Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy
Summary
Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.
Abstract
Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.
Introduction
Craniofacial morphogenesis is a complex multi-step process that requires coordinated interactions between multiple cell types. The majority of the craniofacial skeleton is derived from neural crest cells, many of which must migrate from the dorsal neural tube into transient structures called pharyngeal arches1. As with many tissues, morphogenesis of the craniofacial skeleton is more complicated than can be understood by static images of embryos at specific developmental time points. Although it is time-consuming to perform, in vivo time-lapse microscopy provides a continuous look at a developing embryo's cells and tissues. Each image in a time-lapse series lends context to the others, and helps an investigator move toward deducing why a phenomenon occurs rather than deducing what is occurring at that time.
In vivo imaging is thus a powerful descriptive tool for experimental approaches to deconstruct the pathways that guide morphogenesis. The zebrafish Danio rerio is a popular genetic model of vertebrate embryonic development, and is particularly well suited for in vivo imaging of morphogenesis. Modern, convenient methods for transgenesis and genomic modification are rapidly advancing the number of tools available to zebrafish researchers. These tools enhance already robust methods for genetic manipulation and microscopy. In vivo imaging of almost any tissue in almost any desired genetic context is closer to reality than fantasy.
Morphogenetic movements of the pharyngeal arches are guided by signaling interactions between the neural crest and the adjacent epithelia, both ectoderm and endoderm. There are numerous signaling molecules expressed by the epithelia that are necessary to drive the morphogenesis of craniofacial skeletal elements. Among these signaling molecules, Sonic Hedgehog (Shh) is critically important for craniofacial development2-8. Shh is expressed by both the oral ectoderm and pharyngeal endoderm2,6,9,10. The expression of Shh in the endoderm regulates morphogenetic movements of the arches10, patterning of neural crest within the arches10, and growth of the craniofacial skeleton11.
Bmp signaling is also critically important for craniofacial development12 and may alter morphogenesis of the pharyngeal arches. Bmp signaling regulates dorsal/ventral patterning of crest within the pharyngeal arches13,14. Disruption of smad5 in zebrafish causes severe palatal defects and a failure of the Meckel's cartilages to fuse appropriately at the midline15. In addition, the mutants also display reductions and fusion in the ventral cartilage elements, with the 2nd, 3rd, and sometimes 4th pharyngeal arch elements fused at the midline15. These fusions strongly suggest that Bmp signaling directs the morphogenesis of these pharyngeal elements.
Pdgf signaling is necessary for craniofacial development, but has unknown roles in pharyngeal arch morphogenesis. Both mouse and zebrafish Pdgfra mutants have profound midfacial clefting16-18. At least in zebrafish this midfacial clefting is due to a failure of proper neural crest cell migration16. Neural crest cells continue to express pdgfra after they have entered the pharyngeal arches. Additionally, Pdgf ligands are expressed by facial epithelia and within the pharyngeal arches16,19,20, thus Pdgf signaling could also play a role in morphogenesis of the pharyngeal arches following migration. However, analyses of the morphogenesis of the pharyngeal arches in pdgfra mutants have not been performed.
Here, we demonstrate in vivo confocal microscopy of pharyngula-stage transgenic zebrafish and describe the morphogenesis of the pharyngeal arches within this period. We further demonstrate tissue behaviors that are affected by mutations that disrupt the Bmp, Pdgf, and Shh signaling pathways.
Protocol
Representative Results
Discussion
Time-lapse confocal microscopy is a powerful tool for the analysis of development. Here, we demonstrate the method's usefulness in studying pharyngeal arch morphogenesis in zebrafish that are mutant for important signaling pathways using a transgenic that labels neural crest cells. In addition to tissue-level analyses, time lapse analyses are also applicable to analyses at a cellular scale28. Many widely used zebrafish methods can also be incorporated into time-lapse microscopy experiments, including micro…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Melissa Griffin and Jenna Rozacky for their expert fish care. PDM thanks EGN for writing assistance, generosity, and patience. This work was supported by NIH/NIDCR R01DE020884 to JKE.
Materials
| 6 lb. test monofilament line | Cortland Line Company | SLB16 | |
| Agarose I | Amresco | 0710 | |
| Argon laser | LASOS Lasertechnik GmbH | LGN 3001 | |
| Calcium chloride | Sigma-Aldrich | C8106 | |
| Capillary tubing, 100 mm, 0.9 mm ID | FHC | 30-31-0 | |
| Clove oil | Hilltech Canada, Inc. | HB-102 | |
| High vacuum grease | Dow Corning | 2021846-0807 | |
| Isotemp dry-bath incubator | Fisher Scientific | 2050FS | |
| Laser scanning microscope | Carl Zeiss AG | LSM 710 | |
| Magnesium sulfate hexahydrate | Sigma-Aldrich | 230391 | |
| Microscope cover glass, 22×22-1 | Fisher Scientific | 12-542-B | |
| Microscope cover glass, 24×60-1 | Fisher Scientific | 12-545-M | |
| Potassium chloride | Fisher Scientific | M-11321 | |
| Potassium phosphate dibasic | Sigma-Aldrich | P3786 | |
| Sodium chloride | Fisher Scientific | M-11624 | |
| Sodium phosphate dibasic | Sigma-Aldrich | S7907 | |
| TempController 2000-2 | PeCon GmbH | ||
| Tricaine-S | Western Chemical, Inc. |
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