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Drosophila embryo live cell imaging

Drosophila embryo development or embryogenesis is a fast process during the life cycle of the d. melanogaster, completed in 24h after egg fertilization, and followed by three larval stages. Key steps in the body plan formation of the future adult fly occur during this stage.

Drosophila embryo fluorescently

Figure 1, Drosophila embryo imaged by fluorescent microscopy

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Drosophila embryo development

Early embryogenesis occurs in a syncytium, where no cell divisions but nuclear divisions with very rapid DNA replication take place. After migration of 6000 nuclei to the periphery of the syncytium, beginning of cellularization occurs, generating the syncytial blastoderm. Then, in a process called gastrulation, cells change shape and migrate establishing the germ layers of the future body plan: mesoderm, endoderm and ectoderm. The process of cell fate determination is driven by underlying patterned gene expression, which is established prior to gastrulation, at the blastoderm stage. Patterned gene expression is determined by protein gradients originated from maternal mRNAs [1].

Seminal discoveries in patterning of gene expression leading to embryonic development by E. Wieschaus, E.B. Lewis and C. Nüslein-Volhard were awarded the Nobel Prize in 1995 [2]. The following video illustrates drosophila embryogenesis explained by E. Wieschaus:

The deciphering of the developmental steps leading to the formation of the different tissue structures from the syncytial embryo has been possible through the multiple genetic tools available for the fruit fly model organism. Live imaging of the fly embryo has been as well an invaluable resource for the understanding of these processes.

Schematic representation of fruit fly embryo development

Figure , Schematic representation of drosophila embryo development

Live imaging of D. melanogaster embryo

Confocal microscopy allows for the imaging of fluorescently labelled tissues during the different stages of drosophila development. Live imaging of d. melanogaster embryos requires non-invasive mounting procedures. Size (wt are 511±21 μm in length), thickness (wt are 184±5 μm in width), and optical properties are elements to take into consideration to properly mount the embryos for imaging [3]. Any mounting method requires a prior step of dechorionation of embryos, followed by placement of the embryos in the desired orientation depending on the experiment (dorsolateral, ventrolateral, posterior or anterior side).

The choice of the imaging mounting method depends on several factors, according to the biological process being studied: duration of the timelapse, is gas permeability needed or not, is squeezing of the embryo suitable or not. For instance, for morphogenesis studies, methods which require embryo squeezing would not be the most suited [8]. Dehydration, hypoxia and tissue deformation are important factors to be taken into consideration.

Many of the mounting methods require the use of halocarbon oil. Halocarbon oil prevents embryo dehydration and is oxygen permeable. Additionally, due to its physical properties, it helps to prevent motion of the embryos during time lapse imaging.

Some commonly used mounting methods for live imaging of fly embryo are:

1. Mounting between a glass slide and a glass coverslip: embryos in halocabon oil are placed in a sandwich between a glass slide and coverslip. Different methods allow to create imaging chambers by using spacers of the desired thickness (eg: glass coverslips, double sided tape).

2. Glass bottom Petri dish: embryos are mounted on the dish with halocarbon oil [4]. The main advantage of this method is the possibility of adding big volumes covering the embryo, which prevents evaporation and allows for longer imaging. Glass bottom petri dishes with different types of coating are commercially available.

3. Chambered coverslips: The main advantage of these chambers is the possibility of imaging multiple chambers on a single glass coverslip (high resolution imaging compatible), allowing for multiplexed experiments. As in the case of Petri dishes, these chambers allow the addition of big volumes, thus preventing evaporation. Multichambered coverglasses are commercially available.

4. Permeable membranes and stainless-steel imaging slides: embryos are placed between a membrane surface and the glass coverslip. The main advantage of this mounting method is the gas permeability provided by the membrane (made of materials like Teflon or lummox), allowing for long imaging with no risk of hypoxia for the tissues [6,7]. Petri dishes with a membrane-bottom and compatible with high resolution imaging are commercially available. Alternatively, stainless-steel imaging slides or Petri dishes with a membrane bottom can be prepared in the laboratory [5, 7].

5. Hanging drop protocol: it consists on a humid chamber where the embryo is placed on a halocarbon oil drop with no squeezing [8]. It doesn’t require compression of the embryos. Compatible with upright microscope setups.

The CherryTemp temperature controller is compatible with mounting methods 1,2 and 5. and we provide alternative solutions for compatibility with method 4 (gas permeability). (See link: our protocols)

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Reference

[1] Hales, KG. Genetics (2015) https://www.ncbi.nlm.nih.gov/pubmed/26564900
[2] https://en.wikipedia.org/wiki/Eric_F._Wieschaus
[3] Mavrakis M. et al. Curr Protoc Cell Biol. (2008).  https://www.ncbi.nlm.nih.gov/pubmed/18551421
[4] Jankovics F. ¬Dev Cell. (2006) https://www.ncbi.nlm.nih.gov/pubmed/16908221
[5] Kiehart DP et al. Methods Cell Biol. (1994) https://www.ncbi.nlm.nih.gov/pubmed/7707969
[6] Kiehart DP et al., J Cell Biol. (2000) https://www.ncbi.nlm.nih.gov/pubmed/10769037
[7] Reed BH et al. Curr Biol. (2004) https://www.ncbi.nlm.nih.gov/pubmed/15028211
[8] Reed BH et al. J Vis Exp. (2009)  https://www.ncbi.nlm.nih.gov/pubmed/19287353


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