Deimling SJ et al. (2015), Understanding early organogenesis using a simpl...

XB-ART-50072

J Vis Exp 2015 Jan 12;95:e51526. doi: 10.3791/51526.

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Understanding early organogenesis using a simplified in situ hybridization protocol in Xenopus.

Deimling SJ , Halabi RR , Grover SA , Wang JH , Drysdale TA .

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Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: astl2c astl3a.1 etv2 hand1 hba1 mmp15 pax2 pax8 tnni3

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	Figure 1: Examples of the whole mount in situ hybridization on Xenopus embryos. The blue staining from an in situ hybridization experiment can clearly delineate developing structures of the early Xenopus embryo. (A) An anterior view of a stage 26 embryo highlighting the hatching gland as demarcated by the expression of uvs.2 (B) A ventral view of an embryos showing the location of early myeloid cells at stage 20 using the expression of myeloperoxidase15 as a marker. (C) The early heart at stage 28-30 is visualized by the expression of cardiac troponin I . A clear advantage of using this method is that all of these gene expression patterns were visualized using different probes but the protocol used is identical in all cases.
	Figure 2: Different levels of bleaching can be used to visualize staining in the embryo. (A) This blue staining along the bottom of this embryo shows the expression of hemoglobin in the ventral blood islands at about stage 36. This is a region of the embryo that is only lightly pigmented and thus the embryo was not bleached for a long time as can be seen by the tan colored pigment in the eye and along the flank of the embryo. Being able to see the pigmentation allows for better visualization of the stage of the embryo. If the staining is in a region with greater natural pigmentation, such as the nervous system and the flank of the embryo, greater bleaching will help view the in situ as seen in B. (B) Here the expression of pax8 in the forming kidney, pronephric duct and hindbrain is best visualized after bleaching has removed almost all endogenous pigment. Please click here to view a larger version of this figure.
	Figure 3: Manipulation of the agarose base can help with orientation of the embryos. Imaging specific regions of the embryo can be difficult because they tend to take up particular positions in the dish. (A) At tadpole stages the embryo will lie on its side. By cutting a fine channel in the agarose (black arrows) the embryo can be viewed from the ventral side, here showing hemoglobin expression at stage 36, with enough stability to capture a good image. (B) This ventral view of the hand1 expression at stage 20 outlines the lateral plate mesoderm 6. The embryo is placed in a small hole that stabilized its position with the ventral side up. Please click here to view a larger version of this figure.
	Figure 4: Internal organs can be viewed in both opaque, uncleared and in cleared embryos. In an uncleared embryo stained for pax2 at stage 34 (A), the optic stalk (yellow arrow) can be visualized relatively easily as can the staining down the neural tube. However, details are not sharp. By clearing the embryo (B) the boundaries of expression sites, including the optic stalk (yellow arrow) are sharper. The extent of clearing is shown by the ability to see both eyes in this cleared embryo. Also note that some staining has accumulated in the internal cavities (purple arrow), a common problem when viewing cleared embryos.
	Figure 5: A representative double in situ hybridization on a Xenopus embryo. Expression of the lateral plate marker, hand1, is visualized by the light blue staining at stage 26. Expression of etv2 [ETS variant transcription factor 2] within the developing vasculature is visualized by dark blue purple stain. Expression of hand1 is usually very intense and thus it was utilized for the weaker fluorescein-labelled probe and BCIP combination. The utilized the digoxigenin-labelled probe and BM-purple combination for the weaker etv2 expression.

References [+] :

Carroll, Molecular regulation of pronephric development. 1999, Pubmed, Xenbase