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Characterization of the role of morphogens during growth of the Zebrafish pectoral fin using advanced biophysical tools

Final Report Summary - MORFING (Characterization of the role of morphogens during growth of the Zebrafish pectoral fin using advanced biophysical tools)

During development an organ grows to a final sise and shape and its cells acquire information about their position and differentiate according to their location. Morphogens play an important role in this process by coordinating growth and patterning of developing tissues. In this project, the role of morphogens during the growth of the pectoral fin of the zebrafish, a vertebrate, was studied, using a multidisciplinary, quantitative, biophysical approach. Such an approach has been successfully used by the Gonzalez-Gaitan lab to study growth in the wing imaginal disk of fruit fly larvae. In vertebrates, however, growth control by morphogens is poorly understood and therefore three questions were addressed by this project:

1) Are the growth principles found in fruit fly wings generally applicable?
2) Does it work in a similar manner in vertebrates?
3) How is a mesenchymal, three-dimensional (3D) architecture of the tissue affecting these principles?

The results of this project can be subdivided into two parts:

1) establishing a light-sheet microscope, conditions for long-term imaging of zebrafish pectoral fin growth, and data analysis algorithms / tools for cell detection / tracking;
2) location of a morphogen source and gradient in the developing pectoral fin, quantification of its signal transduction response and its relation to tissue size / volume.

1) To image the developing and growing pectoral fin of a 30 hpf zebrafish embryo for over 24 hours on a conventional upright two-photon microscope, we designed and built a special sample holder. Using this sample holder, we are able to image the pectoral fin for a maximum of 32 hours, after which the growth of the pectoral fin is affected by the imaging. We then imaged H2A-GFP transgenic zebrafish, in which GFP is expressed in every cell nucleus and measured the volume of the pectoral fin over time. In parallel, we determined cell cycle lengths at different stages of growth as well as in different locations in the pectoral fin, by doing immunofluorescent stainings against Phosphohistone 3 (PH3). We found that cell division by itself cannot explain the growth of the pectoral fin and that therefore there has to be an external source of cells to account for the observed volume increase.

To determine the movement of all cells in the pectoral, we then developed an automated nucleus detection and tracking algorithm, which has a detection reliability of over 90 %. We, however, found that the data obtained with the two-photon microscope did not have the required spatial and temporal resolution, and we decided to build a light-sheet microscope. After visiting the Huisken lab (MPI-CBG, Dresden) a design for a single plane illumination microscope (SPIM) was made and built.

In anticipation of the results that will be obtained with the SPIM, we developed an algorithm (based on the speed correlation index developed by C. Bouzigues and M. Dahan, 2007) to determine if the movement of a particle (or a cell nucleus) is purely random, or if there is directionality in the movement. We successfully applied this method to show that in zebrafish dorsal epiblast cells, the anthrax toxin receptor 2a exerts a torque on the spindle to align it along the animal / vegetal axis preparing it for oriented cell division.

2) As was shown previously by our lab, Dpp plays an important role in controlling the final sise of the wing imaginal disk of fruitfly larvae. Its homolog(s) in zebrafish, Bmp2a, Bmp2b and Bmp4, might play a similar role. For this purpose, we used a transgenic line, obtained from Beth Roman (Department of Biological Science, University of Pittsburgh), which has a fluorescent reporter for Bmp activity (BRE-GFP). It was shown that BRE-GFP expression exactly overlaps with pSMAD1/5/8 (homolog of drosophila pMAD; MAD gets phosphorylated upon binding of Dpp to its receptor Thickveins) and is therefore a direct readout of Bmp signalling.

Preliminary results indicate that there is, as expected, Bmp signalling in the zebrafish pectoral fin. Bmp signalling is first visible in 33 hpf embryos, and a gradient with a decay length of 12 µm was observed. At 52 hpf, a Bmp signalling gradient is still visible, although now with a decay length of 16 µm. This is an indication that the gradient scales with the sise of the pectoral fin, comparable to what was found in the fruit fly wing imaginal disk. Currently, we are investigating the Bmp signalling gradient more thoroughly.

These, and future results will enable us to give a full description of the growth of the pectoral fin. Moreover, we will establish which role morphogens play in controlling the sise and shape of this organ and this will allow us to compare it to our findings in the drosophila wing imaginal disk.