Faculty Bibliography

Asymmetric cell division can produce daughter cells with different developmental fates and is often accompanied by a difference in cell size. A number of recent genetic and in vivo imaging studies in Drosophila and Caenorhabditis elegans have begun to elucidate the mechanisms underlying the rearrangements of the cytoskeleton that result in eccentrically positioned cleavage planes. As a result, we are starting to gain an insight into the complex nature of the signals controlling cytoskeletal dynamics in the dividing cell. In this commentary we discuss recent findings on how the mitotic spindle is positioned and on cleavage site induction and place them in the context of cell size asymmetry in different model organisms.

Since its introduction into heterologous organisms as a marker of gene expression, the green fluorescent protein (GFP) has led a dramatic revolution in cell, developmental and neurobiology. By allowing breathtaking visualization of fluorescent fusion proteins as they move within and between cells, GFP has fundamentally transformed the spatial analysis of protein function. Now, new GFP technologies allow far more than simple observations of fusion protein localization. The growing family of fluorescent protein variants is enabling more sophisticated studies of protein function and illuminating wide-ranging processes from gene expression to second-messenger cascades and intercellular signalling. Together with advances in microscopy, new GFP-based experimental approaches are forging a second GFP revolution.

Cell-fate diversity is generated in part by the unequal segregation of cell-fate determinants during asymmetric cell divisions. In the Drosophila pupa, the pI sense organ precursor cell is polarized along the anterior-posterior axis of the fly and divides asymmetrically to generate a posterior pIIa cell and an anterior pIIb cell. The anterior pIIb cell specifically inherits the determinant Numb and the adaptor protein Partner of Numb (Pon). By labelling both the Pon crescent and the microtubules in living pupae, we show that determinants localize at the anterior cortex before mitotic-spindle formation, and that the spindle forms with random orientation and rotates to line up with the Pon crescent. By imaging living frizzled (fz) mutant pupae we show that Fz regulates the orientation of the polarity axis of pI, the initiation of spindle rotation and the unequal partitioning of determinants. We conclude that Fz participates in establishing the polarity of pI.

The asymmetric segregation of cell-fate determinants and the generation of daughter cells of different sizes rely on the correct orientation and position of the mitotic spindle. In the Drosophila embryo, the determinant Prospero is localized basally and is segregated equally to daughters of similar cell size during epidermal cell division. In contrast, during neuroblast division Prospero is segregated asymmetrically to the smaller daughter cell. This simple switch between symmetric and asymmetric segregation is achieved by changing the orientation of cell division: neural cells divide in a plane perpendicular to that of epidermoblast division. Here, by labelling mitotic spindles in living Drosophila embryos, we show that neuroblast spindles are initially formed in the same axis as epidermal cells, but rotate before cell division. We find that daughter cells of different sizes arise because the spindle itself becomes asymmetric at anaphase: apical microtubules elongate, basal microtubules shorten, and the midbody moves basally until it is positioned asymmetrically between the two spindle poles. This observation contradicts the widely held hypothesis that the cleavage furrow is always placed midway between the two centrosomes.

In the Drosophila central nervous system, cellular diversity is generated through the asymmetric partitioning of cell fate determinants at cell division. Neural precursors (or neuroblasts) divide in a stem cell lineage to generate a series of ganglion mother cells, each of which divides once to produce a pair of postmitotic neurons or glial cells. An exception to this rule is the MP2 neuroblast, which divides only once to generate two neurons. We screened for genes expressed in the MP2 neuroblast and its progeny as a means of identifying the factors that specify cell fate in the MP2 lineage. We identified a P-element insertion line that expresses the reporter gene, tau-beta-galactosidase, in the MP2 precursor and its progeny, the vMP2 and dMP2 neurons. The transposon disrupts the neurogenic gene, mastermind, but does not lead to neural hyperplasia. However, the vMP2 neuron is transformed into its sibling cell, dMP2. By contrast, expression of a dominant activated form of the Notch receptor in the MP2 lineage transforms dMP2 to vMP2. Notch signalling requires Mastermind, suggesting that Mastermind acts downstream of Notch to determine the vMP2 cell fate. We show that Mastermind plays a similar role in the neurons derived from ganglion mother cells 1-1a and 4-2a, where it specifies the pCC and RP2sib fates, respectively. This suggests that Notch signalling through Mastermind plays a wider role in specifying neuronal identity in the Drosophila central nervous system.

We developed a system for time-lapse observation of identified neurons in the central nervous system (CNS) of the Drosophila embryo. Using this system, we characterize the dynamics of filopodia and axon growth of the motorneuron RP2 as it navigates anteriorly through the CNS and then laterally along the intersegmental nerve (ISN) into the periphery. We find that both axonal extension and turning occur primarily through the process of filopodial dilation. In addition, we used the GAL4-UAS system to express the fusion protein Tau-GFP in a subset of neurons, allowing us to correlate RP2's patterns of growth with a subset of axons in its environment. In particular, we show that RP2's sharp lateral turn is coincident with the nascent ISN.

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Here we show that Staufen interacts in vivo with a segment of the prospero 3' UTR. Staufen protein and prospero RNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase. Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Furthermore, Miranda colocalizes with Staufen protein and prospero mRNA during neuroblast divisions, and neither Staufen nor prospero RNA are localized in miranda mutants. Thus Miranda, which localizes Prospero protein, also localizes prospero RNA through its interaction with Staufen protein.