muscle segment homeobox



MSH mRNA is first detected at about stage 5, in a bilateral series of segmentally repeated bands of dorsal/lateral ectoderm cells. Eventually eight bands are formed that subsequently fuse and extend the full length of the germ band [Images]. Expression is next seen in segregating neuroblasts. msh expression is restricted to the lateral column on neuroblasts in the developing CNS (D'Alesso, 1996). In a third stage of expression, msh is expressed in mesoderm, in clusters of cells. More ventrally located segmental groups of mesodermal cells that express msh from stage 11 onwards give rise to the fat body, and thus msh appears to be one of the earliest markers for fat body precursors (D'Alessio, 1996). By stage 14 msh expression declines in the mesoderm but continues in the ventral nerve cord (CNS) and brain (Lord, 1995).

msh expression also prefugures invagination centers of the stomatogastric nervous system. In mutants for wingless, which form only one, rather than three, invaginations, msh expression is absent in the stomatogastric nervous system anlage (D'Alessio, 1996).

An early and important step in the generation of neural diversity in Drosophila is the specification of individual neuroblasts according to their position. The msh gene is likely to play a role in this process. The msh/Msx genes are one of the most highly conserved families of homeobox genes. During vertebrate spinal cord development, Msx genes (Msx1-3) are regionally expressed in the dorsal portion of the developing neuroectoderm. The neuroectoderm is divided into three longitudinal columns, with medial (ventral) and lateral (dorsal) columns flanking a central column. In Drosophila, msh is expressed in two longitudinal bands that correspond to the lateral (dorsal) portion of the neuroectoderm, and subsequently in many dorsal neuroblasts and their progeny. The neuroectodermal expression of msh is first initiated at stage 5 as discontinuous patches in several segments, which later extend and merge to form bilateral stripes that run along the length of the embryo. From this region, four S1 NBs of the lateral column delaminate. Strong MSH mRNA is detected in only one of the four lateral NBs, although the other three NBs also appear to express the transcript at low level. The expression is transient and disappears by late stage 10. Expression is reinitiated in many dorsal S3-S5 NBs at stage 10. At stage 16, some positive cells, identified as glial cells, migrate either medially or laterally. These cells include six longitudinal glia (derived from longitudinal glioblasts), two cell body glia (derived from NB6-4) and some channel glia (derived from NB7-4) (Isshiki, 1997).

Drosophila msh loss-of-function mutations lead to cell fate alterations of neuroblasts formed in the dorsal aspect of the neuroectoderm, including a possible dorsal-to-ventral fate switch. In msh embryos, longitudinal glioblasts form and conduct their first cell division, but further cell division and migration is found to be abnormal. Abnormality in the migration and/or morphology of cell body glia is observed in 71% of hemisegments. Duplication of even skipped positive GMC4-2a and RP2 neurons indicates a possible dorsal-to ventral NB change. Ectopic expression of msh in the entire neuroectoderm severely disrupts the proper development of the midline and ventral neuroblasts. The results provide the first in vivo evidence for the role of the msh/Msx genes in neural development, and support the notion that they may perform phylogenetically conserved functions in the dorsoventral patterning of the neuroectoderm (Isshiki, 1997).

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

The somatic musculature in the abdominal hemisegments of Drosophila consists of 30 uniquely identifiable muscle fibers. Previous studies have suggested that the muscle diversity originates in a special class of myoblasts, called muscle founders, which are formed by the division of muscle progenitors. However, the mechanisms that locate and specify the muscle progenitors/founders are largely unknown. A novel marker, rP298-LacZ, was used to chart the development of muscle progenitors/founders during the formation of distinct groups of mature muscles. rP298-LacZ expression is first detected in a small number of large cells that arise sequentially during stage 11/12. These cells then divide to give rise to smaller cells that occupy specific positions in the somatic mesoderm by mid-stage 12. Shortly after, these cells fuse with the neighboring myoblasts to form the muscle precursors. During this process, additional cells begin to express rP298-LacZ upon fusion with the cells that had earlier expressed the reporter gene. This results in a rapid increase in the number of the rP298-positive nuclei, toward the end of the germ band shortening. At stage 16, when the mature pattern of muscle fibers is achieved, rP298-LacZ is detected in the nuclei of the 30 differentiated muscle fibers. These features of rP298-LacZ expression suggest that it marks the progenitors and founders of all muscle fibers during early development of the mesoderm. The early onset of rP298-LacZ expression and its persistence until later stages when individual muscle fibers can be uniquely identified, allows for tracing the origin of the majority of muscle fibers in abdominal segments A2-A7. Based on such analysis, the progenitors/founders can be divided into four domains based on their dorsal, dorsolateral, lateral or ventral origins (Nose, 1998).

The dorsal domain is located at the dorsal-most region of the somatic mesoderm, just anterior to the ectodermal engrailed (en) expressing stripe and includes rP298-LacZ-positive putative progenitors/founders that give rise to the four dorsal muscles (1, 2, 9 and 10). Some of the rP298-positive cells express msh and contribute to muscles 9 and 10, which occupy the external layer of the dorsal musculature. The dorsolateral domain includes the progenitors/founders of the six dorsolateral muscles (3, 4, 11 and 18-20) and is located between the tracheal placode and the En protein stripe. The progenitors in this domain are among the first to exhibit rP298-LacZ expression. The progenitors in the lateral domain group originate in the lateral region of the somatic mesoderm, near the boundary of the neuroectoderm. One of them divides to form four smaller cells that migrate ventrally and contribute to ventral external muscles 26, 27 and 29. The other two progenitors in this domain divide to produce four founders that will later form the lateral muscles 21-24. The progenitors/founders in the ventral domain group are found interior to the developing central nervous system (CNS), and give rise to the ventral internal muscles (6, 7, 12-17 and 30). Progenitors of three other muscles (5, 8, 25) also arise in this domain but follow distinct fates. Another progenitor appears to migrate dorsally and forms the segment border muscle 8. It should be noted that a majority of the muscles differentiate near the position where their progenitors are initially formed. Thus, in general, their final location corresponds to the domain of their origin (e.g. dorsal muscles are derived from the D domain). However, several muscles migrate during their formation, and their final location differs from that of their origin (e.g. ventral external muscles 26, 27 and 29 originate from the L domain) (Nose, 1998).

The function of the muscle segment homeobox gene, with reference to the rP298-LacZ marker, was determined during the process of myogenesis. msh is expressed in the dorsal and lateral domains of muscle progenitors, but not dorsolateral or ventral domains, and is required for the specification of the progenitor cells. Ectopic expression of msh in the entire mesoderm inhibits the proper development of the normally msh-negative muscle progenitors in the dorsolateral domain. These results suggest that msh plays a role in regional specification of muscle progenitors/founders (Nose, 1998).

The expression and function of msh during myogenesis and during neurogenesis show a number of similarities. During neurogenesis, msh is expressed in neural progenitors (neuroblasts) that form in the dorsal portion of the developing neuroectoderm. In msh mutant embryos, dorsal neuroblasts that normally express msh fail to produce their appropriate lineage (Isshiki et al., 1997). Similarly, during myogenesis, msh is expressed and required in the muscle progenitors that form in specific regions of the somatic mesoderm. In both cases, msh is not required for the initial formation of the progenitors, but for the subsequent events leading to the formation of particular neural or muscular cells. The expression of msh in the progenitors is preceded by its expression in the overlying ectoderm -- initially, this occurs in the dorsal neuroectoderm, during early neurogenesis and later in two ectodermal patches, during late neurogenesis and myogenesis. Ectopic expression of msh interferes with the proper development of normally msh-negative neural or muscular progenitor cells. Thus, msh appears to function in the same manner during both neurogenesis and myogenesis to determine the positional identities of the progenitor cells (Nose, 1998).

ventral nervous system defective, msh, and intermediate neuroblast defective regulate dorsoventral patterning of the procephalic neuroectoderm. vnd, msh, and ind are each expressed in the procephalic ectoderm: Vnd in a ventral domain, Ind in three small clusters of cells at intermediate positions, and Msh in a dorsal domain. There are two differences in gene expression and regulation in the procephalic region compared with the thoracic and abdominal neuroectoderm: (1) Vnd and Msh share an extensive border, only interrupted by two small islands of Ind+ cells. In vnd embryos, Msh expands into the ventral domain of the procephalic neuroectoderm, showing that Vnd is required to repress msh expression in the head. Consistent with this result, misexpression of vnd leads to repression of msh. (2) The Ind+ anterior cell cluster 1 appears to coexpress Vnd; coexpression of Vnd and Ind is never observed in the thoracic and abdominal neuroectoderm. Surprisingly, vnd embryos show a loss of the Ind+ cluster 1, and misexpression of vnd does not affect Ind expression in cluster 1; thus, in this domain of the embryo, Vnd is required for the development of the Ind+ cluster 1. Because the Ind+ cells of cluster 1 are primarily restricted to neuroblasts, one possibility is that loss of vnd in the neuroectoderm leads to a failure of neuroblast formation and thus to a loss of Ind+ cells, rather than that Vnd directly activates ind transcription in this domain. The remaining two Ind+ cell clusters (2 and 3) are expressed and regulated in a manner consistent with the thoracic and abdominal neuroectoderm. Both Ind+ cell clusters 2 and 3 directly abut Vnd+ cells but do not express Vnd. In vnd embryos, the Ind+ cluster 3 expands ventrally into the domain normally expressing vnd, whereas Ind+ cluster 2 appears unaffected. Misexpression of vnd represses ind expression in clusters 2 and 3. Thus, vnd can both activate ind (cluster 1) or repress ind (clusters 2 and 3) depending on the position within the procephalic neuroectoderm (McDonald, 1998).

The expression patterns of the murine genes Lhx2 and Msx1 and their Drosophila orthologs apterous and muscle-segment homeobox are described and compared. Lhx2 and Msx1 show complementary patterns of expression in most tissues, including the neural and cranial epithelium, pituitary gland, olfactory organs, and neural tube; in contrast, Lhx2 and Msx1 are coexpressed in the developing limbs. Strikingly, the spatial relationship between ap and msh expression in Drosophila is very reminiscent of the expression of their murine orthologs. ap and msh show complementary expression in the leg and antennal imaginal discs and brain and ventral ganglion of the central nervous system (CNS), but both are coexpressed in the wing imaginal disc. These observations suggest conservation in the regulation of these genes between Drosophila and mice (Lu, 2000).

Lhx2 and Msx1 are found to be coexpressed in the progress zone of the developing mouse limbs. However, Lhx2 is excluded from the tip of the limb bud corresponding to the apical ectodermal ridge (AER), whereas Msx1 is expressed in this area. The Drosophila genes msh and ap also exhibit overlapping expression patterns in the wing imaginal disc, particularly within the dorsal compartment. msh is also expressed in the anterior mesopleura where ap is not expressed. Similar expression profiles are observed in haltere discs (Lu, 2000).

The spatial relationship between Lhx2 and Msx1 expression was examined in other embryonic areas. In contrast to the limbs, Lhx2 and Msx1 have reciprocal expression patterns in other regions, including mutually exclusive domains within the same tissue and juxtaposed domains in adjacent tissues. For instance, Msx1 and Lhx2 are both expressed in the olfactory epithelium, however Msx1 is restricted to the anterior region, while Lhx2 is in the posterior region that precisely fills the Msx1-negative area. Sagittal views reveal that Msx1 is expressed in the medial and lateral nasal processes, whereas Lhx2 seems to label the epithelium of the vomeronasal organ. In addition, Msx1 and Lhx2 are both expressed in the dorsal neural tube, however, Msx1 is prominent at the roof plate, whereas Lhx2 is directly lateral to the Msx1-positive zone in the marginal layer of the dorsal commissures (Lu, 2000).

The second type of reciprocal expression is that seen in adjacent tissue layers. For instance, Msx1 is expressed in the developing anterior pituitary, called Rathke's pouch, while Lhx2 is expressed in the base of the diencephalon and its infundibular evagination, which will form the posterior pituitary. In addition, Msx1 is expressed in the cranial epithelium, whereas Lhx2 is expressed in the underlying neural epithelium. Indeed, Lhx2 is absent in the roof between the telencephalic hemispheres, which is a region strongly labeled by Msx1 (Lu, 2000).

ap and msh reciprocal expression has been found in other Drosophila tissues. ap is expressed in a ring-like domain corresponding to the presumptive fourth tarsal segment of the leg discs, whereas msh is expressed in two arc-like domains that flank the ap territory. In the eye-antennal disc, msh is broadly expressed in the eye portion of the disc and in several ring-like domains within the antenna region, the stronger of which is found in the second antennal segment. In contrast, ap is specifically expressed in the center of the antenna disc, where it shares discrete areas of overlapping and complementary expression with msh. It was also found that ap and msh have mutually exclusive domains within the brain and ventral ganglion. Thus the data provide previously unreported expression profiles for msh in Drosophila larval stages, and uncover a conserved spatial relationship between the expression of msh/ap and Msx1/Lhx2 genes during evolution (Lu, 2000).

Expression of the DV patterning genes in the brain

The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, a comprehensive analysis was carried out of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, evidence is provided indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deuto-cerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed (Urbach, 2003).

In addition to the segment polarity genes, the dorsoventral patterning genes ventral nervous system defective (vnd), intermediate neuroblast defective (ind) and muscle segment homeobox (msh) have been shown to confer positional information to the truncal neuroectoderm, which also contributes to the specification of NBs. For the head and brain, a detailed analysis of the expression of these genes has not yet been undertaken. In order to elucidate their putative role in patterning the head and brain, the expression of vnd, ind and msh was analyzed in the procephalic ectoderm and NBs in the early embryo (until stage 11). Although the data are consistent with their role in dorsoventral patterning being principally conserved in the procephalon, significant differences are found in their patterns of expression compared with the trunk (Urbach, 2003).

At the blastodermal stage, Ventral nervous system defective protein (Vnd) is expressed in bilateral longitudinal stripes corresponding to the most ventral neuroectodermal column, and is by stage 11 detected in all ventral and two intermediate NBs of the ventral nerve cord. Interestingly, the latter co-express en and are located in the posterior compartment of each truncal neuromere. At gastrulation the ventral longitudinal vnd domain reaches anteriorly across the cephalic furrow into the procephalic neuroectoderm. By stage 9, vnd maps in the ventral neuroectoderm of the prospective intercalary, antennal and ocular segment and is observed in ventral NBs of the antennal (Dv2, Dv3, Dv6) and ocular neuromere (Pcv1, Pcv3, Pcv6, Ppv2). It appears as if the dorsal part of the Vnd-positive antennal neuroectoderm partly co-expresses ind at that stage, but the NB Dd1, which emerges from this ectodermal region expresses only ind and not vnd. This is possibly due to the transient expression of vnd in most parts of both the ventral antennal ectoderm and corresponding NBs: by stage 10 Vnd is detected in the ventral Dv2, Dv4 and Dd5, but is already downregulated in Dv3 and Dv6, and by stage 11 it is confined to Dd5 and the new Dv8. As a consequence of the downregulation of vnd, some ventral deutocerebral NBs, which delaminate between stage 9 and 11 from this domain were not observed to express vnd (e.g. Dv1, Dv5, Dv7). By stage 11 Vnd is seen in four tritocerebral NBs (Tv2, Tv3, Tv4, Tv5), in two deutocerebral NBs (Dd5, Dv8), and in a cluster of about 13 protocerebral NBs. Interestingly, vnd expression expands along the posterior border of the en intercalary stripe (en is), and is also significantly extended dorsally into the en antennal stripe; the NBs delaminating from there. The fact that vnd and en are co-expressed in Tv5 and in Dd5, Dv8 is in agreement with findings in the ventral nerve cord, where these genes are co-expressed in two intermediate NBs. This indicates that vnd demarcates the ventral part of the posterior border in trunk as well as in brain neuromeres. Furthermore, the posterior border of the ocular vnd domain (including the NBs Pcv1, Pcv2, Pcv3, Ppv1, Ppv2, Ppv3) abuts dorsally the En-positive NBs Ppd5 and Ppd8 (deriving from the en head spot), supporting the view that these NBs demarcate the posterior border of the ocular neuromere (Urbach, 2003).

intermediate neuroblast defective (ind) is expressed in the blastoderm in a bilateral longitudinal column (intermediate column neuroectoderm) just dorsal to the vnd domains. In the trunk, at stage 9 (when ind mRNA is no longer present in the neuroectoderm), it is expressed in all intermediate NBs and finally, at stage 11, it is confined to the NB 6-2. In the head, at stage 9, ind is detected in an intermediate longitudinal ectodermal domain in the intercalary segment, and weakly in an intermediate ectodermal patch in the antennal segment as well as in the deutocerebral NB Dd1 which develops from this patch. At the same stage, a further signal is observed in a dorsal ectodermal patch of the ocular region. The ectodermal ind patches in the intercalary, antennal and ocular segments are both separate from each other and from the ind domain in the trunk. Interestingly, ind mRNA is significantly present longer in the ectoderm of the intercalary and mandibular segment, when compared with the antennal segment and the trunk ectoderm. This presumably mirrors the delayed onset of neurogenesis in both segments. Until stage 10, five NBs derive from the three ind patches: Td1, Td2, Td3, from the intercalary, Dd1 from the antennal and Ppd13 from the ocular ind patch. Subsequently, the ocular ind patch enlarges but never reaches the ocular vnd domain, and by stage 11 about four additional Ind expressing NBs (Pcd7, Pcd13, Ppd6, Ppd9) are identifiable (Urbach, 2003).

muscle segment homeobox (msh) expression is first detected at the blastoderm stage in discontinuous patches in the dorsolateral part of the neuroectoderm that later extend and form a bilateral longitudinal stripe; this domain gives rise to the lateral NBs of the ventral nerve cord. At stage 7 msh expression is detected anterior to the cephalic furrow, which expands until stage 9 to cover, as a broad domain, the dorsal ectoderm of the intercalary and the antennal segment. As evidenced by Msh/Inv double labelling during stage 9 and stage 11, the anterior border of the msh domain coincides with the posterior border of the en hs. This suggests that msh expression in the pregnathal region is restricted to the intercalary and antennal segments, and matches the border between the antennal and ocular segment. This is further supported by Msh/hh-lacZ double labelling in stage 11 embryos, using hh as a marker for the posterior border of the ocular segment. All identified brain NBs delaminating from the dorsal intercalary and antennal neuroectoderm express msh. This suggests that during early neurogenesis, msh controls dorsal identities of the procephalic neuroectoderm and brain NBs, as was shown for the ventral nerve cord. In the ventral nerve cord, most glial precursor cells (glioblasts and neuroglioblasts) derive from the dorsal neuroectoderm, and express msh. In the intercalary segment of the early brain, two glial precursors (Td4 and Td7) were identified. Interestingly, both precursors are also located dorsally and express msh. At least until stage 11 no msh expression is found in the preantennal segments (Urbach, 2003).

In Drosophila the DV patterning genes subdivide the trunk neuroectoderm into longitudinal columns; vnd is required for the specification of the ventral neuroectodermal column and NBs; ind and msh have analogous functions in the intermediate and dorsal neuroectodermal columns and NBs, respectively. Remarkably, homologous genes are found to be expressed in the vertebrate neural plate and subsequently in the neural tube. In the neural tube the order of expression along the DV axis is analogous to that of Drosophila: like vnd, the vertebrate homologs of the Nkx family are expressed in the ventral region; the ind homologs, Gsh-1/2, are expressed in the intermediate region; and the msh homologs, Msx-1/2/3, are expressed in the dorsal region of the neural tube (Urbach, 2003).

Thus, in the brain msh is confined to more posterior regions, and vnd expression extends into anterior regions of the brain. Moreover, the expression border of msh and vnd coincide with neuromeric borders. A comparison of the anteroposterior sequence of DV patterning gene expression in the early brain of Drosophila, with that published for the early mouse brain, reveals striking similarities. Msx3, which presumably represents the ancestral msh/Msx gene, becomes restricted to the dorsal neural tube during later development (in contrast to Msx1/2). The anterior border of the Msx3 domain is positioned within the rostral region of the dorsal rhombencephalon, thus showing the shortest rostral extension of all vertebrate DV patterning genes. This displays analogy to msh, the expression domain of which coincides with the anterior border of the dorsal deutocerebrum, thus representing the shortest anterior extension of DV patterning genes in Drosophila. Mouse Nkx2.2 extends ventrally into the most rostral areas of the forebrain. vnd is expressed ventrally in anterior parts of the ocular and labral protocerebrum. Thus, the expression of the respective homologs in both species displays the most anterior extension among DV patterning genes. Moreover, Nkx2.2 expression in the mouse forebrain suggests that Nkx2.2 may be involved in specifying diencephalic neuromeric boundaries. Similarly, in Drosophila, dorsal expansions of the vnd domain appear to correspond to the tritocerebral and deutocerebral neuromeric boundaries (Urbach, 2003).

Furthermore, Drosophila ind and its mouse homologue Gsh1 show similarities in their expression in the early brain. In the posterior parts of the Drosophila brain, ind is expressed in intermediate positions between vnd and msh. Likewise, in the posterior part of the mouse brain, Gsh1 appears to be expressed in intermediate positions, dorsally to Nkx2.2, and in the hindbrain ventrally to Msx3. Gsh1 has been shown to be expressed in discrete domains within the mouse hindbrain, midbrain (mesencephalon) and the most anterior domain in the posterior forebrain (diencephalon). Correspondingly, in Drosophila ind expression in restricted domains within the gnathocerebrum, the tritocerebrum, deutocerebrum and ocular part of the protocerebrum, demonstrating that the anteriormost extension of ind (and Gsh1) expression lies between that of msh and vnd (Urbach, 2003).

Taken together, considering these similarities, it is suggested that in the Drosophila and vertebrate early brain the expression of DV patterning genes is to some extent conserved, both along the DV axis (as suggested for the truncal parts of the Drosophila and mouse CNS) and along the AP axis. Furthermore, in Drosophila large parts of the anterodorsal procephalic neuroectoderm and NBs (more than 50% of all identified brain NBs) lack DV patterning gene expression. Likewise, in the vertebrate neural tube, gaps between the expression domains of DV patterning genes have been described, raising the possibility that other genes might fill in these gaps. How DV fate is specified in the anterior and dorsal part of the Drosophila procephalic neuroectoderm, and if other genes are involved, remains to be clarified (Urbach, 2003).

Segment-specific requirements for dorsoventral patterning genes during early brain development in Drosophila

An initial step in the development of the Drosophila central nervous system is the delamination of a stereotype population of neural stem cells (neuroblasts, NBs) from the neuroectoderm. Expression of the columnar genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh) subdivides the truncal neuroectoderm (primordium of the ventral nerve cord) into a ventral, intermediate and dorsal longitudinal domain, and has been shown to play a key role in the formation and/or specification of corresponding NBs. In the procephalic neuroectoderm (pNE, primordium of the brain), expression of columnar genes is highly complex and dynamic, and their functions during brain development are still unknown. These genes (with special emphasis on the Nkx2-type homeobox gene vnd) have been investigated in early embryonic development of the brain. At the level of individually identified cells it is shown that vnd controls the formation of ventral brain NBs and is required, and to some extent sufficient, for the specification of ventral and intermediate pNE and deriving NBs. However, significant differences were uncovered in the expression of and regulatory interactions between vnd, ind and msh among brain segments, and in comparison to the ventral nerve cord. Whereas in the trunk Vnd negatively regulates ind, Vnd does not repress ind (but does repress msh) in the ventral pNE and NBs. Instead, in the deutocerebral region, Vnd is required for the expression of ind. In the anterior brain (protocerebrum), normal production of early glial cells is independent from msh and vnd, in contrast to the posterior brain (deuto- and tritocerebrum) and to the ventral nerve cord (Urbach, 2006).

Expression of Vnd protein is first detectable in the blastoderm in bilateral longitudinal columns along the ventral neuroectoderm of the trunk and the ventral procephalic neuroectoderm (pNE), covering the prospective ventral parts of the trito-(TC), deuto-(DC) and protocerebrum (PC). Although in the trunk Vnd expression is maintained within a continuous ventral neuroectodermal column during subsequent stages, it becomes more complex and diverse among head segments. By stage 8 (before first brain neuroblasts have developed0, Vnd becomes downregulated in parts of the procephalic domain. Until stage 10, Vnd has largely vanished in the anterior pNE and NBs of the DC, but its level remains high in the ventral pNE and delaminating NBs of the TC, posterior DC and PC. During stages 10/11, Vnd becomes downregulated at the ventral border between TC and DC. Accordingly, by late stage 11, Vnd expression is restricted to separate domains at the posterior border of the TC, DC and PC, respectively. Whereas the number of Vnd-positive NBs in the domains of the TC and DC is rather small, a large population of about 13 NBs is found in the PC. Thus, in contrast to the situation in the trunk, Vnd expression in the early brain is highly dynamic and becomes progressively confined to three separate ventral domains, encompassing different numbers of NBs and their progeny in the posterior compartments of the TC, DC and PC (Urbach, 2006).

In vnd mutant embryos, it was found at embryonic stages 9 and 11 that ventral NBs in the TC, DC and PC are largely absent, although at different frequencies. Analogously, in the primordium of the VNC of vnd embryos a significant loss of ventral NBs has been reported. In the absence of Vnd, an increase in cell death, which contributes to the loss of ventral brain NBs, was found. Apoptosis acts at the level of both pNE progenitor cells and NBs. It is not yet clear whether the reduction of ventral NBs is solely due to cell death, or whether it also involves activity of proneural genes. In the truncal neuroectoderm, proneural genes of the AS-C complex promote NB formation. There is evidence that vnd interacts with proneural genes, but also that it has additional function in promoting NB formation apart from activating proneural genes. The latter assumption is supported by the finding that, in vnd embryos, lethal of scute (l'sc) can still be expressed in the ventral proneural clusters of, for example, NB5-2, although the respective NB is missing. In the pNE, genes of the AS-C complex are expressed in large proneural domains, of which those of achaete, but especially of l'sc, seem to overlap with the vnd expression domain, suggesting a possible genetic interaction. However, in vnd embryos, no substantial differences were observed in the expression pattern of l'sc transcript compared with the wild type. Thus, similar to the situation in the trunk, Vnd does not appear to exert proneural function through activation of l'sc. However, the data propose a possible interaction between vnd and the proneural gene atonal. In vnd mutants, expression of atonal is often missing in proneural clusters of the sensory organ precursors of the hypopharyngeal-latero-hypopharyngeal organ. Clearly, further investigations are required to clarify in how far interactions between vnd and proneural genes play a role in the formation of ventral brain NBs (Urbach, 2006).

In vnd mutants, not only ventral, but also intermediate brain NBs in the TC and DC show defects in their formation or specification, comparable with the situation in the trunk. As intermediate brain NBs do not express vnd (but ind), these defects appear to be non-cell-autonomous. Another, more likely explanation is that determination occurs at the blastodermal stage, when Vnd is transiently expressed in a much larger population of cells in the pNE, which presumably include progenitors of intermediate NBs. A similar proposal was made for intermediate NBs in the trunk. Furthermore, early commitment of ventral neuroectodermal cells and cell-autonomous expression of ventral and intermediate NB fates has been demonstrated by heterotopic transplantations of neuroectodermal cells from ventral to dorsal sites at the early gastrula stage (Urbach, 2006).

In the trunk, a segmentally reiterated combinatorial code of genes expressed within each particular proneural cluster specifies the individual identity of the NB it gives rise to. These include DV patterning genes and segment polarity genes, which provide positional information in the neuroectoderm, as well as a number of other factors. Most of these genes are also expressed in specific domains of the pNE before NBs delaminate, although in a segment-specific manner. The present data show that Vnd influences the expression of such site-specific marker genes ('NB identity genes') already in the pNE, before NBs are formed. In vnd embryos, a derepression of dorsal-specific genes occurs in the ventral pNE (e.g. of msh and ems in the intercalary and antennal segment, and dac in the ocular segment) and in the descending NBs, and conversely, a loss of ventral-specific gene expression (e.g. lbe in the PC). The altered expression of 'NB identity genes' in vnd mutants reflects a ventral-to-dorsal transformation of ventral pNE and residual NBs. Further evidence for such a transformation is the production of (ectopic) glial cells by these ventral NBs, which normally is a trait specific to dorsal NBs. By contrast, in the trunk, absence of Vnd results in a ventral-to-intermediate transformation, owing to the derepression of ind (instead of msh in pNE), which induces specification of intermediate NB fates (Urbach, 2006).

Together, these data in the vnd loss- and gain-of-function backgrounds indicate that vnd is required, and is at least partially sufficient, for the induction of ventral fate in brain NBs through the activation of genes specific for the ventral pNE, and through the repression of genes specific for dorsal pNE (Urbach, 2006).

This analysis revealed differences in the regulation of DV patterning genes among the intercalary (IC), antennal (AN) and ocular (OC) head segments, giving rise to the TC, DC and PC, respectively. Overexpression of vnd leads to repression of ind within the IC, but loss of vnd-function does not seem to cause ventral expansion of the ind intercalary spot. Unexpectedly, ind is completely absent in the AN of vnd mutants, suggesting that in this segment vnd is necessary for activation and/or maintenance of ind (rather than repression). This is supported by the finding that the ind antennal spot transiently co-expresses Vnd, which is unique in the neuroectoderm, and by the present finding that in vnd gain-of-function background the ind antennal spot is almost unaffected. In the OC, however, ind expression is partially repressed upon Vnd overexpression, and ventrally expanded in the absence of Vnd, similar to the situation in truncal segments. However, because, in wild type the ind ocular spot does not adjoin the ocular vnd domain, its expansion in vnd embryos cannot be due to a cell-autonomous effect (Urbach, 2006).

Overexpression of vnd abolishes Msh almost completely in the neuroectoderm of all body segments. Yet, absence of Vnd reveals segment-specific differences in the regulation of msh. Owing to insulated ind expression in the IC and lack of ind in the AN of vnd mutants, Msh (instead of ind) is found in the ventral pNE of these segments, which is unique in the CNS anlagen, except for the mandibular segment, which exhibits equivalent expression (Urbach, 2006).

Among the pregnathal segments, the degree of conservation with regard to the expression and interactions of DV patterning genes seems to be highest in the posterior IC (TC) [ind and msh being repressed by (ectopic) vnd, and msh by ind]. In the anterior head, endogenous Msh expression in the dorsal pNE reaches the segmental border between AN (DC) and OC (PC), but does not cross it. Ectopic Msh in vnd mutants does also not cross this border, which suggests interference with regulatory factors acting in AP axis (Urbach, 2006).

Significant differences between the anterior head segments and the trunk have also been reported for the initial mode of activation and cross-regulatory interactions of segment-polarity genes (Urbach, 2006).

In the pNE, vnd is necessary for the formation and specification of brain NB. It remains to be shown whether ind and msh exert analogous functions. However, more than 50% of the identified brain NBs do not express any of the three DV patterning genes. Most of these NBs derive from pNE of the preantennal head, which implies that further factors are involved in DV patterning of the anterior pNE and brain. Several other genes have been reported to be crucial for DV patterning in the truncal neuroectoderm, such as the EGF-receptor homolog Egfr, the Sox genes SoxNeuro and Dicheate, and Nk6. For most of them it has been shown that they are involved in formation and/or specification of truncal NBs. Egfr, both Sox genes and Nk6 are also expressed in the pNE, before and during the phase of NB formation. However, in Egfr mutant embryos the number and pattern of brain NBs is unaffected. How far the Sox genes and Nk6 contribute to the formation and/or specification of brain NBs awaits further investigation (Urbach, 2006).

Most of the glial cells in the VNC derive from dorsal NBs (neuroglioblasts or glioblasts), which depend on msh for proper specification. Accordingly, glial cells deriving from these progenitors are missing or improperly differentiated in msh mutants, as well as in sca-vnd embryos. Likewise, in the TC and DC, first glial cells are closely associated with dorsal NBs that descend from Msh-expressing pNE. In the TC, some dorsal NBs have been identified as glial progenitors, e.g. the neuroglioblast Td4 and the glioblast Td7, which are putative serial homologs of the truncal neuroglioblast NB5-6 and the glioblast LGB, respectively. In absence of Vnd, the number of glial cells in the TC, and especially in the DC, were found to be to be increased. This is most probably due to the segment-specific early derepression of Msh in the ventral pNE and NBs of the TC and DC. In the truncal segments, however, ind instead of Msh is derepressed in the ventral NE, and the number of glial cells is not significantly affected in the VNC of vnd mutants. Furthermore, in msh mutants, glial development in the TC and DC is almost completely abolished, which parallels the phenotype observed upon vnd overexpression (leading to repression of msh in the dorsal pNE and NBs). Thus, comparable with the situation in the VNC, Msh promotes glial fate in the TC and DC. However, in the PC, glial development must be regulated differently (at least in its early phase). Until stage 12 no Msh was detected in this part of the brain, and in msh mutants the number of glial cells in the PC is normal. Glial cell fate in the PC is also not affected by loss of vnd, although it remains repressable by ectopic Vnd. Therefore, as opposed to the TC and DC, and to the VNC, normal production of early glial cells in the PC does not depend on msh, nor indirectly on vnd or ind (Urbach, 2006).

There are striking similarities in the spatial order of expression of vnd, ind and msh in the Drosophila neuroectoderm and homologous genes in the neural plate and neural tube of vertebrates: vnd homologs of the Nkx2 family are expressed in ventral regions; the ind homologs Gsh1 and Gsh2 are expressed in the intermediate regions; the msh homologs Msx1, Msx2 and Msx3 are expressed in the dorsal region of the neural tube. This dorsoventral order of expression is conserved not only in the anlagen of the truncal CNS but also in those that form the posterior part of the brain (in Drosophila, TC and DC; in vertebrates, hindbrain). Moreover, the anterior borders of the expression domains of these columnar genes correspond in the early brains of Drosophila and mouse: expression of vnd/Nkx2 extends most rostrally (mouse ventral forebrain), followed by ind/Gsh1 and, finally, msh/Msx3 expression. Thus, the expression of columnar genes in the brain is, to some extent, evolutionarily conserved both along the DV axis and along the AP axis (Urbach, 2006).

This study has presented evidence that in Drosophila vnd mutant embryos a large fraction of ventral brain NBs is missing, and that ventral pNE and residual ventral NBs show significant traits of a ventral-to-dorsal transformation owing to derepression of msh (as opposed to ind in the VNC). Again, this displays obvious similarities to findings made in mice carrying a deletion of Nkx2.1. Consistent with the pattern of expression in wild type, in the mutant embryonic brain a substantial loss of ventral (especially forebrain) structures has been observed. Moreover, ind/Gsh2 expression is not expanded in Nkx2.1 mutants, and residual basal (ventral) pallidal structures become transformed into dorsal striatal structures. Thus, in both Drosophila and mouse, loss of vnd/Nkx2 in the brain leads to a transformation of ventral into dorsal structures, rather than into intermediate structures, which has been shown to be the case in the truncal CNS of both species. Therefore, in the developing brains of Drosophila and vertebrates, vnd/Nkx2 is crucial for the formation and specification of ventral brain structures, and interacts with other dorsoventral patterning genes in a region-specific manner (Urbach, 2006).

Effects of Mutation or Deletion

Severe muscle defects occur as a result of either epidermal or mesodermal overexpression of msh, especially the latter. Overexpression in the nervous system results in behaviorial abnormalities. What has yet to be clarified is the effect of msh mutation on the expression of mesodermal genes such as nautilus, mef2, tinman and stripe (D'Alessio, 1996).

lottchen (ltt) is a novel gene whose loss of function causes a change in the identity of at least one NB as well as cell fate transformations within the lateral glioblast lineage. lottchen is known to code for the protein Muscle segment homeobox. The Drosophila embryonic central nervous system (CNS) develops from a stereotyped pattern of neuronal progenitor cells called neuroblasts (NB). Each NB has a unique identity that is defined by the time and position of its formation and a characteristic combination of genes it expresses. Each NB generates a specific lineage of neurons and/or glia. In wildtype embryos, the parental NB of the motoneuron RP2 is NB4-2. ltt embryos are distinguished by an additional RP2-like neuron, which appears later in development. The two RP2 neurons are derived from two distinct GMC4-2a-like cells that do not share the same parental NB, indicating that a second NB has acquired the potential to produce a GMC and a neuron: this potential is normally restricted to the NB4-2 lineage. Moreover, the ltt mutations lead to a loss of correctly specified longitudinal glia; this coincides with severely defective longitudinal connectives. Therefore, lottchen plays a role in specifying the identity of both neuroblast and glioblast lineages in the Drosophila embryonic CNS. ltt may act to differentiate NB identity along the medial lateral axis (Buescher, 1997).

It has been shown that NB identity is specified by positional cues, which occur prior to NB delamination. So far, two examples have been described in which the loss of a single gene product transforms the identity of a particular row of NBs into that of a different row of NBs. Loss of gooseberry (gsb) function leads to a transformation of row 5 NBs into row 3 NBs. Conversely, ectopic expression of gsb can lead to a row 3 into row 5 transformation. The protein Wingless (Wg) is secreted from row 5 neuroectoderm and NBs. Loss of wg function alters the fate of adjacent NBs in rows 4 and 6. gsb and wg are segment polarity genes and act to specify NB identity along the anterior-posterior axis. Genes that differentiate between the identities of NBs in medial and lateral columns of NBs are as yet unknown. ltt is a likely candidate for a gene that functions in medial-lateral specification (Buescher, 1997 and references).

ltt mutations affect the longitudinal glioblast (LG) lineage. Six LGs are derived from the LGB which forms at stage 10 in the lateral-most row of NBs at the anterior margin of each segment. Repo expression can be detected in the LGB shortly before its first division. At early stage 11, the LGB divides along the apical/basal axis to generate two progeny of approximately equal size. The dorsal cell is positive for nuclear Prospero (Pros) while the ventral cell remains negative for nuclear Pros. Both daughter cells migrate medially and anteriorly. During stages 11/12 the LGB progeny undergo further divisions that result in six Pros/Repo double positive cells, which are arrayed in a characteristic rhomboid pattern. At stage 15, eight to ten Repo-positive glia form two rows on the dorsal surface of the neural connectives; six of these cells are also positive for nuclear Pros. The ltt mutation causes a loss of pros expression in the LGB lineage. In ltt mutants, the division of the LGB occurs during stage 12, no further formation and/or maintenance of the longitudinal connectives is observed and frequently the longitudinal connectives are lost. Loss of pros expression alone cannot account for the LG phenotype: it has been shown previously that in pros loss-of-function mutants the six LG are formed, although these mutant LG appear spatially disorganized and fail to undergo terminal differentiation. Nevertheless, in pros loss-of-function mutants, the LG do express repo and can be identified unambigiously. Since in ltt embryos the six LG are either not present or fail to express Repo, it is concluded that the ltt mutation must cause defects in the LGB lineage, in addition to the loss of pros expression. Interestingly, in wild type embryos, many glial precursor cells do express Pros and the ltt mutation does not abolish pros expression in these cells. In contrast to the LG, the post-mitotic progeny of these glial precursors are Pros-negative. This suggests that pros expression is regulated differently within the LGB lineage and other glial lineages and that the ltt gene product is required for pros expression in the LGB lineage but not in other glial lineages (Buescher, 1997 and references).

The lack of correctly specified LG in ltt mutants coincides with a reduction of 22C10 expression in early MP2 neurons and severe defects of the longitudinal axon tracts. However, the causal relationship between these defects is difficult to assess. The presence of correctly specified pros-expressing LG may be an absolute requirement for axon pathfinding and loss of the ltt function within the LGB lineage may be sufficient to cause a lack of longitudinal connectives. Alternatively, the neurons whose axons contribute to the longitudinal connectives may be affected by the mutation and may not be able to recognize the positional cues required for axon pathfinding. These scenarios are not mutually exclusive (Buescher, 1997).

Thus ltt mutation causes a duplication of the RP2 neuron and a lack of correctly specified LG. These results suggest that ltt function is required to restrict the number of RP2 neurons to one per hemisegment and to ensure that six Pros-positive LG per hemisegment are formed. The strongest ltt allele causes a duplication of RP2 in approx. 70% of the hemisegments but the LG are affected in all hemisegments. This observation suggests that the ltt function may be indispensable for the formation of the LG but may be partially redundant with respect to RP2 formation. These results raise the interesting possibility that ltt may belong to a class of genes that acts to differentiate NB identities between medial and lateral columns of NBs (Buescher, 1997).

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the DrMio allele is coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis reveals that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the DrMio mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae is sufficient to arrest the morphogenetic furrow in the eye imaginal disc and results in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins (Mozer, 2001).

Loss-of-function alleles can frequently be recovered as intragenic suppressors (revertants) of a dominant gain-of-function mutation following secondary mutagenesis. A previous study of the Dr mutants has suggested that the dominant eye phenotype is not due to a gain-of-function mutation in stg, but rather identifies a second distal gene. However, genetic analysis of the distal gene in the Dr revertants was complicated by the presence of an additional lethal lesion in stg on the mutant chromosomes. In order to identify the affected gene and the consequence of genetic reversion, EMS-induced revertants of DrMio were isolated and tested for complementation with Df(3R)KE, a chromosomal deletion which removes the distal 99A region. The deficiency complements all stg mutant phenotypes, including the recessive bristle and eye defects associated with Dr mutant alleles. Thus, the phenotype of Dr/Df(3R)KE heterozygotes reveals the allelic state of the distal gene independent of stg mutant effects (Mozer, 2001).

The absence of stg expression in the DrMio eye imaginal disc suggests that msh overexpression interferes with events anterior to the furrow, which may be required for its progression. Furthermore, the results of ectopic expression using different Gal4 drivers are consistent with this hypothesis, since msh overexpression behind the furrow (gmr driver) results in a normal-sized rough eye while ubiquitous expression (hsp/70 driver) arrests the furrow. Inhibition of the furrow by ectopic msh could involve the transcriptional repression of genes required for furrow progression or the up-regulation of those genes that serve as negative regulators. Two HLH transcription factors, emc and hairy, have been implicated in the negative regulation of morphogenetic furrow progression; however, hairy expression (as monitored by the C93 enhancer trap) is not affected in the DrMio mutant, suggesting that it is not a target of ectopic msh. The absence of furrow progression in the Dr mutant eye disc is not associated with a change in wg expression, suggesting that msh misexpression interferes with hh-mediated transcription of dpp or some other process. Ectopic msh may interfere with the function of the early eye patterning gene, eyes absent (eya), which is required for dpp transcription in the furrow and whose absence leads to cell death (Mozer, 2001).

Drosophila limbs develop from imaginal discs that are subdivided into compartments. Dorsal-ventral subdivision of the wing imaginal disc depends on apterous activity in dorsal cells. Apterous protein is expressed in dorsal cells and is responsible for (1) induction of a signaling center along the dorsal-ventral compartment boundary; (2) establishment of a lineage restriction boundary between compartments, and (3) specification of dorsal cell fate. The homeobox gene msh (muscle segment homeobox) acts downstream of apterous to confer dorsal identity in wing development (Milán, 2001).

Four structural features distinguish the dorsal and ventral surfaces of the adult wing: (1) bristle morphology in the anterior wing margin; (2) the presence or absence of bristles in the alula; (3) the surface on which the veins are corrugated, and (4) the location of certain sensory organs. The anterior wing margin (AWM) is composed of three rows of bristles, two located in the dorsal surface and one in the ventral. The dorsal wing margin differentiates a row of thick, densely aligned, mechanosensory bristles and a second row of thinner, curved, chemosensory bristles. The dorsal AWM produces one chemosensory bristle per five mechanosensory bristles. The ventral row is composed of thin bristles interspersed with chemosensory bristles in every fifth position. The alula is located in the posterior compartment. It produces a single row of long thin bristles along the margin on the ventral surface. The dorsal surface of the alula lacks bristles. The adult wing differentiates five longitudinal veins. L1 is located on both dorsal and ventral sides of the wing margin and L2-L5 veins are located in the wing blade. Veins L2-L5 are asymmetrical on the dorsal and ventral surfaces of the wing. One side contains more rows of tightly packed cells ('corrugated vein'). The opposite side is thinner ('ghost vein'). Corrugated veins consist of three rows of strongly pigmented and densely packed cells. Ghost veins consist of a single row of cells. Longitudinal veins L3, L5 and the distal tip of L4 are dorsally corrugated. Veins L2 and proximal L4 are ventrally corrugated (Milán, 2001).

The msh gene belongs to the msh/Msx family of homeobox genes involved in dorsal cell fate specification in the Drosophila neuroectoderm. Since msh is expressed in the dorsal compartment of the wing disc, an investigation was carried out to see whether msh is also involved in dorsal identity specification in the Drosophila wing. For this purpose, msh mutant clones were generated in the wing and the DV identity of the bristles located along the AWM, in the alula and the DV corrugation of longitudinal veins in mutant cells, was assessed. Clones mutant for msh have no aberrant phenotype in the ventral surface of the wing. When mutant for msh, the dorsal anterior wing margin differentiates ventral bristles. A single row of thin bristles interspersed with chemosensory bristles in every fifth position is observed. Thus, the anterior wing margin differentiates a ventral pattern of bristles symmetrically on both surfaces (Milán, 2001).

When covered with mutant cells, the dorsal surface of the alula differentiates bristles. This reflects transformation to a ventralized cell fate. Absence of msh activity also induces a change in the pattern of corrugation of the longitudinal veins. In wild-type wings, veins L2 and L4 differentiate as 'ghost veins' on the dorsal surface. When mutant for msh, these veins are corrugated and differentiate three rows of strongly pigmented cells, thus mimicking a ventral-like pattern. Veins L3 and L5 are normally corrugated on the dorsal surface. When mutant for msh, they lose pigmentation and consist of a single row of aligned cells. Thus veins differentiate ventral characteristics in the dorsal surface when mutant for msh. It is concluded that msh is required in the dorsal compartment of the Drosophila wing to confer dorsal cell identity. In the absence of msh, symmetric wings are observed that differentiate ventral characteristics on both surfaces (Milán, 2001).

Apterous is expressed in dorsal cells and is required to confer dorsal cell identity. It was therefore necessary to determine whether msh expression in the dorsal compartment is regulated by Apterous activity. MSH mRNA and msh-lacZ reporter genes are expressed in the dorsal compartment of the wing disc. MSH mRNA is expressed at a low level throughout the dorsal compartment, except in the region of the anterior margin where it is expressed at higher level. Ectopic expression of Apterous in the ventral compartment under control of dppGal4 induces ectopic expression of MSH mRNA at a level comparable to the overall low dorsal level. In apterous mutant discs, msh expression is lost from dorsal cells of the reduced wing pouch, but expression in the anterior mesopleura and hinge region remains. Finally, overexpression of dLMO, a repressor of Apterous activity in the Drosophila wing, represses expression of the msh-lacZ reporter gene. These results indicate that msh is indeed a target of Apterous. Additional studies show that ectopic expression of msh in the ventral surface is sufficient to confer dorsal identity on ventrally located cells (Milán, 2001).

The results presented thus far indicate that mshis necessary and sufficient to specify dorsal identity in the Drosophila wing. A dominant mutation Dlw1 has been identified that shows partial dorsalization of the AWM. Both surfaces of Dlw1/+ AWMs have dorsal bristles, similar to what is observed when msh is ectopically expressed in the ventral compartment. Interestingly, Dlw alleles are associated with breakpoints located 30-90 kb upstream of the msh gene, raising the possibility that Dlw alleles may be regulatory mutants of msh. Indeed, a lethal allele of msh, mshDelta68, has proved to be lethal when heterozygous with Dlw1 and the recessive lethal alleles Dlw3 and lw4. Dorsal clones mutant for Dlw3differentiate ventral structures. Genetic evidence is provided that supports the proposal that the msh gene is expressed in an Apterous-independent manner in Dlw1 wings (Milán, 2001).

msh mRNA levels are reduced throughout the wing pouch in discs heterozygous for Dlw1. The low level of msh expression in the Dlw1 background may explain the loss of function characteristics exhibited by the Dlw1 allele in homozygous mutant clones. Dlw1/Dlw1 mutant clones located in the dorsal surface of the wing differentiate ventral structures. Thus, Dlw1 causes a dominant transformation of ventral cells to dorsal identity when heterozygous and an opposite transformation of dorsal cell to ventral identity when homozygous mutant in clones. Interestingly, the dominant mutation Drop, which affects eye development, has been recently shown to be a gain-of-function allele of msh (Mozer, 2001). Drop mutants contain lesions in the same region as Dlw mutants (i.e. upstream of the msh transcription start site) and ectopic expression of msh in the eye phenocopies the Drop phenotype. However, Mozer was not able to find detectable misexpression of msh in Drop mutants. Thus, undetectably low levels of msh misexpression in eye and wing seem to be associated with the dominant adult phenotypes associated with the Dlw and Drop alleles of msh (Milán, 2001).

Apterous activity is required to confer dorsal identity and dorsal-type signaling properties. Fringe and Serrate expression in dorsal cells induce a cascade of short-range interactions between dorsal and ventral compartments that lead to the expression of the organizing molecule Wingless along the DV compartment boundary. The results reported in this study suggest that msh confers dorsal identity without affecting DV signaling. In order to verify that this is the case, the ability of msh to restore dorsal identity and dorsal signaling properties in the absence of Apterous activity was examined. In apGal4/apUGO35 flies, the wing margin is reduced and the wing is considerably smaller than normal owing to reduced Apterous activity. When present, margin bristles have ventral identity in this genotype. Expression of msh in apGal4/apUGO35;uas-msh flies does not restore outgrowth of the wing. The few margin bristles observed in the dorsal surface of these wings have dorsal identity. Growth and wing margin formation can be restored in the apGal4/apUGO35 mutant background by expression of Fringe under apGal4 control. In these wings, both surfaces differentiate ventral structures: the AWM and the alula differentiate ventral bristles on both surfaces and the pattern of vein corrugation is ventral. Co-expression of msh with EP-fringe confers dorsal differentiation in the bristles of the dorsal AWM in these rescued wings. It was also noted that overexpression of msh in dorsal cells reduces the size of the dorsal wing pouch, induces differentiation of ectopic bristles in the wing blade and affects vein differentiation. This was also observed in apGal4/+; uas-msh/+ flies and presumably reflects defects caused by higher than normal Msh levels in dorsal cells. Note that the endogenous levels of msh expression in the wing pouch are quite low. These results suggest that developmental regulation of Msh protein levels may be crucial for proper wing development and differentiation of patterning elements. All these results indicate that msh confers dorsal identity without affecting dorsal signaling properties (Milán, 2001).

Two apterous homologs, Lmx1 and Lhx2, have been implicated in vertebrate limb development. Interestingly, these two genes appear to have separable functions in conferring dorsal identity and limb outgrowth. Lmx1 is expressed in the dorsal compartment of vertebrate limbs and is necessary and sufficient to confer dorsal identity. Lhx2 induces Radical-fringe expression in the apical ectodermal ridge, which is required for limb outgrowth. This contrasts with the situation in Drosophila where Apterous is responsible for both dorsal fate specification and for establishing the Fringe-dependent signaling center at the DV boundary. The findings reported here implicate msh as the principle target gene through which Apterous confers dorsal cell fate. msh is necessary and sufficient to induce dorsal cell fate, but has no role in DV boundary signaling. Intriguingly, the msh/Msx family of homeobox genes is also differentially expressed along the DV axis of the embryo and msh is required in the Drosophila neurectoderm to specify dorsal fate (Milán, 2001).

In Drosophila embryos, founder cells that give rise to cardiac precursors and dorsal somatic muscles derive from dorsally located progenitors. Individual fates of founder cells are thought to be specified by combinatorial code of transcription factors encoded by identity genes. To date, a large number of identity genes have been identified; however, the mechanisms by which these genes contribute to cell fate specification remain largely unknown. Regulatory interactions of ladybird (lb), msh and even skipped (eve), the three identity genes specifying a subset of heart and/or dorsal muscle precursors, have been analyzed. Deregulation of each of them alters the number of cells that express the other two genes, thus changing the ratio between cardiac and muscular cells, and the ratio between different cell subsets within the heart and within the dorsal muscles. Specifically, mutation of the muscle identity gene msh and misexpression of the heart identity gene lb leads to heart hyperplasia with similar cell fate modifications. In msh mutant embryos, the presumptive msh-muscle cells switch on lb or eve expression and are recruited to form supernumerary heart or dorsal muscle cells, thus indicating that msh functions as a repressor of lb and eve. Similarly, overexpression of lb represses endogenous msh and eve activity, hence leading to the respecification of msh and eve positive progenitors, resulting in the overproduction of a subset of heart cells. As deduced from heart and muscle phenotypes of numb mutant embryos, the cell fate modifications induced by gain-of-function of identity genes are not lineage restricted. Consistent with all these observations, it is proposed that the major role of identity genes is to maintain their restricted expression by repressing other identity genes competent to respond positively to extrinsic signals. The cross-repressive interactions of identity genes are likely to ensure their localized expression over time, thus providing an essential element in establishing cell identity (Jagla, 2002).

Ectopically expressed lb has been shown to inhibit eve in the founder cell of the DA1 muscle. This effect may be due to either a specific inhibition of eve by lb or a more general regulatory mechanism of fate specification. Data presented here favour the latter possibility, showing that the gain of lb function affects expression of several identity genes and consequently influences fates of cells in which these genes are expressed. Specifically, embryos that ectopically express lb have an increased number of tin-positive heart cells with a concomitant reduction of dorsal muscles. To demonstrate that the supernumerary cardiac cells result from cell fate switches, rather than from additional proliferation, mshDelta mutants, displaying heart hyperplasia similar to that observed in embryos overexpressing lb, were used. In this particular msh mutant, the presumptive msh-positive muscle cells monitored by lacZ start to express cardiac markers. This suggests that switches from muscular to cardiac fates contribute to heart hyperplasia induced by deregulation of identity genes. Interestingly, the ectopic expression of lb and msh leads to reciprocal phenotypes, and indicates that the identity genes specifically expressed in the heart promote dorsal mesodermal cells to enter the cardiogenic pathway, while the muscle identity genes promote the myogenic pathway. However, more detailed analysis shows that ectopic lb promotes only specific cardiac fates and ectopic msh only specific muscle identities, thus indicating that the identity genes instruct dorsal mesodermal cells to adopt the specific cardiac or muscular fates, rather than make a choice between cardiac and muscular development. This property is particularly well illustrated by the phenotypes generated by the ectopic eve, which is involved in the specification of a subset of heart and dorsal muscle cells and when ectopically expressed promotes specification of supernumerary cells of both types. Moreover, deregulated heart and dorsal muscle identity genes preferentially affect fates of mesodermal cells located in dorsal but not in ventral regions, thus suggesting that the identity gene action is instructive only in a permissive context (Jagla, 2002).

This observation is in complete agreement with the model of competence domain. According to this concept, the high level of Wg and Dpp signals present in the anterodorsal region (under the intersection of Wg and Dpp epidermal domains) provides a major cue that direct mesodermal cells into cardiac or dorsal muscle development. In relation to this model, these data design a new regulatory mechanism that provides a paradigm of how the intrinsic transcription factors and extrinsic signaling molecules converge to specify cell fates (Jagla, 2002).

The findings suggest cross-repressive interactions that occur between transcription factors that specify adjacent and non-overlapping populations of muscle and heart cells. Most likely, in normal development, these interactions have a functional relevance once the progenitor cells segregate, and then continue to play an important role in the next step of cell fate diversification, namely in founder cells. The gain- and loss-of-function experiments presented indicate that the identity genes may function as repressors starting from the progenitor stage onwards. However, the earliest activation of inappropriate identity gene as a result of the loss of function of repressor (in mshDelta embryos) was documented in founder cells (Jagla, 2002).

It is proposed that cross-repressive interactions allow the refinement of the potentially imprecise pattern of identity gene expression induced by the interplay of Wg and Dpp signaling pathways. Wg and Dpp create a permissive context for the development of cardiac and dorsal muscle precursors. In such a context, the transcription factors that specify these two types of cells (e.g. lb, eve and msh) are expected to be activated in all dorsal mesodermal cells. The local restriction of identity gene expression is, however, provided by a combinatorial signaling code mediated by two receptor tyrosine kinases, the Drosophila epidermal growth factor receptor and the Heartless (Htl) fibroblast growth factor receptor. Transient localized activity of these two mesodermal signaling pathways is thought to subdivide the large competence domain into small clusters of equivalent cells from which individual progenitors segregate. Depending on the combination of RTKs activities, the individual identity genes are activated only in a defined equivalence group and in the resulting progenitor. This study defines an additional step to the aforementioned model. It is proposed that the major role of identity genes is to maintain their restricted expression in progenitors and subsequently in founder cells by repressing other identity genes competent to respond positively to Wg and Dpp signals. These cross-repressive interactions are likely to ensure constant localized identity gene expression over time, thus providing a crucial element in establishing cell identity (Jagla, 2002).

muscle segment homeobox: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of mutation | References

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