Identification of neural stem cells in the Drosophila larval brain

Summary

The balance between self-renewal and differentiation must be tightly regulated in somatic stem cells to ensure proper tissue generation and to prevent tumor-like overgrowth. A Drosophila larval brain lobe consists of the central brain and the optic lobe, and possesses three well-defined neural stem cell lineages that generate differentiated cells in a highly reproducible pattern. Unambiguous identification of various cell types in these stem cell lineages is pivotal for studying the regulation of neural stem cells and progenitor cells at a single-cell resolution. This chapter will describe the methodology for collection and processing of larval brains for examination by fluorescence confocal microscopy.

1. Introduction

The central brain occupies the medial half of a fly larval brain lobe, and contains neural stem cells (called neuroblasts) that undergo repetitive asymmetric divisions to self-renew and to generate a neural progenitor cell with limited developmental potential (Sousa-Nunes et al., 2010). Two distinct larval brain neuroblast lineages (types I and II) can be unambiguously identified based on the progenitor progeny generated and the combination of cell fate markers expressed (Weng and Lee, 2011). A type I neuroblast divides asymmetrically to self-renew and to generate a neural progenitor cell called a ganglion mother cell (GMC), which divides once to produce two post-mitotic neurons (Doe, 2008; Egger et al., 2008; Knoblich, 2008). In contrast, a type II neuroblast divides to self-renew and to produce an immature intermediate neural progenitor cell (INP), which acquires restricted developmental potential during maturation and undergoes limited rounds of asymmetric divisions to regenerate and to produce GMCs (Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008). While all neuroblasts express the molecular marker Deadpan (Dpn), a type I neuroblast also expresses Asense (Ase) whereas a type II neuroblast expresses PointedP1 (PntP1) (Bowman et al., 2008) (Komori and Lee, unpublished). An immature INP expresses a high level of PntP1, but following maturation, an INP expresses Dpn and Ase. Finally, a GMC shows nuclear localization of Ase and Prospero (Pros) whereas an immature neuron expresses Pros only.

The optic lobe occupies the lateral half of a fly larval brain lobe, and contains two single-cell layers of neuroepithelial stem cells that form the inner and the outer proliferation center (Sousa-Nunes et al., 2010; Weng and Lee, 2011). Neuroepithelial cells in the outer proliferation center are located on the surface of the optic lobe, and initially divide symmetrically to expand their population (Egger et al., 2007). In third larval instar, these neuroepithelial cells progressively differentiate into lamina precursor at the lateral edge and into medulla neuroblasts at the medial edge (Egger et al., 2010; Ngo et al., 2010; Reddy et al., 2010; Wang et al., 2011; Yasugi et al., 2010; Yasugi et al., 2008). The apical complex protein PatJ specifically labels all neuroepithelial cells while the expression of the Notch reporter E(spl)mγ-GFP labels differentiating neuroepithelial cells at the medial edge and medulla neuroblasts. Medullaneuroblasts express molecular markers including Dpn and Ase.

2. Materials

2.1. Reagents

1. Fix solution: 4% formaldehyde, 0.1M PIPES (pH=6.9), 0.3% TritionX-100, 20mM EGTA, 1mM MgSO₄. Fix solution should be prepared fresh every time.

2. 10XPBS: 1.37M NaCl, 27mM KCl, 100mM Na₂HPO₄, 20mM KH₂PO₄. Dissolve and adjust pH to 7.4 with concentrated HCl. Bring the volume up to 1 L with dH₂O and sterilize. Store at room temperature.

3. 10% Triton X-100: 100% Trition X-100 diluted in sterilized water. 3. PBST (500 ml): 1X PBS, 0.3% TritonX-100. Store at room temperature.

4. 10X Glycine: 1M Glycine, 2% sodium azide, 1XPBS.

5. Block solution: 1X PBST, 0.1% Normal goat serum, 1X Glycine. Prepare fresh and keep on ice.

6. 70% Glycerol: 100% Glycerol diluted in sterilized water.

7. Prolong Gold Anti-fade mounting medium (Invitrogen) 8. Schneider’s Insect Medium (Sigma-Aldrich)

2.2 Equipment

1. 22 × 22 mm coverslips, #1 thickness

2. 24 × 40 mm coverslips

3. Dissection dishes

4. Fine-tipped forceps (2 pairs)

5. Fine micro knife with thickness about 0.15 mm

6. Microfuge tubes (0.5 μl)

7. Microscope slides

8. Nutator or rocker

9. Pipettes & sterile tips

3. Methods

3.1. Dissection of Larval Brains

1. Fill wells of dissection dishes with 200–400 μl cold Schneider’s medium.

2. Dissect larvae by rolling them onto their dorsal side so the denticle belts are facing up.

3. Using a pair of forceps, gently grasp the larva just posterior of the midpoint. With the second pair of forceps, grasp the anterior end of the larva with one tip pushing mouth hook inwards and the other tip outside on the cuticle.

4. Carefully tear the cuticle at the tip of second pair of forceps while slowly drawing the body away from the mouthpart. The brains will remain attached to the head and be clearly visible among the gut and salivary glands. Remove excess tissues, but leave the brains attached to the mouth hooks. (see Note 1–2)

5. Place the brains in a 0.5 ml tube containing cold Schneider’s medium. (see Note 3)

3.2. Fixation and Staining

• 1Remove Schneider’s medium from the tube containing the brains.
• 2Add 500 μl fixative to the brains and incubate with rocking for 23 minutes at room temperature.
• 3Quickly wash the brains for three times in ~500 μl of PBST.
• 4Incubate the brains in primary antibodies diluted in PBST for 3 hours at the room temperature or overnight at 4°C. (see Note 4–7)
• 5Quickly wash the brains three times in PBST.
• 6Incubate the brains in secondary antibodies overnight at 4°C. Secondary antibodies are typically diluted 1:200–500 in PBST. Wrap the tube with foil to keep the brains protected from light after this point. (see Note 4–7)
• 8Quickly wash the brains three times in PBST.
• 9Equilibrate the brains in Prolong Gold at the room temperature. Samples can be stored in the dark at room temperature or 4°C.

3.3. Mounting Samples

3.3.1. Taking a Z-stack from posterior to anterior side

1. Adhere two 22 × 22 mm coverslips to a slide with a small amount of 70% glycerol, leaving a ~5 mm space between them. (see Note 8)

2. Transfer the brains to a slide using a pipette with the tip cut off.

3. Remove all excess tissues including discs from each brain with forceps.

4. Orient the brains so that the ventral side is down. Arrange the brains in an array for easy tracking during confocaling. (see Note 9)

5. Place a 24 × 40 mm coverslip over the samples and back-fill the space between the slide and coverslip by pipetting a small amount of mounting medium along the edge of the coverslip.

3.3.2. Taking a Z-stack from lateral to medial side

1. Adhere two 22 × 22 mm coverslips to a slide with a small amount of 70% glycerol, leaving a ~5 mm space between them.

2. Transfer one brain to the slide using a pipette with the tip cut off. (see Note 10)

3. Remove all excess tissues including the discs from each brain with forceps.

4. Using one pair of forceps to hold the brain and cut the brain lobes apart from the ventral nerve cord using a micro knife. (see Note 11)

5. Place a 24 × 40 mm coverslip over the samples and back-fill the space between the slide and coverslip by pipetting a small amount of mounting medium along the edge of the coverslip. Do not seal the coverslip with nail polish. (see Note 12)

Figure 1. Neuroblast lineages in the fly larval central brain.

(A) A cartoon summarizes the expression profile of molecular markers in the type I neuroblast and type II neuroblast lineage. Imm INP: immature INP. (B–C) A third instar larval brain was stained with antibodies against Dpn, Ase and PntP1. The cell cortex was marked by phalloidin. White arrows indicate type I neuroblasts whereas arrowheads indicate type II neuroblasts and asterics indicate immature INPs. The scale bar is 50μm.

Figure 2. Neuroepithelial cells and neuroblasts in the fly larval optic lobe.

(A) A cartoon summarizes the expression profile of molecular markers in neuroepithelial cells and neuroblasts. Non-diff NEC: non-differentiating neuroepithelial cells. Diff NEC: differentiating neuroepithelial cells. imm neurob: immature neuroblasts. Neurob: neuroblasts. (B) A third instar larval optic lobe was stained with antibodies against PatJ, E(spl)mγ-GFP and Dpn. Arrows indicate non-differentiating neuroepithelial cells whereas arrowheads indicate differentiating neuroepithelial cells and asterics indicate immature neuroblasts. The scale bar is 50μm.

Figure 3. Comparative analyses of four distinct primary antibody staining protocols.

(A–D) Third instar larval brains were stained with Dpn, Pros and Ase. Arrows indicate neuroblasts. The scale bar is 50μm.