Mapping by VESGEN of Wing Vein Phenotype in Drosophila for Quantifying Adaptations to Space Environments

The fruit fly, Drosophila melanogaster, is a major model organism for space experiments (Benguria et al., 1996; Ikenaga et al., 1997; Le Bourg, 1999; Marcu et al., 2011; Taylor et al., 2014) because of its susceptibility to targeted mutation, ease of culture, small size, short life span, ability to fly, and sequenced genome (Adams et al., 2000). Approximately 75% of known human genetic diseases can be matched with genes of the fruit fly (Pandey and Nichols, 2011; Reiter et al., 2001). Only a handful of higher organisms (e.g., the fruit fly, mouse (Mus musculus), zebrafish (Danio rerio), transparent nematode (Caenorhabditis elegans), and thale cress (Arabidopsis thaliana)) have been identified that are relatively convenient for targeted single mutations. All of these model organisms that are of critical importance to terrestrial biomedical and developmental biology research are also important in space biology for investigating genetic and physiological response to space stressors, such as microgravity, radiation, and engineered environmental factors, such as temperature and light.

Venation patterning in the wings of Drosophila is highly stereotyped (i.e., identical among individual flies). Venation development is highly responsive to genetic manipulation and results from precise, multi-step coordination of signaling by the Notch, epidermal growth factor receptor (EGFR), Decapentaplegic (Dpp), Hedgehog (Hh), Wnt, Wingless (Wg), and possibly other pathways during the overall process of Drosophila development (reviewed by Blair, 2007; De Celis, 2003). We therefore hypothesize the Drosophila wing and its venation phenotypes may provide sensitive, quantified readouts of the effects of environmental factors encountered during spaceflight when mapped and quantified by NASA’s VESsel GENeration Analysis (VESGEN) software. In general, Drosophila development is a highly choreographed process requiring the precise expression of a series of temporally and spatially expressed cues. Modified venation phenotypes may reflect gene expression responses to environmental alterations. Thus, we investigated the potential relevance to wing venation patterning from gene expression changes reported in Drosophila larvae and female adults from previous spaceflight data (Marcu et al., 2011). While it is yet to be tested whether these levels of gene expression changes induced by travel to low Earth orbits will result in changes in wing vein patterning after spaceflight, future longer-duration deep space missions, coupled with significantly higher backgrounds of ionizing radiation, could result in much greater perturbations to biological organisms. Therefore, model organisms such as Drosophila will be important to assess the effects of these novel spaceflight environments as a precursor to long-term deep space exploration by humans. Miquel and Philpott (1978) observed blistered and tattered wings on Drosophila specimens returned from space on a Soviet Satellite Cosmos 936, and Drosophila flight remains an active area of research (Costa et al., 2014; Fry et al., 2003).

A series of increasingly severe pheno-types in Drosophila wing venation (Johannes and Preiss, 2002) was mapped and quantified by the VESGEN software (Chen et al., 2013; Vickerman et al., 2009; Zamanian-Daryoush et al., 2013) as a step toward developing methods suitable for testing whether gene expression changes seen in spaceflight could affect wing vein phenotypes. Abnormal ectopic veins that resulted from overexpressing the H-C2 construct of Notch antagonist Hairless (H) \, which contains a deletion of the binding domain to Suppressor of Hairless [Su(H)], were quantified using our software. The VESGEN software was originally developed to analyze human and vertebrate vascular remodeling according to specific physiological rules, such as vessel bifurcation and tapering, but is now being expanded to the analysis of vessel patterning in major tissues of other experimental organisms, such as the Drosophila wing and Arabidopsis leaf.

Increasingly severe ectopic venation in the adult Drosophila wing resulting from H-C2 overexpression are compared to stereotyped venation in the wild type with vascular maps generated by VESGEN (Figure 1). Vascular parameters measured by the software within the vascular maps confirm that abnormal ectopic venation is greatly increased in the Class 5 H-C2 phenotype, compared to wild type (Table 1). Interestingly, results by VESGEN also demonstrate that in contrast to ectopic venation, the stereotyped venation patterning is quite equivalent in the wild type and Class 5 phenotype. For example, A_v and L_v for stereotyped Class 5 vessels are 1.03× and 1.13× relative to wild type (e.g., L_v=0.0250 and 0.0257 px-px^-2 for stereotyped vessels, respectively). In the Class 5 phenotypic category, only the stereotyped PCV is incomplete. However, for ectopic veins in the wild type compared to Class 5 H-C2, N_v increased from 1 to 18 and L_v increased from 0.0004 to 0.0095 px-px². A_v, L_v, and N_v for ectopic vessels in Class 5 H-C2 are 24×, 42×, and 18× greater than wild type. Johannes and Preiss (2002) then used this ectopic-vein phenotype to identify several more genes involved in Notch and EGF signaling by screening for genetic modifiers of the phenotype. Therefore, VESGEN provides a sensitive tool that can be used to analyze Drosophila wing vein phenotypes of genetic mutants terrestrially. We hope to use VESGEN in future spaceflight studies to investigate whether wing vein patterns are altered, since data from a previous spaceflight mission indicates significant changes in expression of genes that influence wing vein development and patterning.

Microarray data analyzed by us from both larvae and adult flies returned from space suggest significant changes in genes related to wing vein development that include the EGFR, Notch, Hh, Wg, and Dpp signaling pathways (Table 2 and Table 3), compared to ground control samples. Expression of Smoothened, a gene that possesses Hh receptor activity, was significantly down-regulated in space-returned adult flies (-0.8 fold change; p-value-0.00). Similarly, expression of rhomboid 7 (-0.7 fold; p-value-0.00) and aveugle (-0.8 fold; p-value-0.00) was significantly down-regulated in space-returned adult flies, compared to ground control. For the case of space-returned larvae, however, expression of ash2 (absent, small, or homeotic discs 2) was significantly up-regulated (+0.6 fold; p-value-0.00).

The goals of our Methods study are twofold. Our first goal is to demonstrate the mapping and quantification methodology of normal stereotyped and abnormal ectopic vessel patterning in the Drosophila wing by VESGEN analysis. Our second goal is to justify the relevance of future studies on the response of Drosophila wing venation patterning to the stresses of space environments, in part by analyzing gene expression data from our previous spaceflight experiment.

The first finding by VESGEN for our Methods study is that the stereotypical patterning of Drosophila wing venation was not significantly altered in successively severe ectopic phenotypes (Figure 1; Table 1). Quantification of both stereotyped and abnormal ectopic venation in the Drosophila wing were generated by VESGEN from increasingly severe phenotypes produced previously by varying expression conditions of H-C2 construct of the Notch antagonist Hairless (Johannes and Preiss, 2002). Insightful quantification by VESGEN confirms observations by Johannes and Preiss that the stereotyped patterning of wing venation was preserved as an essentially equivalent patterning in all H-C2 specimens, despite the increasingly severe phenotypes of additional ectopic venation. Our second finding with the VESGEN analysis was that increasingly severe phenotypes of ectopic wing venation increased quantitatively in both vessel number and density, as was previously observed qualitatively by Johannes and Preiss.

Our third finding was that many of the genes altered by spaceflight, and previously reported by one of the authors in our present study (Marcu et al., 2011), are also known to be involved in the development of wing venation (Blair, 2007). Developmental programs of Drosophila respond sensitively to environmental factors, in which normal veins can be lost due to failure in maintenance. Blistered and tattered wings upon return of Drosophila specimens from spaceflight have been reported previously (Miquel and Philpott, 1978). Imaginal wing discs are defined during embryogenesis and form mature discs during larval development. As summarized previously, the main regulators of wing disc development are epidermal growth factor receptor (EGF), Notch, Hedgehog (Hh), Wingless (Wg), and the Decapentaplegic (Dpp) signaling pathways (Blair, 2007).

We report here significant changes in genes related to these specific pathways from microarray data of both larvae and adult flies returned from space, compared to ground control samples (Tables 2 and 3). Although most of these genes play a vital role in wing disc-derived wing morphogenesis and wing vein morphogenesis, their exact role in vein patterning as phenotypic responses to spaceflight environments is not yet clear. However, vein pattern formation starts in the imaginal disc and progressively depends on Hedgehog, EGF, and DPP signaling pathways (Bier, 2000). For instance, Hedgehog pathway regulates the positioning of longitudinal veins, such as L3 and L4 (Blair, 2007). Expression of the gene Smoothened, which possesses Hedgehog receptor activity, was significantly down-regulated in space-returned adult flies. Expression of rhomboid-7 and aveugle was also significantly down-regulated in space-returned adult flies compared to ground control; rhomboid and aveugle are critical in EGF-regulated stereotypical vein patterning. Previous studies have shown that mutations in the rhomboid gene inhibit vein development and disrupt vein patterning (Brentrup et al., 2000). In the case of space-returned larvae, expression of ash2 (absent, small, or homeotic discs 2) was also significantly up-regulated, suggesting possible changes in intervein cell fate that determines intervein patterning.

Our spaceflight data further indicate that several genes whose expression patterns are important for wing vein patterning are altered at different stages of development during spaceflight. While genomic data from the prepupal stage are not available, and the prepupae is thought to be a phenocritical stage for wing vein patterning, data from spaceflight returned adult flies and late third instar larvae indicate changes in expression of key components of the Hedgehog, EGF, and related pathways. Therefore, it is possible that in future spaceflight missions different wild type and sensitized mutant background lines could be flown to investigate the use of altered wing venation in offspring developed in space as a phenotypic measure/readout by VESGEN of spaceflight-induced stress. The VESGEN software will allow a careful analysis of the response of Drosophila wing venation to space environments and other factors, such as space radiation, since these effects have not yet been systematically mapped and quantified. Other interesting software measures wing venation landmarks and wing shape related to sexual dimorphism (Kunkel and Bettencourt, 2011) or Drosophilid species (Houle et al., 2003). The VESGEN approach is distinguished by an automated, insightful grouping of vascular characteristics that have been used extensively to map other similar patterns.

Previously perturbed vascular patterns mapped by VESGEN include: mouse coronary and intestinal vessels (Liu et al., 2009; Parsons-Wingerter and Reinecker, 2012; Vickerman et al., 2009), Arabidopsis leaf venation (Parsons-Wingerter et al., 2014), the avian chorioallantoic membrane (Vickerman et al., 2009), and the human and mouse retinas (Parsons-Wingerter et al., 2010; Vickerman et al., 2009). We propose that VESGEN mappings of healthy or pathological adaptations in vascular patterning to space environmental factors by major genetic organisms, such as Drosophila, offer sensitive, quantifiable phenotypic read-outs that help to integrate the many molecular signals generated by complex, interacting genetic pathways. Furthermore, analysis by VESGEN of the response of wing venation patterning in the Drosophila model to various types of terrestrial environmental stressors, as well as studies of different genetic mutants, can be useful for terrestrial research on developmental, environmental, and other biomedical applications.