InteractiveFly: GeneBrief

Pox meso: Biological Overview | References

BIOLOGICAL OVERVIEW

The Pax gene Pox meso (Poxm) was the first and so far only gene whose initial expression was shown to occur specifically in the anlage of the somatic mesoderm, yet its role in somatic myogenesis remained unknown. This study shows that it is one of the crucial genes regulating the development of the larval body wall muscles in Drosophila. It has two distinct functions expressed during different phases of myogenesis. The early function, partially redundant with the function of lethal of scute [l(1)sc], demarcates the 'Poxm competence domain', a domain of competence for ventral and lateral muscle development and for the determination of at least some adult muscle precursor cells. The late function is a muscle identity function, required for the specification of muscles DT1, VA1, VA2 and VA3. These results led to a reinterpretation of the roles of l(1)sc and twist in myogenesis and to the proposal of a solution of the 'l(1)sc conundrum' (Duan, 2007).

The development of the complex pattern of the larval body wall muscles of Drosophila provides an excellent paradigm of how a final pattern is established through precise genetic control. Each of the abdominal hemisegments A2-A7 has 30 identifiable individual muscles that develop from the somatic mesoderm. This process is initiated when the invaginated mesoderm migrates dorsolaterally under the ectoderm and is prepatterned by the segmentation genes: the product of sloppy paired (slp), whose activity is maintained by the ectodermal Wingless (Wg) signal, restricts high levels of the bHLH transcription factor Twist (Twi) to the mesodermal regions below the posterior portions of the ectodermal parasegments. These high levels of Twi function as a myogenic switch, separating the posterior somatic and cardiac mesoderm from the anterior visceral mesoderm and fat body. When the dorsal migration of the mesoderm is complete, these metamerically repeated Slp or high Twi domains are further subdivided along the dorsoventral axis by the ectodermal signal Dpp. This signal restricts transcription of tinman (tin) to the dorsal mesoderm, where its homeodomain protein specifies heart and dorsal somatic mesoderm. However, the determinant of the non-dorsal somatic mesoderm remains largely unknown. It appears that Pox meso (Poxm) expression is restricted to the ventral part of the high Twi domain by Dpp (Staehling-Hampton, 1994) to define the lateral and ventral somatic mesoderm anlage. The characterization of the role of Poxm in somatic myogenesis is therefore expected to fill an important gap in understanding of the gene network regulating this process (Duan, 2007).

Soon after this subdivision of the mesoderm, the proneural gene lethal of scute [l(1)sc] begins to be expressed in at least 19 promuscular clusters of cells within the high Twi domain. From these clusters, muscle progenitors are singled out by lateral inhibition through Notch (N) and Ras signaling and are specified by the expression of muscle-identity genes. Cells not singled out begin to express the zinc finger protein Lame duck (Lmd; also known as Minc), which specifies them as fusion-competent myoblasts (FCMs). The progenitors divide to generate different muscle founders, a muscle founder and an adult muscle precursor, or a founder and a cell producing either two adult muscle precursors or two pericardial cells. Each founder forms an individual syncytial muscle precursor by fusing with neighboring FCMs. One of the key steps in muscle pattern formation is the specification of a muscle founder by the expression of a specific set of muscle identity genes. Although an increasing number of these genes have been identified in recent years, the mechanisms that activate their transcription are still poorly understood. Hence, it is important to identify the genes whose products directly regulate the muscle identity genes (Duan, 2007).

This study describes the functional characterization of the Poxm gene. Poxm belongs to the Pax gene family whose members encode transcription factors, including a paired domain (Bopp, 1989) (reviewed by Noll, 1993). The temporal and spatial expression patterns of Poxm and its loss- and gain-of-function phenotypes reported in this study demonstrate that it is required for most ventral and lateral abdominal muscles to develop properly in all segments and for the activation of muscle identity genes. In addition, Poxm acts itself as muscle identity gene in a few muscles and thus plays a dual role in somatic myogenesis (Duan, 2007).

The Poxm gene has been cloned on the basis of its homology to the paired box of the paired (prd) and gooseberry (gsb) genes (Bopp, 1986), and was mapped to chromosomal band 84F11-12 (Bopp, 1989). It extends over more than 20 kb that include two exons and many cis-regulatory elements located in the upstream region and the large intron (Bopp et al., 1989). The Poxm protein, predicted from the longest open reading frame, consists of 370 amino acids and includes a paired domain close to its N terminus and an octapeptide in its C-terminal moiety. Except for its first 10 base pairs, the open reading frame is encoded entirely by exon 2. The paired domain of Poxm belongs to the Pax1/9 class (Bopp, 1989; Noll, 1993) and displays 88% identity and 92% similarity to mammalian Pax1/9-type paired domains (Fu, 1997; Duan, 2007 and references therein).

In agreement with earlier results (Bopp, 1989), Poxm protein is localized in the nucleus and first detectable in the somatic mesoderm at early stage 10. During stage 10, Poxm becomes expressed in segmentally repeated mesodermal 'stripes' underlying the ectodermal parasegments 2-14, in the cephalic mesoderm, the proctodeal anlage and a group of ectodermal cells in the clypeolabrum, which presumably corresponds to part of the esophageal anlage. At this stage, the posterior boundaries of mesodermal Poxm coincide with those of ectodermal Gsb (Bopp, 1989), which largely coincide with the parasegmental borders. Consistent with these calibrations along the anteroposterior axis and those of others (Riechmann, 1997), it was found that Poxm is expressed in cells of the high Twi domain in the ventral and lateral mesoderm. Since Poxm is repressed in the dorsal portion of each segment by the ectodermal signal Dpp (Staehling-Hampton, 1994), the number of Poxm-expressing cells is reduced with decreasing distance from the dorsal margin, thus forming a triangular pattern. At this stage, Tin expression is not yet completely restricted to the dorsal mesoderm. Whereas high levels of Tin in the dorsal region and Poxm are expressed in complementary patterns, Poxm is coexpressed with low Tin levels in the ventral and lateral regions. During stage 11 Poxm is restricted to fewer cells, some of which will form subsets of muscle progenitors and cells of the promuscular clusters, as evident from its partial co-localization with L(1)sc. During germ band retraction, Poxm disappears from the most anterior mesodermal stripe and the telson. By stage 12, Poxm expression is maintained only in six cells each of the abdominal segments A1-A7, identified as founders of muscles DO3, DT1 and VA1-VA3, and as ventral adult muscle precursor (VaP) by double-staining of Poxm and Slouch (Slou). At this time, it becomes apparent that more cells express Poxm in the ventral regions of the thoracic segments than of the abdominal segments. This study focuses on the role of Poxm in myogenesis of abdominal segments A2-A7 (Duan, 2007).

As myoblast fusion proceeds during stage 13, the number of Poxm-positive nuclei increases. These coincide with the precursors of muscles DT1 and VA1-3, identified by double-staining of Poxm and MHC-tauGFP (Myosin heavy chain-tauGFP). During stage 15, Poxm expression begins to be reduced in the ventral clusters and is diminished in the dorsolateral region, from which it disappears during stage 16. By stage 17, Poxm is no longer detectable in the mesoderm or any of its derivatives. Outside the mesoderm, particularly striking is its expression in the developing gut ectoderm and midgut, where it is maintained at high levels throughout embryogenesis (Duan, 2007).

In summary, Poxm expression in the ventral and lateral portions of the high Twi domain colocalizes with weak Tin expression and is complementary to the high levels of Tin in the dorsal region. Subsequently, its mesodermal expression is confined to fewer cells, some of which will form promuscular clusters and muscle progenitors. Poxm persists in some, but not all of the muscle founders derived from the Poxm-expressing progenitors and is ultimately expressed in four muscle precursors. After the formation of muscle fibers, Poxm disappears. This time course of Poxm expression in developing muscles suggests that Poxm functions in somatic myogenesis (Duan, 2007).

To further analyze the nature and fate of Poxm-expressing cells during early and late myogenesis, lacZ was expressed under the indirect control of different Poxm upstream regions by the use of the Gal4/UAS system. Because of the perdurance of ß-galactosidase (ß-gal) resulting from (1) the amplification and delay of ß-gal inherent in the Gal4/UAS system and (2) the considerably enhanced stability of both Gal4 and ß-gal proteins as compared to that of Poxm, the fate of cells expressing Poxm can be followed during earlier embryonic stages by examining ß-gal expression at later stages (Duan, 2007).

Under the control of a 1.8 kb upstream fragment of Poxm, ß-gal is expressed in a pattern similar, but not identical, to that of early Poxm in the mesoderm, presumably because of the temporal delay in expression of the Gal4/UAS system. A similar early expression pattern is observed when lacZ is expressed under the control of an 8.4 kb upstream fragment. ß-gal expression was examined under the direct control of the 1.8 kb and 8.4 kb Poxm enhancers. In both cases, ß-gal and Poxm are coexpressed during early embryonic stages and no ectopic ß-gal is detectable (Duan, 2007).

Patterns of ß-gal expression were then examined at later stages in differentiating muscles. At stage 16, the 8.4 kb fragment supports strong lacZ expression in muscles DT1 and VA1-3, in agreement with late Poxm expression, which is restricted to these muscles. In addition, however, muscles VL1-4, VO1-6, frequently LT3 and LT4, and occasionally muscle SBM are labeled by ß-gal, although at moderate or considerably lower intensities. By contrast, when lacZ is expressed under control of the 1.8 kb fragment, it is not detected in muscle DT1 and only at low or moderate levels in muscles VA1-3. It follows that late Poxm expression is under the control of sequences present in the 8.4 kb but not the 1.8 kb fragment. Owing to perdurance, when expressed only under control of the early enhancer, ß-gal is also observed at moderate or low levels in the ventral muscles VL1-4, VO1-6, frequently in the lateral muscles LT3, LT4, LL1, LO1, SBM and rarely in LT2 and VT1 (Duan, 2007).

These results indicate that cells expressing Poxm early during myogenesis are those from which ventral and lateral muscle progenitors are selected. However, since muscle fibers form by fusion of founders with FCMs, ß-gal patterns observed in differentiating muscles may result from the perdurance of ß-gal in founders or FCMs. To rule out the possibility that this perdurance is derived exclusively from expression in FCMs, the expression of nuclear GFP was examined under indirect control of the 1.8 kb fragment in lmd¹ or Dmef2^22-21 mutants, in which fusion is blocked and muscle founders were marked by the dumbfounded enhancer trap chromosome rP298-lacZ. Because of the perdurance of GFP, the fate of cells expressing early Poxm can be followed by examining their expression of GFP at later stages. In lmd¹ embryos, GFP is expressed only in the ventral and lateral portions of each segment at stage 15. Since in the absence of myoblast fusion most founders, marked by ß-gal, also express GFP at least weakly, it is concluded that cells expressing Poxm early during myogenesis will give rise to most founders of the ventral and lateral muscles. In addition, Poxm is expressed early in mesodermal cells that are not selected as progenitors, as evident from the perdurance of GFP in many mesodermal cells different from founders. Similar results were obtained for Dmef2^22-21 mutants (Duan, 2007).

The expression patterns of Poxm suggest that it plays a crucial role in myogenesis. Assuming that absence of Poxm functions results in lethality, a collection of 1,400 lethal P-element insertions on the third chromosome was performed for lack of complementation with the deficiency Df(3R)dsx^D+R5, which uncovers Poxm (Bopp, 1989), and subsequently for complementation with Df(3R)dsx^M+R29, whose distal break point is located proximal to Poxm, at 84F6-7. One lethal insertion, P282, was identified that had inserted into the neighboring gene, 5 kb downstream of the second exon of Poxm. Embryos homozygous for P282 did not show any muscle defects. Imprecise excision of this P element produced a deficiency, Df(3R)159, whose distal breakpoint is located about 10 kb upstream of the Poxm transcription start site. Its proximal breakpoint maps distal to the more proximal deficiency Df(3R)dsx^M+R29, with which it complements. Embryos homozygous for Df(3R)159 show severe defects in the larval somatic musculature (Duan, 2007).

Since Df(3R)159 deletes, in addition to Poxm, at least another gene, the observed muscle phenotype might result from the absence of more than just Poxm functions. Therefore, eight EMS-induced embryonic lethal mutants, obtained in a screen for genes on the third chromosome affecting neuromuscular connectivity, that showed defects in muscle patterning were tested for complementation with Df(3R)159. One of these mutants, R361, failed to complement and showed the same larval muscle phenotype as Df(3R)159, in homozygous and transheterozygous conditions. No Poxm protein was detectable in either mutant. Sequencing of R361 genomic DNA identified, in Poxm, a single point mutation, Poxm^R361, that converts a glutamine codon at position 7 of the N-terminal paired domain into an amber stop codon and hence is expected to result in a truncated N-terminal Poxm peptide of 14 amino acids. It follows that Poxm^R361 is a null allele of Poxm (Duan, 2007).

To investigate the effects of Poxm on muscle development, embryos homozygous or transheterozygous for Df(3R)159 and Poxm^R361 were examined after visualizing their somatic muscles by staining with anti-MHC. These mutants all displayed the same severe defects in the formation of larval muscles. In this analysis, which focused on abdominal segments A2-A7, Poxm was considered to be required for the proper development of a specific muscle if that muscle did not form normally in a significant fraction of hemisegments in Poxm null mutants. It does not imply that this muscle never forms normally, as the penetrance of the phenotype may not be 100% (Duan, 2007).

In the ventral region of Poxm mutant embryos, usually muscles VO4-6 are absent, whereas muscles VA1-3 are still present in most segments but are poorly developed, lacking their normal shape and attachment sites. Further analysis revealed that muscles VL3 and VL4 are frequently abnormal or missing, whereas muscles VL1 and VL2 are occasionally or rarely affected. Also muscles VO2 and VO1 are strongly and moderately disturbed, respectively (Duan, 2007).

In the dorsolateral region, muscle DT1, in most cases, is missing or abnormal, whereas muscle DO3, which is derived from the same progenitor, is mostly duplicated or abnormal and very rarely missing. Two additional muscles, DA3 and DO4, are occasionally abnormal, whereas the two most posterior lateral muscles, LO1 and LT4, are frequently missing and abnormal, respectively. By contrast, all dorsal muscles remain unaffected (Duan, 2007).

Ordering the muscles along the abcissa according to decreasing severity of their Poxm mutant phenotype reveals a striking correlation with the early triangular Poxm expression pattern. Muscles located more ventrally or more posteriorly in a segment are always more strongly affected as compared to muscles located roughly at the same anteroposterior or dorsoventral positions, respectively. For example, muscle VL4 is affected more severely than its dorsal neighbor VL3, which is again more frequently abnormal than VL2 or VL1. Similarly, the phenotype of muscle LT4 is stronger than that of its anterior neighbors LT1-3. This phenotype suggests that it might be affected by a function that depends on a dorsoventral as well as an anteroposterior gradient, on which indeed the early Poxm expression pattern depends, namely on Dpp (Staehling-Hampton, 1994) and Wg, and which explains its characteristic triangular shape (Duan, 2007).

To test whether Poxm can determine muscle development, it was expressed ectopically and effects on myogenesis were examined. 24BGal4 was used to drive expression of UAS-Poxm in the entire mesoderm beginning at mid stage 10. Ectopic Poxm produces a severely altered muscle pattern, which varies among different segments and embryos. The most striking defects occur in the dorsal and dorsolateral muscles, where Poxm is normally absent or present at low levels. In the dorsal region, which includes four muscles in wild-type embryos, ectopic muscles are generated in most segments. Ectopic muscles similar in shape and orientation to muscle DA3 occupy the dorsolateral region, which is largely free of muscles in wild-type embryos. Usually several muscles with abnormal shape occur at the position of muscle DT1, whereas muscles LL1, DO4 and DO5 exhibit aberrant shapes or are missing in some segments. In addition, some of the lateral muscles are abnormally shaped. By contrast, the ventral muscles, all of which exhibited a strong early Poxm expression, remain largely unaffected, although some muscle fibers appear enlarged (Duan, 2007).

Since adult muscle precursors derive from progenitors that also generate founders of larval muscles, it was suspected that Poxm also affects adult muscle precursors. To test this hypothesis, the effects of loss-of-function and ectopic expression of Poxm on the expression of Twi, which is present in all adult muscle precursors but not in larval founders after germ band retraction, was analyzed. In stage 14 wild-type embryos, adult muscle precursors appear in four groups with a single precursor each in the ventral and dorsal groups (VaP and DaP) and two each in the dorsolateral and lateral groups (DLaPs and LaPs) (Duan, 2007).

In the lateral mesoderm of embryos expressing Poxm ubiquitously, in most segments DLaPs are missing and only one of the two LaPs is present. The reverse situation was found in Poxm mutants. The number of LaPs increases to four to seven cells in each abdominal hemisegment, and more than two DLaPs are present in 20% of the segments. Therefore, in the lateral portions of the abdominal segments, Poxm acts to prevent the formation of supernumerary adult muscle precursors and, when ectopically expressed, can inhibit the formation of normal adult muscle precursors (Duan, 2007).

In the dorsal region, after mesodermal ubiquitous expression of Poxm, on average two DaPs instead of one are present in about half of the segments. This result correlates with the appearance of ectopic dorsal muscles and hence suggests that ectopic expression of Poxm leads to the production of supernumerary adult muscle precursors and muscle founders in the region where normally only a very low level of Poxm is expressed at early embryonic stages. In embryos lacking Poxm, however, DaPs remain largely unaffected. In the ventral region, the number of VaPs is hardly changed not only in the presence of mesodermal ubiquitous Poxm but also in the absence of Poxm (Duan, 2007).

Early expression of slou, one of the well-studied muscle identity genes, occurs in a subset of muscle progenitors and their offspring founders, some of which also express Poxm. This raises the possibility of an epistatic relationship between these genes. Early Slou-expressing cells are arranged in three groups of muscle founders: group I will generate muscles LO1 and VT1; group II, muscles VA1-3 and the VaP; and group III, muscles DO3 and DT1. After stage 13, Slou remains expressed only in the precursors of muscles DT1, VT1 and VA2, two of which, DT1 and VA2, also express Poxm. In Poxm^R361 embryos, Slou protein is expressed in groups I and II, yet is absent from group III in most, though occasionally observed in more posterior, abdominal segments during late stage 12. After stage 13, Slou is detectable only in the precursor of muscle VT1 but no longer maintained in that of VA2 in abdominal segments. Therefore, Poxm is essential for the activation of slou in the progenitor of muscle DT1 and for its maintenance in the precursor of muscle VA2 (Duan, 2007).

In 24BGal4/UAS-Poxm embryos, in which Poxm is ubiquitously expressed in the mesoderm, additional muscles expressing Slou were found in the dorsolateral portion of some segments, which suggests that in these cells ectopic Poxm suffices to activate slou and corroborates the observation that Poxm acts upstream of the muscle identity gene slou (Duan, 2007).

Since Poxm is expressed during early myogenesis in cells that later give rise to progenitors of most of the ventral and lateral muscles, it may play an important role in the initiation of muscle patterning. To investigate which part of the Poxm^R361 muscle phenotype results from the loss of this early Poxm function, a transgene expressing Poxm only during the early myogenic stages, um1-2-Poxm, was introduced into Poxm^R361 embryos. In these embryos, the phenotypes of muscles VO4-6, VL2-VL4, VO2, VO1, LO1, LT4 and VT1 are efficiently rescued. The only muscles affected in Poxm mutants that are only slightly rescued by early Poxm are DT1, DO3 and VA1-3, in which Poxm is also expressed during later stages in their founders and/or muscle precursors. These results strongly suggest that Poxm exerts an early function, demarcating a mesodermal domain of competence for ventral, lateral and dorsolateral somatic muscle development (Duan, 2007).

The partial penetrance of the Poxm muscle phenotype suggests that the early Poxm function is largely redundant with that of other genes, an argument also raised to explain the weak muscle phenotype of l(1)sc mutants. The l(1)sc gene encodes a bHLH transcription factor the function of which is thought to be required for the selection of muscle progenitors. Therefore, the effect was examined of Poxm and l(1)sc mutations on larval muscle development in single and double mutant embryos (Duan, 2007).

In agreement with earlier studies, l(1)sc mutants exhibit a weak muscle phenotype, which deviates only slightly from that of wild-type embryos. Although Poxm^R361 embryos show a considerably stronger muscle phenotype, most lateral and dorsal muscles are normal. Assuming that Poxm and l(1)sc act independently in muscle development, it was expected that the probability of a muscle being wild-type in Df(1) l(1)sc¹⁹/Y; Poxm^R361 embryos is the product of the probabilities of the muscle being wild-type in the single mutants. Conversely, if significantly enhanced probabilities are found for muscle defects in double mutants, it may be concluded that Poxm and l(1)sc exhibit partially redundant functions during muscle development. Applying this test, it was found that most muscles are affected independently or nearly independently, with some notable exceptions. These concern muscles VL1-3, SBM, VO1, VO2, DT1, LT3, LT4 and VA3 that are more often absent. Some muscles are strongly affected in Poxm null mutants, such as muscles VO4-6 or muscles VA1-3. Among the other muscles, the more ventral and the more posterior a muscle is located within a segment, the more probable it is that it will show an enhanced phenotype in double mutants. Clearly, there is some redundancy between Poxm and l(1)sc functions in the somatic mesoderm, which is restricted largely to ventral and posterior muscles (Duan, 2007).

In Poxm mutants, only muscle DO3 is frequently duplicated. This duplication results from the transformation of muscle DT1 to DO3, as previously observed for muscles derived from the same progenitor in the absence of a muscle identity gene that is asymmetrically expressed in the two founders and muscle precursors. Thus, late expression of Poxm in the precursor of muscle DT1, but not of DO3, is crucial for their distinction and hence serves a muscle identity function. However, a more detailed analysis shows that muscle DT1 is missing in only two thirds (23/34) of all cases in which muscle DO3 is duplicated. In the remaining 11 cases, muscle DT1 is normal (4), abnormal (6) or duplicated (1). This finding suggests that the late Poxm function is necessary in about 10% (11/108) of all cases to prevent an additional division that generates a second founder of muscle DO3. Absence of Poxm in their founders results in abnormal muscles VA1-3 that cannot be rescued by the early Poxm function, which suggests that their proper specification also depends on the late function of Poxm (Duan, 2007).

These results have demonstrated that the development of larval body wall muscles depends on distinct Poxm functions during two phases. The early function of Poxm specifies, within the high Twi or Slp domain, a subdomain of competence for lateral and ventral muscle development, the 'Poxm competence domain'. This function appears to be analogous to that of tin, which specifies competence for heart and dorsal muscle development in the complementary part of the Slp domain. Poxm and tin thus subdivide the posterior Slp domain into ventral and dorsal subdomains in a manner similar to the partitioning by serpent and bap of the anterior Eve domain into the ventral fat body and the dorsal visceral mesoderm anlagen. After selection of muscle progenitors, proper development of a few muscles still depends on Poxm, which is expressed in muscles DT1 and VA1-3. This late function of Poxm participates in founder specification and muscle differentiation, as is characteristic for muscle identity genes. Finally, the findings suggest a solution to a conceptual problem of the current model of somatic myogenesis, the l(1)sc conundrum (Duan, 2007).

The muscle phenotype of Poxm mutant embryos and its rescue by early Poxm expression shows that the early Poxm function is crucial for the proper development of many ventral and lateral muscles. In addition, the generation of ectopic dorsal and dorsolateral muscles by ectopic Poxm suggests that Poxm has the ability to change cell fates and render cells competent for myogenesis. Therefore, it is proposed that early Poxm demarcates a ventral and lateral domain of competence for somatic myogenesis (Duan, 2007).

The partial penetrance of the Poxm mutant phenotype implies the existence of other competence domain genes performing partially redundant functions. Poxm and L(1)sc partially co-localize in the promuscular clusters and muscle progenitors. In addition, a detailed analysis of l(1)sc and Poxm single and double mutants demonstrates that their functions are partially redundant. Since the muscle phenotype of l(1)sc; Poxm double mutants still shows partial penetrance, additional competence domain genes should be expressed in the Slp domain. One of them is probably tin, which is initially expressed in the entire early mesoderm, because tin mutants affect muscle development in the dorsal as well as lateral and ventral Slp domain. Another candidate is D-six4 (Clark, 2006), which is required for the development of specific muscles that arise from the dorsolateral and ventral regions (Duan, 2007).

Thus, after the initial subdivision of the mesoderm, the high Twi domain is further subdivided by competence domain genes, which specify domains that become competent to select progenitors of distinct subsets of somatic muscles and/or of myocardial and pericardial cells by responding to spatially restricted extracellular signals. These competence domain genes act in a cooperative manner to determine the identities of specific muscles by regulating the expression of the muscle identity genes. When one of them is inactivated, in some cells active competence domain genes can partially compensate for the inactive gene by activating its target genes such that these sometimes, but not always, exceed the threshold levels required for normal development. Hence, muscles derived from these cells exhibit a mutant phenotype with partial penetrance. For other cells, active competence domain genes can compensate completely for the missing gene activity such that these cells will adopt the proper fate and the muscles develop normally. This illustrates that competence is not a matter of 'all' or 'nothing' for muscle development. The deeper reason for this is thought to be that the genetic program regulating myogenesis is not organized in a hierarchical fashion but rather as a complex gene networkthat has an integrated function which is much more stable against mutations within the network than a hierarchical regulation would be (Duan, 2007).

Muscle identity genes usually encode transcription factors, such as Slou, Nau, Ap, Vg, Kr, Eve, Msh, Lb, Run and Kn, that are expressed in subsets of muscle progenitors and founders and determine in a combinatorial fashion the identity of each muscle founder and its subsequent differentiation into a specific muscle of defined size, shape, attachment sites, and innervation. It is envisioned the activation of these genes in promuscular clusters or, after lateral inhibition, in muscle progenitors by Twi and/or the products of competence domain genes and through combinations of localized extracellular signals from the ectoderm and mesoderm. During asymmetric division of progenitors, expression of a muscle identity gene may be maintained in one or both of the two sibling founders, or it may persist in the founder when division generates a founder and an adult muscle precursor. Late expression of Poxm illustrates all three cases. It is expressed in progenitors P26/27 and P29/VaP, which are derived from promuscular cluster 10 and give rise to the founders of muscles VA1 (F26) and VA2 (F27), and to the founder of muscle VA3 (F29) and the ventral adult precursor VaP. Poxm is also expressed in the progenitor derived from cluster 13, P11/18, which generates the founders of muscles DO3 (F11) and DT1 (F18). Although Poxm expression persists in F29 and F18 but not in their siblings, it is maintained in both sibling founders F26 and F27 (Duan, 2007).

The late function of Poxm is identified as a muscle identity function by the high correlation between absence of muscle DT1 and corresponding duplication of muscle DO3 in Poxm mutants. If Poxm was the sole determinant discriminating between F11 and F18, mesodermal ubiquitous expression of Poxm would be expected to transform muscle DO3 into DT1. The results confirm the presence of additional muscles in the region of muscle DT1. It is possible that one of these originates from a transformed F11, but it is impossible to tell whether muscle DO3 is missing because additional muscles have been recruited (Duan, 2007).

It has been shown that in the process of muscle diversification, identity genes may repress or activate other identity genes in progenitors and founders. This study found that the muscle identity gene slou fails to be activated in P11/18 of Poxm mutants. The simplest explanation of this result is that activation and maintenance of slou expression depend on Poxm in P11/18 and its offspring founders. In addition, slou expression is not maintained in F27 of Poxm mutants despite its initial activation in P26/27. It therefore appears that in P26/27 and its offspring F26 and F27, in addition to Kruppel (Kr), Poxm is necessary for the maintenance of slou expression. Although Poxm expression is maintained in both F26 and F27, slou expression is restricted to F27 because Kr is repressed in F26 by N signaling. Apparently, Kr is the crucial determinant that distinguishes F26 from F27, as F27 is altered to F26 in Kr or numb mutants (Duan, 2007).

Since Poxm is expressed in both F26 and F27, whereas its expression is restricted to F18 and not maintained in F11, its late expression in F26 and F27 must be regulated differently from that in F11 and F18 where it appears to be subject to asymmetric N signaling repressing Poxm in F11 (Duan, 2007).

These considerations imply that slou is part of the same gene network as Poxm, a conclusion consistent with the proposed gene network hypothesis since, in the first test of this hypothesis, slou had been isolated as a PRD 9 gene on the basis of its homology to the prd gene (Duan, 2007).

The mechanism of progenitor selection from the somatic mesoderm depends on a process of lateral inhibition very similar to that of neuroblast or sensory organ precursor (SOP) selection in the neuroectoderm from proneural clusters expressing the proneural genes. Because of this similarity, a search among proneural genes for 'promuscular' genes expressed in the somatic mesoderm was performed. This search identified a single proneural gene, l(1)sc, a member of the achaete-scute complex (AS-C), that is expressed in promuscular clusters of the somatic mesoderm. It was, therefore, attractive to consider its function in myogenesis to be analogous to that of proneural genes in neurogenesis. However, whereas proneural genes confer on neuroectodermal cells the ability to become neural precursors rather than epidermal cells, which is their default fate, l(1)sc does not seem to confer on mesodermal cells the ability to undergo somatic myogenesis instead of becoming part of the visceral mesoderm, heart or fat body. When L(1)sc was expressed in the entire mesoderm from stage 8 onward, other mesodermal tissues could not be transformed into somatic mesoderm, whereas a deficiency of l(1)sc resulted in only minor defects of somatic muscle development. In addition, as the l(1)sc muscle mutant phenotype can be rescued by ubiquitous mesodermal L(1)sc expression, its expression in clusters is not decisive for the formation of promuscular clusters and, therefore, l(1)sc cannot play the decisive role in the development of larval body wall muscles that has been proposed. Thus, although l(1)sc serves as an excellent marker for promuscular clusters, it lacks a property expected to be crucial for a promuscular gene. Are there genes that might qualify as promuscular genes and thus extend the close evolutionary relationship of progenitor selection between myogenesis and neurogenesis (Duan, 2007)?

There is indeed a gene that is homologous to proneural genes and expressed in the somatic mesoderm, in the absence of which somatic myogenesis is seriously disturbed. This gene is twi, whose function at stages 10 and 11 more closely corresponds to that of a promuscular gene and which, like l(1)sc, encodes a bHLH transcription factor. Although Twi is also expressed earlier when it is required for mesoderm specification during gastrulation, this early function can be distinguished from its later 'promuscular' function in temperature-sensitive mutants. In these mutants, only high levels of Twi activity, necessary for the formation of the somatic mesoderm, are abolished and no normal somatic muscles develop. Moreover, ubiquitous expression of high levels of Twi in the mesoderm blocks all other mesodermal fates, transforming them to somatic mesoderm. Since the subsequent patterning of somatic muscles depends critically on the relative levels of the products of twi and the proneural gene da, it seems appropriate to consider them both as promuscular genes (Duan, 2007).

In addition to its strict requirement for somatic myogenesis, the proposed promuscular function of twi may be subject to lateral inhibition by N signaling, in further analogy to proneural functions in neurogenesis. This is apparent from experiments demonstrating that the restriction of high Twi levels to the Slp domain during stage 9 depends on N signaling, which downregulates twi in the mesoderm underlying the anterior regions of parasegments where Slp does not override it. Since this process acts directly on an identified twi enhancer during stages 9 and 10, it is conceivable that this enhancer also responds to N signaling during the subsequent lateral inhibition. An alternative, though not mutually exclusive, mechanism for the downregulation of twi implicates the Gli-related zinc finger transcription factor Lmd (Minc), whose expression is maintained by N signaling and in the absence of which twi is not downregulated in fusion-competent myoblasts (Duan, 2007).

During lateral inhibition, loss of Twi precedes that of L(1)sc in the promuscular clusters. It is therefore possible that l(1)sc expression in these cells also depends on high levels of Twi, i.e. on Twi homodimers. Consistent with this interpretation, shifting the equilibrium between Twi homodimers and Twi-Da heterodimers in favor of the latter represses l(1)sc. Since early Poxm expression also depends on Twi, Poxm would be similarly repressed in promuscular clusters through lateral inhibition, either indirectly by repression of twi and/or directly by Twi/Da heterodimers. Such a mechanism might apply generally to both competence domain genes and muscle identity genes during lateral inhibition of promuscular clusters (Duan, 2007).

Thus, twi satisfies two criteria considered to be crucial for a promuscular gene in analogy to those of proneural genes in neurogenesis. However, a third criterion is not fulfilled by twi: its expression, in contrast to that of proneural genes in the neuroectoderm, is ubiquitous rather than restricted to promuscular clusters although this criterion is not a crucial property of proneural genes. Yet promuscular clusters from which the myogenic progenitors are selected exist, as evident from the pattern of l(1)sc expression. These promuscular clusters depend on combinations of the long-range ectodermal signals Wg and Dpp and the localized activities of the EGF signal Spi in the mesoderm and the FGF signals Pyr and Ths in the ectoderm. These signals, together with Twi and/or products of competence domain genes depending on Twi, determine the promuscular clusters by activating specific combinations of muscle identity genes. The identity of the promuscular clusters depends not only on the combination of these signals but, in the case of MAPK signaling elicited by FGF and/or EGF, also on their intensity. In addition, multiple positive and negative feedback loops of the coupled MAPK and N signaling networks ensure a stable selection and specification of muscle progenitors not only within, but also beyond, the limits of a promuscular cluster. Such a conclusion implies that these clusters are not a priori determined, but depend on the range and intensities of the MAPK activating signals, in agreement with the assumption that it is not the expression of l(1)sc that determines the promuscular clusters. In fact, it may be the absence of such a priori determined clusters of equivalent cells in the somatic mesoderm that necessitates such a complex N and Ras signaling circuitry (Duan, 2007).

Therefore, it is proposed that twi and da, instead of l(1)sc, function as promuscular genes by regulating the activities of competence domain genes, which in turn regulate the combinatorial activities of muscle identity genes and thereby specify the fates of muscle progenitors and founders. It is nevertheless surprising that l(1)sc appears to be expressed in all promuscular clusters even though its function is not necessary in most of them. It is possible that this expression pattern is an evolutionary remnant of an atavistic promuscular function of l(1)sc that was later replaced by the promuscular function of twi on whose expression l(1)sc activity depends (Duan, 2007).

Search PubMed for articles about Drosophila pox meso

Bopp, D., Burri, M., Baumgartner, S., Frigerio, G. and Noll, M. (1986). Conservation of a large protein domain in the segmentation gene paired and in functionally related genes of Drosophila. Cell 47: 1033-1040. PubMed ID: 2877747

Bopp, D., Jamet, E., Baumgartner, S., Burri, M. and Noll, M. (1989). Isolation of two tissue specific Drosophila paired box genes, Pox meso and Pox neuro. EMBO J. 8: 3447-3457. PubMed ID: PubMed ID

Clark, I. B. N., Boyd, J., Hamilton, G., Finnegan, D. J. and Jarman, A. P. (2006). D-six4 plays a key role in patterning cell identities deriving from the Drosophila mesoderm. Dev. Biol. 294: 220-231. PubMed ID: PubMed ID; Online text

Duan, H., Zhang, C., Chen, J., Sink, H., Frei, E. and Noll, M. (2007). A key role of Pox meso in somatic myogenesis of Drosophila. Development 134: 3985-3997. PubMed ID: PubMed ID; Online text

Fu, W. and Noll, M. (1997). The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye. Genes Dev. 11: 2066-2078. PubMed ID: 9284046

Noll, M. (1993). Evolution and role of Pax genes. Curr. Opin. Genet. Dev. 3: 595-605. PubMed ID: 8241771

Riechmann, V., Irion, U., Wilson, R., Grosskortenhaus, R. and Leptin, M. (1997). Control of cell fates and segmentation in the Drosophila mesoderm. Development 124: 2915-2922. PubMed ID: PubMed ID; Online text

Staehling-Hampton, K., Hoffmann, F. M., Baylies, M. K., Rushton, E. and Bate, M. (1994). dpp induces mesodermal gene expression in Drosophila. Nature 372: 783-786. PubMed ID: 7997266

date revised: 2 January 2023

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