Visualization and Quantification of Drosophila Larval Ovaries

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This version of the chapter has been accepted for publication, after peer review and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1007/978-1-0716-2970-3_2 Visualisation and quantification of Drosophila larval ovaries 1 2 Alicia E. Rosales-Nieves1, 2, *, #, Miriam Marín-Menguiano1, Alejandro Campoy-Lopez1, and Acaimo 3 González-Reyes1, * 4 5 1Centro Andaluz de Biología del Desarrollo, 6 CSIC/Universidad Pablo de Olavide/JA, 7 Carretera de Utrera km 1, 8 41013 Sevilla, Spain 9 10 2Current address: Instituto de Biomedicina de Sevilla (IBiS) 11 Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla 12 41011 Seville, Spain 13 14 *Correspondence to: AER-N ([email protected]; phone: +34-955923048 and AG-R 15 ([email protected]; phone +34-954 348672) 16 17 #Lead contact 18 19 Running head: Larval and Pupal Ovary staining 20 21 Key words: Drosophila, Larvae, Gonad, Ovary, dissection, staining, image analysis 22 23 Rosales-Nieves et al 2 ABSTRACT 24 The morphogenesis of the ovarian germline stem cell (GSC) niche during larval stages in Drosophila 25 provides the initial cellular and molecular basis for female gamete production in the adult. During 26 larval instars, the Drosophila female gonad matures gradually from a round structure enclosing 27 primordial germ cells (PGCs) and somatic cells into a functional organ containing GSC populations in 28 their niches that later in adult stages support oogenesis. Here we describe a technique for dissecting, 29 staining and analysing gonads from female Drosophila larvae and early pupae, offering the possibility 30 of a direct view of the morphogenesis of an ovarian niche. 31 32 Rosales-Nieves et al 3 1 INTRODUCTION 33 Gamete production in the Drosophila ovary relies on populations of somatic and germline stem cells 34 (GSCs) housed in the germarium, a well-studied structure present at the tip of adult ovaries. Somatic 35 support cells constitute the cellular component of the GSC niche and are essential for ovary 36 homeostasis and gamete production, emphasizing their importance for proper female reproduction. 37 This well-characterized ovarian niche develops from somatic and germline precursors that 38 proliferate during larval instars and populate the larval gonad. The future GSC niche forms during 39 the third larval instar (L3), starting at ~4 days after the egg was laid. In addition to the primordial 40 germ cells (PGCs) or germline precursors, the main somatic cell types present in early L3 (EL3) gonads 41 are the anterior sheath cells (ShCs), follicle stem cell precursors (FSCPs), intermingled cells (ICs) and 42 swarm cells (SwCs) (Fig. 1a). As gonads develop into the middle (ML3) and late L3 (LL3) stages, six 43 somatic cell types are found, which include terminal filament cells (TFCs), cap cells (CpCs), ShCs, 44 SwCs, FSCPs and ICs (Fig. 1b-c) [1-7]. 45 These cell types are organized into structured units that will function as GSC niches by a 46 number of signalling and cellular events. First, a wave of morphogenetic cell rearrangements and 47 cell recruitment is directed by an early ecdysone peak starting between EL3 and ML3. As a 48 consequence, individual terminal filaments are gradually formed along the medial-lateral axis. In 49 addition, 2-3 ICs located juxtaposed to the base of the terminal filaments are incorporated into each 50 of the units and adopt a CpC fate. Next, the PGCs that directly associate with the developing niches 51 become GSCs and begin to self-renew. Whereas PGCs not in direct contact with CpCs differentiate 52 by a process controlled by hormone pulses and cell signalling [2, 8]. Importantly, for adult 53 Rosales-Nieves et al 4 gametogenesis, a number of the cell types specified in larval stages are maintained into adulthood, 54 including GSCs, TFCs, CpCs, FSCPs and ICs, which will give rise to escort cells. 55 In this protocol, we describe a method for dissecting and staining Drosophila third instar 56 larval and pre-pupal gonads. In addition, we provide some morphological cues than can assist in the 57 classification of the developmental stage of the gonads. Specifically, third instar gonads are grouped 58 into EL3, ML3 or LL3 based on the presence and numbers of mature terminal filaments and 59 associated CpC clusters and GSCs (Fig. 1). 60 Finally, because the larval gonad is a disk-shaped structure with hundreds of cells arranged 61 in precise patterns that change with time, an accurate description of the different cell types and 62 their organization could be cumbersome. To aid in this, we have developed an ImageJ-based method 63 to quantify cell numbers in fixed gonads. This method, which requires immunofluorescence staining 64 and gives consistent results, can be adapted to different fluorescent signals and cellular localizations 65 of antigens, thus providing a versatile and efficient tool to analyze larval gonads. 66 67 2 MATERIALS 68 Diligently follow all rules and regulations for waste disposal. 69 70 2.1 Gonad Dissection 71 1. Ringer’s solution: 128 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 4 mM MgCl2, 35.5 mM Sucrose, 5 72 mM Hepes pH 6.9. 73 2. # 5 forceps. 74 3. Dissecting microscope (see Note 1). 75 Rosales-Nieves et al 5 4. Fly culture with larvae of the desired age. Avoid crowded cultures to improve the yield of 76 well-fed larvae; Fig. 2a; see Note 2). 77 5. Dissecting dish: 9-well glass plates (85 x 100 mm, 22 mm O.D. x 7 mm deep; Fig. 2b). 78 79 2.2 Gonad staining 80 1. 6-well plastic plates (127.76 mm x 85.46 mm). 81 2. 40 µm cell strainer (25 mm diameter; Fig. 2c). 82 3. 5% formaldehyde in Ringer’s solution, diluted from 10% formaldehyde, methanol-free, 83 electron microscopy grade. 84 4. Orbital rotator. 85 5. 1% PBT solution: 1% Triton x-100 in phosphate buffered saline (PBS). 86 6. 0.3% PBTB (blocking solution): 0.3% Triton X-100 and 1% bovine serum albumin (BSA) in PBS 87 7. Fetal bovine serum (FBS). 88 8. 0.3% PBT: 0.3% Triton X-100 in PBS. 89 9. Primary and secondary antibodies. 90 10. Nutator mixer or mini-shaker. 91 11. DNA stain (see Note 3). 92 12. Pipettes and pipette tips. 93 94 2.3 Gonad mounting 95 1. Shallow dissecting dish: Microscope slides with cavities 76 x 26 mm, 15 mm O. D. x 0.8 mm 96 deep (Fig. 2d). 97 Rosales-Nieves et al 6 2. Nickel plated pin holder and stainless steel minutien pins 0.15 mm diameter, 12 mm long. 98 3. Microscope slides (25 mm × 75 mm non-treated surface). 99 4. Coverslips (22 mm x 22 mm, 0.13–0.16mm thick). 100 5. Mounting medium (see Note 4). 101 6. Nail polish. 102 103 2.4 Gonad counting 104 1. ImageJ. 105 2. Workstation. 106 107 3 METHODS 108 3.1 Dissect larval fat body 109 1. Collect larvae of desired stage (see Note 2) by either flooding a food vial with tap water or by 110 directly picking the larvae with forceps from the walls of the vial (Fig. 2a). 111 2. Place the selected larvae in a dissecting dish containing cold Ringer’s solution (Fig. 2b). 112 3. Under a dissecting microscope, separate female from male larvae. Larval gonads are clear 113 spheres embedded in fat body and located about one-third from the posterior end of the 114 larvae (Fig. 3a, a’). Female gonads can be distinguished because they are significantly smaller 115 than male gonads (Fig. 3a’). 116 4. Transfer the selected larvae into a shallow dissecting well (Fig. 2d) containing Ringer’s 117 solution and remove the head by pulling with one pair forceps while holding with another 118 (Fig. 4a). Grasp the remaining of the larva with one pair of forceps and slowly turn the cuticle 119 Rosales-Nieves et al 7 inside out from the posterior spiracles towards the anterior end with the other pair of forceps 120 (Fig. 4b). Ideally, the now exposed fat body should be recovered intact (see Note 5). 121 5. Transfer all the collected fat bodies to a cell strainer placed in a 6-well plate filled with 122 Ringer’s solution (Fig. 2c). 123 124 3.2 Stain larval fat body 125 Transfer the cell strainer containing the fat bodies into new wells with the appropriate solutions for 126 fixation and washing steps (see Note 6). 127 1. Fix the dissected larval fat bodies in 5% formaldehyde in Ringer’s solution for 20 min on an 128 orbital rotator at 35 revolutions per minute (rpm) at room temperature (RT). 129 2. Wash for 5, 10 and 45 min with 1% PBT (35 rpm, RT). 130 3. Block with 0.3% PBTB for 1 h (35 rpm, RT). 131 4. After blocking, with a cropped pipette tip, transfer the fat bodies to 0.2 ml tubes and incubate 132 with the desired primary antibody diluted in 0.3% PBTB overnight at 4oC with gentle agitation 133 on a nutator or mini-shaker (see Note 7). 134 5. The following day, transfer with a cropped pipette tip the fat bodies back to the cell strainer 135 and wash three times, 30 min each, with 0.3% PBTB (35 rpm, RT). 136 6. Block with 0.3% PBTB supplemented with 5% fetal bovine serum for 1 h (35 rpm, RT). 137 7. After blocking, transfer with a cropped pipette tip fat bodies to 0.2 ml tubes and incubate 138 with the secondary antibodies for at least 2 h in blocking solution with gentle agitation at RT 139 (see Note 8) on a nutator or mini-shaker. 140 Rosales-Nieves et al 8 8. Using a cropped pipette tip, return fat bodies to the cell strainer. Wash three times, 30 min 141 each, with 0.3% PBT (see Note 9). 142 9. In case of staining the gonads with the DNA dye Hoechst, add Hoechst (1/1000 dilution of a 143 5mg/ml stock) after 20 min in the third wash in 0.3% PBT, and incubate for 10 minutes. 144 10. Wash three times, 5 min each, in 0.3% PBT (see Note 9). 145 146 3.3 Mount gonads 147 1. Transfer 2-3 fat bodies to a shallow dissecting dish with a cropped 0.2 mL pipette and 148 examine thoroughly each of the fat bodies looking for the gonads. They are translucent 149 spheres surrounded by a “flower-shaped” fat body (Fig. 3b). Exert extreme caution with the 150 tweezers to avoid damaging the gonads during their manipulation. 151 2. Separate the fat body area containing the gonad from the main fat body using the minutien 152 pins (Fig. 4c). Be careful to leave enough fat body still attached to the gonad to facilitate 153 transfer to a mounting slide. 154 3. Add a drop of mounting medium to a slide and transfer the gonads. With the help of two pin 155 holders remove excess fat body surrounding the gonad (Fig. 4d). This can be done by shearing 156 the fat body with the two minutien pins (Fig. 4d). 157 4. Repeat steps 3.3.1 to 3.3.3 until all the processed gonads are in the mounting medium with 158 the fat bodies removed. 159 5. Position the cleaned gonads at the centre of the drop of mounting medium and remove the 160 fat body debris using the pins (we use the same slide from the previous step). Once all the 161 Rosales-Nieves et al 15 8. Gilboa, L., and Lehmann, R. (2004). 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Bioinformatics 32, 3532-3534. 294 295 296 Lateral Anterior ML3 LL3EL3 a b c TF precursor/ TFCCC IC PGC/GSC SwC differentiating GC ShC FSCP Fig. 1 ab c d Fig. 2 a a’ b Anterior Posterior Fig. 3 ab d Anterior Posterior Anterior Posterior Turn the cuticle inside out Hold the posterior of the larva Remove the head Hold the body c Anterior Posterior Slide Fig. 4 a b c de Original image + 3d Gaussian blur + Top hat + Morph. opening + CLAHE + 3D Odj. counter f Fig. 5