A novel genetic tool for clonal analysis of fourth chromosome mutations

Abstract

The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FRT). Here we introduce a method that overcomes these limitations and allows for the generation of single Minute haplo-4 clones of any fourth chromosome mutant gene in tissues of developing and adult flies.

Introduction

The success of Drosophila as a model organism relies on the ability to manipulate its genome. These manipulations include reverse and forward genetics approaches to study the functions of individual genes. Forward genetics approaches are feasible because mutations that cause a phenotype of interest can be readily mapped and the responsible gene identified, and somatic clones of loss-of-function alleles can be induced in a spatially and temporally controlled manner to allow analysis of their effects in any tissue and cell type. However, not all regions of the Drosophila genome are equally amenable to these types of approaches. A particularly troublesome region is the fourth chromosome, which has been resistant to forward genetic technologies for almost a century, primarily due to the lack of meiotic crossover that prevents meiotic mapping, but also due to the poor availability of deletions with defined molecular limits. The barrier to routine mapping of genes on the fourth chromosome has been partly overcome by the generation and characterization of deletions with molecularly mapped breakpoints.^1-3 However, a general method for systematically analyzing the loss-of-function phenotypes of fourth chromosome genes in somatic mosaics has remained elusive for a number of reasons. Lack of recombination prevents the introduction of necessary markers onto fourth chromosomes carrying FLP-recombinase recognition target sites (FRT). The older way of inducing mitotic recombination, by ionizing radiation, results in haploidy or aneuploidy of the fourth chromosome at a very low frequency.⁴ Other strategies employed to generate somatic clones of mutants on the fourth chromosome rely on the use of mutations that cause chromosomal loss and allow the generation of clones of haplo-4 cells. Although haplo-4 cells and tissues are viable and develop normally, the frequency of these events is low, on the order of 1–2% (Sousa-Neves, unpublished observation).⁵ Another difficulty posed by chromosomal loss strategies is the inability to control the time of clone induction and the size of somatic clones.

So far, the most efficient technology currently available to generate somatic clones of fourth chromosome mutations relies on cloning and transferring wild-type copies of genes from this chromosome to other regions of the genome, where they can then be lost by standard FLP/FRT-based mitotic recombination methods to form a clone of cells in which a constitutionally homozygous fourth chromosome mutation has been uncovered.⁶ The high efficiency of this method stems from the use of the yeast FLP-FRT system.⁷ However, this strategy requires generation of the appropriate transgenic material for each individual gene of interest, which is time consuming and can be challenging for large and complex genes.

Here we report a new tool that allows the efficient generation of somatic mosaics of mutations in virtually any fourth chromosome gene. Our method enables the generation of somatic clones using the FLP-FRT system. Clones can be visualized with green fluorescent protein (GFP), by utilizing components of the previously reported mosaic analysis by repressible cell marker (MARCM) system.⁸ We believe that this method should greatly simplify the functional analyses of fourth chromosome mutations.

Results

Genotypes and chromosomes

The genetic system to generate somatic mosaics of fourth chromosome mutants involves several genetic components (outlined visually in Fig. 1A). On the X chromosome there is a heat-inducible FLP-recombinase transgene (FLP) on a yellow white background.⁷ One of the second chromosomes bears a FLP-recombinase recognition target site (FRT42D) located close to the centromere of chromosome 2R, a yellow⁺ transgene, a ubiquitous source of Gal80, and terminally, a reciprocal translocation that links all genes of the fourth chromosome to the right arm of the second chromosome (2R).^9-11 This chromosome is referred to as FRT Yellow Translocation (FYT). The other second chromosome bears a UAS-GFP transgene on the left arm, and FRT42D and a ubiquitous source of Gal4 on the right.^12,13 This chromosome is referred to as GFP-FRT-ActinGal4 (GFA). The fourth chromosomes are modified as well. One is the complementary half of the FYT reciprocal translocation. The other fourth chromosome bears the mutation of interest.

Figure 1. Schematic overview of the system. (A) Schematic of a somatic cell in a FYT/GFA fly. One copy of the second chromosome bears FRT42D, yellow⁺, Gal80, and a translocation of the entire fourth chromosome. The other bears UAS-GFP on the left arm. The right arm bears a FRT42D, upstream of Actin > Gal4. One copy of the fourth chromosome bears a translocation of the second (reciprocal to the translocation of the fourth on the second). The other carries the mutation of interest. This cell appears wild type and grows normally, as it has two second chromosomes, two fourth chromosomes, Gal4 repressed by the Gal80, and the mutation on the fourth covered by the translocation. The X chromosome is yellow white, and contains a heat shock inducible FLP-recombinase (not shown). (B) Schematic of the above cell after heat shock. Under heat shock conditions, a heat shock FLP-recombinase on the first chromosome (not shown) is activated. In cells undergoing mitosis, the FLP-recombinase will target the FRT sites on the second chromosome, causing recombination of the second chromosomes' transgenic right arms and the translocation. This will lead some mitotic cells to yield two different daughter cells, both homozygous for the different right arms of chromosome two. (C) Schematic of the genotypes of daughter cells. One of the resultant daughter cells will be haploid for most of the second arm due to the translocation of the fourth, which is a cell lethal condition (gray). The other daughter cell will now lack the Gal80 which repressed the Gal4, and as such will now fluoresce with GFP (green). It also will lack the copy of yellow⁺, marking the cuticle. Most importantly, it will have lost the wild-type translocated fourth chromosome, leaving only the fourth chromosome with the recessive mutation.

Clone induction and marking

Heterozygous FYT/GFA animals do not express GFP, as Gal80 represses Gal4. However, induction of FLP-recombinase in dividing cells produces randomly distributed clones of cells in which the chromosome arm carrying y⁺, Gal80 and the linked fourth chromosome gene complement has been lost, and thus, are also hemizygous for the structurally normal fourth chromosome containing the mutation of interest (Figs. 1B and 2). In any living tissue, the descendants of these cells can be directly visualized by their GFP expression and in the sclerotized adult cuticle are marked by y^- (Fig. 1C). Haplo-4 tissues and even entire haplo-4 individuals develop into normal adults, except for their dominant Minute phenotype due to the haploinsufficiency of the RpS3 ribosomal protein gene, which slows the rate of cell growth and proliferation, causing slowed development, late emergence of adult flies and thinning of the bristles.^14,15

Figure 2. FYT-generated clones in developing and adult tissues. (A) Wild-type eye from a FYT and spa individual subjected to heat shock as a larva. (B) The second eye from the same individual as in (A). Notice the clone patch which has been made Haplo-4 for a fourth chromosome bearing sparkling (outlined region). (C) Wing disc of a heat shocked GFA/FYT larvae. The haplo-4 fourth chromosome of clone cells is wild type but cells fluoresce due to unsuppressed Actin > Gal4 activation of UAS-GFP on the second chromosome. (D) Wing of an adult heat shocked during development marked with kojak and the haplo-4 patch is devoid of trichomes (outlined region). Notice the distal edge of the wing within the clone patch is concave due to the Minute phenotype. (E) Patch of clone tissue in an adult thorax using the size corrected FYT system. The fourth chromosome is wild type but lacks the yellow⁺ on the second chromosome, altering pigmentation of the cuticle and bristles (outlined region).

To test the efficacy of this method in uncovering recessive mutations on the fourth chromosome, we induced clones in larvae heterozygous for the recessive mutation sparkling^pol (spa^pol), a recessive viable mutation that causes eye roughening. As expected, the somatic clones of this mutation in the adult eye exhibited disorganized ommatidia and eye roughening (compare the wild-type eye of Fig. 2A with the eye of the same animal with a spa^pol clone in Fig. 2B), while the heterozygous tissue appears wild type.

Wild-type haplo-4 clones labeled with GFP were also obtained in imaginal discs (Fig. 2C) and in adult wings using the marker kojak (koj) (Fig. 2D, see Materials and Methods for the genotype used).

Due to the growth retardation (Minute) phenotype of haplo-4 cells, haplo-4 clones compete with neighboring faster growing euploid cells, resulting in smaller clones. To minimize the growth competition that favors the wild-type tissue, we made a variant of FYT that contains a mutation in a Minute gene on the right arm of the second chromosome [M(2)53¹]. Under these conditions, the growth of all cells in the entire body is slowed due to the heterozygosity for M(2)53¹. However, when FYT^– clones are generated they gain two wild-type copies of M(2)53¹ and lose one copy of RpS3, and thus, have a growth advantage relative to their euploid neighbors, enabling them to become substantially larger (Fig. 2E).

We also generated pangolin-cubitus interruptus Dominant (pan-ci^D) clones marked with kojak in wings (Fig. 3). Pan acts downstream of Wingless (wg), which is normally required along the wing margin. pan-ci^D is an inversion between pan and ci in which the promoter of pan directs the expression of ci in both the anterior and posterior compartments of imaginal discs. The visible phenotype of heterozygotes ci^D/+ is the interruption of the posterior vein 4, but no defects in the anterior compartment. pan-ci^D is also mutant for pan and embryonic lethal. Here, we show a clone pan-ci^D in the anterior compartment. Like previously reported,⁶ we note that the loss of pan does not affect the pattern of the wing blade, but removes the sensilla along the margin that requires wg signaling.

Figure 3.FYT induced clones of pan-ci^D on the wing. (A) pan-ci^D/+ wing and a clone pan-ci^D/0. (B) Higher magnification view of the pan-ci^D/0 clone shown in (A). The limits of the clone are marked by kojak which exclusively removes trichomes on the wing surface. The removal of pan on the wing surface does not disrupt patterning. However, mutant cells reach the wing margin where wg is normally expressed and required. In this position they cause the loss of sensilla (arrow) and the appearance of sensilla with abnormal polarity (arrowhead). (C) a pan-ci^D/+wing without a clone shown for comparison. (D) Higher magnification view of (C), in the region of the clone in (B).

Haplo-4 tissues can be obtained in larval and adult tissues including the brain (Fig. 4A-H). In this case, we generated clones of the semi-lethal mutant dati¹ that impairs locomotion and female behavior, and compared the mRNA levels within a clone, the neighboring diploid wild-type tissue and adjacent neuropile. Direct fluorescence intensity levels measurements show the reduction of dati mRNA in these clones (Fig. 4G and H).

Figure 4.FYT induced clones in the adult brain and measurement of RNA levels by multiplex in situ hybridization. (A–F) Brain from a 3 d-old female heterozygous FYT/GFA and the dati¹ mutation on the fourth chromosome. Clones were induced via heat shock between second and third instar. (A) Nuclei stained with DAPI. (B) Neuronal cell bodies stained with anti-Elav fluorescent antibody. (C) Haplo-4 cells for dati¹ mutation marked with GFP. (D) Neuropil stained with nc82. (E) Merged image of elav neuronal stain in red and GFP in green. Colocalization of GFP with Elav distinguishes mutant neurons from other cell types, such as glia. (F) Merged image of nc82 neuropil stain in magenta and haplo-4 cells in green showing position of clones. Note the clusters of clones on both the left and right anterior ventral lateral protocerebrum (arrowheads) as well as the optic lobe (arrow). (G) The antennal lobe (AL) of another female containing a dati¹ mutant clone (right, below), stained with DAPI (blue), Elav (not shown), GFP (green) and a dati probe (gray). The Elav channel is omitted to reduce the complexity of the figure. The red line indicates the position and direction from which the pixel intensity values of the three visible channels (gray, blue and green) were collected and normalized. (H) The normalized pixel intensity levels of all three channels. On the left most part of the graph (0 to ~40 μms) we observe diplo-4 nuclei (blue) and normal levels of dati mRNA (gray). From ~40 to ~80 μms we note the strong GFP signal from the clone, which coincides with a drop in the dati mRNA. From 80–120 μm, the pixel intensity collected comes from neuropile which has no nuclei and mostly background levels can be detected on the three channels. From ~120–160 μms the signals of the three channels come from diplo-4 cell bodies and the levels of dati mRNA and DAPI rise again.

Frequency of clone recovery

To estimate the frequency of clone recovery, we generated clones in which both parents were balanced for the second chromosome and one of them (i.e., the carrier of GFA) was heterozygous for ci^D spa or dati. In this setting, 1/3 of the larval progeny is heterozygous for FYT and GFA, and carries either ci^D spa or dati. We next screened larvae for GFP and found that 13% exhibited clearly distinguishable clones (n = 138). By these counts, we note that nearly half of the expected number of clones could be recovered. However, when we analyzed the non-ci^D adults capable of generating clones (i.e., FYT/GFA), without pre-selecting for GFP, we observed that 21/24 (87%) had clones in the brain. This data suggests that ci^D spa are more difficult to recover than dati clones and that the frequency of clone generation is equal or greater than 87% in individuals that carry FYT and GFA.

In summary, the fourth chromosome contains several important genes predicted to be involved in nervous system development and behavior. The FYT technique reported here will enable researchers to test these predictions and systematically investigate any role these genes play in neurodevelopment, physiology and behavior.

Discussion

The strategy of generating somatic clones has been crucial to the study of the biological functions of individual genes in Drosophila, but a generally applicable method of doing so for any fourth chromosome mutation has not been available. Application of the most advanced and efficient methods for producing somatic clones of fourth chromosome mutations requires prior knowledge of what gene is affected and laborious introduction of wild-type transgenes onto one of the other autosomes. While the number of genes on the fourth chromosomes are few and the challenges to studying them have been great, many fourth chromosome genes are of vital importance to several fields of study. In the field of development, the examples are numerous. Pan has been identified as a member of the Wg/Wnt signaling pathway.¹⁶ ci is a well-known downstream effector in the Hedgehog signaling cascade.^17-19 In visual system development specifically, the fourth chromosome hosts spa, ey and toy, the first being a homolog of Pax2, and the latter two homologs of Pax6.^20-22 Also well represented on the fourth chromosome are genes related to neuronal patterning and function. The semaphorins plexA and plexB have roles in axon guidance in the developing brain.^23,24 The fourth chromosome also contains both Ephrin and its associated receptor tyrosine kinase, which have roles in patterning the mushroom bodies and the visual ganglia.^25,26 Unc-13 encodes a synaptic vesicle fusion protein essential for neurotransmission in all neurons.²⁷ DmGluRA encodes a metabotropic receptor for the excitatory neurotransmitter glutamate.²⁸

As such, the genes of the fourth chromosome are indispensable to our understanding of development and neural functioning of Drosophila. However, prior to the development of this system, the methods available to clonally study these genes were time-intensive, inefficient or both. The generation of haplo-4 clones using mitotic loss inducer is reported to occur at a frequency of only 0.013.⁵ Clones of fourth chromosome genes have also been performed by translocating the gene of interest to the second chromosome, but this is relatively labor intensive and must be repeated for every gene of interest.⁶ This system, however, readily incorporates any allele of any gene generated on the fourth chromosome. The advent of RNAi technology has led to much greater access to knock-downs of many genes, but can be prone to disruption of off-target genes, insertion effects, incomplete knockouts, and is incapable of investigating an allele of interest. Our method, on the other hand, generates the most extreme knockout of an allele short of deletion: the haploid condition of a recessive allele.

The generation of clones is quite simple and only two stocks are required: (1) the clone inducer FYT stock (y w FLP; SM/FRT42D y+ Gal80 T(4;2)X-4; T(2;4)X-4/spa) and (2) the mutation receiver GFA ci^D (y w FLP; SM/ UAS-GFP FRT42D Actin > Gal4; ci^D spa/Df(4)BH). The mutations of interest are received by the GFA stock and the male offspring ci^D mated to FYT. Clones are analyzed in heterozygotes FYT/GFA.

There are several considerations worthy of note for those interested in using this system to generate haplo-4 clones. The first consideration is that the loss of a fourth chromosome leads to the loss of one copy of the RpS3 ribosomal protein, a Minute gene, and as such clones will be predicted to grow more slowly than surrounding tissues. As alluded to in the results section, this effect can be mitigated by a variant of FYT in which the right arm of the second chromosome contains a mutation in M(2)53¹. In this case overall development is slowed, and clone tissues should grow faster than surrounding tissues. In either case, a deviation from the normal rate of growth should be recognized and accounted for. The FLP-recombinase-induced mitotic recombination event in the FYT/GFA heterozygotes, which is the crux of the system, will, upon segregation, yield one daughter cell that will be haplo-4 (and triplo for a number of genes on the 2R that are linked to the fourth chromosome centromere of the complementary half of the FYT reciprocal translocation). We have not observed any adverse effect in terms of clone recovery and survival with the triplo condition for this segment. The other daughter cell will be haploid for the part of the right arm of chromosome 2, which is cell lethal. Thus, the haplo-4 clone will not have a counterpart twin clone. This is a consideration in the design of certain experiments, such as those involving asymmetric cell division or lineage tracing. With these considerations in mind, this system provides a much-needed general tool for efficient clonal analysis of any fourth chromosome mutation. It incorporates all of the most advanced capabilities currently available for efficient production and analysis of genetic mosaics and should readily allow incorporation of any further improvements.

Materials and Methods

Fly culture conditions and genotypes used to generate haplo-4 clones

Flies were reared at 18 and 25°C depending on the experimental setting. Unless otherwise stated, the stocks and genotypes can be found in Flybase and references elsewhere dati¹ was previously described. All constructs were introduced to FYT and GFA chromosomes by meiotic recombination in multiple steps. A detailed description of these steps can be obtained upon request. Wild-type haplo-4 clones marked with y- were obtained from the genotype y w FLP; FRT42D w+/FRT42D y+ T(4;2)X-4; T(2;4)X-4/+. The translocation used is a reciprocal translocation in which region 102A-F of the fourth chromosome is translocated to position 56 D-F of the 2R. Wild-type haplo-4 clones in the wing surface were induced and marked with kojak^VAI51 (AKA shavenoid), a marker that eliminates trichomes (FBal0099828). These clones were induced in the genotype y w FLP; FRT42D koj^VAI51 w+/ FRT42D y+ T(4;2)X-4 ; T(2;4)X-4/+. Clones in wing discs were generated in y w FLP; UAS-GFP FRT42D Actin > Gal4/ FRT42D y+ Gal80 T(4;2)X-4 ; T(2;4)X-4/+. Clones corrected for size were generated in the y w FLP; FRT42D w+/FRT42D y+ Gal80 M(2)53¹ T(4;2)X-4; T(2;4)X-4/+ genotype. sparkling clones were generated in y w FLP; FRT42D w+/FRT42D y+ Gal80 T(4;2)X-4; T(2;4)X-4/spa^pol (FBal0015991). Finally, dati clones in the brain were generated in y w FLP; UAS-GFP FRT42D w+ Actin > Gal4/FRT42D y+ Gal80 T(4;2)X-4; T(2;4)X-4/dati (FBal0217973).

Heat shock regimens

Larvae between first and third instar were heat shocked for 1 h at 37°C in a water bath with the cotton plug pushed down to the level of the water surface. After heat shock they were allowed to recover and develop at 25°C. Larvae were removed from media in late third instar and washed twice in 1X PBS. Larvae were then sorted using a Leica DMI6000 inverted fluorescent microscope and individuals with clones in the developing central nervous system were placed into individual vials and allowed to grow until adulthood.

Brain dissections, multiplex in situ and confocal microscopy

Double protein and in situ stainings were performed according to Kosman et al., (2004) with some modifications.²⁹ Adult brains were dissected in cold PBT, fixed for 20 min in PBT 4% formaldehyde, rinsed three times in PBS and stored in 100% methanol at -20°C before processing. Next they were washed three times in 100% ethanol, incubated for 1 h at room temperature in a 1:1 ethanol:xylene solution and then washed in 100, 80, 50, 30% ethanol and finally water. After these washes the brains were incubated in acetone for 10 min at -20°C, washed in PBT and fixed again in 4% formaldehyde for 20 min at room temperature and washed five times in PBT to remove any trace of formaldehyde. They were then washed in 1:1 PBT:Hybe solution, and washed three times in Hybe solution before being incubated in Hybe for 90 min at 55°C. After the incubation, a DNP-labeled probe for dati mRNA was added to the solution at 1:100, and left to hybridize overnight at 55°C. The next day, the brains were washed four times in Hybe solution 15 min each, at 55°C. After a wash in 1:1 PBT:Hybe, they were washed four times 15 min each in PBT. The brains were then blocked with 5% PBT-Western blocking solution for 1.5 h and incubated overnight at 4°C with the primary antibodies. The primary antibodies used and their dilutions were as follows: rat anti-Elav (1:1000), chicken anti-GFP (1:1000), and either rabbit anti-DNP (1:1000, for in situs) or mouse anti-nc82 (1:50, for protein stainings). After the incubation of the primaries, the brains were washed four times in PBT, for 15 min per wash. The brains were then incubated with DyLight donkey anti-chicken 488 (1:1000) (Jackson ImmunoResearch), Alexa Fluor goat anti-rat 555 (1:1000) (Invitrogen), and either Alexa Fluor donkey anti-rabbit 647 (1:1000, for in situs) (Invitrogen) or Alexa Fluor donkey anti-mouse 647 (1:1000, for protein stainings) (Invitrogen) for 2 h at room temperature. Following secondary antibody incubation, the brains were again washed for 15 min in PBT, and then incubated in 1:1000 solution of DAPI for 15 min. Brains were then washed twice more for 15 min in PBT, then added to Slow Fade Gold antifade reagent (Invitrogen) overnight. The next day, the brains were mounted in Prolong Gold (Invitrogen) and stored in 4°C, until being imaged using a Zeiss LSM 700 confocal microscope.

Fluorescence signal normalization

To normalize the expression of dati mRNA in euploid and haplo-4 cells, and the signals of DAPI and GFP, we collected the pixel intensities of each point along a line in one channel and subtracted these values from the lowest value collected from that channel. The corrected value of each channel was then divided by the maximum pixel intensity value of that channel and multiplied by 100. This procedure was applied to all channels. This normalization allows the visualization of fluorescent signals that vary in intensity in a scale in which their percentages can be compared.

Glossary

Abbrevations:

FRT

FLP-recombinase recognition target

FLP

FLP-recombinase

GFP

green fluorescent protein

MARCM

mosaic analysis by repressible cell marker

FYT

FRT-yellow-translocation

GFA

GFP-FRT-ActinGal4

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.