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First published online 20 September 2006
doi: 10.1242/dev.02586

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Cardioblast-intrinsic Tinman activity controls proper diversification and differentiation of myocardial cells in Drosophila

Stéphane Zaffran^1,*,, Ingolf Reim^1,*, Li Qian², Patrick C. Lo¹, Rolf Bodmer² and Manfred Frasch^1,

¹ Brookdale Department of Molecular, Cell and Developmental Biology, Box 1020, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
² The Burnham Institute, Center for Neurosciences and Aging, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

View larger version (47K): [in a new window]   Fig. 1. Generation of mutants lacking specific phases of tin expression. (A-C) Schematic embryo drawings illustrating main areas and phases of tin expression and their assignment to enhancer elements, color-coded as in D. (D) tin locus with its enhancers tinA-tinD (white boxes: introns) (Yin et al., 1997). Constructs used in transgenes are shown below. As expected, tinD did not have any rescuing activity in tin- because the Dpp response requires the presence of Twist-dependent Tin (Xu et al., 1998). (E) tin-null mutant embryos expressing Tin from tin-AB show uniform mesodermal Tin expression at stage 9. (F) tin-null mutant embryos with tin-ABD show dorsal mesodermal Tin expression at stage 10.

View larger version (127K): [in a new window]   Fig. 2. Early mesodermal expression of tin is sufficient to specify the dorsal mesoderm. Wild-type (left) and tin-null mutant embryos [homozygous for Df(3L)GC14] carrying the transgene tin-AB (right); (A-D) Lateral views; (E-H) Dorsal views. (A,B) Bin protein in visceral muscle progenitors of stage 10 control and tin mutant embryos carrying tin-AB. (C,D) Detection of bkh mRNA (green), Eve (red) and Tin (blue) protein. In late stage 11 control embryos (C), bkh-expressing cardioblasts (white arrowheads) and pericardial progenitors (red arrowhead, bkh+Eve+) are detected within the Tin domain. In corresponding tin-AB, tin mutant embryos (D), tin-AB-derived Tin has vanished but most bkh- and Eve-expressing cardiac progenitors are formed. (E,F) Staining for Odd, Zfh1 and Eve proteins identifies Odd-(Odd-pc, yellow) and Eve-pericardial cells (Eve-pc, pink) in stage 16 embryos. In the mutant (F), both types of pericardial cells are present, albeit arranged irregularly. The lymph gland (lg) is strongly reduced. (G) Mef2 staining of wild-type embryo at stage 16 (dv, dorsal vessel; sm, somatic musculature). (H) Mef2-expressing cardioblasts are present in tin-AB, tin-, although at reduced numbers (arrowheads indicate interruptions in the dv, which can occur at variable AP positions).

View larger version (168K): [in a new window]   Fig. 3. Formation and differentiation of the dorsal vessel in the absence of cardiac Tin. Dorsal views of stage 15-16 embryos, with the left column showing wild-type control embryos and the right column homozygous tin-null mutant (tin346) embryos carrying tin-ABD. (A) Control embryo stained for mid RNA and for Mef2 protein. (B) tin-ABD, tin- embryo with normal mid and Mef2 expression in cardioblasts. (C) Wild-type expression of Hand mRNA in Mef2+ cardioblasts (cb), Mef2- pericardial cells (pc), and the lymph gland (lg). (D) tin-ABD, tin- embryo expressing Hand mRNA in the developing dorsal vessel. Hand in cardioblasts is present, albeit at reduced levels when compared with pericardial cells and wild-type cardioblasts. (E) Control embryo doubly stained for Mef2 and Tropomyosin (posterior portion of the dorsal vessel). (F) Tropomyosin is present in myocardial cells of tin-ABD, tin- embryo. (G) Staining for the transmembrane protein Toll and Mef2 shows a characteristic alternation between more cubical `working' myocardial cells and elongated pairs of ostial cells (brackets) in the `heart' region of the dorsal vessel. (H) tin-ABD, tin346 mutant embryo stained as in G showing disorganized tube formation and lack of the correct alternation of cell shapes in the `heart' (bracket). Mef2 channels in G,H to identify cardioblasts were omitted in the higher magnification insets.

View larger version (157K): [in a new window]   Fig. 4. Markers for distinct cardioblast sub-types reveal a requirement of tin for cardial cell diversifications. Dorsal views of stage 15-16 wild-type (left column) and tin-ABD, tin346 mutant embryos (right column). (A,B) Staining with antibodies against Mef2 (green) and Doc2+3 proteins (red/red arrowheads). Doc2+3 expands into all cardioblasts in mutant embryos that lack tin expression in the dorsal vessel. (C) Ladybird (Lbe) is detected in two cardioblasts per hemisegment (green arrowheads) just posterior to the Doc-positive pair in the wild type. (D) Cardiac Lbe expression is absent in tin-ABD, tin mutants except for the outflow region (*). (E) In normal stage 16 embryos, Sur mRNA is detected at high levels in Tin cardioblasts and at very low levels in Doc+ cells (red arrowheads). (F) Sur mRNA is barely detectable in mutants lacking cardiac tin expression. (G) In the wild type, ß3-Tubulin (ß3-Tub) is detected in Tin+/Doc- cells of the dorsal vessel (bracket) and in somatic muscles. (H) ß3-Tubulin is maintained with a slightly irregular segmental pattern (bracket) in cardioblasts of tin-ABD, tin346 mutants. (I,J) Detection of svp mRNA and Tin protein. svp is normally expressed in the Tin-negative cardioblasts (I, green arrowheads). In tin-ABD, tin mutant embryos (J) svp expression retains its normal pattern (red arrow indicates sporadic pericardial cell expression of Tin in tin-ABD, tin mutants; rg, ring gland).

View larger version (150K): [in a new window]   Fig. 5. Tin and Doc function as mutual repressors within cardioblasts. Stage 15-16 embryos carrying the svp-lacZ enhancer trap insertion AE127 heterozygously (except for C, where it is homozygous) stained for ß-galactosidase (svp-LacZ), Doc2+3 and Tin as indicated. (A) In the control, svp-LacZ and Doc2+3 are co-expressed in the Tin-negative cells (yellow arrows). rg: ring gland. (B) In a tin346 mutant embryo carrying tin-ABD, Doc but not svp-LacZ expression expands into all cardioblasts. (C) In a tin346, svpAE127 double mutant embryo carrying tin-ABD, expression of Doc is still expanded but svp-LacZ (yellow arrows) is not. (D,E) Expanded cardiac expression of tin via S59-Mef2-HtD-Gal4 leads to Doc repression in many Svp cardioblasts (turquoise and blue arrows in D and E, respectively). Only cardioblasts lacking Tin because of variable driver activity retain Doc (E shows the embryo from D without the green channel). (F) Analogous ectopic svp1 expression causes a reduction of Tin in cardioblasts along with expansion of Doc (yellow and red arrows) and ectopic svp-lacZ in some of those cardioblasts (yellow arrows). pc, Tin+ pericardial cells. (G) Misexpression of Doc2 in the dorsal vessel reduces or abolishes tin expression (blue arrows), particularly in posterior cardioblasts. (H) Df(3L)DocA/Df(3L)29A6 embryo, in which levels of Doc2 and Doc3 are reduced and Doc1 is absent, show svp-lacZ and tin co-expression in numerous cardioblasts (turquoise arrows).

View larger version (87K): [in a new window]   Fig. 6. Control of Wg expression in ostial cardioblasts and summary of regulatory interactions in the heart region. Detection of Wg (green), Doc2+3 (red) and Tin (blue) in the dorsal vessel of stage 16 embryos. (A) In the wild type, Wg marks the three posterior pairs of Doc+/Tin- cardioblasts as ostia (green arrows). (B) Wg and Doc proteins are not detectable in the dorsal vessel of homozygous svpAE127 mutants, whereas Tin is expanded. (C) Embryo expressing svp1 ectopically throughout the dorsal vessel. All cardioblasts of the `heart' region express Doc and Wg (bracket). (D) Misexpression of Doc2 causes less efficient ectopic activation of Wg when compared with svp (arrow). (E) tin346 mutant and (F) tin346, svpAE127 double mutant embryos carrying tin-ABD. Wg is expressed in all Doc-labeled cardioblasts of the posterior dorsal vessel (brackets). (G) Top: schematic representation of the dorsal vessel with corresponding epidermal segment numbers and expression domains of homeotic selector proteins. Middle: in the wild type, tin is activated in cardioblasts downstream of the Tbx20-homolog mid. This activation is blocked by the COUP-TF-homolog Svp (which itself depends on Hh inputs during stages 11-12) in presumptive ostial cells. Doc is expressed by default in these cells and contributes to the repression of tin. Myocardial Tin represses Doc and prevents wg expression. Wg is expressed only in Doc-positive ostial cells that also express Abd-A. These cells, which feature an elongated shape, differentiate into inflow valves. Bottom: in embryos that lack cardiac tin expression (either owing to the absence of the required cis-regulatory element tinC or because of the missing tin trans-activator Mid), all cardioblasts express Doc independently of Svp and wg is activated in all cardioblasts of the Abd-A domain. (H) Misexpression of tin, which causes repression of Doc in Svp+ cardioblasts, leads to loss of Wg in those cells (red arrows; green arrows are as in A). (I) Forced expression of mouse Nkx2.5 in the dorsal vessel using S59-Mef2-HtD-Gal4 and UAS-Nkx2.5 leads to repression of Doc and wg in the heart, similar to UAS-tin.

View larger version (128K): [in a new window]   Fig. 7. Loss of tinman in larval and adult hearts severely compromises its structure and function. (A,B) F-actin (phalloidin) staining of regions of third instar larval dorsal vessels. In the wild type (A, posterior aorta; A', heart), the myofibrils are arranged in a helical network, whereas in tin-ABD, tin346/tinEC40 mutant animals (B, posterior aorta; B', heart), the myofibrils run largely parallel to the AP axis and are highly irregular in the posterior heart region. (C-F) -Actinin staining of 2-day-old adult hearts at low (C,D) and high magnifications (E,F). The tin mutant hearts (arrows in D, F; tin-ABD; tin-ABD, tin346/tinEC40) are much narrower because of severe hypotrophy and have less intensely -actinin-stained myofibrils than do heterozygous controls (C,E). Spiral myofibrils are lacking in the cardiac tin mutant (F). We also observe a much-reduced contractility of these hearts (data not shown). (G) Pacing-induced failure rates for flies with absent cardiac Tin (tin-ABD, tin346/tin346 and tin-ABD; tin-ABD, tin346/tinEC40; 2- to 3-day-old adults) paced by external electrical stimuli to 6 Hz for 30 seconds. Failure rates are dramatically increased in flies lacking cardiac tin expression beginning at mid-embryonic stages to adulthood. (H) Recovery rate from pacing-induced heart failure is dramatically decreased in flies with absent cardiac tin function. The flies with heart failure (arrested or fibrillating) were monitored for recovery from failure to a regular heartbeat for 2 minutes after pacing (recovery rate). (I) Demographic survivorship of flies with ablated cardiac tin expression showing a much-reduced lifespan of these flies.

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