Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Summary

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Abstract

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Introduction

The brain, comprised of a vast number and diverse types of neurons, endows animals with the ability to perceive, process, and respond to challenges from the external world. Neurons of the adult Drosophila central brain are derived from a limited number of neural stem cells, called neuroblasts (NBs), during development^1,2. Most of the NBs participating in brain neurogenesis in Drosophila undergo asymmetric division to generate self-renewing NBs and ganglion mother cells (GMCs), and the GMCs then go through another round of division to produce two daughter cells that differentiate into neurons³ (Figure 1A). Because of the intricacy of neuronal morphology and the challenges associated with identifying specific neurons, a positive mosaic labeling technology, mosaic analysis with a repressible cell marker (MARCM), was invented to enable the visualization of a single neuron or a small subset of neurons out of the population of surrounding, unlabeled neurons⁴.

MARCM utilizes the flippase (FLP)/FLP recognition target (FRT) system to mediate site-specific recombination between homologous chromosomes within a dividing precursor cell carrying a heterozygous allele of a repressor gene, whose expression normally inhibits the expression of a reporter gene⁴. After the mitotic division, the recombinant chromosomes are segregated into twin cells, such that one cell contains homozygous alleles of the repressor gene and the other cell has no repressor gene-the expression of the reporter in that cell (and its descendants) is no longer blocked⁴. Three clonal patterns are usually found in a MARCM experiment when FLP is stochastically induced in NBs or GMCs: single-cell and two-cell-GMC clones, which depict neuronal morphology at single-cell resolution, and multi-cellular-NB clones, which reveal entire morphological patterns of neurons derived from a common NB (Figure 1B). The MARCM technique has been widely applied in Drosophila neurobiological studies, including in neuronal type identification for reconstructing brain-wide wiring networks, neural lineage analyses for disclosing the developmental history of neurons, phenotypic characterization of gene functions involved in cell fate specification, and neuronal morphogenesis and differentiation studies^5,6,7,8,9,10. Because conventional MARCM only labels one of the two daughter cells (and lineages) after the induced mitotic recombination event, potentially useful information from the unmarked side is lost. This limitation precludes the application of the basic MARCM system to high-resolution analyses of many neural lineages that switch cell fates in fast tempo or to precision analyses of gene functions in identical neurons of different animals^11,12.

Twin-spot MARCM (tsMARCM) is an advanced system that labels neurons derived from a common progenitor with two distinct colors, which enables the recovery of useful information from both sides of the twin cells, thus overcoming the limitation of the original MARCM system¹¹ (Figure 2A-2C). In the tsMARCM system, two RNA interference (RNAi)-based suppressors are situated at trans-sites of homologous chromosomes within a precursor cell, and the expression of those suppressors independently inhibit the expression of their respective reporters (Figure 2B). Following site-specific mitotic recombination mediated through the FLP/FRT system, the two RNAi-based suppressors become segregated into twin cells to permit the expression of distinct reporters (Figure 2B). Two clonal patterns, single-cell associated with single-cell clones and two-cell-GMC associated with multi-cellular-NB clones, are typically seen in a tsMARCM experiment (Figure 2C). Information derived from one side of the twin cells can be utilized as the reference for the other side, enabling high-resolution neural lineage analyses, such as birth-dating the labeled neurons, and phenotypic analyses of identical neurons in different animals for the precise investigation of neural gene function^11,12. Here, we present a step-by-step protocol describing how to conduct a tsMARCM experiment, which can be used by other laboratories to broaden their studies of neural development (as well as the development of other tissues, if applicable) in Drosophila.

Protocol

1. Build tsMARCM-ready Flies Using the Required Transgenes^11,13

1. Generate the original version of tsMARCM-ready flies from transgenes that are carried by individual flies¹¹ (see Table 1). Conduct standard fly genetic crossing schemes, which have been described previously, by putting multiple transgenes in the same fly stocks¹³.
   1. In one parental line, assemble the transgenes of FRT^40A, UAS-mCD8::GFP (the first reporter, mCD8::GFP; expression of the transgene is under the control of the upstream activation sequence promoter, which produces a membrane-tethered reporter of mouse cluster of differentiation 8 protein fused with green fluorescent protein), and UAS-rCD2RNAi (the suppressor that inhibits the expression of the second reporter; the RNAi against the expression of rat cluster of differentiation 2, rcd2) on the left arm of the 2^nd chromosome. Place tissue-specific-GAL4 driver on the X or 3^rd chromosome, if possible.
   2. In the other parental line, assemble the transgenes of FRT^40A, UAS-rCD2::RFP (the second reporter, rCD2::RFP; the other membrane-tethered reporter consisting of rCD2 protein fused with red fluorescent protein), and UAS-GFPRNAi (the suppressor that inhibits the expression of mCD8::GFP; the RNAi against the expression of gfp) on the left arm of the 2^nd chromosome. Place heat-shock (hs)-FLP or tissue-specific-FLP on the X or 3^rd chromosome, if possible.
2. Generate the new version of tsMARCM-ready flies from transgenes that are carried by individual flies¹⁴ (see Table 1). Conduct standard fly genetic crossing schemes, which have been described previously, by putting multiple transgenes in the same fly stocks¹³.
   1. In one parental line, assemble the transgenes of an FRT site and UAS-mCD8.GFP.UAS-rCD2i (a combined transgene of mCD8::GFP and the suppressor that inhibits the expression of rCD2::RFP) together in the same arm of the 2^nd or 3^rd chromosome (appropriate pairs of transgenes of FRT and UAS-mCD8.GFP.UAS-rCD2i are indicated in Table 1). Place tissue-specific-GAL4 driver on chromosomes other than the chromosome of the chosen FRT site, if possible.
   2. In the other parental line, assemble the transgenes of an FRT site and UAS-rCD2.RFP.UAS-GFPi (the other combined transgene of rCD2::RFP and the suppressor that inhibits the expression of mCD8::GFP) in the same arm of the 2^nd or 3^rd chromosome (appropriate pairs of transgenes of FRT and UAS-rCD2.RFP.UAS-GFPi are indicated in Table 1). Place hs-FLP or tissue-specific-FLP on chromosomes other than the chromosome of the chosen FRT site, if possible.

2. Cross tsMARCM-ready Flies to Generate tsMARCM Clones in Their Progeny

1. Cross tsMARCM-ready flies together (e.g., put 10-15 male flies from step 1.1.1 or 1.2.1 and 20-30 virgin female flies from step 1.1.2 or 1.2.2 together) in a fly-food vial with fresh-made yeast paste on the wall of the vial.
2. Maintain the crossed tsMARCM-ready flies at 25 °C for 2 days to enhance the chance of mating before collecting fertilized eggs.
3. Frequently transfer the crossed tsMARCM-ready flies into freshly yeasted vials to avoid crowding (tap down the flies or use carbon dioxide to anesthetize the flies to facilitate the transfer; keep no more than 80-100 fertilized eggs in a 10 mL fly-food vial to avoid too many hatched larvae).
4. Culture the tsMARCM animals (i.e., fertilized eggs; an example of the genotype of tsMARCM animals, as used in Figure 3, is hs-FLP^[22],w/w; UAS-mCD8::GFP,UAS-rCD2RNAi,FRT^40A,GAL4-GH146/UAS-rCD2::RFP,UAS-GFPRNAi,FRT^40A; +; +) that have been laid in the serially transferred vials at 25 °C until they develop to the desired stages (e.g., embryos, larvae, or pupae).
5. Place the transferred vials that contain tsMARCM animals at the same developmental stage to a 37 °C water bath for 10, 20, 30, 40, and 50 min to determine the optimal time of heat-shock that induces the expression of FLP to generate the tsMARCM clones of interest. Skip this step if tissue-specific-FLP is being used in the tsMARCM experiment (hs-FLP is preferred for neural lineage analyses; the reasons for this preference have been described previously¹⁵).
   NOTE: Repeat step 2.5 using the optimal time of heat-shock in future tsMARCM experiments if more tsMARCM clones of interest are wanted; generally, more FLP expression is induced in hs-FLP^[22] compared to hs-FLP^[1] with the same heat-shock time.
6. Culture the heat-shocked tsMARCM animals at 25 °C until they have reached the appropriate developmental stage, and then proceed to step 3.

3. Prepare, Stain, and Mount Fly Brains Containing tsMARCM Clones

1. Prepare forceps-protection dishes. Mix part A (30 g) and part B (3 g) of the Silicone Elastomer Kit with activated charcoal (0.25 g). Pour the mixture into the appropriate dishes.
2. Dissect larval, pupal, or adult brains out of the tsMARCM animals in 1x phosphate-buffered saline (PBS) using forceps on a forceps-protection dish viewed through a dissection microscope. NOTE: A protocol for fly brain dissection has been described previously¹⁶.
3. Fix the fly brains in a glass spot plate with 1x PBS containing 4% formaldehyde at room temperature for 20 min. Process the fly brains in this glass spot plate for the remainder of the steps.
4. Rinse the fly brains three times with 1% PBT (1x PBS containing 1% Triton X-100) and wash them three times for 30 min each in 1% PBT (rinse: remove PBT and add new PBT quickly; wash: remove PBT, add new PBT, and incubate the fly brains in the new PBT using an orbital shaker).
5. Incubate the fly brains with the mixture of primary antibodies overnight at 4 °C or for 4 h at room temperature. An example of the mixture of primary antibodies is rat anti-CD8 antibody (1:100), rabbit anti-DsRed antibody (1:800), and mouse anti-Bruchpilot (Brp) antibody (1:50) in 1% PBT containing 5% normal goat serum (NGS).
6. Rinse the fly brains three times with 1% PBT, and then wash them three times for 30 min each with 1% PBT.
7. Incubate the fly brains with the mixture of secondary antibodies overnight at 4 °C or for 4 h at room temperature. An example of the mixture of secondary antibodies is goat anti-rat IgG antibody conjugated with green-fluorescent dye (1:800), goat anti-rabbit IgG antibody conjugated with yellow-fluorescent dye (1:800), and goat anti-mouse IgG antibody conjugated with far-red-fluorescent dye (1:800) in 1% PBT containing 5% NGS.
8. Rinse the fly brains three times with 1% PBT, and then wash them three times for 30 min each with 1% PBT. Mount them on a micro slide with an anti-quenching reagent. Put a micro cover glass on the micro slide to cover and protect the fly brains. Seal the edges of the micro cover glass and micro slide using clear nail polish. Store these mounted and sealed fly brain specimens at 4 °C.

4. Take, Process, and Analyze Fluorescent Images of tsMARCM Clones

1. Capture fluorescent images of tsMARCM clones from the samples created in step 3.8 using the confocal microscopy system of choice.
2. Project stacks of tsMARCM confocal fluorescent images into two-dimensional, flattened images using the image processing software supplied with the confocal microscopy system of choice (see Figure 3B-3E, 3G-3J, 3L-3O, and 3Q-3T for examples of flattened confocal fluorescent images).
3. Count the cell number of the multi-cellular-NB sides of the tsMARCM clones using an appropriate image processing software (e.g., the "Cell Counter" function in ImageJ¹⁷; see Figure 3E, 3J, 3O, and 3T for examples of the cell bodies of neurons).

Results

The tsMARCM system has been used to facilitate neural lineage analyses and gene function studies by retrieving important information on neurons derived from common NBs. The system has been used to identify most (if not all) neuronal types, determine the cell number of each neuronal type, and ascertain the birth-order of these neurons^11,12,18 (see the Discussion Section for detail...

Discussion

Critical Steps within the Protocol

Steps 1.1.1, 1.2.1, 2.3, 2.5, and 3.2 are critical for obtaining good tsMARCM results. Tissue-specific-GAL4 drivers that do not express GAL4 in neural progenitors are preferred for steps 1.1.1 and 1.2.1. Avoid over-crowding the animals grown in the fly-food vials in step 2.3. Because the induction latency, the expression level of FLP upon heat-shock, and the average dividing time of neural progenitors (i.e., NBs and GMCs) are not known, it is b...

Materials

Name	Company	Catalog Number	Comments
Carbon dioxide (CO2)	Local vendor	n.a.	Anesthetize flies
CO2 pad	Local vendor	n.a.	Sort, cross and transfer flies
10x PBS pH 7.4	Uniregion Bio-Tech (or other vendors)	PBS001	Dissect, rinse, wash and immunostain fly brains
Formaldehyde	Sigma-Aldrich	252549	Fix fly brains
Triton X-100	Sigma-Aldrich	T8787	Rinse, wash and immunostain fly brains
Normal goat	Jackson ImmunoResearch	005-000-121	Immunostain fly brains
serum
Rat anti-mouse CD8 antibody	Invitrogen	MCD0800	Immunostain fly brains
Rabbit anti-DsRed antibody	Clonetech	632496	Immunostain fly brains
Mouse anti-Brp antibody	Developmental Studies Hybridoma Bank	nc82	Immunostain fly brains
Goat anti-rat IgG antibody conjugated with Alexa Fluor 488	Invitrogen	A11006	Immunostain fly brains
Goat anti-rabbit IgG antibody conjugated with Alexa Fluor 546	Invitrogen	A11035	Immunostain fly brains
Goat anti-mouse IgG antibody conjugated with Alexa Fluor 647	Invitrogen	A21236	Immunostain fly brains
SlowFade gold antifade reagent	Molecular Probes	S36936	Mount fly brains and protect quenching of fluorescence
PYREX 9 depression glass spot plate	Corning	7220-85	Dissect, rinse, wash and immunostain fly brains
Sylgard 184 silicone elastomer kit	World Precision Instruments	SYLG184	Make black Sylgard dishes to protect forceps during brain dissection
Activated charcoal	Sigma-Aldrich	242276-250G	Make black Sylgard dishes
Dumont #5 forceps	Fine Science Tools	11252-30	Dissect and mount fly brains
Micro slide	Corning	2948-75x25	Mount fly brains
Micro cover glass No. 1.5	VWR International	48366-205	Mount fly brains
Nail polish	Local vendor	not available	Seal micro cover glass on micro slides
Incubator	Kansin Instruments	LTI603	culture flies at 25 °C
(or other vendors)
Water bath	Kansin Instruments	WB212-B2	Induce heat-shock in flies at 37 °C
(or other vendors)
Orbital shaker	Kansin Instruments	OS701	Wash and immunostain fly brains
(or other vendors)
Dissection microscope	Leica	EZ4	Sort, cross and transfer flies; Dissect, rinse, wash and immunostain fly brains
Confocal microscope	Zeiss (or other vendors)	LSM 700 (or other models)	Image tsMARCM clones
image-processing software 1 (e.g., Zeiss LSM image browser)	Zeiss	not available	Project stacks of confocal images
image-processing software 2 (e.g., ImageJ)	not available	not available	Count cell number of tsMARCM clones