, Chen et al., 2012, Neef et al., 2007, Zhu et al., 2005). The third member, Kif20b, is the least understood member of the Kinesin-6 family, partly because it does not have an ortholog in invertebrates. It is a plus-end directed motor protein that has microtubule-binding, bundling and sliding properties in vitro. It has been shown that Kif20b localizes to the midzone during furrowing and accumulates at the midbody; it is nuclear during interphase. In addition, RNAi knockdown of Kif20b in a cell line caused failure of abscission in ∼40% of the cells (Abaza et al., 2003).

In two independent ENU screens for neural development mutants, two microcephalic mouse mutants were isolated with mutations in Kif20b (Dwyer et al., 2011, Garcia-Garcia et al., 2005, Janisch et al., 2013). In the Kif20bmagoo mutant, the protein is reduced below detection level. The Kif20bmcbarker mutant carries a point mutation in loop 11 of the motor domain. Both mutants show reduced cerebral cortex size and thickness, with increased apoptosis. A series of experiments demonstrated that while mitosis appears normal in cortical neuroepithelial progenitors, cytokinetic abscission is disrupted (Janisch et al., 2013). These mice are tools to study the specialized requirements for cytokinesis of cortical neuroepithelial progenitors.

Here we present the step-by-step method we developed to investigate the effects of Kif20b mutations on cytokinesis of cortical neuroepithelial progenitors within the developing brain. This method takes advantage of the fact that neuroepithelial progenitors undergo mitosis and cytokinesis on the apical (ventricular) side of the developing cortex. Therefore, instead of cutting conventional brain slices, we dissect neocortical “slab” whole mounts. These cortical slabs can be immunostained for a wide variety of markers and the apical surface can be imaged with high resolution (Figure 1(C)). In Section 1.2, we show examples of cytokinesis markers we used successfully in mouse cortex. Images of cortical neuroepithelial progenitors at the apical surface can be used to quantify an array of mitotic and cytokinetic parameters as described here in Section 1.4 step 2. Examples of previous and new data analyses can be found in Section 2. This method is for imaging immunostained markers on fixed brain tissue, but can be adapted for live imaging with fluorescent markers.

Section snippets

Cortical Slab Preparation

This method can be used for mouse embryos ranging from E10.5–E15.5. Prepare glass slides and humidity chambers prior to starting the brain dissection. For each embryo, stick one slide well onto a clean glass slide according to the following guidelines: for ages <E14.5, use 0.25-μm slide well; for ages >E14.5, use 0.5-μm slide well. Keep a 12-well plate and two 10-cm Petri dishes with fresh phosphate-buffered saline (PBS) on ice.

  • 1.

    Euthanize the pregnant female mouse according to IACUC protocol and

Results/Discussion

Dissection, immunostaining, and imaging of embryonic cortical slabs can generate data about apical membrane structures of hundreds of cells across wide fields of intact neuroepithelium. Using this novel method to analyze cytokinesis structures, we showed that a microcephalic mouse mutant with a loss-of-function mutation in the gene encoding the Kinesin-6 family member, Kif20b, has abnormal cytokinetic abscission of cortical neuroepithelial progenitors (Janisch et al., 2013). First, we found

Conclusion

The cortical dissection, imaging, and analysis protocol presented here can be adapted to any mouse mutant for cortical neuroepithelial cell division studies, other brain regions, or other epithelial types. It can also be adapted for live imaging if combined with live fluorescent markers. With the method presented here, we are able to investigate cytokinesis in whole tissue during embryonic development. The advantage is the preservation of the integrity of the polarized cortical tissue

Acknowledgments

We thank Vita Vock for developing an earlier version of this protocol and Ayushma Shrestha for comments on this manuscript. We also thank Joe Dardick for his help with quantifying cilia index and comments on this manuscript. This work was supported by National Institute of Neural Disorders and Stroke grant R01NS076640 to N.D.D.

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