Introduction

The generation of asymmetry within a single cell, or polarity establishment, is fundamental to a diversity of biological processes, and is often important in early development.  In many zygotes, polarity is set in place early and ultimately determines the patterning and morphogenesis of the organism.  Zygotes of the marine brown algae in the genera Fucus and Silvetia (formerly known as Pelvetia) have long been used as model organisms to better understand the general mechanisms underlying developmental polarity establishment. 

Fucoid algae grow attached to rocks in the intertidal zones on both coasts of the United States, as well as in Asia and Europe.  The unfertilized egg is radially symmetric and has no known developmental polarity.  In zygotes, polarity establishment occurs early and generates a developmental axis, along which growth occurs several hours later.  Polarity is first established at fertilization, with the site of sperm entry defining the future growth pole of the axis.  Young zygotes subsequently monitor their local environment and if they perceive vectorial information, they abandon their default sperm axes and generate new axes in accordance with the spatial cues.  In nature, unidirectional light is likely the most important signal and the new growth pole assembles on the shaded hemisphere (photopolarization).  This growth axis, defined and expressed in first few hours, orients the first division perpendicular to the growth axis and generates daughter cells of different developmental fates.  The polarly growing rhizoid cell will give rise to a holdfast, which anchors the alga to the rocky substratum.  The opposite thallus cell will give rise to leaf-like photosynthetic and reproductive structures, the stipe and fronds.  Thus, the asymmetry established early in the first cell cycle defines the pattern of divisions and differentiation in the mature alga.

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Fucoid zygotes are well-suited for investigating cellular and physiological mechanisms of polarity establishment.  The major advantages of this experimental system are that 1) eggs and sperm can be easily harvested in large numbers (several thousand), 2) fertilization and subsequent development of a population can be synchronized, 3) developmental polarity is established in the first cell cycle, free of maternal signals, 4) eggs and zygotes are large, ~ 60 - 100 micrometers in diameter, facilitating physiological studies, 5) the growth axes of an entire population of zygotes can be easily manipulated, and 6) the first zygotic division is an invariant, asymmetric division that defines lineages. 

You will be investigating the role of the cytoskeleton in two developmental events: germination and cytokinesis in Silvetia compressa (native to the California coast).  Microtubules form distinct arrays during the cell cycle, nucleating uniformly off the nuclear envelope, then nucleating from centrosomes to form a spindle during mitosis, and then nucleating off daughter nuclei toward the cytokinetic plate.  To test the requirement for microtubules in development, you may depolymerize them with nocodazole or stabilize them with taxol.  During germination and tip growth, a cone of filamentous actin extends from the rhizoid nucleus to the subapex of the tip, and just prior to cytokinesis a plate of actin forms between daughter nuclei.  The actin plate predicts the site of membrane and cell wall deposition to form the two daughter cells.  You may test the requirement for actin using latrunculin B to depolymerize actin or jasplakinolide to stabilize actin filaments.

Choose one of the following treatments:

A) Stabilize MTs with taxol; taxol also nucleates new MTs  [NOTE]
B) Depolymerize MTs with oryzalin  [NOTE]
C) Stabilize actin filaments with jasplakinolide; jasplakinolide also nucleates new actin filaments  [NOTE]
D) Depolymerize actin filaments with latrunculin B  [NOTE]