Abstract
Drosophila imaginal discs are epithelial tissues perfectly suited to use them as a playground to define the functional contribution of genes to epithelial development and organ morphogenesis. The more we know about the discs and the mechanisms directing their development, the best prepared we are to assign specific “functions” to individual genes based on phenotypic observations. Conversely, and thinking from the perspective of the gene, the more we know about its function, the best inferences we could make about the mechanisms underlying imaginal disc development. This reciprocal relationship, coupled to the arsenal of possible experimental approaches available in Drosophila genetics, genomics and cellular biology, makes these tissues excellent systems to address biological problems with biomedical relevance. In this review, an overview of three interconnected aspects related to the use of Drosophila imaginal discs as an experimental system to analyze gene function is given: (i) imaginal discs biology, with a focus in the genetic mechanisms involved in pattern formation; (ii) concepts and available experimental tools for the analyses of gene function and (iii) uses of Drosophila and the imaginal discs for addressing biomedical problems.
Keywords
- Drosophila
- genetic analysis
- growth control
- pattern formation
- imaginal disc
1. Introduction
Imaginal discs are epithelial tissues that grow within the larva of holometabolous insects and differentiate most of the external parts of the adult during metamorphosis [1]. They are named after the adult structures they form, for example, the wing imaginal disc makes the wing and the thorax, while the leg discs develop the leg appendages and the pleura. Each disc has a characteristic size, shape, histology and fate map, and they are connected to the larval epidermis and to the tracheal system of the larvae [1]. Imaginal discs are a favorite subject of study for developmental and cell biologists, and the analysis of their characteristics has shaped key concepts in developmental biology, including the notions of cell determination, cell autonomy and positional information [2]. The study of imaginal discs is also contributing to identify and characterize the cellular and biochemical mechanisms underlying these concepts.
A key peculiarity that in part account for the success of the imaginal discs as experimental model systems is that they retain a considerable developmental plasticity during larval development. Thus, when let unperturbed, each imaginal disc will undergo with a clock-like precision cell divisions, growth and territorial specification, forming a fixed inventory of cuticular structures during differentiation. Simultaneously, the discs remain extremely plastic and reactive to experimental manipulations during most of their development. This developmental plasticity is particularly manifested when the discs are cut and transplanted into adult host, where disc fragments can reconstitute the missing parts (“regeneration”) and even alter their identity (“transdetermination”) [2, 3]. The ability to regenerate has been more recently observed
Imaginal discs are also extremely reactive to genetic manipulations, and altering the expression of genes encoding a wide variety of proteins related to epithelial development in imaginal cells results in precise adult phenotypes [6]. The responsive nature of imaginal discs to genetic and other experimental manipulations is one of the reasons explaining why imaginal discs have been repetitively used in developmental biology. In fact, they have traditionally been either drivers or early adopters of novel experimental approaches directed to unravel the genetic and cellular basis of epithelial biology and organ morphogenesis [2]. In this manner, imaginal discs not only played a key role in the transition from experimental embryology to developmental genetics but also in the posterior move from formal mechanistic interpretations to increasingly detailed molecular and cellular descriptions.
Another aspect that explains the success of imaginal discs as experimental tools at different historical periods is the richness of biological processes participating in their development and differentiation. Thus, most common developmental operations, including cell proliferation and death, cell growth and differentiation, pattern formation and tissue mechanics and morphogenesis, as well as their underlying molecular mechanisms, can be analyzed in the discs. Because these processes are common to the development of all multicellular organisms and also regulated by conserved genes and molecular interactions, the discs are a most convenient system to dissect genetically complex developmental mechanisms. In this review, some key aspects of imaginal disc biology are summarized, and the experimental tools available to analyze the contribution of genes to the development of imaginal disc development are described. We will also summarize how to capitalize on the knowledge we have about the discs to address problems with biomedical impact.
2. Genetic regulation of imaginal disc development
Imaginal discs are versatile, responsive and modular tissues for genetic experimentation. Internal regulatory processes not only determine their development but they also communicate and interact with other larval organs to influence growth and developmental timing [7, 8, 9, 10]. In the discs, patterning and growth are interconnected aspects driven by conserved signaling pathways and complex transcriptional regulatory networks that coordinate gene expression along a field of epithelial cells. Although each imaginal disc has its own peculiarities, their modes of development share multiple common aspects, including regulated cell proliferation and the progressive generation of gene expression domains. Thus, a common feature of all discs is the existence of a continuous deployment and refinement of gene expression patterns that culminate in the allocation of cellular fates to individual cells or fields of cells. This process is linked to the position that each cell occupies in the epithelium, but it is mostly a consequence of each cell developmental history, as defined by the gene regulatory networks that were operating in its progenitors. The mechanistic links between gene activity and the patterned distribution of differentiated cells make possible to use phenotypic approaches to identify and characterize the function of individual genes through genetic analysis.
The origin of imaginal cells is the embryonic ectoderm. It is in this epithelial layer where a set of gene regulatory events defines the position of groups of cells as imaginal precursors [1, 11] (
Figure 1
). The specification of each imaginal primordium follows the same logic of gene regulatory events that will direct their subsequent development. Thus, the segmented embryonic ectoderm contains a Cartesian system of positional information along the antero-posterior (A/P) and dorso-ventral (D/V) axes defined by the expression of ligands belonging to the Hedgehog (Hh), wingless (Wg), decapentaplegic (Dpp) and epidermal growth factor receptor (EGFR) pathways [1, 12, 13, 14, 15] (
Figure 1
). The restricted expression of these ligands results in the generation of overlapping signaling domains in which pathway-specific transcription factors are expressed or active (
Figure 1
). These transcription factors act through
Complementary to the A/P and D/V coordinate systems, which are common to all embryonic segments, the ectoderm also bears a segment-specific code of homeotic genes resulting from the differential expression of the
The examples of the leg and wing discs illustrate how signaling-transcription networks coupled to the increase in the size of the epithelium have been adapted to generate diverse expression patterns associated to cell fate allocations. The leg and wing discs originate from the same primordium located in the ventral region of each hemisegment of the mesothorax (parasegment 5). These early primordia can first be recognized as a group of ~30 cells in each thoracic hemisegment that express the homeobox gene
From this point onwards, both groups of cells follow their development independently, using the information already present in the primordium to drive subsequent developmental steps. In the case of the wing disc, the first subdivision imposed over the antero-posterior compartment initial subdivision is between proximal and distal cells (
Figure 2
). Proximal cells express the EGFR ligand
In the case of the leg, the restricted expression of Dpp and Wg in dorsal and ventral domains of the leg imaginal disc directs the formation of the proximo-distal (P/D) axis. Initially, high levels of Wg and Dpp activate
In summary, as the discs grow in size, its pattern is progressively established and prefigurates as expression domains the position where different structures, such as veins, sensory organs and tarsal joints, that will differentiate during metamorphosis. This process relies upon regulatory mechanisms that link signaling with transcriptional regulation along the epithelium. Subsequently, each disc will initiate its differentiation and morphogenesis during pupal development, in a course that includes extensive morphogenetic movements and fusion between imaginal discs. The culmination of imaginal disc development is the generation of precise patterns of differentiation. As these patterns are under strict regulation of genes encoding transcription factors and signaling components, changes in the expression pattern or activity of these genes result in precise alterations of the pattern of cell differentiation and organ morphogenesis. These alterations, the mutant phenotypes, can be used as diagnostic criteria to define the requirements of the gene and to annotate its function in relation to the processes affected. Most of the impact of
3. Genetic analysis in Drosophila
When genetic analysis was first used to characterize the contribution of a gene to a particular process, the definition of “gene function” was abstract, referring more to the requirement of the gene than to the actual biochemical function of the protein it encoded, which was for the most part unknown. Thus, genes encoding transcription factors or signaling molecules could be classified as “segmentation genes,” because they displayed mutant phenotypes affecting the segmentation of the embryo [44]. By looking at the particularities of the mutant phenotype, these genes could be further classified into discrete classes that later were shown to correspond to the different levels in the hierarchy of regulatory interactions driving segmentation [44]. During most of the 20th century, methods to analyze a gene were blind to a large extent. In this manner, mutations generated randomly were selected because they failed to complement a particular allele or gene deficiency [45, 46] or because they displayed or modified a phenotype in the tissue of interest [47, 48, 49, 50]. Favorite methods to generate mutations were chemical (EMS), physical (ionizing radiations) and later through the mobilization of transposable elements [51, 52].
The availability of the
In more recent years, the adaptation of the CRISPR/Cas9 technology to the fly is allowing an unprecedented level of precision and easiness with which a gene can be targeted [60, 61, 62, 63]. This method is based on the use of the nuclease Cas9 guided by small RNAs (gRNAs) to generate double-strand breaks (DSB) at a target genomic locus, allowing its use as a highly efficient and specific system for gene edition. Using CRISPR/Cas9 allows targeted manipulation of a given gene in different manners. Thus, the CRISPR/Cas 9 system can be employed to generate sequence-specific DSB to disrupt the target locus when a single gRNA is used, resulting in the generation of small insertion or deletions (In/Dels) through the error-prone process of non-homologous end-joining (NHEJ) repair in the coding sequence. This approach can be used to disrupt coding genes, leading to an array of mutations ranging from hypomorphs to amorph alleles (
Figure 3
), caused by frameshifts in the reading frame, premature stop codons or triplet insertions or deletions [63, 64, 65]. The NHEJ repair system can also be directed by two gRNAs to delete a specific fragment flanking the targeted sequences [63, 66]. The resulting DNA change consists in a deletion of a longer sequence, which could include an entire open reading frame, an exon or also non-coding sequences such as candidate regulatory regions, being also an useful approach to analyze transcriptional regulation
A second application of the CRISPR-Cas9 system is to stimulate HDR (
More interestingly, when stimulating HDR using longer homologous repair dsDNA (homology arms), the removed genomic region can be replaced with site-specific recombinase sequences such as attP sites [67], allowing subsequent integrations at this genomic position of modified versions of the gene or orthologous genes and sequences encoding for tagged proteins (
Figure 3
). Creation of a DSB dramatically increases the frequency of homologous recombination [70], allowing the expression
Beyond genome engineering, CRISPR/Cas9 has also been used to regulate endogenous gene expression in both cells and organisms without causing any mutation. For this approach, a nuclease-dead or inactive Cas9 is fused to a transcriptional activator or repressor domain and can be recruited to specific target DNA by its gRNAs, allowing activation through CRISPR or repression by CRISPR interference (
Figure 3
) [71, 72, 73]. In addition, an inactive Cas9 co-expressed with a gRNA can also be used for immunoprecipitation of specific DNA regions as a variant form of engineered DNA binding-mediated chromatin immunoprecipitation (enChIP), and associated proteins can be subsequently identified by mass spectrometry (enChIP-MS) [74]. In summary, CRISPR/Cas9 technology is providing a precise and efficient method for sequence-specific targeting of Cas9, resulting in genomic alterations, changes in gene expression and even the isolation of protein complexes bound to specific DNA regions. The application of this technology is triggering an unprecedented level of precision in genetic and genomic analysis in
4. Uses of Drosophila and the imaginal discs to address biomedical problems
The precision by which the genome can be modified, combined with the knowledge we have about the cellular and molecular fundaments of imaginal disc development, justify the use of these epithelial tissues as experimental models to address a variety of biological questions, including biomedical ones. According to the Homophila database, around 75% of human disease genes cataloged in OMIM (On-line Mendelian Inheritance in Man) database have close homologs in the fruit fly [75]. This high degree of evolutionary conservation makes even more compelling the use of
The generation of
Apart from offering a convenient experimental system to identify the function of a gene or the consequences of the expression of relevant protein variants,
5. Conclusions
Several characteristics converge to sustain and reinforce the use of
Acknowledgments
We are very grateful to A. López-Varea and N. Esteban for excellent technical support. This work was supported by MINECO grant BFU2015-64220-P to JFdC. Institutional support from Fundación Ramón Areces and Banco de Santander is also acknowledged.
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