Protocol

Microinjection of Helobdella (Leech) Embryos

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA

¹Corresponding author (weisblat{at}berkeley.edu)

This article is also available in Emerging Model Organisms: A Laboratory Manual, Vol. 1. CSHL Press, Cold Spring Harbor, NY, USA, 2009.

INTRODUCTION

One advantage of using Helobdella (leech) embryos as an experimental system is their amenability for microinjection. Blastomeres ranging in size from the zygote (400 µm diameter) down to micromeres and primary blast cells (~20 µm diameter) can be injected by pressure under a dissecting microscope. Smaller cells can be injected by iontophoresis under a compound microscope. Microinjection is useful for studying embryonic development. For example, developmental fates of a cell can be followed by injecting a lineage tracer. A specific cell can be killed by injecting a toxic substance. Furthermore, cells can be killed at a given developmental stage by directing intense fluorescence illumination or a blue laser beam on fluorescein-labeled cells. Finally, gene expression can be manipulated in leech embryos by injecting zygotes or selected blastomeres with synthetic mRNA, morpholino antisense oligo, or a plasmid construct. Molecules <1500 Da can diffuse freely among early blastomeres via gap junctions. When intercellular diffusion of an injected substance is undesirable, small molecules should be conjugated to larger molecules such as dextran (10 kDa). Different commercial microinjection setups can be adopted for Helobdella embryos. This article describes how to microinject embryos using a versatile homemade pressure injection system. Under a dissecting microscope, embryos are immobilized by suction in a custom-fabricated chamber with the target cell facing upward. Cells are visualized using transillumination via a long-working-distance, dark-field condenser. The tip of a micropipette is brought into the target cell with a micromanipulator, and the injectant is delivered into the cell by pressure.