The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm

Abstract

Blood progenitors arise from a pool of pluripotential cells (“hemangioblasts”) within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos, is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters (“cardiogenic clusters”) that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts.

Introduction

Cells of the blood, vascular and excretory system are developmentally related in all animals. In vertebrates, the lateral plate mesoderm gives rise to the progenitor cells of endothelia and blood cells (hemangioblasts) (Al-Adhami and Kunz, 1977; Coffin and Poole, 1988; Medvinsky et al., 1993; Cleaver et al., 1997; Eichmann et al., 1998; Gering et al., 1998; Crosier et al., 2002; Hartenstein, 2006; Xiong, 2008; Bertrand and Traver, 2009). Adjacent to the lateral plate is the intermediate mesoderm that produces the nephrocytes of the excretory system (pro-, meso-, metanephros). Hemangioblasts leave the lateral plate and migrate dorsally to differentiate into a system of endothelial tubes that pioneer the major blood vessels, including the aorta and cardinal veins. Progenitors of adult blood cells are specified within the walls of these primitive vessels; they are budded off into the vessel lumen and eventually settle in the blood forming organs (“definitive hematopoiesis”). In addition to these adult blood progenitors, blood cells populating the embryo are formed within the ventral blood islands and yolk sac (“primitive hematopoiesis”).

In Drosophila, progenitors of the blood, vascular and excretory system originate from a lateral domain of the mesoderm, called the cardiogenic mesoderm (Hartenstein et al., 1992; Bate, 1993; Rugendorff et al., 1994; Zaffran et al., 2002; Mandal et al., 2004). The cardiogenic mesoderm is comprised of ten paired, segmentally repeated clusters of cells, three thoracic and seven abdominal clusters. Each cluster gives rise to a set of cardioblasts which assemble into a contractile, myo-endothelial tube, the dorsal vessel (or “heart”) of the fly (Bodmer et al., 1997). In addition, the abdominal cardiogenic mesoderm produces groups of nephrocytes (“pericardial nephrocytes”) which form part of the excretory system (Crossley, 1985; Rugendorff et al., 1994; Ward and Skeath, 2000). Progenitors of adult blood cells (prohemocytes) arise within the three thoracic cardiogenic mesodermal clusters. These prohemocytes aggregate together and form the so called “lymph gland” that flanks the anterior tip of the dorsal vessel in the late embryo and larva (Rugendorff et al., 1994; Mandal et al., 2004). Prohemocytes proliferate during larval stages and are released into the hemocoel (the body cavity of the insect) during metamorphosis (Lanot et al., 2001; Unpublished data). Aside from this late embryonic/postembryonic “definitive” phase of blood development, there exists, just as in vertebrates, an early, “primitive” phase of blood formation that takes place in the mesoderm of the head of the early embryo (Tepass et al., 1994; Holz et al., 2003; Evans et al., 2003b; deVelasco et al., 2006).

Molecular factors specifying cells of the blood-vascular and nephrocyte lineages, as well as the signaling mechanisms controlling the expression of these factors, are highly conserved among all animals (reviewed in Zaffran et al., 2002; Evans and Banerjee, 2003a; Hartenstein and Mandal, 2006; Hartenstein, 2006; Crozatier and Meister, 2007; Martinez-Agosto et al., 2007). Hemangioblasts of the vertebrate lateral plate and Drosophila cardiogenic mesoderm are induced from naive mesoderm by several signaling pathways that include Bone morphogenetic protein (BMP)/Decapentaplegic (Dpp) and Fibroblast Growth Factor (FGF)/Heartless (Htl). The activity of these signals is required for upregulating blood and vascular/heart determinants such as the GATA factors/Serpent (Srp) or Nkx2.5/Tinman (Tin), and thereby specifies and maintains the fate of hemangioblasts (Baron, 2003; 2005; Crosier et al., 2002; Maeno, 2003). Notch signaling plays an important role in specifying blood progenitors from the bi-potential (vascular/blood) hemangioblasts. Thus, Notch activation in the endothelial hemangioblasts of vertebrate embryos, results in the expression of blood cell determinants such as GATA2, Stem Cell Leukemia (SCL) and Acute Myeloid Leukemia 1 (AML1), placing the Notch signaling pathway high up in the molecular network initiating hematopoiesis (Kumano et al., 2003; Hadland et al., 2004; Robert-Moreno et al., 2005; Burns et al., 2005). Again, the same switch between vascular cells and blood progenitors is under the control of Notch signaling in Drosophila: Loss of Notch activity during the phase when the fate of cardiogenic mesodermal cells is specified results in the absence of blood progenitors and an excess of cardioblasts (Mandal et al., 2004). Whereas the important role of Notch signaling for blood progenitor fate in Drosophila, as in vertebrates, is well established, the signal that activates the Notch receptor, and the mechanism by which the signal itself becomes active in the restricted spatial pattern that guaranteeing the proper number and distribution of blood progenitors, has not so far been elucidated. In this paper we address these questions in Drosophila, taking advantage of the fact that the Drosophila cardiogenic mesoderm is comprised of only a few cells, many of which can be individually marked by molecular reagents.

The Drosophila mesoderm arises as a single cell layer during gastrulation. Subsequently, mesodermal cells undergo two parasynchronous mitotic divisions (Campos-Ortega and Hartenstein, 1985; Bate, 1993). During this early phase, genes which later demarcate specific mesodermal lineages, such as tin or Myocyte Enhancer Factor 2 (Mef2), are expressed in the entire mesoderm (Azpiazu and Frasch, 1993; Bodmer, 1993; Lilly et al., 1994; Taylor et al., 1995), suggesting that mesodermal cells are equipotent and have not yet split up into discrete lineages. The specification of different lineages is initiated by a series of signaling steps, which include the Wingless (Wg), Notch, Dpp, Epidermal Growth Factor (EGF), and FGF pathways (Bate, 1993; Zaffran et al., 2002; Hartenstein, 2006). As a result, the mesoderm of each segment becomes subdivided into four quadrants (Borkowski et al., 1995; Riechmann et al., 1997; Mandal et al., 2004; Martinez-Agosto et al., 2007; Fig. 1): High levels of Tin expression, dependent on both Fibroblast Growth Factor Receptor (FGFR) activity and an ectodermally derived Dpp signal, define the dorsal mesoderm. The anterior, Wg-positive dorsal mesoderm forms the cardiogenic mesoderm while the Wg-negative posterior dorsal mesoderm becomes the visceral mesoderm. The two ventral quadrants give rise to the somatic musculature (Wg-positive, anteriorly) and fatbody (Wg-negative, posteriorly). The expression of cell type specific genes at this stage indicates that fat body, somatic mesoderm and visceral mesoderm have become molecularly distinct entities. By contrast, the cardiogenic mesoderm at this stage still represents a mixed population of cells with different fates. These fates include cardioblasts, pericardial nephrocytes and at least two other, different types of “pericardial” cells (Even-skipped (Eve) and Tin-positive pericardial cells), blood progenitors, and dorsal muscle cells (Ward and Skeath, 2000; Knirr and Frasch, 2001).

Fig. 1.

Partitioning of the mesoderm by the Wingless (Wg) and Decapentaplegic (Dpp) signals. cm cardiogenic mesoderm; fb fat body sm somatic mesoderm; vm visceral mesoderm. Combined Notch and MAPK activity defines smaller clusters (“myogenic and cardiogenic clusters”) in the somatic and cardiogenic mesoderm (light colored ovals).

Cell-cell interactions depending on Notch, EGF and FGF signaling single out from within the Wg-positive somatic and cardiogenic mesoderm 19 small clusters of cells, called C1-C19 (Carmena et al., 1995; 1998; 2002; Buff et al., 1998; Fig. 5A). These are revealed by the expression of the bHLH transcription factor Lethal of scute (L’sc), as well as the expression of phosphorylated Mitogen Activated Protein Kinase (pMAPK), a reporter for EGF and FGF signaling activity. Clusters of the somatic mesoderm (C1, C3-C13, C17) give rise to the so-called muscle founder cells, which act as pioneers to specify the pattern of somatic muscles (Bate, 1993; Carmena et al., 1995). While C18 and C19 have not been as thoroughly characterized as the rest, it is known that C18 expresses higher levels of L’sc in thoracic than in the abdominal segments, while C19 is found only in thoracic segments (Carmena et al., 1995). We show, in this study, that the dorsal clusters (C2; C14-C16) constitute the definitive cardiogenic mesoderm that gives rise to cardioblasts, blood progenitors and nephrocytes. As the cardioblasts emerge they accumulate high levels of the Notch ligand Dl, which is required to restrict the number of cells adopting the cardioblast fate. Loss of Dl converts all cells of the cardiogenic mesoderm into cardioblasts. Activation of the MAPK pathway by EGFR and FGFR is required for the specification and maintenance of the cardiogenic mesoderm, and is also likely to be involved in the spatial restriction of the Dl ligand to cardioblasts.

Fig. 5.

Overlapping expression of Dl, L’sc and pMAPK in the cardiogenic mesoderm. A: Map of the pattern of L’sc/pMAPK-positive mesodermal clusters of one segment (adapted from Carmena et al., 1995). B-G: Three pairs of photographs (B, C; D, E; F, G) show a lateral view of the mesoderm of two consecutive trunk segments. Upper panel of each pair (B, D, F) represents early stage 11; lower panel (C, E, G) early to mid stage 12 (stage 12B). B and C show in situ hybridizations, with a probe against l’sc. In D and E, a double labeling of anti-Dl (green) with anti-Phosphorylated MAP kinase (pMAPK, red) is shown. Specimens shown in F and G were labeled with anti-Dl (grey). Note that the dorsal clusters (C2 at stage 11; C14 and C16 at stage 12) that express l’sc (B, C) and pMAPK (D, E) correspond to the anterior (a) and posterior (p) high-Dl clusters in the cardiogenic mesoderm (F, G). A light blue circle represents the area where cluster C2 is found (F). Dark blue circles represent areas where clusters C14 and C16 are located (G). The purple circle represents the area where cluster C15 is located (G).

Abbreviations: dt dorsal tracheal branch; tp tracheal pit. Yellow bracket in D indicates spatial proximity between dorsal tracheal branch and posterior high-Dl cluster (see also Fig. 3). Large numbers in (A) identify the L’sc-positive mesodermal clusters; small numbers underneath indicate number of cells per cluster. Bar: 20μm (B-G).

MATERIALS AND METHODS

Fly Stocks

Delta9p, Df(1)scB57, dppH46, dpp-lacZ, EGFRf2, htlAB42, spitz-lacZ, UAS-EGFR DN, UAS-htl Constitutively activated, UAS-RasV12, UAS-dpp, UAS-wg, wgCX4 (Bloomington Stock Center). 12X Su(H)-lacZ (construct with 12 Su(H) binding sites upstream of a lacZ; Go et al., 1998), Mef2-gal4 (Ranganayakulu et al. 1996), UAS-EGFR activated #3 (UAS-λ top 4.4; Queenan et al. 1997, kindly gifted by T. Schupbach), UAS-l’sc (Carmena et al. 1995), UAS-l’sc Rnai (NIG: Japan). htl-lacZ (Stathopoulos et al. 2004), thisbe-lacZ #2 (Stathopoulos et al. 2004), Df(2R)ths238 and Df(2R)pyr36 (Kadam et al. 2009), wg-lacZ (NIG: Japan N755).

Antibodies

Antibodies used for Immunohistochemistry include: α-Mouse-β-Galactosidase (1:50) (Promega), α-Rabbit-β-Galactosidase (1:2500) (Cappel), α-Guinea Pig-Delta (1:1000)(Huppert et al., 1997), α-Rat-EGFR (1:500) (Kindly gifted by B. Shilo), α-Rat-L’sc (1:800)(Martín-Bermudo et al., 1991, kindly gifted by A. Carmena), α-Rabbit-Mef2 (1:1500) (Nguyen et al., 1994), α-Rabbit-Odd (1:500) (Ward and Skeath, 2000), α-Mouse-Pericardin (1:3) (Developmental Hybridoma Bank), α-Mouse-pERK (pMAPK) (1:50) (Sigma), α-Rabbit-Tinman (1:800) (Venkatesh et al., 2000).

Embryo Fixation

Regular

Flies were placed on fruit juice plates for overnight egg laying collection. Embryos/eggs were treated with 100% bleach for 6 minutes and then washed with water. Embryos were then fixed in a solution of 3.5 ml PEMS, 3.5 ml Heptane and 1.5 mL 37% Formaldehyde for 35 minutes. PEMS (pH 7) is a solution consisting of 100 mL PIPES (0.2 M solution, pH 7), 90 mL of Deionized water, 8 mL EGTA (0.05 M solution) and 2 mL MgSo4 (0.1 M solution). The bottom phase of the solution (PEMS) was then removed and MeOH was added in an amount comparable to the PEMS removed. Vial was shaken vigorously and settled embryos were collected and stored in EtOH at −20 C till use.

Embryo Fixation for pMAPK Staining

Above fixation technique was carried out with the following modification: 10mM of Sodium Orthovanadate and 1:500 of 30% Hydrogen Peroxide (H202) were added to the PEMS solution. Also, for pMAPK staining, fixed embryos were stained immediately with pMAPK antibody.

Staging of embryos

We followed the system of stages introduced by Campos-Ortega and Hartenstein (1985). To recognize smaller steps within stages 11 (late extended germband) and 12 (germband retraction), each of which lasts for about 2 hours, we defined morphological criteria that are easily recognizable without specific markers. Stage 11 and 12 were subdivided into three roughly equal intervals called 11A (early 11), 11B (mid 11), 11C (late 11), 12A (early 12), 12B (mid 12) and 12C (late 12). Defining criteria for these stages:

• 11A: Tracheal pits start to form and progress to hemispherical invaginations

• 11B: Tracheal pits form vertical slits; salivary placode can be distinguished as a thickened part of the ventral ectoderm of the labial segment.

• 11C: Salivary placode starts to invaginate

• 12A: Salivary placode invaginates to form a straight, vertical, tube-shaped primordium. Germband starts to retract (83% to 75%)

• 12B: Salivary primordium elongates and is bent posteriorly at posterior tip. Germband retracts to 50%

• 12C: Stage of rapid germband retraction (50%–0%)

Each of these stages can be further subdivided into intervals of 10–15 min length, each defined by clear (if subtle) morphological changes (V. Hartenstein, unpublished data).

Immunohistochemistry

Regular

Samples were washed with 0.1% PBT three times for 15 minutes each at room temperature while rotating. Samples were then blocked for 30 minutes in a solution of 10% NGS made in 0.1% PBT. Samples then placed in 1 antibody solution made in 10% NGS and left to rotate overnight at 4 C. Samples were then washed and blocked as described above and placed in a 2 antibody conjugated to a fluorescent marker. Samples were left in 2 antibody to rotate overnight at 4 C. Samples were then washed as described above and mounted in Vectashield (Vector Laboratories) containing a 1:500 concentration of Toto-3-iodide (Invitrogen).

L’sc antibody staining

Used Perkin-Elmer Life Sciences Renaissance Tyramide Signal Amplification Biotin System (Catalog # NEL700A).

Insitu Hybridization

L’sc RNA probe synthesis

L’sc clone (RE59335) was received from Berkeley Drosophila Genome Project (BDGP). Plasmid was digested with appropriate restriction enzyme. Then treated with 300 μL of TE or water. Phenol/Chloroform extraction was carried out by adding 150 μL of each agent and then centrifuging DNA twice at 12,000 rpm for 5 minutes each. The third time 300 μL of Chloroform was added and centrifugation was carried out as described above. The upper phase was saved each time and moved to a new centrifuge tube. After final spin, 270 μL of the upper phase was transferred to a new tube and 27 μL of 3M sodium acetate and 210 μL of isopropanol was added. Sample was mixed well between addition of each agent. Sample was then incubated for 20 minutes at room temperature. The sample was then spun at 15,000 rpm for 15 minutes. The supernatant was discarded and the pellet was washed with 500 μL of 70% EtOH. Sample was spun again for 5 minutes at 15,000 rpm. The supernatant was again discarded and the pellet was allowed to air dry for 10 minutes. Pellet was then dissolved in 16 μL TE. Probe was then synthesized by placing 1 μg of prepared template DNA with 4μL of 5X transcription buffer, 2 μL of 100 mM DTT, 2 μL Dig-NTP mix, 2 μL RNA Polymerase, 1 μL RNase inhibitor, 1 μL50 mM MgCl₂ and filling up to 20 μL with ddH₂O. The mixture was incubated for 2 hours at 37°C. 2 μL of DNaseI (RNase free) was then added and sample was incubated for an additional 25 minutes at 37°C. 80 μL of RNase free water was then added. The RNA probe was then purified using a Qiagen RNeasy Kit (Cat # 74106). Protocol from kit was used to purify RNA. Ethanol precipitation was then carried out by adding 6 μL of 3M sodium acetate and 15 μL of 100% EtOH to the sample and leaving it at either −20°C overnight or at −80°C for 30 minutes. Sample was then spun at 15,000 rpm for 15 minutes at 4°C. Supernatant was discarded and pellet was rinsed with 300 μL of 70% EtOH. Sample was spun again at 15,000 rpm for 5 minutes at 4°C. EtOH was then discarded and RNA was dissolved in 40 μL of RNAse free water. The quality of the probe was checked by electrophoresis and 120 μL of hybridization buffer was added to it before it was stored at −20°C.

Embryo preparation and Insitu Hybridization

Embryos fixed as stated above, except in 4% Formaldehyde (EM grade)/PBS with same amount of heptane for 30–45 minutes. Embryos are stored in MeOH. To rehydrate, embryos were treated with 75%, 50%, and 25% MeOH made in PBS for 5 minutes each. Embryos were then washed three times for 5 minutes each in 0.1% PBST. Embryos were then treated with 300 μL of 10μg/mL proteinase K for 4 minutes (did not rotate). Proteinase K was then discarded and glycine (2 mg/ml) made in PBST added twice for 1 minute each. Sample was then fixed again with 4% EM Grade Formaldehyde for 5 minutes. Sample then washed with PBST 5 times for 5 minutes each. Embryos were then pre-hybridized by adding a 1:1 solution of pre-hybridization buffer and PBST to them for 5 minutes. Embryos were then treated with Pre-hybridization buffer alone for 5 minutes. Embryos were then rinsed with 300 μL of hybridization buffer before 300 μL of hybridization buffer was added and embryos were incubated for more than 1 hour at 60°C. To carry out hybridization, diluted 16 μL of labeled probe with 100 μL hybridization buffer. The diluted probe was heated at 80°C for 10 minutes and then chilled on ice. Extra solution was removed from tube containing embryos, leaving only 100 μL of hybridization buffer. Denatured probe was then added to the tube and hybridization was allowed to ensue at 60°C overnight. The embryos are then washed with preheated (60°) washing reagents. Washed initially with 50%Formamide and 2X SSCT twice for 20 minutes each at 60°C. Followed by washing twice with 2X SSCT for 10 minutes each. Finally, washed twice with 0.2X SSCT for 30 minutes each. Immunoreaction was then carried out. Embryos were rinsed with PBST. Embryos were then blocked with 0.2% blocking reagent (Roche) made in PBST for 2 hours at room temperature. Embryos were then placed in anti-DIG antibody made in 0.2% blocking reagent overnight at 4°C or for 2 hours at room temperature. Antibody was then washed 6 times with PBST for 10 minutes each. Embryos then washed twice with AP buffer (with 0.01% Tween20) for 5 minutes each. Finally, to stain NBT/BCIP tablets were dissolved in water and 500 μL of it was added to the embryos. Signal was then developed in the dark. To stop the reaction, embryos were washed 5 times with PBST (short washes initially, followed by a final 5 minute wash). To remove background, PBST containing 1% tween20 was added to embryos overnight at room temperature. embryos were then rinsed and mounted in 50% glycerol/PBS initially and finally in 100% glycerol.

Solutions included: Hybridization buffer (20 mL Formamide, 10 mL of 20X SSC, 40 mg RNA (ribosomal), 200 μL 10mg/mL Heparin, 200 μL 20% Tween20 and ddH₂0 (autoclaved) up to 40 mL), Pre-Hybridization buffer (20 mL Formamide, 10 mL of 20X SSC, 200 μL 10mg/mL Heparin, 200 μL 20% Tween20 and ddH20 (autoclaved) up to 40 mL), 20X SSC (175.3 g NaCl and 88.2 g Sodium Citrate in 1L of ddH₂O with a pH of 7.0), AP buffer (5 mL Tris-Hcl (pH 9.5), 1 mL of 5N NaCl, 2.5 mL of 1M MgCl₂ and up to 50 mL of ddH₂O), AP buffer with Tween20 (added 0.5 μL of 20% Tween20 to each 1 mL of AP buffer), PBST (PBS containing 0.1% Tween20).

Generation of 3D digital models

Staged Drosophila embryos labeled with anti-Delta and other markers were viewed as wholemounts by confocal microscopy (Biorad MRC 1024ES microscope using Biorad Lasersharp version 3.2 software; lenses: 40× oil; 60× oil). Complete series of optical sections were taken at 1mm intervals for at least five individuals per stage. Tif files of the confocal stacks were generated using Image J (Abramoff et al., 2004), and imported into the software package TrakEM2 (Cardona et al., 2010), which allows for efficiently segmenting structures by outlining them or, in case of simple shapes like nuclei, simply clicking them. We used the built in 3D viewer of TrakEM2 to generate three dimensional models of the segmented structures.

Results

Delta Signaling within the Cardiogenic Mesoderm Specifies Blood Precursor Fate

Previous studies had shown that Notch activity is required within the cardiogenic mesoderm during 8–10h after egg laying (AEL) for the correct specification of the different cardiogenic lineages, including cardioblasts (vascular cells), blood progenitors, and pericardial nephrocytes. Specifically, a high level of Notch activity is necessary for the specification of Odd-skipped (Odd)-positive pericardial nephrocytes and blood progenitors (Mandal et al., 2004). Drosophila Notch has two ligands, Dl and Serrate (Ser). To test which one of these acts in the cardiogenic mesoderm, we analyzed their loss of function phenotype. Embryos mutant for the Ser gene did not exhibit any detectable abnormalities among the cell types formed from the cardiogenic mesoderm (data not shown). On the other hand, null mutations in Dl caused a complete loss of pericardial cells and hematopoietic precursors (Fig. 2E–F). In contrast, cardioblast numbers were dramatically increased, phenocopying the Notch mutant phenotype (Mandal et al. 2004; Fig. 2A–D). Two markers, Mef2 and Tin were used to label cardioblasts. Mef2 in addition to being found in cardioblasts is also found in somatic muscle cells (Fig. 2A). Meanwhile Tin is expressed in cardioblasts as well as in the Tin positive pericardial cells (Fig. 2C). Dl mutants labeled with either marker showed an increase in the number of cells being labeled in comparison to their WT counterparts (Fig. 2B, D).

Fig. 2.

Delta (Dl) activity is localized and required in the cardiogenic mesoderm for the specification of blood progenitors and pericardial nephrocytes. A-F: Lateral views of stage 13 (A-B, E-F) and 14 (C-D) embryos. Left column (A, C, E) are wild type, right column (B, D, F) Dl mutants. Upper panels (A-B) are labeled with anti-Dl (green), anti-Mef2 (red) and Sytox (blue). Mef2 labels both cardioblasts (cb) and somatic muscle cells (sm). Middle panels (C-D) are labeled with anti-Tinman (Tin; red) and anti-Delta (green). Tin labels cardioblasts and Tin positive pericardial cells (tpc). Lower panels (E, F) show labeling with Sytox (blue) and anti-Odd skipped (red), which labels the lymph gland progenitors (lg) and the Odd positive pericardial nephrocytes (pcn). Dl is expressed in cardioblasts in wild type embryos (A). In the absence of Dl (B, D), cardioblasts are strongly increased in number, while Odd-positive pericardial nephrocytes and lymph gland blood progenitors are missing (F). G-I: High magnification of the cardiogenic mesoderm of a stage 13 wild-type embryo, showing Dl expression in the cardioblasts (G), and expression of a lacZ gene found downstream of 12 Suppressor of hairless [Su(H)] binding sites [reporter for Notch activity (N. rep.)] in the adjacent pericardial nephrocytes and lymph gland (H, I).

Other abbreviations: de Odd-positive dorsal ectoderm cells

Bars: 25μm (A-D); 10μm (E-G).

A construct with 12X Su(H) binding sites upstream of a lacZ was used to report activated Notch signaling (Go et al., 1998). This in conjunction with Dl expression studies provided further insight into the Notch/Dl-dependent signaling step acting in the cardiogenic mesoderm. As shown in the following section, in more detail, expression of Dl is widespread and dynamic in the mesoderm around the time when the fate of cardiogenic cells is specified (stage 12; 7–9h AEL). Towards the end of this period, expression of Dl becomes restricted to the cardioblasts, where it stays on until stage 14 (approximately 11h AEL; Fig. 2G). 12X Su(H)-lacZ is also expressed in the cardiogenic mesoderm of stage 12–14 embryos, but becomes restricted to the pericardial cells and blood progenitors flanking the Dl expressing cardioblasts (Fig. 2H–I). These findings indicate that the expression of Dl in the cardiogenic mesoderm provides the signal controlling the proper balance between the different cardiogenic lineages.

Origin and morphogenesis of the cardiogenic mesoderm

To learn more about the signaling mechanism that distinguishes between the different cardiogenic fates we next carried out a detailed analysis of the dynamic changes that occur in cell position and proliferation within the mesoderm. We then correlated these changes to the expression pattern of Dl, as well as other factors known to be involved in the control of Dl, notably the proneural gene l’sc, and members of the EGF and FGF signaling pathways.

The different cardiogenic lineages split from each other during embryonic stage 12 (7–8.5h AEL). Prior to the beginning of this phase, the cardiogenic mesoderm, defined by the expression of Tin, forms segmentally reiterated, elongated clusters of approximately 30–35 cells each (“early cardiogenic mesoderm”; Fig. 3A, Q). This number increases to about 60 cells at the end of stage 12 (Fig. 3N, T), following the fourth round of mitosis (Bate, 1993) that takes place during this time period (Fig. 3F, L, O). Based on cell size/shape and the expression levels of Dl, one can distinguish a dorsal and ventral subdomain within the cardiogenic mesoderm of the stage 12 embryo. Cells of the ventral domain are small and express moderate levels of Dl and Tin; dorsally, we see larger cells, expressing higher levels of Tin (Fig. 3E, R). Dl expression is highly dynamic in the dorsal domain; at most time points during stage 12, we see an anterior and posterior cluster of strongly Dl-positive cells, flanking a central cluster with lower Dl levels (Figs.3E′, R; schematically shown in Fig. 4). This central cluster expresses Even-skipped (Eve; Fig. 3F′; Fig. 4). As a matter of fact, Eve-expression defines a dorso-central cluster already in the early cardiogenic mesoderm (Fig. 3B, C and see below); from within this cluster, the pair of Eve-positive pericardial cells is specified (Carmena et al., 1998). The later Eve-positive/low-Dl dorso-central cluster gives rise to a dorsal muscle. The anterior and posterior, high-Dl clusters form the “definitive” cardiogenic mesoderm (cm_d in Fig. 3); as detailed below, they will give rise to the cardioblasts of the dorsal vessel and the Odd-positive blood progenitors and pericardial nephrocytes. The ventral domain showing low levels of Tin and Dl (colored green in Fig. 3Q–T) contribute to the dorsal musculature; a number of these cells seem to undergo apoptosis at a later stage (data not shown).

Fig. 3.

Morphogenesis and cell fate in the cardiogenic mesoderm. Photographic panels show lateral views of embryos at stages indicated to the left (for subdivision of stage 12, see Material and Methods). Each panel shows several consecutive segments of the mesoderm, labeled by anti-Dl (green); segments are indicated at bottom of panels (T1-T3: thoracic segments 1–3; A1-A3: abdominal segments 1–3). In most cases, a low magnification view and a high magnification view of the same specimen are shown in consecutive panels (e.g., E and E′; F and F′). For stage 12A, panel C presents a view of the cardiogenic mesoderm, and panel D shows a more medial section corresponding to the visceral mesoderm. Similarly, for stage 12B, E/E′ and F/F′ present the cardiogenic mesoderm, I and J the visceral mesoderm. In all panels of left column, the second marker used besides anti-Dl is anti-Tinman (Tin; red) which labels the cardiogenic mesoderm. Embryos shown in the panels of the second column, as well as panels C and D of the third column, were labeled with anti-Phosphohistone3 (PH3), a marker for mitotic cells, as well as anti-Even-skipped (Eve; blue). In the third column, Seven-up (Svp)-positive cells are labeled by svp-Gal4 > UAS-GFP (red). In these embryos, the lumen of the segmental tracheae and the demarcating segmental boundaries, are rendered in blue. Panels on the right (Q-T) show 3D digital models of the cardiogenic mesoderm at stages indicated at the top of each panel. Individual cells of cardiogenic mesoderm are shown as colored spheres, with colors representing different transient and definitive cell fates: cb cardioblasts (red in S-T); cm_d definitive cardiogenic mesoderm (orange in Q; comprises anterior (a) and posterior (p) high-Dl cluster); cm_e early cardiogenic mesoderm (light orange/green in Q); cb_svp Svp-positive cardioblasts (pink in T); Dl^- low-Dl clusters (pale yellow in R); eve-pc Even skipped-positive pericardial cells (brown in R-T); lg blood progenitors of the lymph gland (magenta in T); pcn pericardial nephrocytes (magenta in T); pre-lg/pcn precursors of blood progenitors and pericardial nephrocytes (yellow in S); vm_e early visceral mesoderm (blue in Q); vm_l late visceral mesoderm (cyan and dark blue in R-T, corresponding to Tin-positive and Tin-negative late visceral mesoderm cells, respectively).

Other abbreviations: dt dorsal tracheal branch; dlt dorsal longitudinal trunk of the trachea; tp tracheal pit;

Bars: 25μm (A-P); 10μm (E′, F′, H′; K′-P′).

Fig. 4.

Specification of different cell fates in the cardiogenic mesoderm. Schematic depiction of cardiogenic mesodermal cells (colored spheres) of two adjacent thoracic segments (left) and two abdominal segments (right) at stage 11 (top), mid stage 12 (middle) and stage 13 (bottom). Different cell types are annotated and colored in the same way as in the models shown in Fig. 3.

Individual lineages derived from the cardiogenic mesoderm segregate from each other and begin to differentiate during the second half of stage 12 (12B and 12C; 8–9h AEL). Morphogenetic events that unfold during this time interval are complex; they include cell division (the fourth mesodermal mitosis), cell movements within each segment, as well as cell movements occurring as part of germ band retraction. The anterior and posterior high-Dl cluster of each segment move closer together (compare Fig. 3E/E′ with 3K/K′), while the central, low-Dl cluster moves “out of the way”, migrating laterally and ventrally (compare Fig. 3F/F′ with 3L/L′). During the phase when this cell rearrangement takes place and most cells undergo their final (4^th) mitosis, Dl expression becomes restricted to the cardioblasts. In the thoracic segments, each of the high-Dl clusters forms two cardioblasts (Fig. 3K/K′, 3N/N′; Fig. 4). In abdominal segments, three cardioblasts arise per cluster. However, only two cells of each abdominal cluster maintain a high level of Dl and Tin. The third cell down-regulates these genes and expresses Seven-up (Svp; Gajewski et al., 2000; Ward and Skeath, 2000). Expression of Svp is already apparent during the beginning of stage 12 when the high-Dl cells form a separate anterior and posterior cluster in each segment (Fig. 3G). Like most other cells within the high-Dl clusters, the Svp-positive cells also divide once, forming an anterior and posterior pair in each segment (Fig. 3G, 3H/H′). When high-Dl clusters are pushed together during germ band retraction, the posterior Svp-positive pair of a given segment meets the anterior Svp-positive pair of the posteriorly adjacent segment (Fig. 3M/M′; 3P/P′; schematically shown in Fig. 4). The dorsal sibling of each Svp-positive pair will become a cardioblast; the ventral sibling a pericardial nephrocyte (Ward and Skeath, 2000; see below). In this manner, the pattern of alternating sets of four Tin/Dl-positive and two Svp-positive cardioblasts characteristic of the abdominal dorsal vessel is generated (Fig. 3P/P′).

Pericardial nephrocytes and blood progenitors also become discernable by size, position and expression of the gene odd by late stage 12. Based on our detailed reconstructions of embryos expressing odd in combination with tin or Dl we propose that nephrocytes and blood progenitors derive from the high-Dl clusters (Figs.3, 4). Abdominal segments produce four pairs of nephrocytes; two pairs are svp-positive (siblings of the Svp-positive cardioblasts; see above), the other two are Svp-negative. Meanwhile, the three thoracic segments each give rise to approximately eight to ten pairs of blood progenitors, all of which are Svp-negative. Similar to the abdominal nephrocytes, a considerable fraction of the blood progenitors are also siblings of the cardioblasts (Mandal et al., 2004). What accounts for the higher number per segment of blood progenitors vs. nephrocytes? The high-Dl clusters of the thoracic segments of stage 12B embryos (prior to separation of cardioblasts and blood progenitors) are not significantly larger than their abdominal counterparts (Fig. 3F/F′; Fig. 4). However, the fraction of cells undergoing a fourth round of mitosis appears to be higher in the thoracic segments than the abdominal segments (Fig. 3F/F′), which may be in part responsible for the higher number of blood progenitors. A second contributing factor may be the fact that the two pairs of cells that express Svp and produce cardioblasts and nephrocytes in the abdominal segments, generate only blood progenitors in the thoracic segments.

To sum up, we show that two high-Dl clusters located within the lateral mesoderm of the stage 11/early 12 embryo, called “cardiogenic clusters” from hereon onward, represent the definitive cardiogenic mesoderm from which cardioblasts, blood progenitors and pericardial nephrocytes are derived. Notch/Dl dependent interactions within the cardiogenic clusters separate these cell types, whereby the signal Dl becomes restricted to cardioblasts, and high levels of Notch activity appear in the adjacent blood progenitors/nephrocytes.

The cardiogenic clusters are defined by the co-expression of Dl, l’sc and activated MAPK

Careful mapping studies of the mesoderm, using a probe for the l’sc gene, had revealed nineteen clusters (C1-C19) defined by (transiently) high levels of L’sc. (Carmena et al., 1995; Fig. 5A). These same clusters also co-express pMAPK, a reporter for MAPK signaling. We can identify two of these clusters, C14 and C16, as the high-Dl, definitive cardiogenic mesoderm described in the previous section. C2 and C15 correspond to the Eve-positive clusters. Thus, in situ and antibody stainings revealed a cluster (C2) of L’sc positive cells in the center of the early cardiogenic mesoderm of the stage 11 embryos (Fig. 5B), and three clusters covering the definitive cardiogenic mesoderm. The latter three included the two clusters (C14, C16) and the late Eve-cluster (C15) of the early stage 12 embryo (Fig. 5C). Using double labeling with an antibody against Dl and pMAPK we confirmed that the same clusters also express pMAPK during stage 11 (C2) and stage 12 (C14-16; Fig. 5D, E). These findings suggest that both the MAPK pathway as well as L’sc may act in the cardiogenic mesoderm to maintain/focus the expression of Dl, and thereby provide necessary input for the development of pericardial nephrocytes and blood progenitors. To test this hypothesis we carried out loss and gain of function studies for members of both pathways.

MAPK activity, mediated by the EGF and FGF pathways, is required for the specification of blood progenitors and pericardial nephrocytes

Past studies had already shown that Ras, a member of the MAPK pathway, is capable of activating Dl expression. Constitutive activation of Ras leads to a persistence of Dl expression in the clusters C2 and C15 of L’sc positive cells present in the cardiogenic mesoderm (Carmena et al. 1998; 2002). We wanted to investigate whether this pathway is also active in the cardiogenic mesoderm, that is, clusters C14 and C16. Overexpression of a constitutively active form of Ras (RasV12) using a Mef2-gal4 driver, expressed globally in the somatic and cardiogenic mesoderm (Taylor et al., 1995; Ranganayakulu et al., 1996) resulted in an increase in cardioblast number (Fig. 6B). Both blood progenitors and pericardial nephrocytes were also increased in number. In this experiment, high levels of Dl expression were also seen in Mef2 positive muscle cells, whereas in WT embryos no such expression was apparent (data not shown).

Fig. 6.

Cardiogenic phenotypes in EGFR and FGFR pathway mutants. A-C: dorsal view of stage 16 embryos. Pericardial nephrocytes (pcn) and blood progenitors in the lymph gland (lg) are labeled by anti-Odd (red) and anti-Pericardin (green); Pericardin labels odd positive pericardial nephrocytes as well as Seven-up (Svp) positive cardioblasts (cb). Sytox (blue) labels all nuclei. In embryo where mesodermally expressed Mef2-Gal4 drives an activated Ras construct (B), all cardiogenic cells, and in particular cells of the lymph gland and pericardial nephrocytes, are strongly increased in number. The same phenotype results from driving a constitutively active FGF receptor (FGFR (htl); C). D, E: dorsal views of stage 16 embryos. Anti-Tinman (Tin; red) labels cardioblasts and Tin-positive pericardial cells (tpc) in wild type embryos (D). Cardioblasts that appear negative for Tin are the Svp positive cardioblasts (D). In embryos carrying a null allele of the EGF receptor gene (EGFR; E), cardioblasts are absent. F: lateral view of stage 12 EGFR-mutant embryo. Labeling with anti-Dl (green) and anti-Caspase 3 (Casp), a marker for apoptotic cell death. Note clusters of Casp-positive cells in dorsal mesoderm (large arrowheads). Small arrowheads point out artefactual labeling of lumen of tracheae. G-I: lateral view of stage 13 embryos labeled with antibodies against Odd (red) and Dl (green). Note normal pattern of Odd-positive pericardial nephrocytes, lymph gland, and epidermal stripes (ep) in wild-type embryo (G). Nephrocytes and lymph gland cells are strongly reduced in loss of function mutants for both EGFR (H) and FGFR (Htl; I). J-L′: lateral views of stage 11 embryos labeled with anti-Dl (green), anti-Mef2 (red) and Sytox (blue). Upper row (J-L) shows sections of cardiogenic mesoderm, located more superficially in the embryo (right underneath the ectoderm). Lower row (J′-L′) represent section of deeper (more medial) mesoderm of same embryos as shown in upper row, including (in wild type; J′) the visceral mesoderm (vm), fat body mesoderm and somatic mesoderm (msfb/sm). Note strong expression of Dl and Mef2 in early cardiogenic mesoderm (cme) of wild type embryo (J, J′). In an EGFR mutant (K, K′), the early cardiogenic mesoderm (based on Dl levels and number of Mef2-positive nuclei) is strongly reduced or absent. In an FGFR (htl) mutant (L, L′), at this early stage, the cardiogenic mesoderm is still present, even though irregularities in the size and shape of cardiogenic mesoderm clusters are apparent.

Bars: 20μm (A-F); 25μm (G-L′).

Ras is a downstream activator of both the EGF and FGF receptor tyrosine kinase pathways. In order to determine which of these was acting in the cardiogenic mesoderm, we carried out gain and loss of function studies for both the EGFR [Drosophila homolog: faint little ball (flb)] and FGFR [Drosophila homolog: heartless (htl)] pathways. Mef2-Gal4-driven expression of a constitutively active FGFR construct (Fig. 6C) or EGFR construct (not shown) mimicked the phenotype of Ras expression, in terms of a strongly increased number of blood progenitors/nephrocytes and cardioblasts. EGFR loss of function mutants showed a highly reduced level of Dl in the entire mesoderm of stage 11/12 embryos (Fig. 6K). Labeling of late mutant embryos with anti-Odd, anti-Pericardin or anti-Mef2 demonstrated that blood progenitors, nephrocytes and cardioblasts were virtually absent in EGFR mutants (Fig. 6E, H). Since in other tissues (Kurada and White, 1998; Protzer et al., 2008), EGFR is an important factor for cell survival, we also carried out anti-Caspase staining to monitor cell death in the cardiogenic mesoderm. Significant amounts of cell death were seen in the cardiogenic mesoderm of stage 11/12 EGFR loss of function mutants (Fig. 6F). These data support the idea that EGFR activity is required for the maintenance of Dl expression and survival of the cardiogenic mesoderm.

It had been previously established that mutations in the FGFR gene, htl, result in a loss of the dorsal vessel and blood progenitors in late embryos (Beiman et al., 1996; Gisselbrecht et al., 1996; Shishido et al., 1997; Mandal et al., 2004). In order to follow the fate of the cardiogenic mesoderm-derived lineages and Dl expression levels in embryos lacking FGFR, we did a developmental series of FGFR mutants labeled with anti-Dl and markers for the different cardiogenic lineages. FGFR mutant embryos at stages 11 and 12 showed an abnormally shaped mesoderm; however, metameric clusters of Dl-positive cells were still visible in the cardiogenic mesoderm (Fig. 6L). By stage 14, these cells were no longer present (Fig. 6I), indicating that FGFR signaling, like EGFR, is required for the maintenance of cardiogenic lineages, but acts at a later phase than EGFR.

The bHLH gene l’sc is required for all cell types derived from the cardiogenic mesoderm

In order to identify a possible role of l’sc in the cardiogenic mesoderm, we visualized the cardiogenic lineages in embryos carrying the deficiency Df(1)scB57, which removes the entire achaete-scute complex (AS-C) (Fig. 7). Homozygous mutants showed a highly significant decrease in the number of pericardial cells and lymph gland blood progenitors, as well as a small decrease in the number of cardioblasts. A similar reduction in blood progenitors/nephrocytes and cardioblasts was observed in embryos where a l’sc-Rnai was driven with a Mef2-Gal4 driver. In neither the deficiency nor the RNAi knock-down experiment did we see a significant reduction in Dl expression in the cardiogenic mesoderm (data not shown). These findings indicate that l’sc is required for the appropriate number of cells within all cardiogenic lineages to form, even though it does not noticeably alter expression levels of the Notch ligand, Dl.

Fig. 7.

Reduction in cardiogenic cell lineages in embryos deficient for l’sc. A: dorsal view of a stage 16 Df(1)scB57 (AS-C deficient) embryo labeled with anti-Odd skipped (Odd; red) and anti-Pericardin (green). Labeling of nuclei by Sytox (blue). Number of lymph gland blood progenitors (lg) and pericardial nephrocytes (pcn) is significantly reduced (see histogram in B); cardioblasts are mildly reduced. Arrowhead in A points at wide gap in row of pericardial nephrocytes. B: For all of following, two samples were counted per organism. Lg (WT: n=8 lobes, mutant: n=4 lobes; p value: 4.85E-06); Pcn (WT: n=6, mutant: n=7; values shown are for 4 hemisegments; p value: 3.34E-05); Cb (WT: n=4, mutant: n= 6; values shown are for 4 hemisegments; p value: 0.0197). P values calculated via Student T-test (1 tailed, equal variance). Bar: 20μm.

Controlling the Spatial Domains of Activity of Dl, l’sc, EGFR and FGFR

We next addressed the mechanisms operating upstream of Dl, l’sc and EGFR/FGFR to set up the spatial domains in which these factors are active. Previous data had shown that the cardiogenic mesoderm as a whole depends on the combined presence of high levels of Wg and Dpp (Lockwood and Bodmer, 2002). Removal of either of these genes results in the complete absence of all cardiogenic lineages (Mandal et al., 2004; Fig. 8A). Similarly, the early cardiogenic mesoderm, which is strongly positive for Mef2 in wild type, (Fig. 6J) does not become specified in a wg mutant (Fig. 8E). Both Dpp and Wg act upstream of Dl; loss of dpp (data not shown) or wg causes the failure to upregulate Dl or L’sc expression in the mesoderm. Thus, the cardiogenic clusters C14 and C16 which are strongly Dl-positive and L’sc positive in the wild type, (Fig. 8D), are not apparent in a wg LOF mutant (Fig. 8E). We confirmed that Wg, detected by an antibody and lacZ reporter construct, indeed coincides with the cardiogenic mesoderm. Throughout the early stages of mesodermal development (up to stage 10; 5h AEL), Wg is expressed in a stripe-like domain covering the central part of the ectoderm and the underlying mesoderm of each segment. The early cardiogenic mesoderm forms the lateral part of the Wg-positive mesodermal domain (Fig. 8B). At the point when the visceral mesoderm separates from the cardiogenic mesoderm, the cells within the latter rearrange, spreading out in the longitudinal axis to cover almost the entire length of a segment. During early stage 12, the time of appearance of C14/C16, Wg signal can be detected in these clusters (Fig. 8C).

Fig. 8.

Expression and function of Wingless (Wg) in the cardiogenic mesoderm.

A: Dorsal view of stage 16 wg mutant embryo, labeled with anti-Odd-skipped (red), anti-Pericardin (green), and Sytox (blue). All cardiogenic lineages are absent. B, C: lateral views of stage 11 (B) and early-mid stage 12 (C) wild-type embryos labeled with anti-Dl (green), and expressing a wg-lacZ reporter construct (Wg; red). Wg is expressed in segmentally reiterated stripes that overlap with the early cardiogenic mesoderm (cme in B). The wg reporter is expressed in all cells of the definitive cardiogenic mesoderm (cmd), which, by this stage, has lengthened in the ap-axis, to encompass the anterior and posterior high-Dl clusters (a, p). D, E: Lateral view of early stage 12 wild-type (D) and wg mutant (E) embryo labeled with anti-Dl (green), anti-L’sc (red), and Sytox (blue). Note anterior and posterior high-Dl clusters (a, p) of definitive cardiogenic mesoderm, which also contain L’sc-positive cells in wild type (D). Strongly Dl-positive cells ventrally adjacent to cardiogenic mesoderm are the emerging dorsolateral trunks and dorsal branches of the tracheae (dlt). In a wg mutant (E), the cardiogenic mesoderm is absent; the smooth epithelial layer dorsal of the Dl-positive tracheal cells (dlt) corresponds to the dorsal ectoderm (dec), which in the absence of dorsal mesoderm, folds far medially. F, F′: Lateral view of early stage 12 wg mutant embryo at a more superficial (F) and a deeper (more medial) level (F′). Labeling is with anti-Dl (green) and anti-Mef2 (red). Loss of Dl/Mef2 in (F) confirms absence of cardiogenic mesoderm.

Other abbreviations: ep epidermis; mg midgut; tp tracheal placodes; vec ventral ectoderm; vm/fb visceral mesoderm/fat body.

Bars: 20μm (A-C); 20μm (D-F′).

The expression of Dl, as well as activation of the MAPK pathway, becomes highly polarized in presumptive cardioblasts. This polarization may be important for Dl to exert its proper function, that is, to modulate Notch activation in the C14/C16 clusters such that the proper balance of cardioblasts vs. pericardial nephrocytes/blood progenitors is achieved. We wondered whether a spatially restricted expression of the ligands and/or receptors of the EGFR and FGFR pathways may help explain the localized activity of MAPK, which in turn maintains/polarizes Dl expression. The EGF receptor and FGF receptor (Htl) are widely expressed within the cardiogenic mesoderm (Fig. 9A-A′, C). Likewise, Spitz (Spi), one of the ligands capable of activating the EGFR signal is expressed throughout the cardiogenic mesoderm in stage 11–13 embryos (Fig. 9B). However, the FGFR ligands, Thisbe (Ths) and Pyramus (Pyr) (Strathopoulos et al., 2004; Klingseisen et al., 2009; Kadam et al., 2009), are expressed in clusters of ectodermal cells that appear in areas overlying the cardiogenic mesoderm (Fig. 9D–E′). This directional signal could be involved in triggering signaling asymmetries in the underlying C14/C16 clusters, resulting in the eventual accumulation of higher MAPK activity (followed by Dl expression) in the dorsal-most cells of the clusters which will become cardioblasts.

Fig. 9.

EGFR and FGFR ligands in the cardiogenic mesoderm. A-C: Lateral view of early stage 12 embryos labeled with anti-EGFR (red in A; white in A′) and anti-Delta antibody (A; green), spitz-lacZ reporter (B; green), and FGFR (htl) reporter (C; green). All of these are expressed widely in the cardiogenic mesoderm and beyond. Arrows in A and A′ point at Dl+ cardiogenic clusters that upregulate EGFR. D: lateral view of early stage 11 embryo showing Pyramus (Pyr; blue) expression via in situ. Pyr is expressed in segmental ectodermal clusters (Kadam et al., 2009) overlying the cardiogenic mesoderm (white box outline). Figure adapted from Kadam et al., 2009. E, E′: lateral view of early stage 12 embryo at superficial level of dorsal ectoderm (E) and level medially adjacent to ectoderm, showing cardiogenic mesoderm (E′). Labeling with lacZ reporter of Thisbe (Ths; green), the other FGFR (Htl) ligand. Mesoderm cells are labeled by anti-Mef2 (red). Expression of Ths, similar to that of Pyr shown in D, occurs in cells forming part of the dorsal-most row of the ectoderm, adjacent to underlying cardiogenic mesoderm. F: Dorsal view of stage 16 embryo mutant for pyr, labeled with anti-Odd skipped (red) and Sytox (blue). Note significant reduction of lymph gland cells (lg) and pericardial nephrocytes (pcn).

Other abbreviations: cm cardigenic mesoderm; tp tracheal placodes.

Bars: 20μm (A-C); 20μm (D-E).

Previous genetic studies had already shown that loss of pyr resulted in a reduction of the Eve-positive cells derived from mesodermal cluster C2 (Klingseisen et al., 2009; Kadam et al., 2009). We confirmed that the same mutation had significantly reduced numbers of pericardial nephrocytes and blood progenitors, indicating that the Pyr-mediated signal plays a definitive role in activating the MAPK pathway (Fig. 9E). By contrast, loss of ths, the second FGFR ligand, did not result in any abnormalities in the cardiogenic lineages (data not shown).

Discussion

In this study we pursued two goals: to elucidate the precise location and cellular composition of the cardiogenic mesoderm, and to analyze the mechanism by which Notch becomes activated in the restricted subset of these cells that become blood progenitors. Our findings show that the cardiogenic mesoderm is comprised of segmentally reiterated pairs of clusters (cardiogenic clusters) defined by high expression levels of Dl, L’sc and activated MAPK. The MAPK pathway, activated through both EGFR and FGFR signaling, is required for the specification (EGFR) and maintenance (EGFR and FGFR) of all cardiogenic lineages. As shown previously, the default fate of all cardiogenic cells is cardioblasts (Hartenstein et al., 1992; Mandal et al., 2004). Notch activity triggered by Dl is required for the specification of blood progenitors (thoracic cardiogenic clusters) and nephrocytes (abdominal cardiogenic clusters), respectively. One of the downstream effects of MAPK signaling is to maintain high levels of Dl in the cardiogenic clusters, and to help localize Dl expression towards a dorsal subset of cells within these clusters, which will become cardioblasts. Dl stimulates Notch activity in the surrounding cells, which triggers the blood progenitor/nephrocyte fate in these cells.

Cardiogenic and myogenic clusters: variations on a common theme in Drosophila mesodermal development

The cardiogenic clusters form part of a larger population of mesodermal cells defined by high expression levels of l’sc (Carmena et al., 1995). Based on l’sc in situ hybridization, these authors mapped 19 clusters of l’sc expressing cells within the somatic mesoderm. Many of these clusters (called “myogenic clusters” in the following) give rise to one or two cells that transiently maintain high levels of l’sc, whereas the remaining cells within the cluster lose expression of l’sc. The l’sc-positive cells segregate from the mesoderm to a more superficial position, closer to the ectoderm, undergo one final mitotic division, and differentiate as muscle founder cells (Carmena et al., 1995). Dl/Notch mediated lateral inhibition was shown to act during the singling-out of muscle founders from the myogenic clusters. Loss of this signaling pathway caused high levels of L’sc to persist in all cells of the myogenic clusters, with the result that all cells developed as muscle founders. Interestingly, loss of l’sc had only a mild effect, consisting of a slight reduction in muscle founders. This is similar to what we find in this paper in l’scdeficient embryos, which show only a mild reduction in cardioblasts and other cardiogenic lineages.

The developmental fate of most of the L’sc-positive clusters within the dorsal somatic mesoderm is different from that of the ventral and lateral myogenic clusters discussed above, even though several parallels concerning the morphogenesis, proliferation, and dependence on Dl/Notch signaling are evident. The somatic (anterior, Wg-positive) mesoderm is divided into a dorsal and ventral domain based on the expression of Tin. Initially expressed at high levels in the entire mesoderm, this gene is maintained only in the dorsal mesoderm, as a result of Dpp signaling from the dorsal ectoderm. (Staehling-Hampton et al., 1994; Frasch, 1995). The dorsal somatic mesoderm, which we here call “early cardiogenic mesoderm”, includes four L’scpositive clusters, C2 and C14-C16. The development of C2 has been described in detail by Carmena et al. (1998; 2002) and Buff et al. (1998). C2 gives rise to a progenitor that divides twice; two of the progeny become the Eve-positive pericardial cells. Meanwhile, C15 which appears later at the same position as C2, seems to behave like a “normal” myogenic cluster. It produces a progenitor that divides once and forms the founders of the dorsal muscle DA1. As shown in this paper, the two remaining dorsal clusters, C14 and C16, give rise to the cardioblasts. We note that the Eve-positive progenitors, as well as the cardioblasts, resemble the muscle founders derived from the typical myogenic clusters in three aspects. First, they segregate towards a superficial position, close to the ectoderm, relative to the remainder of the cells within the clusters. Secondly, they undergo one (in case of C2: two) rounds of division right after segregation. And third, they are restricted in number by Dl/Notch signaling: in all cases, they are increased in number following Dl or Notch loss of function.

Cardiogenic clusters, like myogenic clusters, also depend on the MAPK signaling pathway. Past studies have shown that in the Eve-positive C2 and C15 clusters, Ras, is capable of inducing the formation of additional Eve-positive progenitors (Carmena et al., 2002). Ras is a downstream activator of both the EGFR and FGFR tyrosine kinase pathways, both of which have been seen to be important for the formation of the Eve-positive progenitors. With a loss of the FGFR pathway, Eve-positive progenitors of both the C2 and C15 cluster are lost; by contrast, the EGFR pathway affects only C15. The balanced activity of MAPK and Notch, which in part depends on reciprocal interactions between these pathways, determines the correct number of C2/C15 derived progenitors. Ras-induced MAPK activation upregulates the expression of other MAPK signaling pathway members (autoregulatory feed-back loop), but also stimulates the antagonist Argos, as well as Dl. Dl-activated Notch, in turn, inhibits MAPK signaling.

We show here that both Dl/Notch and MAPK signaling are active in the C14 and C16 clusters, which constitute the definitive cardiogenic mesoderm. MAPK activity is required for the maintenance of all lineages derived from these clusters, as shown most clearly in the EGFR LOF phenotype that entails a lack of cardioblasts, blood progenitors, and pericardial nephrocytes. Overexpression of Ras results in an increased number of all three cell types, which indicates that the C14/C16 clusters attain a larger size, possibly by an additional round of mitosis. The phenotype seen in embryos suffering from loss- or overexpression of Dl/Notch pathway members can be interpreted in the framework of a classical lateral-inhibition mechanism: Dl is upregulated in the C14/C16 derived cardioblast progenitors (analogous to the Eve-progenitors of C2/C15), from where it activates Notch in the remainder of the C14/C16 cells; these cells are thereby inhibited from forming cardioblasts, and instead become nephrocytes/blood progenitors. The level of Notch activity affects the expression of tin (low Notch) and the GATA homolog srp (high Notch), which triggers the fate of cardioblasts and blood progenitors/nephrocytes, respectively (Mandal et al., 2004).

MAPK is required for the initial activation of Dl in the cardiogenic clusters (just as in the myogenic clusters). Input from the pathway is most likely also instrumental in the subsequent restriction of Dl to the cardioblast progenitors. The positive interaction between MAPK and Notch signaling could occur at several levels. A mechanism shown for the ommatidial precursors of the eye disc involves Ebi and Strawberry notch (Sno), which are thought to act downstream of EGFR signaling and lead to an upregulation of Dl through the Su(H) and SMRTER complex (Tsuda et. al. 2002).

Generally, when one progenitor cell is seen to give rise to two different cell types it is accomplished in one of two ways. One: there is an asymmetric division, where a factor expressed by the progenitor is segregated into only one daughter cell; two: a non-uniformly expressed extrinsic signal effects one cell, but not its neighboring sibling. Ward and Skeath (2000) had demonstrated that in the posterior (abdominal) segments of the Drosophila cardiogenic mesoderm, inhibition of Notch by Numb accounts for the asymmetric activity of Notch in a small set of cardiogenic mesoderm cells, the Svp-positive cells. If Numb function is removed, these cells, which normally produce two cardioblasts and two pericardial nephrocytes, instead give rise to four cardioblasts. However, multiple nephrocytes per segment remain in numb loss-of-function mutations; furthermore, loss of numb does not cause any defect in the blood progenitors, where asymmetrically dividing Svp-positive cells are absent. This suggests that in addition to the numb-mediated mechanism, directional activation of Notch by one of its ligands is required for the majority of nephrocytes and all of the blood progenitors. We propose here that the spatially restricted upregulation/maintenance of Dl in nascent cardioblasts acts to activate Notch in the remainder of the cells within each cardiogenic cluster, which promotes their fate as blood progenitors and nephrocytes.

The Notch signaling/bHLH gene cassette in vertebrate blood/vascular development

The Notch signaling pathway is typically associated with members of two different types of bHLH transcription factors. One type act as activators, while the other act as repressors (Fisher and Caudy, 1998). In the context of lateral inhibition, best studied in Drosophila neurogenesis (Heitzler and Simpson, 1991; Campos-Ortega, 1993), activating bHLH transcriptions factors, including genes of the AS-C like l’sc, are expressed at an early stage in clusters of ectodermal or mesodermal cells, where they activate genetic programs that promote differentiative pathways such as neurogenesis, or myogenesis/cardiogenesis (see above). Subsequently, Notch ligands initiate the Notch pathway in these clusters; cells with high Notch activity turn on members of the Hairy/E(spl) (HES) family of bHLH genes which act as repressors and abrogate the transcriptional programs that had been set in motion by the activating bHLH factors (Heitzler et al., 1996). As shown in this paper, the gene cassette consisting of the Notch signaling pathway, as well as activating and repressing bHLH factors, operates in the cardiogenic mesoderm to determine the balance between cardioblasts and blood progenitors/nephrocytes. As discussed in the following, the same cassette also appears to be centrally involved in the specification of vascular endothelial cells and hematopoietic stem cells in vertebrates, which adds to the list of profound similarities between Drosophila and vertebrate blood/vascular development.

Even prior to the appearance of hemangioblasts, the lateral mesoderm of vertebrates is prepatterned by sequentially activated signaling pathways and transcriptional regulators similar to those that act in flies. The Wnt/Wg pathway, for example, separates subdomains of the mesoderm in vertebrates and Drosophila, as well as more ancestral ecdysozoans (Eriksson et al., 2009). Notch signaling plays an essential role in generating boundaries between segmental, as well as intra-segmental, subdomains within the ectoderm and mesoderm (McGrew and Pourquie, 1998; Qiu et al., 2009; Tapanes-Castillo and Baylies, 2004). The FGF signaling pathway predates the appearance of Bilaterians and plays a highly conserved role in early mesoderm patterning (Vasiliauskas and Stern, 2001; Wilson and Leptin, 2000; Böttcher and Niehrs, 2005; Rebscher et al., 2009). Likewise, specific sets of transcriptional regulators are the targets of these signaling pathways (e.g., twist: Furlong, 2004; Vernon and LaBonne, 2004; Yeo et al., 2009); zfh: Su et al., 1999; van Grunsven et al., 2006; Kittelmann et al., 2009; myostatin: Lo and Frasch, 1999; Artaza et al., 2005; Grade et al., 2009) and play a role during the establishments of cell fate in the mesoderm. It appears, therefore, that the bilaterian ancestor featured a mesodermal subdomain, the “cardiogenic/lateral mesoderm”, in which signals of the Wg, BMP, Notch, and FGF pathways and conserved sets of transcriptional regulators established boundaries and cell fate in the mesoderm.

The vertebrate gene encoding an activating bHLH factor with sequence similarity to the Drosophila AS-C genes is SCL (Begley et al., 1989). SCL expression in the lateral mesoderm marks the first appearance of hemangioblasts (Gering et al., 1998; Davidson and Zon, 2000; note that SCL is also expressed widely in the developing vertebrate CNS; Van Eekelen et al., 2003). In Zebrafish, from their site of origin in the lateral mesoderm, SCL-positive hemangioblasts migrate dorso-medially and form the intermediate cell mass (ICM). The ICM is the site of primitive endothelial blood vessel and hematopoietic cell specification. Gain of function studies carried out in zebrafish embryos have shown SCL to be one of the genes important in specifying the hemangioblast from the posterior lateral plate mesoderm. The specification of hemangioblast here comes at the expense of other mesodermal cell fates, namely the somitic paraxial mesoderm (Gering et al., 2003; Xiong, 2008; Gering and Patient, 2008; Dooley et al. 2005) In mice, Lack of SCL affects blood and vascular development as SCL mutants are bloodless and show angiogenesis defects in the yolk sac (Xiong 2008; Robb et al 1995; Shivdasani et al 1995; Visvader et al 1998).

Vertebrate homologs of the repressive Drosophila Hairy/E(spl) family of bHLH genes are the Hes and Hey (hairy/Enhancer-of-split related with YRPW motif) genes (Fischer and Gessler 2007; Iso et al. 2003, Kokubo et al. 1999, Satow et al. 2001, Leimeister et al. 1999, 2000; Steidl et al. 2000). A well studied member of the Hey family in zebrafish is gridlock, which is required for the specification of hematopoietic progenitors from the ICM. Hey 2 mutations in mice lead to severe congenital heart defects (Fischer et al. 2007). In addition, the Hes protein plays a role in hematopoiesis as it is a positive regulator of Hematopoietic Stem Cell (HSC) expansion (Kunisato et al. 2003).

Genetic studies of the Notch receptors and their ligands in vertebrates supports the idea that this pathway does indeed play a crucial role in the initial determination of hematopoietic stem cells (Bigas et al., 2010). The yolk sac and the para-aortic splanchnopleura (P-Sp)/AGM (aorta gonad mesonephros) of Notch null mouse embryos lack HSC’s (Kumano et al 2003). A similar phenotype is observed in mutants of Jagged 1, one of the Notch ligands (Robert-Moreno et al., 2005; 2008). Notch is thought to be the deciding factor between hematopoietic and endothelial cell fates when the two originate from a common precursor or hemangioblast. In murine mutants exhibiting lower Notch1 mRNA levels, a lack of hematopoietic precursors is seen and is accompanied by an increase in the number of cells expressing endothelial cell markers (Robert-Moreno et al. 2005). Likewise, in Drosophila, loss of Notch is associated with an increase in cardioblast number and a loss of blood precursor cells (Mandal et al 2004).

Research Highlights.

• Analysis of the origin and morphogenesis of the cardiogenic mesoderm in Drosophila.

• Delta required for specification of blood cell fate in hemangioblast progenitors.

• Dl expressed in early cardiogenic clusters but becomes restricted to cardioblasts.

• MAPK activity required for maintenance of Dl and cardiogenic mesoderm survival.