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First published online 17 October 2007
doi: 10.1242/dev.008821

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A key role of Pox meso in somatic myogenesis of Drosophila

Hong Duan^1,*,, Cheng Zhang^1,, Jianming Chen^1,, Helen Sink², Erich Frei¹ and Markus Noll^1,

¹ Institute for Molecular Biology, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
² Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Expression of Poxm protein in developing larval body wall muscles during embryogenesis. (A-L) Whole-mount wild-type embryos were stained with purified anti-Poxm antiserum. Entire embryos (A,C,E,G,I,K) and enlarged views of parasegments 6-8 (B,F,H), 7 and 8 (D), or 6-9 (J,L) at early stage 10 (A,B), late stage 10/early stage 11 (C,D), mid stage 11 (E,F), stage 12 (G,H), stage 13 (I,J) and stage 15 (K,L) are shown. Note that the characteristic triangular pattern of early Poxm expression (D) in parasegments 2-14 is obscured in the overviews (C,E) because not all Poxm-expressing cells are in focus. Therefore, embryos were unfolded and flattened to show enlarged views of parasegments at extended germ band stages (B,D,F) and during germ band retraction (H). Embryos are oriented with their anterior to the left and dorsal side up. At stage 12, Poxm is expressed in the founders of muscles DO3, DT1, VA1-3 and in the ventral adult precursor (VaP; H), and later in the precursors of these muscles except DO3 and VaP (J,L). (M-O) Muscles that express Poxm were identified by double-labeling of embryos from P{Mhc-tauGFP}/TM6B parents with anti-GFP and anti-Poxm to reveal the muscle pattern (M), the late Poxm expression (N) and the merged image (O). Lateral and ventral muscles in four abdominal hemisegments of a stage 15 embryo with its anterior to the left and dorsal side up are shown. Note that muscles VA1 and VA2 overlap dorsally. For muscle nomenclature, see Bate (Bate, 1993) or Fig. 4J,N.

View larger version (101K): [in this window] [in a new window]   Fig. 2. Early ventral and lateral Poxm expression complementary to dorsal high Tin expression in somatic mesoderm precedes its expression in progenitors of ventral and lateral somatic muscles and in a specific subset of muscle founders. (A-C) Early Poxm expression is restricted to the ventral and lateral somatic mesoderm. Poxm expression (B) and high levels of Twi (A), which mark the somatic mesoderm (Dunin Borkowski et al., 1995; Baylies and Bate, 1996) can be seen to coincide in the ventral and lateral regions of the somatic mesoderm (C), as observed by confocal microscopy. A ventral view of four abdominal parasegments of a late stage 10/early stage 11 embryo, oriented with their anterior to the left. (D-F) Complementary expression patterns of Poxm and high levels of Tin in the somatic mesoderm. Poxm expression in the ventral and lateral regions (E) abuts high expression levels of Tin in the dorsal region (D), but coincides with lower levels of Tin (F) in an early stage 11 embryo shown in a ventral view. (G-I) Poxm is expressed in cells of promuscular clusters and muscle progenitors. Poxm (G) and L(1)sc (H) expression coincide in many ventral and lateral muscle progenitors (I), as observed by double-labeling of Poxm and L(1)sc. Some cell clusters and single progenitors that coexpress L(1)sc and Poxm are indicated by arrows and arrowheads, respectively. Three abdominal parasegments, oriented with their anterior to the left, are shown on both sides of the ventral midline of a mid stage 11 embryo. (J-L) Poxm is expressed in specific muscle founders and in the ventral adult precursor. Lateral views of four abdominal hemisegments of a stage 12 embryo double-labeled for late Poxm (J) and Slou (K) expression, oriented with its anterior to the left, revealed by confocal microscopy. In the hemisegment where muscle founders are marked by arrows, expression of late Poxm and Slou coincide (L) in the founders of muscles DO3, DT1, VA2, VA3, and in the ventral adult muscle precursor VaP, whereas Slou has disappeared from the VA1 founder that continues to express Poxm (Carmena et al., 1995; Dohrmann et al., 1990). Expression of Slou in the founders of VT1 and LO1, which do not express Poxm, is also clearly visible (Carmena et al., 1995; Dohrmann et al., 1990).

View larger version (98K): [in this window] [in a new window]   Fig. 3. Most ventral and lateral somatic muscle founders are recruited from cells expressing early Poxm. (A-E) Early and late Poxm expressions in the somatic mesoderm are regulated by different enhancers. Whole-mount transgenic Poxm1.8-Gal4/UAS-lacZ (A,B) and Poxm8.4-Gal4/UAS-lacZ (C,D) embryos were stained with rabbit anti-ß-galactosidase antiserum. A map of the Poxm upstream region (B, BamHI; R, EcoRI; X, XbaI), which delimits the 1.8 kb and 8.4 kb fragments used as enhancers in combination with the hsp70 minimal promoter to drive Gal4 expression in the Poxm-Gal4 transgenes, is shown in E. Overviews of late stage 11 embryos (A,C) and enlarged ventral and lateral views of abdominal segments A2-A4 (B) or A4-A6 (D) of stage 16 embryos are shown with anterior to the left and dorsal up. Muscle patterns (B,D) were visualized from the interior (B) or exterior (D) after staining for ß-gal, by dissecting the embryos in halves along the dorsal and ventral midlines, removing tissue below the muscles, and mounting the ectoderm with the attached muscles for bright-field microscopy in a Zeiss Axiophot. The moderate to low ß-gal levels observed at late stages after early activation by the 1.8 kb enhancer result from perdurance (B), whereas the high ß-gal levels observed after activation by the 8.4 kb enhancer mimic late stage Poxm expression (D). For muscle nomenclature, see Bate (Bate, 1993) or Fig. 4J,N. (F,G) Absence of late Poxm expression of a Poxm transgene driven by the early enhancer. Homozygous Poxm361 embryos, rescued by the um1-2-Poxm transgene that includes only upstream cis-regulatory sequences up to the XbaI site (E) and no intron, exhibit a wild-type early Poxm pattern (stage 11; F) but no late Poxm expression (stage 16; G). Confocal micrographs of embryos with their anterior to the left and dorsal side up are shown. (H-J) Cells expressing early Poxm give rise to most ventral and lateral muscle founders. Cells expressing early Poxm were labeled by nuclear GFP (H) and their fate was followed by confocal microscopy in rP298-lacZ; Poxm1.8-Gal4/UAS-GFPnls; lmd1 embryos, in which founders are marked by ß-gal (I) and their fusion with FCMs is blocked. Most ventral and lateral muscle founders that are labeled by ß-gal are also marked by GFP (J), many of which are marked by white arrowheads in two of the three abdominal segments of a stage 15 embryo shown in H and I.

View larger version (96K): [in this window] [in a new window]   Fig. 4. Muscle phenotypes of Poxm and l(1)sc mutants. (A-I) Muscle phenotypes resulting from loss of, or ectopic, Poxm. Dorsal (A-C), lateral (D-F) and ventral (G-I) muscles were visualized using an anti-MHC antiserum in three abdominal hemisegments of stage 16 wild-type (A,D,G), PoxmR361 (B,E,H), and 24BGal4/UAS-Poxm (C,F,I) embryos, oriented with anterior to the left and dorsal up. In PoxmR361 mutants, positions of missing muscles DT1 (white arrowheads) or an abnormal muscle DT1 (*) and of a missing muscle DO3 (black arrowhead) are indicated in E, ventral muscles VO4-6 are missing (H), and ventral muscles VA1-3* have lost their normal shape and attachments (H), as evident from a comparison with the wild-type ventral muscle pattern (G). Muscle VL4 is also frequently absent, as evident from inspection of a plane of focus interior to and below that shown in H. A detailed analysis of the PoxmR361 muscle phenotype is summarized in Fig. 5A. Ectopic ubiquitous mesodermal expression of Poxm (C,F,I) generates ectopic dorsal and lateral muscles (marked by asterisks in F) and enlarges some ventral muscles (arrows in I), the number and positions of which are not altered. (J) Schematic external view (dorsal up and anterior to the left) of larval muscles in abdominal segments A2-A7 (Ruiz-Gómez et al., 1997), with external muscles in red and more internal muscles in blue and yellow; muscles are designated and numbered according to Bate (Bate, 1993) and in parentheses according to Crossley (Crossley, 1978). (K-M) Muscle phenotypes of Poxm mutants rescued by early Poxm, and of l(1)sc; Poxm double and l(1)sc single mutants. Muscle phenotypes were visualized using an anti-MHC antiserum in three abdominal hemisegments of PoxmR361 embryos rescued by two copies of the um1-2-Poxm transgene (K), of Df(1)sc19 embryos (L), and of Df(1)sc19; PoxmR361 embryos (M) at stage 16. Anterior is to the left and dorsal up. A detailed analysis of these phenotypes is summarized in Fig. 5B-D. Some muscles that are abnormal in shape and/or position are marked by asterisks, duplicated muscles DT1 (K) and DO3 (M) are labeled, and missing muscles VT1 (L), DT1 and LO1 (M) are indicated by black arrowheads. Ventral muscles VO4-6 that are missing in nearly all segments of Poxm single or double mutants (Fig. 5A,D) are also absent but not marked (M). (N) Schematic internal view of a hemisegment opposite to that shown in J (Ruiz-Gómez et al., 1997) of the muscle phenotype attributable to the absence of the early Poxm function, in which each muscle is colored in a graded fashion from red (0%) to yellow (100%) corresponding to the fraction of normal muscles observed (Fig. 5A). Muscles DO3, DT1 and VA1-3 are not colored as the contribution to their phenotype of the missing early Poxm cannot be estimated because they are also affected by the late Poxm function, and muscle VO3 is not colored because it has not been recorded in Fig. 5A.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Larval body wall muscle phenotypes of Poxm and l(1)sc single and double mutants, and of Poxm mutants rescued by early Poxm. (A-D) The somatic muscle patterns of PoxmR361 embryos (A), PoxmR361 embryos rescued by two copies of the um1-2-Poxm transgene (B), of Df(1)sc19/Df(1)sc19 or Y (C), and Df(1)sc19/Df(1)sc19 or Y; PoxmR361 (D) embryos and of y w control embryos (not shown) were analyzed at stage 16 under bright-field optics in a Zeiss Axioplan 2 microscope after staining with anti-MHC antiserum, dissection along the ventral midline, and removal of internal tissues. Each muscle plotted on the abscissa was scored for absence (red), abnormality (yellow), duplication (purple), or wild-type appearance (green) in each of 108 (A-C) or 168 (D) hemisegments in abdominal segments A2-A7, and the resulting fractions were plotted on the ordinate. Muscles of the y w control embryos were usually normal in all 108 hemisegments scored, with the occasional absence, duplication or abnormality of a single muscle. (E) The fractions of normal muscles shown in A-D are plotted for Poxm mutants rescued by early Poxm (purple), Poxm (red) and l(1)sc (yellow) single mutants, and for l(1)sc; Poxm (green) double mutants, whereas muscles are ordered along the abscissa with decreasing abnormality of Poxm mutant phenotypes.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Loss of Poxm and ectopic Poxm change the number of adult muscle precursors. (A-C) Expression of Twi in abdominal hemisegments of wild-type (A), PoxmR361 (B), and 24BGal4/UAS-Poxm (C) stage 14 embryos was visualized by staining with an anti-Twi antiserum (anterior to the left and dorsal up). Only precursors of adult muscles and the alary cells express Twi at this stage [alary cells, marked by black arrows, are located on the segment margins in the dorsal and dorsolateral region; see Bate et al. (Bate et al., 1991)]. The adult muscle precursors are arranged in dorsal (D), dorsolateral (DL), lateral (L) and ventral (V) groups. The staining of the trachea is caused by a crossreactivity of the anti-Twi antiserum. (D) Analysis of number of adult muscle precursors in embryos mutant for Poxm or expressing ubiquitous mesodermal Poxm. The number of adult muscle precursors was analyzed in 90 hemisegments each of PoxmR361 and 24BGal4/UAS-Poxm embryos. The total numbers for 90 hemisegments are shown for the dorsal (DaP), dorsolateral (DLaP), lateral (LaP) and ventral (VaP) group of adult muscle precursors with the numbers expected for wild-type embryos in parentheses.

View larger version (138K): [in this window] [in a new window]   Fig. 7. Altered Slou expression in Poxm mutants or in the presence of ectopic Poxm. Slou expression in wild-type (A,C,E), PoxmR361 (B,D), and 24BGal4/UAS-Poxm (F) embryos at stage 12 (A,B), 14 (C,D) and 16 (E,F) was visualized by staining with an anti-Slou antiserum. Ventrolateral views of whole embryos (C,D) and enlarged ventrolateral (A,B) and dorsolateral (E,F) views of parasegments 4-10 (A,B) and 6-7 (E,F) are shown with anterior to the left and dorsal up. At late stage 12, Slou is expressed in three groups, I-III, of muscle founder cells of each abdominal segment of wild-type embryos (A), whereas it is expressed only in groups I and II of PoxmR361 mutants (B). At stage 14, Slou is expressed in the precursors of dorsal muscle DT1 and of ventral muscles VT1 and VA2 of each abdominal segment of wild-type embryos (C), whereas it is detectable only in the precursor of ventral muscle VT1, but not of muscles DT1 and VA2, in PoxmR361 embryos (D). After ectopic expression of Poxm, additional muscles express Slou (arrows in F) in a dorsolateral region where Slou is expressed only in muscle DT1 of stage 16 wild-type embryos (E).

View larger version (20K): [in this window] [in a new window]   Fig. 8. Regulatory network of somatic myogenesis. The scheme depicts the interactions among the major genes and/or their products that regulate the development of larval body wall muscles from gastrulation to the specification of muscle founder cells, as explained in detail in the text. After the establishment of the high Twi domain in the mesoderm underneath the posterior portions of parasegments by Slp, domains competent for somatic myogenesis are specified by `competence domain genes', such as l(1)sc, tin (the hypothetical role of tin as competence domain gene is indicated by parentheses) and Poxm, regulated by Twi and the ectodermal signals Wg and Dpp. These signals, in combination with the localized EGF (Spi) and FGF (Pyr, Ths) signals and through remote inhibition of Spi signaling by Aos (Freeman, 1997), determine the promuscular clusters, which express l(1)sc and activate the MAPK signaling pathway. Singling out of muscle progenitors and separation from fusion-competent myoblasts (FCMs) occurs by lateral inhibition, which is mediated by N signaling that is coupled to MAPK signaling through multiple feedback loops. At this stage, or subsequently, in muscle founder cells generated from progenitors by asymmetric division mediated by N signaling, muscle identity genes, such as Poxm, Kr and slou, are activated by the integration on their enhancers of competence domain gene products with the effectors of the mentioned signals (for references, see text). Hypothetical interactions are indicated by dashed lines.

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