Abstract
For coordinated circulation, vertebrate and invertebrate hearts require stereotyped arrangements of diverse cell populations. This study explores the process of cardiac cell diversification in the Drosophila heart, focusing on the two major cardioblast subpopulations: generic working myocardial cells and inflow valve-forming ostial cardioblasts. By screening a large collection of randomly induced mutants we identified several genes involved in cardiac patterning. Further analysis revealed an unexpected, specific requirement of EGF signaling for the specification of generic cardioblasts and a subset of pericardial cells. We demonstrate that the Tbx20 ortholog Midline acts as a direct target of the EGFR effector Pointed to repress ostial fates. Furthermore, we identified Edl/Mae, an antagonist of the ETS factor Pointed, as a novel cardiac regulator crucial for ostial cardioblast specification. Combining these findings we propose a regulatory model in which the balance between activation of Pointed and its inhibition by Edl controls cardioblast subtype-specific gene expression.
Introduction
The heart consists of a variety of cells with distinct molecular and physiological properties in both vertebrates and invertebrates. A complex regulatory network of transcription factors and signaling pathways orchestrates the specification of these different cell populations and their proper arrangement within a regionalized working myocardium or other functional structures such as valves, inflow and outflow tracts (reviewed in (Greulich, Rudat, & Kispert, 2011; Miquerol & Kelly, 2013; Rana, Christoffels, & Moorman, 2013); for the invertebrate Drosophila heart see e.g. (Rolf Bodmer & Frasch, 2010; Lehmacher, Abeln, & Paululat, 2012; Lovato & Cripps, 2016; Reim & Frasch, 2010)). For example, the vertebrate T-box gene Tbx20 promotes working myocardial fate by restricting Tbx2 expression and enabling the expression of chamber myocardium-specific genes (Cai et al., 2005; M. K. Singh et al., 2005; Stennard et al., 2005). By contrast, Tbx2 and Tbx3 repress working myocardium-specific gene expression and chamber differentiation in the non-chamber myocardium and thus contribute to the formation of endocardial cushions and structures of the conduction system (Christoffels et al., 2004; Hoogaars et al., 2007; R. Singh et al., 2012). Normal endocardial cushion formation also requires COUP-TFII, an orphan nuclear receptor transcription factor that regulates cell fate decisions in several tissues (Lin et al., 2012; S. P. Wu, Yu, Tsai, & Tsai, 2016). In the embryonic mouse myocardium, COUP-TFII is restricted to atrial cardiomyocytes, a pattern consistent with a function in promoting atrial over ventricular fate (Lin et al., 2012; S. P. Wu et al., 2013). This function appears to involve the up-regulation of Tbx5 (S. P. Wu et al., 2013), another T-box gene with non-uniform cardiac expression and a fundamental role in heart development and human cardiac disease (Basson et al., 1997; Bruneau et al., 1999; Bruneau et al., 2001; Ghosh, Brook, & Wilsdon, 2017; Steimle & Moskowitz, 2017). Furthermore, FGF-mediated receptor tyrosine kinase (RTK) signaling upstream of the cardiogenic transcription factor Nkx2-5 was recently shown to sustain ventricular chamber identity of cardiomyocytes in zebrafish (Pradhan et al., 2017). As emphasized below, spatial restriction of cardiac transcription factors as well as precisely controlled RTK signaling activities are not only important in vertebrate but also invertebrate hearts ((Gajewski, Choi, Kim, & Schulz, 2000; Lo & Frasch, 2001; Zaffran et al., 2006) and this work).
The Drosophila heart (dorsal vessel) comprises several types of cardiomyocytes (in the embryo called cardioblasts, CBs) and non-contractile pericardial cells (PCs) (Rolf Bodmer & Frasch, 2010; Lovato & Cripps, 2016). The progenitors of these cells are specified in segmentally repeated heart fields located at the intersection of BMP/Dpp and Wg/Wnt signaling activities (Frasch, 1995; Reim & Frasch, 2005; X. Wu, Golden, & Bodmer, 1995). Subsequent specification of the definitive cardiogenic mesoderm depends on a conserved group of transcription factors, most importantly those encoded by the Nkx2-5 ortholog tinman (tin), the Gata4 ortholog pannier (pnr) and the Dorsocross1-3 T-box genes (three Tbx6-related paralogs that also share features with Tbx2/3/5; in the following collectively called Doc) ((Alvarez, Shi, Wilson, & Skeath, 2003; Azpiazu & Frasch, 1993; R. Bodmer, 1993; Gajewski, Fossett, Molkentin, & Schulz, 1999; Junion et al., 2012; Reim & Frasch, 2005; Reim, Lee, & Frasch, 2003); reviewed in (Reim & Frasch, 2010; Reim, Frasch, & Schaub, 2017)).
While the identification of cardiogenic factors has greatly improved our understanding of early specification events, much less is known about the mechanisms that lead to the diversification of cardiac cell subpopulations. In this study, we mainly focus on the development of the two major cardioblast subpopulations: generic cardioblasts (gCBs), which make up the main portion of the contractile tube ("working myocardium"), and ostial cardioblasts (oCBs), which form bi-cellular valves (ostia) for hemolymph inflow. Due to Hox gene inputs, ostial progenitor specification is limited to the abdominal region ((Lo, Skeath, Gajewski, Schulz, & Frasch, 2002; Lovato, Nguyen, Molina, & Cripps, 2002; Ponzielli et al., 2002; Ryan, Hoshizaki, & Cripps, 2005), reviewed in (Monier, Tevy, Perrin, Capovilla, & Semeriva, 2007)). Current research suggests that each abdominal hemisegment generates at least seven distinct progenitors that give rise to six CBs (4 gCBs + 2 oCBs) and several types of PCs (Tin+/Even-skipped(Eve)+ EPCs, Tin+ TPCs, and Odd-skipped(Odd)+ OPCs; (Rolf Bodmer & Frasch, 2010) and references therein). Whereas gCBs (a.k.a. Tin-CBs) maintain expression of tin, oCBs (a.k.a. Svp-CBs) specifically express the COUP-TFII ortholog seven-up (svp) and Doc (Gajewski et al., 2000; Lo & Frasch, 2001; Ward & Skeath, 2000; Zaffran et al., 2006). Previous work has shown that Doc is repressed in gCBs in a tin-dependent manner (Zaffran et al., 2006). Robust tin expression in turn depends on the Tbx20 ortholog midline (mid/nmr2). The mid gene is first activated in gCB progenitors, but later, like its paralog H15/nmr1, becomes expressed in all cardioblasts (Miskolczi-McCallum, Scavetta, Svendsen, Soanes, & Brook, 2005; Qian, Liu, & Bodmer, 2005; Reim, Mohler, & Frasch, 2005). In oCBs, svp represses tin expression thereby permitting continued Doc expression in these cells (Gajewski et al., 2000; Lo & Frasch, 2001; Zaffran et al., 2006). In the abdomen, gCBs and most PCs are preceded by a precursor that undergoes symmetric division, whereas oCBs and half of the OPCs are derived from common, asymmetrically dividing CB/PC progenitors (Alvarez et al., 2003; Han & Bodmer, 2003; Ward & Skeath, 2000).
The process of progenitor specification in the somatic and cardiogenic mesoderm involves the antagonistic actions of RTK/Ras/MAPK and Delta/Notch signaling (Carmena et al., 2002; Grigorian, Mandal, Hakimi, Ortiz, & Hartenstein, 2011; Hartenstein, Rugendorff, Tepass, & Hartenstein, 1992). Two types of RTKs, the fibroblast growth factor (FGF) receptor Heartless (Htl) and the epidermal growth factor (EGF) receptor EGFR, act positively on progenitor selection via MAPK signaling, although they are used by different progenitors to different extents (Buff, Carmena, Gisselbrecht, Jimenez, & Michelson, 1998; Carmena et al., 2002; Michelson, Gisselbrecht, Zhou, Baek, & Buff, 1998). Htl and its FGF8-like ligands Pyramus (Pyr) and Thisbe (Ths) have a dual function as regulators of mesodermal cell migration and cell specification, with progenitors of the Eve+ lineage as the most prominent example for the latter (reviewed in (Bae, Trisnadi, Kadam, & Stathopoulos, 2012; Muha & Müller, 2013)). The exact contribution of EGFR signaling to Drosophila heart development has been less clear until now, but it was shown that EGFR loss of function results in a severe reduction of the numbers of cardioblasts, pericardial nephrocytes, and blood progenitors (Grigorian et al., 2011). Molecularly, the predominant EGFR ligand in the embryo, Spitz (Spi), relies on the protease Rhomboid (encoded by rho) and the chaperon Star (S) for its conversion from a membrane-bound into its active form (reviewed in (Shilo, 2014)). Among the most important downstream effectors of RTK/Ras/MAPK pathways are the ETS transcription factors PntP2 (encoded by pointed/pnt) and Yan/Aop (encoded by anterior open/aop). While PntP2 becomes an active transcriptional activator upon phosphorylation by MAPK, the transcriptional repressor Yan is negatively regulated by MAPK (Gabay et al., 1996; O'Neill, Rebay, Tjian, & Rubin, 1994). Unlike PntP2, a shorter isoform encoded by pnt, PntP1, is constitutively active but was shown to require activated MAPK for its transcriptional activation at least in some cell types (Brunner et al., 1994; Gabay et al., 1996; Klämbt, 1993; O'Neill et al., 1994). Notably, chordate Pnt orthologs (ETS1/2) were shown to contribute to cardiac progenitor formation in the tunicate Ciona and during transdifferentiation of human dermal fibroblasts into cardiac progenitors (Davidson, Shi, Beh, Christiaen, & Levine, 2006; Islas et al., 2012). During early Drosophila cardiogenesis, Pnt favors expression of eve over that of another homeobox gene, ladybird (lbe, expressed in mesodermal cells immediately anterior of the Eve+ cluster and later in TPCs and two of the four gCBs per hemisegment; (K. Jagla et al., 1997)) (Liu et al., 2006). In addition, Pnt promotes pericardial cell development and antagonizes CB fate, especially that of oCBs (Alvarez et al., 2003).
Despite the progress in the understanding of cardiac progenitor specification, the mechanisms that diversify progenitors of the oCB and gCB lineages have remained elusive. We have performed an unbiased large-scale mutagenesis screen to identify genes that regulate cardiac development in Drosophila embryos and found several mutants that show CB subtype-specific defects. On this basis we discovered a novel and rather unexpected function of the EGF pathway in specifying the gCBs of the working myocardium, thus revealing an intimate link between cardioblast specification and diversification. Furthermore we identified ETS domain lacking (Edl a.k.a. Modulator of the activity of ETS, Mae) as a crucial regulator of the specification of inflow valve-forming oCBs. Edl possesses a SAM domain, which mediates binding to the SAM domain-containing ETS factors PntP2 and Yan, thereby inhibiting their activity as a transcriptional activator or repressor, respectively (Baker, Mille-Baker, Wainwright, Ish-Horowicz, & Dibb, 2001; Qiao et al., 2006; Qiao et al., 2004; Tootle, Lee, & Rebay, 2003; Vivekanand, Tootle, & Rebay, 2004; Yamada, Okabe, & Hiromi, 2003). Our data imply that Edl enables svp expression and thus oCB fate by limiting the activity of PntP2, thereby blocking subsequent activation of important downstream targets such as pntP1 and mid. Collectively, our data provide the basis for an elaborated model of cardiac cell fate diversification that links MAPK signaling, Pnt activity and the cell type-specific expression patterns of key cardiac transcription factors.
Results
Novel EMS-induced mutants reveal a specific requirement of EGF signaling for the specification of generic cardioblasts
In order to identify genes involved in heart and muscle development in an unbiased manner we have performed an EMS mutagenesis screen for chromosome 2 in Drosophila melanogaster embryos (Hollfelder, Frasch, & Reim, 2014). Several of the isolated mutants display a partial loss or irregular alignment of cardioblasts (CBs). Such defects may potentially result from mutations in genes that regulate the specification or differentiation of all CBs or only a particular CB subtype. In the latter case, disturbances in the characteristic "2+4" CB pattern of two ostial cardioblast (oCBs; Doc+/Tin-) and four generic CBs (gCBs; Doc-/Tin+) per hemisegment are to be expected. To analyze the cardiac pattern of mutants in more detail, we performed immunofluorescent double stainings for Doc and H15 (or alternatively Mef2) to label oCBs and all CBs, respectively. We then genetically and in part also molecularly mapped the mutations responsible for CB pattern anomalies (for details see the Materials and Methods section and Table S1). The class of mutants characterized by a loss of CBs contained several novel alleles of genes involved in RTK/Ras/MAPK signaling, which is consistent with the assumed role of this pathway in cardiac progenitor selection or maintenance (Carmena et al., 2002; Grigorian et al., 2011). However, no specific role for the specification of a particular cardioblast subtype or diversification of gCB versus oCB progenitors had been previously attributed to RTK/Ras/MAPK signaling. Our phenotypic analysis now shows that diminished EGF/EGFR but not FGF/Htl signaling leads to a preferential reduction of gCB numbers. Embryos with partially reduced FGF/Htl signaling, i.e. mutants lacking both copies of the FGF-encoding gene pyr and one copy of its paralog ths, as well as hypomorphic htl mutants, show an about equal reduction of gCB and oCB numbers (Figure 1B, for quantification see Figure 1M; additional examples in Figure 1-figure supplement 1B,C). This CB reduction can be sufficiently explained by uneven spreading of the early mesoderm to Dpp-receiving areas. By contrast, several mutations mapped to EGF signaling components feature a preferential loss of gCBs. In strong Egfr mutants very few CBs can be found (Figure 1C, Figure 1-figure supplement 1E). Remarkably, the overwhelming majority of the residual CBs express Doc. The few remaining Doc-negative CBs are usually located towards the anterior and thus are possibly remnants of the oCB-free anterior aorta. In spitz, rhomboid and Star loss-of-function mutants the number of Doc-/Tin+ CBs is strongly reduced while that of ostial Doc+/Tin- CBs is nearly normal or in some cases even increased by a few cells (Figure 1D-G,M, Figure 1-figure supplement 1E,F, Figure 1-figure supplement 2A-C). CBs apparently do not require activity of the ostial marker gene svp to develop and survive independently of EGF, since total CB numbers are similar in Star single and Star svp double mutants (compare Figure 1H to 1G; quantification in Figure 1M).
Previous studies in EGF pathway mutants suggested that incorrectly specified mesodermal progenitors undergo apoptosis (Buff et al., 1998; Grigorian et al., 2011). Using TUNEL and anti-activated caspase stainings we could not reliably detect signs of apoptosis in the Doc-labeled cardiogenic mesoderm of Star mutants, while numerous signals were observed in other tissues (Figure 1-figure supplement 3 and data not shown). If the baculoviral apoptosis inhibitor p35 (Zhou et al., 1997) is artificially expressed in the mesoderm of S mutants the number of CBs slightly increases in comparison to S mutants without p35 (Figure 1I,M). Although this is consistent with a pro-survival function of EGF signaling, it does not fully account for the gCBs missing in S mutants and suggests that the presumptive gCB progenitors largely adopt other fates at reduced EGFR activity. Collectively, these phenotypes imply that EGF signaling plays a major role in the correct specification of gCBs.
Generic CBs and a subset of Odd+ pericardial cells require spatially and temporally coordinated EGF signals
Because EGF signaling is involved in multiple processes during embryogenesis we next asked whether its impact on gCB specification is directly linked to signaling activity within mesoderm cells. Indeed, mesoderm-specific attenuation of the pathway by expression of a dominant-negative EGFR variant resulted in essentially the same phenotype as with the spitz group mutants (Figure 1J,M). Activation of the EGF pathway in mesoderm cells appears to be largely controlled by the spatially restricted expression of rho (Bidet, Jagla, Da Ponte, Dastugue, & Jagla, 2003; Grigorian et al., 2011; Halfon et al., 2000). In the wild type, rho expression is first seen in the Eve+ progenitor P2 (Buff et al., 1998; Carmena, Bate, & Jimenez, 1995; Halfon et al., 2000) followed by expression in the adjacent CB progenitor-containing clusters C14 and C16 ((Bidet et al., 2003; Grigorian et al., 2011); see also Figure 1-figure supplement 4A-D). Overexpression of rho with the pan-mesodermal how24B-GAL4 driver has been previously reported to affect the number of tin-expressing pericardial cells (Bidet et al., 2003), but CBs and their subtypes were not unambiguously labeled in these experiments. We extended these experiments using also other drivers.
Consistent with a mesoderm-autonomous function, overexpression of rho in the dorsal ectoderm (via pnrMD237-GAL4) has no significant effect on CB number or pattern (Figure 1M and data not shown). By contrast, all mesodermal rho overexpression setups increase the gCBs:oCBs ratio in comparison to the wild type (Figure 1K-M and data not shown). The impact on the absolute CB numbers depends on the timing and strength of transgene expression. The later rho is activated in mesodermal cells (with following drivers according to their temporal order and progressive spatial restriction: twist-GAL4, how24B-GAL4 and tinD+tinCΔ4-GAL4) the larger the total number of CBs (Figure 1K-M and data not shown).
Since half of the odd-expressing pericardial cells (OPCs) are siblings of oCBs we also analyzed PCs in EGF-related mutants by Odd/Eve double-stainings (Figure 2A-C and data not shown). Consistent with the results of previous studies on Eve+ progenitor derivatives (Buff et al., 1998; Carmena et al., 2002; Su, Fujioka, Goto, & Bodmer, 1999), we detected EPCs in almost normal numbers in spi group mutants and in embryos with pan-mesodermal dominant-negative EGFR, whereas spi-dependent Eve+ DA1 muscles were largely absent. OPCs are strongly reduced in these loss-of-function backgrounds. Our quantification revealed that about half of the OPCs were lost in rho7M43/L68 mutants (-45%, n=4). A converse phenotype with many extra OPCs is generated by rho overexpression with tinD+tinCΔ4-GAL4 (Figure 2D). Notably, the number of oCB-sibling OPCs (as identified by svp-lacZ reporter analysis) is not significantly reduced in Star mutants if compared to the wild type (Figure 2E,F), thus implying that the EGF signaling-dependent OPCs are those derived from symmetrically dividing OPC progenitors.
Altogether, these data demonstrate that EGF pathway activity is required in the mesoderm specifically for the specification of the symmetrically dividing gCB and OPCs progenitors, but is largely dispensable or even detrimental for the specification of the svp-expressing oCB/OPC progenitors.
The SAM domain protein Edl promotes specification of ostial cardioblasts by blocking Pointed activity
Our EMS screen also yielded mutants in which the number of ostial cardioblasts was specifically reduced. One such complementation group consisting of three alleles was mapped to the numb gene (alleles listed Table S1), which is consistent with its well-known function as a Notch suppressor during asymmetric cell division in the oCB lineage (Gajewski et al., 2000; Ward & Skeath, 2000). Preferential reduction of oCBs was also observed in the mutant line S0520. We found that its cardiac phenotype was caused by loss of the gene ETS domain lacking (edl) as part of a multi-gene deletion and named this mutant Df(2R)edl-S0520 (Figure 3A, Table S2). We identified edl as the gene responsible for the oCB losses by obtaining phenocopies with other edl mutants (Figure 3A-D and data not shown). The lacZ enhancer trap insertion allele edlk06602 was used in most edl loss-of-function experiments since its cardiac phenotype is indistinguishable from that of Df(2R)edl-S0520 and Df(2R)edl-L19 (Figure 3C,D and data not shown), and we detected in this strain a small deletion that specifically destroys the edl gene (Figure 3A, Table S2). Furthermore, we were able to rescue the cardiac phenotype of edl by introducing a genomic edl transgene ((Yamada et al., 2003); Figure 3E) and by artificially expressing edl in the dorsal mesoderm cells or in cardioblasts (Figure 3F,G), demonstrating that Edl is required directly within these cell types. In accordance, edl mRNA is found within the cardiogenic region during stages 10 to 11 and in CBs during stage 12 (Figure 3-figure supplement 1A-C). Thereafter edl expression shifts to the pericardial region, where it persists until stage 15 (Figure 3-figure supplement 1D and data not shown).
A distinctive feature of edl mutants is that the normal "2+4" pattern of 2 Doc+ CB + 4 Doc- CBs is often transformed into a "1+5" pattern (e.g. bracket in Figure 3D), indicating a fate switch from ostial to generic CBs. However, Edl is not a direct activator of Doc expression because Doc is found in CBs of edl double mutants with CB-specific ablation of tin (Figure 3I), a phenotype reminiscent of that of CB-specific tin single mutants (Figure 3H; (Zaffran et al., 2006)). This suggests that edl normally contributes to the activation of Doc in oCBs via suppression of tin. This role of edl in CB patterning is further supported by the observation of some CBs with low levels of Tin and Doc in edl mutants (Figure 3K; compare to the strictly complementary distribution of Doc and Tin in the wild type, Figure 3J).
Next we analyzed Edl function by ectopic expression. Consistent with a mesoderm-autonomous function, overexpressing edl in the dorsal ectoderm via pnrMD237-GAL4 has no significant effect on cardiogenesis (data not shown). By contrast, overexpression of edl in the entire mesoderm via twist-GAL4 results in an increase of CB numbers (Figure 4A) and a decrease of OPCs (described in the next subsection). The increase in Doc+ CBs is disproportionately high. The extra Doc+ CBs in the heart proper also activate ostial cell differentiation markers such as wg (data not shown). In agreement with the proposed function of Edl as a negative regulator of PntP2 (Yamada et al., 2003), our overexpression phenotypes of edl are very reminiscent to that of pntP2-specific (pntRR112 reported in (Alvarez et al., 2003) and pntMI03880 shown in Figure 4B) and amorphic pnt mutants (pntΔ88, pnt2; see Figure 4E,I and (Alvarez et al., 2003)). Accordingly, overexpression of constitutively active PntP2VP16 (Figure 4C) or PntP1 (not shown) via tinD+tinCΔ4-GAL4 causes a phenotype similar to that of edl loss-of-function mutants (Figure 3C,D). By contrast, analogous overexpression of the potential Edl target Yan/Aop leads to a loss of heart cells irrespective of their subtype. These losses may result from a more general block in cell specification and differentiation since Yan has been related to such functions in several other types of MAPK-dependent progenitors (Bidet et al., 2003; Caviglia & Luschnig, 2013; Halfon et al., 2000; Rebay & Rubin, 1995). If the predominant function of Edl during CB specification is the inhibition of Pnt, edl pnt double mutants should mimic pnt mutants. In principle, this is what we observed (Figure 4E,F; quantifications in Figure 4I). By contrast, edl aop double mutants show an additive combination of aop and edl single mutant phenotypes (compare Figure 4H with 4G and 3D; see also quantifications in Figure 4I). Amorphic aop mutants display a reduction in CB number irrespective of CB subtype, which we ascribe to a permissive function during CB development that is probably linked to its well-documented role in restricting eve expression in the early dorsal mesoderm ((Bidet et al., 2003; Halfon et al., 2000; Liu et al., 2006; Webber, Zhang, Mitchell-Dick, & Rebay, 2013)). Importantly, and in contrast to edl and pnt activity changes, manipulating aop activities does not lead to significant shifts in the oCBs:gCBs ratio (Figure 4I). Thus we suggest that Edl acts mainly via negative modulation of PntP2 activity during cardioblast diversification.
Edl and Pnt regulate ostial fate by controlling seven-up expression
The population of oCBs is characterized by expression of svp. In svp mutants all oCBs are converted into Tin+/Doc- CBs due to de-repression of tin ((Gajewski et al., 2000; Lo & Frasch, 2001; Zaffran et al., 2006); Figure 5-figure supplement 1A). Therefore we tested the possibility that Edl promotes oCB fate by regulating svp. In the wild type, expression of svp is recapitulated by the enhancer trap svpAE127-lacZ (Figure 5A; Lo, 2001 #1072). In edl mutants, svp-LacZ expression is strongly reduced in cardiac cells (Figure 5B,D). The reduction in numbers of both svp-LacZ+ oCBs and OPCs at late stages (Figure 5D cf. 5C) suggests that edl already affects the fates of their common progenitors. Consistent with a function in promoting svp expression and oCBs fates, mesodermal overexpression of edl leads to larger numbers of svp-LacZ+ cardiac cells, particularly of CBs, where svp expression correlates with expanded Doc expression (Figure 5E,F). As shown for Doc expression, svp expression can be suppressed by PntP2 hyperactivity (green asterisks in Figure 5H). These observations and further evaluation of the epistatic relations between svp and edl (Figure 5-figure supplement 1) demonstrate that edl affects CB patterning by blocking Pnt activity upstream of svp.
Cardioblast subtype-specific expression of the PntP1 isoform is regulated by PntP2 and Edl
Proposing a gCB-specific function of Pnt, we next analyzed its cardiac expression. Boisclair Lachance et al. previously reported that the expression of a fully functional genomic pnt-GFP transgene mirrors the combined expression of all Pnt isoforms (Boisclair Lachance et al., 2014). The authors detected Pnt-GFP fusion protein in nearly all cells of the cardiac region, but highest levels were observed in two Yan-negative clusters per hemisegment flanking Eve+ cells. We confirmed and refined these observations showing that high levels of Pnt-GFP are present in the nuclei of gCB progenitors as identified by their position, characteristically enlarged size, presence of only low levels of Doc, and absence of svp-LacZ expression (Figure 6A). We attribute these high total Pnt levels largely to a gCB-specific expression of the PntP1 isoform since PntP1-specific antibodies (Alvarez et al., 2003) specifically label gCB progenitors (Figure 6B), whereas pntP2 transcripts are present in a rather uniform pattern in the mesoderm including the cardiogenic area ((Klämbt, 1993) and data not shown). We further speculated that PntP2 could activate pntP1 transcription in gCB progenitors for a sustained signaling response as found in other tissues (Shwartz, Yogev, Schejter, & Shilo, 2013). This assumption is indeed supported by our genetic data. First, we detect PntP1 in an expanded pattern in the cardiogenic mesoderm of edl mutants in which PntP2 activity is assumed to increase (Figure 6C). Second, overexpression of edl (i.e. repression of PntP2 function) as well as genetic disruption of pntP2 resulted in a near-complete loss of cardiac PntP1 (Figure 6D,E; note persistent expression of PntP1 in other cells located more laterally). We conclude that the combined activities of Edl and PntP2 lead to the confined pntP1 expression in gCBs. The EGF Spitz appears to be a major, although not necessarily the sole factor for the MAPK-mediated activation of PntP2 in this context, because PntP1 levels are reduced but not eradicated in cardiac cells of amorphic spi mutants (Figure 6F).
The Tbx20 ortholog Midline contributes to Pnt-dependent repression of svp in the working myocardial lineage
According to the common view, we expect Pnt to act as a transcriptional activator also during CB diversification, particularly since overexpression of PntP2 fused to the VP16 activator domain has essentially the same effect on cardiac patterning as PntP1 overexpression (Figure 5H and data not shown). Therefore, its negative impact on svp expression is likely to involve Pnt-dependent activation of a transcriptional repressor. Interestingly, the T-box factor Midline (Mid), like PntP1, shows expression in early gCB progenitors (Figure 6G). We previously reported that mid functions to maintain tin expression in gCBs, thereby restricting Doc expression to oCBs (Reim et al., 2005). Consistent with this function our EMS screen also generated novel mid alleles showing the same CB patterning defects as previously described alleles (Table S1, Figure 6H and data not shown). While a direct regulation of tin by Mid was previously proposed to be responsible for these changes (supported by the gain- and loss-of-function phenotypes of mid; (Qian et al., 2005; Reim et al., 2005)), another non-exclusive scenario could involve repression of svp by Mid. Consistent with the latter, we observe a Doc-like expansion of svp expression in mid loss-of-function mutants (Figure 6I) and a reduction of svp expression upon ectopic overexpression of mid in cardiac cells (Figure 6J). The cardiac pattern phenotype of edl mid double mutants is a composite of the single mutant phenotypes. The number of oCBs (average oCBs: 24.4 ±3.6; n=6) is strongly increased as compared to edl mutants, but reduced in comparison with mid mutants, with total CB numbers being similar to those of edl mutants. In some cases a near wild type pattern is observed (Figure 6K), although many embryos display an asymmetric arrangement of CBs. While the prevalence of many Doc-negative CBs in this background implies that mid is not the only factor that limits oCB fate, it also indicates that edl is normally required in the oCB lineage to restrict mid activity, possibly by blocking a Pnt-dependent activation of mid transcription. This hypothesis is indeed supported by the reversion of ectopic Doc and svp expression in pnt mutants upon forced mid expression (Figure 6L, Figure 6-figure supplement 1C). By contrast, overexpression of the potential Mid target tin in this background only represses Doc, but not svp (Figure 6-figure supplement 1D).
To further test the idea that Mid is a repressor of oCB fate downstream of pnt, we analyzed whether it is a direct target of Pnt. Notably, an enhancer identified as a Tin target and named midE19 (mid180 for a shorter minimal version) was recently shown to drive mid expression specifically in gCBs ((Jin et al., 2013; Ryu, Najand, & Brook, 2011); Figure 7A). Consistent with our assumption that this enhancer is also a target of Pnt, very little midE19-GFP activity is detectable in pnt mutants (Figure 7B), reduced activity is observed in embryos with mesodermal edl overexpression (Figure 7-figure supplement 1A), and expanded activity is seen upon overexpression of PntP1 (Figure 7C; note occasional expansion into CBs with no detectable Tin) or PntP2VP16 (not shown). An observed reduction of midE19-driven GFP levels in many of the retained Tin+ gCBs of rho mutants (Figure 7-figure supplement 1B) corroborates that EGF signaling feeds into mid activation. The idea that mid is a target of Pnt is further supported by the almost complete elimination of reporter activity upon mutating a single ETS binding site within the mid180 minimal cardiac enhancer (Figure 7D,E) as well as the strong reduction of endogenous mid transcription in emerging CBs during germ band retraction stages in pnt mutants (Figure 7F-I). After germ band retraction, endogenous mid is activated independent of pnt in all CBs (Figure 7K) as observed in the wild type (Figure 7J) indicating that distinct mechanisms regulate mid transcription in early gCB progenitors and maturing CBs.
In sum our data lead to the conclusion that EGF signaling contributes to gCB specification by at least two distinct mechanisms, Pnt-independent specification of a subset of cardiac progenitors as well as Pnt-dependent inhibition of ostial cardioblast fate. Modulation by Edl is needed to inhibit Pnt-dependent gene activation and thus enable formation of ostial cardioblasts.
Discussion
The specification and diversification of particular cell types are linked to the establishment of lineage-specific transcriptional programs. The differences in these programs are often prompted by distinct local signaling activities. The cells in the early heart fields of Drosophila acquire their cardiogenic potential by intersecting BMP and Wnt signal activities (Frasch, 1995; Reim & Frasch, 2005; X. Wu et al., 1995), but cell diversification within this area requires additional regulatory inputs. Previous studies established that progenitors of cardioblasts, pericardial cells and dorsal somatic muscles are selected by RTK/Ras/MAPK signaling, whereas lateral inhibition by Delta/Notch signaling activity counteracts this selection in neighboring non-progenitor cells (Carmena et al., 2002; Grigorian et al., 2011; Hartenstein et al., 1992). The progenitors of the definitive cardiogenic mesoderm, which give rise to all cardiac cells except for the somatic muscle lineage-related EPCs, co-express the cardiogenic factors Tin, Doc and Pnr, a unique feature that separates them from other cells (Reim & Frasch, 2005). In addition to limiting the number of progenitors, Notch signaling has a second function during Drosophila cardiogenesis that promotes pericardial (or in thoracic segments, hematopoietic) over myocardial fate (Albrecht, Wang, Holz, Bergter, & Paululat, 2006; Grigorian et al., 2011; Hartenstein et al., 1992; Mandal, Banerjee, & Hartenstein, 2004). Other factors previously reported to impose heterogeneity in the heart field include the cross-repressive activities of the homeodomain factors Eve and Lbe (T. Jagla, Bidet, Da Ponte, Dastugue, & Jagla, 2002) as well as ectoderm-derived Hedgehog (Hh) signals (Liu et al., 2006; Ponzielli et al., 2002). In segmental subsets of cardioblasts, Hh signaling was proposed to act as a potential activator of svp in prospective oCBs (Ponzielli et al., 2002) but whether these are direct or indirect effects of Hh on these cells has not been ascertained.
Based on the findings of our study, we present a novel model of cardioblast diversification that introduces EGF signaling activities and lineage-specific modulation of the MAPK effector Pointed by Edl as crucial factors for the specification of generic working myocardial and ostial cell fates.
A novel model for cardioblast diversification connecting EGF signaling, ETS protein activity and lineage-specific transcription factor patterns
Combining previous findings with our new data we have conceived the regulatory model of cardioblast diversification illustrated in Figure 8. We propose that EGF/MAPK signaling promotes the development of generic working myocardial progenitors (red/left cell in Figure 8) by two mechanisms that differ in their requirement for the ETS protein Pnt:
1) EGF promotes the correct selection and specification of gCB progenitors. This is evident from our loss- and gain-of-function analysis of EGF signaling components. Notably, in most hemisegments of the analyzed EGF pathway mutants the number of gCBs is reduced by even numbers, indicating a defect prior to completion of the final mitotic division at the progenitor stage. This EGF function is obviously independent of pnt, since pnt null mutants display excessive numbers of CBs (with gCB numbers comparable to the wild type or even increased), a phenotype different from that of mutants defective in EGF pathway components upstream of Pnt ((Alvarez et al., 2003) and this study).
2) EGF signals affect the diversification of CB progenitors by impinging on a PntP2-dependent transcriptional cascade that eventually leads to suppression of Tin- oCB and the adoption of Tin+ gCB fates. This function is mediated by stimulating the gCB progenitor-specific expression of regulatory genes such as mid (depicted in red in Figure 8), which in turn will promote transcription of gCB-specific differentiation genes and/or repression of oCB-specific factors (depicted in green in Figure 8).
Basic features of gene regulation in the gCB lineage
We identified mid as a key target gene of Pnt in gCB progenitors based on its early gCB-specific expression, Pnt-dependent transcriptional regulation and its ability to repress the oCB-specific regulator gene svp. Since Svp represses tin expression (Gajewski et al., 2000; Lo & Frasch, 2001), svp suppression provides an important part of the explanation for the previously reported positive role of Mid in maintaining tin expression in gCBs (Qian et al., 2005; Reim et al., 2005), although it does not exclude the possibility that Mid stimulates tin expression also directly. Of note, the vertebrate Mid ortholog Tbx20 is also a promoter of working myocardial fate that can act as transcriptional activator and repressor depending on context (Cai et al., 2005; M. K. Singh et al., 2005; Stennard et al., 2003; Stennard et al., 2005; Takeuchi et al., 2005). While Tin acts as a repressor of Doc via unknown mechanisms in gCBs, it does not repress svp ((Zaffran et al., 2006) and Figure 6-figure supplement 1D). On the contrary, at least in the early cardiogenic mesoderm, it acts as an activator of svp in oCB progenitors (Ryan, Hendren, Helander, & Cripps, 2007). Thus, in the absence of appropriate repressors such as Mid, svp expression can expand into gCBs.
Basic features of gene regulation in the oCB lineage
In prospective oCB progenitors, Pnt activity must be kept in check to permit svp expression and thereby tin repression and Doc activity. Fittingly, we identified edl, a gene linked to negative regulation of MAPK signaling and cell identity determination in several tissues - including the eye (Yamada et al., 2003) and recently in certain somatic muscle progenitors (Dubois, Frendo, Chanut-Delalande, Crozatier, & Vincent, 2016) - as a novel regulator in the context of cardiac cell specification, particularly that of oCB progenitor fate (green/right cell in Figure 8). This function was first hinted at by the over-proportional increase of svp-expressing oCBs in pnt mutants (Alvarez et al., 2003). Our phenotypic analysis demonstrates that Edl is required for svp and Doc gene activity (the latter being due to restriction of tin expression) as well as the restriction of PntP2-dependent PntP1 expression in cardiac progenitors. Molecularly, Edl can modulate the activities of PntP2 as well as Yan (Baker et al., 2001; Qiao et al., 2006; Qiao et al., 2004; Tootle et al., 2003; Vivekanand et al., 2004; Yamada et al., 2003). The comparison of single and double mutant phenotypes, combined with the reproducibility of nearly all aspects of the cardiac pnt phenotype by Edl overexpression, implies that Edl acts primarily by inhibiting Pnt during cardiac cell diversification, although we cannot fully exclude additional interactions with Yan. Our observations further support the function of Edl as an antagonist of Pnt (first demonstrated in the context of eye and chordotonal organ development, (Yamada et al., 2003)) and rule out an initially proposed Pnt-stimulating function (Baker et al., 2001).
Linkage of MAPK and Pnt activities
The involvement of Edl also leads to important conclusions regarding the placement of Pnt function within the cardiac gene regulatory network. Based on the phenotypic discrepancies between pnt and other EGF pathway components (gain and loss of CBs, respectively), Alvarez et al. proposed that PntP2 acts independent of MAPK signaling to limit the number of CBs (Alvarez et al., 2003). Since we found that Edl blocks Pnt activity in oCB progenitors, and Edl is thought to antagonize PntP2 mainly by blocking MAPK-dependent phosphorylation (Qiao et al., 2006), we propose that PntP2 acts downstream of MAPK also during cardiogenesis (see Figure 8). This is further supported by our data demonstrating spi-sensitive cardiac expression of PntP1 and the observation that, if timed properly, both EGF and Pnt activities can lead to expanded gCB and reduced oCBs populations. However, not all MAPK activities require pnt, which is the case for the pro-cardiogenic activities of EGF. Notably, parallel pnt-dependent and pnt-independent MAPK signaling functions take place also during other processes such as epithelial branching morphogenesis (Cabernard & Affolter, 2005).
EGF signaling and cardiac progenitor selection
As discussed above, cardioblast formation as such is independent of pnt. How could this be achieved? Growth factor-activated MAPK can also phosphorylate the repressor Yan thereby diminishing its activity as an antagonist of progenitor selection (Halfon et al., 2000; O'Neill et al., 1994; Rebay & Rubin, 1995). Therefore, it is conceivable that MAPK activity in the context of CB progenitor selection might be primarily required to eliminate the repressive activity of Yan. This would be consistent with the observed reduction of cardiac cells upon aop/yan hyperactivation ((Halfon et al., 2000) and this study). In this context, a minor function of Edl could contribute to the robustness of cardiac progenitor selection and thus total cardioblast and pericardial cell numbers by reducing the repressive Yan activity.
According to our data, EGF signals are the major source for MAPK activation and progenitor specification in the symmetrically dividing progenitors of gCBs and OPCs. By contrast, EGF signals are dispensable (in high doses even unfavorable) for the development of progenitors of oCBs and their sibling OPCs. Thus, EGF signaling appears to be more critical for cell fate specification of cardiac cells than for their mere survival. Our overexpression studies demonstrate that the timing of EGF signals is crucial for this function. In previous studies, earlier functions of MAPK signaling might have obscured its specific impact on gCBs and OPC subtypes. While early pan-mesodermal activation of MAPK signaling or expression of constitutive active Pnt forms via the twi-GAL4 driver reduces the numbers of all cardiac cells except the Eve+ progenitors ((Alvarez et al., 2003; Bidet et al., 2003; Liu et al., 2006) and our own data), later MAPK activation favors formation of the symmetrically dividing OPC and gCB progenitor subpopulations (based on our experiments with tinD-GAL4-driven rho; increased total numbers of OPCs and CBs were also observed after expressing activated forms of Ras or the receptors of FGF and EGF with a Mef2-GAL4 driver, (Grigorian et al., 2011)). We propose that the specification of these progenitors requires the context of the definitive cardiogenic mesoderm, whereas premature MAPK activation in all mesoderm cells negates any pro-cardiogenic effects due to the massive expansion of Eve+ clusters (which are normally the first cells in the heart field to display MAPK and rho activity) at the expense of the cardiac progenitors in the neighboring C14/C16 clusters (Buff et al., 1998; T. Jagla et al., 2002; Liu et al., 2006; Qian et al., 2005); and our own data not shown).
Special features of Pnt-dependent regulation in working myocardial cells
Our model of CB diversification incorporates the observation that the PntP1 isoform is activated specifically in gCB progenitors in a PntP2-dependent and EGF-sensitive fashion. This is reminiscent of the situation in other tissues such as the developing eye where the PntP1 isoform is also activated in a MAPK/PntP2-dependent manner (Gabay et al., 1996; O'Neill et al., 1994; Shwartz et al., 2013). We propose that PntP1 becomes activated at a particular threshold of MAPK/PntP2 activity. This activation marks a point of no return for CB diversification, because PntP1 cannot be inhibited via Edl. The activation of PntP1 also explains why edl overexpression with relatively late acting drivers such as tinD-GAL4 (as used in the edl mutant rescue experiment) does not cause the cardiac phenotypes observed with early pan-mesodermal drivers.
Besides pntP1 and the already discussed mid gene, there are very likely additional target genes activated by PntP2 and/or PntP1 to execute the differentiation program in generic working myocardial cells. Incomplete conversion of gCBs in mid mutants also calls for the existence of additional repressors that contribute to oCB fate suppression. Interestingly, a study investigating Tin target genes found that cardiac target enhancers of Tin are not only enriched for Tin binding sites but also for a motif highly reminiscent of ETS binding sites, termed "cardiac enhancer enriched (CEE) motif" (with the consensus ATT[TG]CC or GG[CA]AAT in antisense orientation) (Jin et al., 2013). Mutation of four CEE sites (one of which overlapping our predicted ETS binding site) in a ca. 600 bp version of the midE19 enhancer nearly abolished reporter activity in that study. Thus, many of the CEE-containing Tin target enhancers might in fact also be targets of Pnt (potentially mediating ETS-dependent activation) or Yan (potentially mediating ETS-dependent repression in the absence of MAPK signals). Therefore, a combination of closely spaced Tin and ETS binding sites might be a key signature in enhancers of working myocardial genes, although additional features must be present in their architecture to distinguish them from Tin+ETS-binding site containing enhancers active in pericardial cells or their progenitors (Halfon et al., 2000). The differences might include elements directly or indirectly regulated by Delta/Notch signaling. Notably, the juxtacrine Notch ligand Delta is upregulated in the CB lineage in an MAPK activity-dependent manner (Grigorian et al., 2011). Hence, it is conceivable that Pnt proteins might stimulate Delta transcription in gCBs to control OPC development in a non-autonomous manner. This would explain both, simultaneous mis-specification of gCB progenitors and non-ostial-related OPCs in EGF mutants as well as phenotypic similarities between pnt mutants and mutants for components of the Delta-Notch signaling pathway. However, Notch pathway mutants do not display a biased increase of oCBs ((Albrecht et al., 2006) and our own data not shown) because of the herein described function of Pnt in suppressing svp transcription and oCB fate.
What is the original signal that discriminates generic and ostial progenitors?
Our model proposes that factors that tilt the balance between PntP2 activity and Edl will have a major impact on CB subtype choice (see Figure 8). Thus any input that modestly increases MAPK/PntP2 activity within the appropriate window of time would favor gCB fate, whereas factors that have the opposite effect should promote oCB specification. This points to activities that impinge on the highly complex and dynamic expression of rho, because the Rhomboid protease is a key determinant in the decision of which cells will activate the more broadly expressed EGF Spitz and thus emanate signaling activity. A candidate would be Hh, which was proposed to be an oCB-promoting factor (Ponzielli et al., 2002). However, its effect on MAPK and rho activity in the dorsal mesoderm was suggested to be positive rather than negative (Liu et al., 2006). This would refute a function favoring oCB fate. The regulation of rho and the role of hh during CB diversification await more detailed analysis in future studies.
Factors that regulate edl expression levels might also determine the outcome of the competition between Edl and Pnt. The edl gene was found to be positively regulated by EGF signaling, and a target of Pnt and Yan, and thus was proposed to provide a negative feedback system for EGF inputs (Baker et al., 2001; Leatherbarrow & Halfon, 2009; Vivekanand et al., 2004; Yamada et al., 2003). Our model therefore includes regulation by Pnt as a possibility (dashed arrows in Figure 8). ChIP-on-chip experiments suggest that edl is also targeted by cardiogenic factors (Junion et al., 2012).
The spatio-temporal dynamics and detailed mechanisms that regulate MAPK and edl activities within the cardiogenic mesoderm remain to be investigated in future studies. Such studies may also help to understand lineage decisions in other tissues and species. Edl/Mae-relatives are also present in non-Dipteran insects (e.g. Tribolium, (Bucher & Klingler, 2005)), echinoderms, and the chordate Ciona. Although no clear ortholog of Edl appears to be present in vertebrates, a SAM domain-only isoform of the human Yan-relative TEL2 as well as Drosophila Edl were shown to inhibit transcriptional stimulation by the mammalian Pnt orthologs ETS1/ETS2 in cell culture (Gu et al., 2001; Vivekanand & Rebay, 2012). Hence, the restriction of ETS protein activities by protein-protein interactions offers an intriguing mechanism to fine-tune MAPK signaling output in developing tissues of both invertebrates and vertebrates.
Materials and Methods
Drosophila melanogaster stocks
The mutants Df(2R)edl-S0520, EgfrS0167, EgfrS2145, EgfrS2307, EgfrS2561, midS0021, midS2961, numbS1342, numbS3992, numbS4439, pyrS3547 (Reim, Hollfelder, Ismat, & Frasch, 2012), spiS3384, StarS4550 were recovered from our EMS screen. The lines mid1, UAS-mid-B2, how24B-GAL4, pnrMD237-GAL4, svpAE127-lacZ (a svp mutant in homozygous condition), UAS-svp.I, 2xPE-twi-GAL4, twi-SG24-GAL4, tinD-GAL4, UAS-tin#2, {tin-ABD}T003-1B1; tinEC40, UAS-p35 were as described previously (Reim et al., 2012; Reim et al., 2005; Zaffran et al., 2006). In addition the following strains were used: aop1=aopIP (Nüsslein-Volhard, Wieschaus, & Kluding, 1984; Rogge et al., 1995), UAS-aop.ACT-IIa (Rebay & Rubin, 1995), edlL19=Df(2R)edl-L19 (edl and some neighboring genes deleted) and UAS-edl-X (both from Y. Hiromi, (Yamada et al., 2003)), P{lacW}edlk06602 (Baker et al., 2001; Török, Tick, Alvarado, & Kiss, 1993), UAS-EgfrDN.B-29-77-1;UAS-EgfrDN.B-29-8-1 (Buff et al., 1998), htlYY262 (Gisselbrecht, Skeath, Doe, & Michelson, 1996), midE19-GFP ((Jin et al., 2013); from M. Frasch), pntΔ88 (Scholz, Deatrick, Klaes, & Klämbt, 1993), pntMI03880 (PntP2-specific; harbors a gene-trap cassette with an artificial splice acceptor followed by stop codons upstream of the pntP1 transcription start site, (Venken et al., 2011)), UAS-pntP2VP16-2 ((Halfon et al., 2000); originally from C. Klämbt), UAS-pntP1-3 and UAS-pntP2-2 (Klaes, Menne, Stollewerk, Scholz, & Klämbt, 1994), PBac{pnt-GFP.FPTB}VK00037 (R. Spokony and K. White, (Boisclair Lachance et al., 2014)), pyr18 and ths759 (Klingseisen, Clark, Gryzik, & Müller, 2009), rho7M43 (Jürgens, Wieschaus, Nüsslein-Volhard, & Kluding, 1984), rhoL68 (Salzberg et al., 1994), rhoEP3704 (Bidet et al., 2003), UAS-rho(ve.dC) (de Celis, Bray, & Garcia-Bellido, 1997), spi1=spiIIA14 (Nüsslein-Volhard et al., 1984), StarB0453 ((Chen et al., 2008), from F. Schnorrer), tinCΔ4-GAL4 ((Lo & Frasch, 2001), from M. Frasch), Df(2R)Exel7157, and about 180 additional deficiencies spanning chromosome 2 (except where noted, all stocks available from the Bloomington Stock Center).
Flies expressing edl+ from a transgene were generated anew by standard P-element transgenesis using the previously described rescue construct edl[+t18] ((Yamada et al., 2003), provided by Y. Hiromi).
Unless noted otherwise, y w or S-18a-13b-16c.1 control (Hollfelder et al., 2014) flies were used as wild type controls. Mutant lines were maintained over GFP- or lacZ-containing balancer chromosomes to allow recognition of homozygous embryos. Flies were raised at 25°C, except for UAS/GAL4-driven overexpression at 29°C.
Isolation and mapping of novel EMS mutants
Novel EMS-induced mutants were obtained from our screen for embryonic heart and muscle defects and mapped to a particular gene through extensive complementation testing analogous to the previously described procedure (Hollfelder et al., 2014). Many alleles were mapped by unbiased complementation tests with a set of chromosome 2 deficiencies and subsequent non-complementation of lethality and embryonic phenotype by previously described alleles. Df(2R)edl-S0520 was mapped by non-complementation of lethality with Df(2R)Exel7157, Df(2R)edl-L19 and Df(2R)ED3636, but the cardiac phenotype was only reproduced in trans with Df(2R)Exel7157, Df(2R)edl-L19 and edlk06602. Novel alleles of Egfr and Star were mapped using a candidate gene approach.
Molecular analysis of mutations and deletions
Several EMS alleles and the unmutagenized S-18a-13b-16c.1 control were analyzed by sequencing of overlapping PCR products covering the coding sequence and splicing sites of the candidate gene as described (Hollfelder et al., 2014). Details about the mutations are provided in Table S1. The area deleted by Df(2R)edl-S0520 and its approximate break points were determined by iterative PCR amplification tests. The insertion of P{lacW}edlk06602 near the edl transcription start site was confirmed by PCR using primers binding to the 5' P end and adjacent genomic DNA. Although the integrity of the both P element ends could be confirmed by PCR, no genomic edl sequences expected next to the 3' P end could be amplified using several primer pairs shown to amplify control DNA. This indicates that P{lacW}edlk06602 is associated with a deletion in edl. Details of the deletion mapping are listed in Table S2.
Generation of reporter constructs for enhancer analysis
The mid180-GFP reporter constructs were generated according to a similar lacZ construct published by Ryu et al. (Ryu et al., 2011). The forward primer 5'-EcoRI-CGTGCCTCCCACTTCAGGGCGG-3' and the backward primer 5'-BamHI-TTAATTTCATTTTTCACTCTGCTCACTTGAGATTCCCCTGCTTTGTCTGCGGCATTTCCGCTTCT-3' were used to amply DNA from y w flies. The predicted ETS binding site matching the antisense sequence of published ETS binding motifs ((Halfon et al., 2000; Hollenhorst, McIntosh, & Graves, 2011), underlined) was mutated by replacing the invariable TCC core (bold) with AAA in the backward primer. Amplicons were cloned into EcoRI/BamHI of pH-Stinger-attB (Jin et al., 2013), sequenced and inserted into the attP2 landing site via nos-driven ΦC31 integrase.
Staining procedures
Embryo fixations, immunostainings for proteins and RNA in situ hybridizations were carried out essentially as described (Knirr, Azpiazu, & Frasch, 1999; Reim & Frasch, 2005). VectaStain Elite ABC kit (Vector Laboratories) and tyramide signal amplification (TSA, PerkinElmer Inc.) were used for detection of RNA and certain antigens (as indicated). The following antibodies were used: guinea pig anti-Doc2+3 (1:2000, TSA) and anti-Doc3+2 (1:1000) (Reim et al., 2003), rabbit anti-H15/Nmr1 (1:2000), guinea pig anti-H15/Nmr1 (1:2000), rabbit anti-Mid/Nmr1 (early stages: 1:250, TSA; late stages: 1:1000 direct) and rabbit anti-PntP1 (1:250, TSA) (all from J. Skeath; (Alvarez et al., 2003; Leal, Qian, Lacin, Bodmer, & Skeath, 2009)), rabbit anti-Mef2 (1:1500) (from H.T. Nguyen), rat anti-Odd (1:600, TSA) (Kosman, Small, & Reinitz, 1998), rabbit anti-Eve (1:3000) (Frasch, Hoey, Rushlow, Doyle, & Levine, 1987), rabbit anti-Tin (1:750) (Yin, Xu, & Frasch, 1997) (all from M. Frasch), rabbit anti-β-galactosidase (Promega, 1:1500), rabbit anti-GFP (Molecular Probes, 1:2000 and Rockland, 1:1000), mouse anti-GFP 3E6 (Life Technologies, 1:100, TSA), anti-cleaved-Caspase-3 (Asp175, Cell Signaling Technology, 1:100, TSA), sheep anti-Digoxigenin (Roche, 1:1000, TSA), monoclonal mouse antibodies anti-β-galactosidase 40-1a (1:20 direct or 1:50 with TSA), anti-Seven-up 5B11 (1:20, TSA) and anti-Wg 4D4 (1:30, TSA) (all from Developmental Studies Hybridoma Bank, University of Iowa), fluorescent secondary antibodies (1:200) (Jackson ImmunoResearch Laboratories and Abcam), biotinylated secondary antibodies (1:500) and HRP-conjugated anti-rabbit IgG (1:1000) (Vector Laboratories). TUNEL staining was performed as described (Reim et al., 2003) using the Millipore ApopTag S7100 kit in combination with TSA.
Digoxigenin-labeled antisense riboprobes against mid, edl, rho and pntP2 were used for whole mount in situ hybridizations. The mid probe was generated as described previously (Reim et al., 2005). T7 promoter-tagged edl, rho and pntP2 (isoform-specific exons) templates for in vitro transcription were generated by PCR (primers edl: CAATCGTGAAAGAGCGAGGGTC, T7-TGACGAGCAGAACTAAGGACTAGGC, pnt: CCAGCAGCCACCTCAATTCGGTC, T7-GCGTGCGTCTCGTTGGGGTAATTG, rho: ATGGAGAACTTAACGCAGAATGTAAACG, T7-TTAGGACACTCCCAGGTCG) from DNA of wild-type flies or flies carrying UAS-rho(ve.dC) or UAS-pntP2, respectively.
Embryos were mounted in Vectashield (Vector Laboratories). Images were acquired on a Leica SP5 II confocal laser scanning microscope and projected using Leica LAS-AF and ImageJ.
Competing interests
The authors declare that no competing interests exist.
Author Contributions
BS, IR conceived, designed and performed the experiments, analyzed and interpreted the data, wrote the manuscript. DH, KS, LH performed the experiments, analyzed and interpreted the data.
Funding
This research was supported by grants from the Deutsche Forschungsgemeinschaft.
Acknowledgements
We are grateful to Manfred Frasch for critical reading of the manuscript, Patrick Lo and Christoph Schaub for their contributions to the EMS screen, Edmar Heyland, Emi Vargatoth, Tanja Drechsler and Angela Bruns for technical assistance. We thank Manfred Frasch, Yasushi Hiromi, James Skeath, Hanh Nguyen, Frank Schnorrer, the Bloomington Stock Center and the Developmental Studies Hybridoma Bank (University of Iowa) for providing fly stocks or reagents.