Within multicellular organisms, tissue displays high degrees further in their spatial orientation. Cells align and display a range of priority over large distances over the course of morphogenesis. To understand how the planar arrangements of epithelia is reached during development, it is crucial to track cell's orientations and growth dynamics with high spacial temporal fidelity in vivo.
The described protocol is designed to image and analyze cell behaviors at global and local levels in the drosophila pupal epidermis. This methodology could provide insights into different theory of research, cell biology, morphogenesis, and planar polarity and can be applied to the analysis of other tissues, structures, and models. All the steps can be performed easily after some practice.
They demand precision. Visualization of this protocol helps achieving optimal results in a shorter time. The pupal dissection is complex.
To begin, use a moistened paintbrush to transfer staged white pre-pupae to a fresh plastic vial, and keep them there for as long as necessary at 25 degrees Celsius. Then, remove each pupae from the wall of the vial with the help of forceps. Glue the ventral side of each pupa on a glass slide covered with double sided sticky tape.
Gently tap on the head spiracles and the dorsal surface of the pupa with the tips of the forceps to assure the adhesion of the pupal case to the tape. Begin dissection under a stereo microscope by gently removing the operculum from the puparium with the forceps. Insert one tip of the forceps in a shallow between the pupal case and the pupa surface through the opercular opening.
Tear the case from head to tail laterally in one or more swings, avoiding pinching the pupa. Fold back the cracked pupal case to the lateral sides as you keep proceeding to the posterior end. Remove the pupa from the opened up pupal case by carefully inserting the forceps under the animal and gently pulling up.
The pupa sticks to the tip of the forceps. Hold the pupa gently by the ventral side, and transfer the pupa to a glass bottomed dish on top of a small drop of gas-permeable halocarbon oil. Roll a piece of wet tissue paper at the edges of the dish and cover the dish to maintain humidity.
Orient the pupa over the oil drop on the glass bottomed dishes according to the domain and the process to be evaluated. For long term live imaging of the early expansion of the dorsal nests, dorsal laterally orient the pupa. To image their late expansion and tissue remodeling, dorsally orient the pupa.
Transfer the glass bottomed dish containing the mounted pupae to the microscope stage and focus on the surface of the abdominal area using transmitted light. To employ mitotic recombination to induce genetic mosaics in the abdominal epithelium, prepare virgin females carrying a heat shock inducible flipase, an FRT site at a specific genomic location, and a recognizable cellular marker distal to the FRT site. Prepare mutant males carrying an FRT site at the equivalent genomic location.
Cross several females and males in a three to one proportion in a plastic vial at 25 degrees Celsius for four to five days. To generate somatic clones in the histoblasts, perform heat shock treatment at the third instar larval stage of the progeny of the cross by submerging the plastic vials containing the animals in a water bath at 37 degrees Celsius for 45 minutes to an hour. In the image J software, use the maximum intensity projection function to protect the Z stack slices acquired by confocal microscopy in 2D.
The nests have a characteristic shape that orients them in a planar coordinate system anterior to the left and dorsal to the top. To achieve optimal results with this analysis, the input image has to be acquired with high resolution. To obtain qualitative orientation values on local cell edges, click on the plug-in menu and choose the orientation J distribution option.
Set the Gaussian window sigma to one pixel, cubic spline to gradient, minimum coherency to 20 percent, and minimum energy to one percent. Employ the color survey option of the plug-in. Set hue to orientation, saturation to coherency, and brightness to input image.
This color codes cell edge orientations relative to the set planar coordinate system. Next, under the plug-in menu, use the orientation J measure option to quantify cellular orientations and directional cell to cell alignment. Generate small, adjacent non-overlapping regions of interest of uniform weight around 20 micrometers times 20 micrometers within the area occupied by the histoblasts.
Pressing measure calculates the dominant local orientation and coherency from the ROIs. In this study, the methodology used is able to generate high resolution movies of the developing pupae for periods of up to 48 hours without significant photo bleaching or photo toxicity. Examples of wild type clones marked by the absence of RFP NLS and their twin spots at 26 hours after puparium formation are shown here.
The clones elongated along the segmental boundaries. Twin clones arranged in parallel or in tandem. Morphology of a wild type clone at 26 hours and 47 hours after puparium formation shows complex border morphology at both stages.
Box and whisker plots for geometrical parameters at 26 hours and 47 hours after puparium formation show the averaged area and perimeter increased significantly in this time window. The clone's orientation was sustained during expansion and remodeling. Shape parameters at 26 hours and 47 hours after puparium formation indicate the roughness, roundness and circularity barely changed in the wild type clones.
Be sure to not damage the pupa, otherwise it will die within a few hours, affecting live imaging. This protocol does not require a sartus reagent or instrument to be employed. Avoid pinching yourself when dissecting.
By applying these methodologies possible to generate high resolution movies for long term periods using different imagine techniques. Focal to photon or spinning disks. Chloral analysis can be employed to explore non-autonomous responses since random cells facilitate in the identification of cross talks or cell communication mechanisms.
These methods can be easily adapted to study a full range of morphogenetic phenomena, including the coordination of epidermal, muscular, and neuro development during metamorphosis.
