A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish

Abstract

BackgroundTissue heating has been employed to study a variety of biological processes, including the study of genes that control embryonic development. Conditional regulation of gene expression is a particularly powerful approach for understanding gene function. One popular method for mis-expressing a gene of interest employs heat-inducible heat shock protein (hsp) promoters. Global heat shock of hsp-promoter-containing transgenic animals induces gene expression throughout all tissues, but does not allow for spatial control. Local heating allows for spatial control of hsp-promoter-driven transgenes, but methods for local heating are cumbersome and variably effective.ResultsWe describe a simple, highly controllable, and versatile apparatus for heating biological tissue and other materials on the micron-scale. This microheater employs micron-scale fiber optics and uses an inexpensive laser-pointer as a power source. Optical fibers can be pulled on a standard electrode puller to produce tips of varying sizes that can then be used to reliably heat 20-100 μm targets. We demonstrate precise spatiotemporal control of hsp70l:GFP transgene expression in a variety of tissue types in zebrafish embryos and larvae. We also show how this system can be employed as part of a new method for lineage tracing that would greatly facilitate the study of organogenesis and tissue regulation at any time in the life cycle.ConclusionThis versatile and simple local heater has broad utility for the study of gene function and for lineage tracing. This system could be used to control hsp-driven gene expression in any organism simply by bringing the fiber optic tip in contact with the tissue of interest. Beyond these uses for the study of gene function, this device has wide-ranging utility in materials science and could easily be adapted for therapeutic purposes in humans.

Heat‐shock–mediated conditional regulation of hedgehog/gli signaling in zebrafish

“…Activation of Hh pathway components by heat‐shock recapitulates known loss or gain of function Hh/Gli signaling phenotypes and remains effective into early adult stages, but becomes less robust with age, perhaps due to changes in heat‐shock promoter activation and/or changes in tissue responsiveness (Murtha and Keller, ). Region‐specific manipulation of Hh signaling using these lines is now possible using a micro‐scale heating device (Hardy et al, ; Placinta et al, ). Two new Hh reporter lines provide a highly sensitive read‐out of Hh signal activity throughout the life cycle.…”

Temporally coordinated signals progressively pattern the anteroposterior and dorsoventral body axes

“…Finally, inducing heat shock results in embryo-wide expression of the transgene. To achieve spatially restricted gene control, one may induce local heat shock by sublethal laser irradiation [254] or a microheater [255]. Alternatively, one may transplant cells from the transgenic line into a background without a heat shock transgene and heat shock the entire embryo after transplantation (cell transplantations are described below).…”

Embryonic exposure to excess thyroid hormone causes thyrotrope cell death

“…We exposed 48-hpf Tg(tshb:EGFP) transgenic embryos to T4 and observed thyrotropes using timelapse microscopy ( Figure 3, B-L and Supplemental Video 1; supplemental material available online with this article; doi:10.1172/ JCI70038DS1). The GFP protein is stable in live cells for 24 to 48 hours (22,23), which allowed us to track thyrotropes even if tshb gene transcription was reduced. Surprisingly, GFP-labeled thyrotropes rapidly disappeared starting 6-8 hours after the addition of 300 nM T4 ( Figure 3F and Supplemental Video 1).…”