BIOLOGICAL OVERVIEW

The neurogenetic role of lethal of scute resembles that of the other three proneural gene members in the achaete-scute complex (achaete, scute and asense). This overview will examine instead the role of lethal of scute in the specification of muscle progenitors.

Muscle development takes place in two phases. First, the pattern of muscle development is laid down by allocation of founder cells, specific cells in the mesoderm, each one serving as a founder for a unique muscle. Second, founder cells recruit neighboring myoblasts to form the syncylial precursors of mature muscle by fusion. Initially l'sc expression is widespread in cell clusters, but becomes allocated to muscle founder cells through the action of the Notch pathway.

The expression of lethal of scute in mesoderm is transient, occuring in twist expressing cells. From late stage 9 until stage 12, there are at least 19 clusters of l'sc expressing cells in each hemisegment. In each cluster, one cell accumulates higher levels of l'sc than the other cells in the cluster. It is this single cell, allocated from a cluster of cells, that moves to a position close to the ectoderm and eventually becomes a muscle founder cell.

Genes coding for three transcription factors, (nautilus, S59 and msh1) are each expressed in small groups of cells destined to differentiate into muscle cells. Another transcription factor, MEF2, is required for myosin expression and the fusion of myoblasts. None of these are selector genes initiating muscle fate. Genes with a decisive role in myogenesis, similar to members of the MYO-D family in vertebrates, have not been found in Drosophila.

In neurogenic mutants, the domains of mesodermal S59 expression are expanded. This suggests the Notch pathway is involved in restricting the expression of l'sc and S59 to single cells, the same way it functions in neuroblast differentiation. l'sc is not the only factor involved in founder cell specification. In some instances no l'sc is found in the founder cell, and l'sc mutation does not completely upset muscle specification.

Founder cell specification is easily comparable to neuroblast specification. The role of l'sc in muscle specification is analagous to the role of achaete-scute genes in neural growth. Specification of muscle founder cells is one of the many different processes involving the Notch pathway, a significant proportion of which involve proneural genes (Carmena, 1994).

Determination of cell fate along the anteroposterior axis of the Drosophila ventral midline

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers during neurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in the lateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is still not well understood. This study shows that most midline cells are determined not at the precursor cell stage, but as daughter cells. After the precursors divide, a combination of repression by Wingless and activation by Hedgehog induces expression of the proneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog and Lethal of scute activate Engrailed in these anterior cells. Engrailed-positive midline cells develop into ventral unpaired median (VUM) neurons and the median neuroblast (MNB). Engrailed-negative midline cells develop into unpaired median interneurons (UMI), MP1 interneurons and midline glia (Bossing, 2006).

The determination of midline cells appears to take place during germband elongation, since by germband retraction most midline subsets can be identified by the expression of unique molecules. The anteroposterior position of midline siblings was determined during germband elongation. Midline precursors were labelled with the lipophilic dye DiD or DiI in embryos expressing GFP in the Engrailed domain (en-GAL4/UAS-tauGFP). After division of the precursors, the daughter cells were followed throughout development, recording their segmental position at stage 10 and stage 11. MP1 interneurons, UMI and MNB neurons each arise from one precursor, and their daughter cells occupy fixed anteroposterior positions during germband elongation. The four daughter cells of the two glial precursors can be located either in the middle of the segment or just anterior to the Engrailed domain. VUM neurons arise from three midline precursors, and the six daughter cells of these precursors are located inside the Engrailed domain and immediately posterior to the domain, in the anterior of the next segment (Bossing, 2006).

In summary, the midline glia and MP1 interneurons are the most anterior midline subsets, followed by a second pair of midline glia and a pair of UMIs, and, finally, the VUM and MNB neurons. DiI labelling cannot resolve whether the MP1 interneurons or the midline glia are the most anterior cells. Since determination of the MP1 interneurons depends on Notch/Delta signalling, it is possible that the anteroposterior position of the most anterior midline cells, the midline glia and MP1 interneurons, is random. Interestingly, four VUM neurons and the MNB neurons seem to arise from the anterior compartment of the next posterior segment. These cells initiate Engrailed expression half-way through germband elongation, and, during germband retraction, they join the adjacent anterior segment to become the most posterior midline subsets (Bossing, 2006).

The separation of midline cells into two compartments is an early and crucial step in midline cell determination. During germband elongation, a second phase of Engrailed expression is initiated at the midline in the anterior cells of the next posterior segment. During germband retraction, these cells join the anterior segment where they develop into posterior midline cells. Expression of late Engrailed depends on Hedgehog signalling and the proneural gene lethal of scute. Lethal of scute precedes Engrailed expression and is also activated by Hedgehog. Hedgehog and Wingless signalling counteract each other to define the position of the Lethal of scute cluster, and to divide the 16 midline daughter cells into eight non-Engrailed- and eight Engrailed-expressing cells (Bossing, 2006).

It has generally been believed that the determination of the different subsets of midline cells occurs before the precursors undergo their simultaneous division at stage 8. This view is challenged by the observation that expression of the proneural gene lethal of scute, and the subsequent expression of Engrailed, is initiated in midline daughter cells at stage 10, about one hour after the precursors divide. In the neuroectoderm, proneural genes confer neural competence to a cluster of ectodermal cells. Lateral inhibition by Notch/Delta signalling then limits the expression of proneural genes to a single cell, which delaminates from the ectoderm and becomes a neural precursor (neuroblast). Because the only neuroblast at the ventral midline (median neuroblast, MNB) originates from the proneural Lethal of scute cluster, it seems likely that the MNB is selected by lateral inhibition from a cluster of midline daughter cells. However, the process of lateral inhibition in the midline differs from that in the adjacent neuroectoderm. In the neuroectoderm, a single cell delaminates and the remaining cells of the cluster cease proneural expression and give rise to the epidermis. The proneural cluster in the midline consists of three pairs of siblings generated by the division of three separate precursors. Labelling of single precursors shows that, during the selection of the MNB, only one of the two labelled siblings enlarges, but both delaminate from the embryo. In contrast to the neuroectoderm, the remaining cells of the midline cluster continue to express Lethal of scute after delamination of the MNB. This extended proneural expression might be necessary to maintain neural competence in the non-delaminating cells that develop into VUM neurons (Bossing, 2006).

The results cannot exclude the possibility that some of the midline subsets are determined as precursors, but at least two of the five midline subsets, the VUM neurons and the MNB, are determined after precursor cell division. There are striking similarities between the development of the ventral midline of Drosophila and grasshopper embryos. In grasshopper, Engrailed expression can be detected in the MNB, its progeny and the midline precursors MP4 to MP6, which each give rise to two neurons with projection patterns comparable to the Drosophila VUM neurons. Hence, the same types of midline cells express Engrailed in grasshopper and Drosophila, but in grasshopper Engrailed expression is initiated in all midline precursors prior to division (Bossing, 2006).

In the ectoderm from stage 10 onwards, Wingless, Engrailed and Hedgehog maintain the expression of one another by a feedback loop: Wingless maintains Engrailed expression, Engrailed is needed for the expression of Hedgehog and Hedgehog maintains Wingless expression. In the developing CNS, Wingless and Hedgehog expression seem to be independent of each other. At the ventral midline there are two separate stages of Engrailed expression: the early phase is maintained by Wingless; the late phase does not require Wingless and is instead activated at stage 10 by Hedgehog signalling and Lethal of scute. In the ectoderm, Wingless and Hedgehog act in concert to maintain Engrailed expression, but at the midline Wingless and Hedgehog act in opposition: Wingless represses and Hedgehog activates Lethal of scute expression (Bossing, 2006).

Wingless may repress Lethal of scute expression indirectly, via its maintenance of early Engrailed. As in the ectoderm, midline Engrailed represses expression of the Hedgehog receptor Patched and the Hedgehog signal transducer Cubitus interruptus. It is possible that early Engrailed-expressing midline cells are not able to receive the Hedgehog signal. However, ectopic expression of Hedgehog is able to induce Lethal of scute in all midline cells, suggesting that Wingless may repress Lethal of scute by a yet unknown mechanism. Recently it has been reported that a vertebrate wingless orthologue, Wnt2b, can maintain the naïve state of retinal progenitors by attenuating the expression of proneural and neurogenic genes (Bossing, 2006).

The differentiation of midline cells was studyed in wingless and hedgehog mutants. Consistent with earlier reports, many midline cells become apoptotic in both mutants. The surviving midline cells are not integrated into the CNS and show no morphological differentiation. The reduction in the number of Engrailed-positive midline cells in hedgehog mutant embryos may be mainly due to the loss of midline cell identity. In hedgehog mutants, midline cells lose the expression of Sim, the master regulator of midline development. As described for sim mutants, the loss of midline identity results in increased cell death and misspecification of the surviving midline cells as ectoderm (Bossing, 2006).

Ectopic expression of Hedgehog in the neuroectoderm and the developing CNS induces the expression of Lethal of scute and, approximately 40 minutes later, the expression of late Engrailed in all midline cells. It seems likely that Lethal of scute is an early target of Hedgehog signalling, and its activation may only require release from repression by the short form of Cubitus interruptus. By contrast, the delay in induction of late Engrailed in the same midline cells indicates that Engrailed activation may not only require release from repression, but also activation by the long form of Cubitus interruptus (Bossing, 2006).

Uniformly high levels of ectopic Hedgehog prevent the differentiation of most midline subsets and cause increased cell death. A single source of ectopic Hedgehog, achieved by cell transplantation, does not result in midline cell death, but reveals that the differentiation of the MP1 interneurons is more sensitive to Hedgehog levels than is the differentiation of midline glia. No other midline subsets are affected. It seems likely that Hedgehog not only activates Lethal of scute and late Engrailed, but also acts as a morphogen to control the differentiation of the MP1 neurons and midline glia (Bossing, 2006).

The phenotypes caused by ectopic Hedgehog are due to the induction of Engrailed in all midline cells. Expression of ectopic Hedgehog and ectopic Engrailed blocks the differentiation of midline glia and MP1 interneurons, and also prevents the formation of the anterior commissure. Labelling single midline precursors enabled examination of cell fates in embryos expressing ectopic Engrailed in the midline. The frequency of clones obtained indicates that ectopic Engrailed expression does not transform non-Engrailed-expressing midline subsets (MP1 interneurons, midline glia and UMI) into Engrailed-expressing subsets (VUM and MNB). Instead, embryos expressing midline Engrailed show increased cell death. In particular, the MP1 interneurons seem to be affected and were never obtained during this analysis. The low frequency of midline glia also points to apoptosis caused by expression of Engrailed. Surviving midline glia are not able to differentiate properly and cannot enwrap the remaining, posterior, commissure. All other midline subsets, including the UMIs, are able to differentiate, but they show a variety of axonal pathfinding defects that may result from the loss of anterior midline subsets and the absence of the anterior commissure (Bossing, 2006).

It is likely that genes other than hedgehog and wingless are crucial for midline cell determination. In these experiments, non-Engrailed-expressing midline subsets are never transformed into Engrailed-expressing subsets, or vice versa. gooseberry-distal may be one of these genes. From the blastoderm stage, Gooseberry-distal is expressed by two midline precursors and their four daughter cells. During early embryogenesis Gooseberry-distal expression at the midline does not depend on Wingless and Hedgehog. The anterior Gooseberry-distal cells also express Wingless and most likely give rise to the UMIs. The posterior Gooseberry-distal pair also express early Engrailed and Hedgehog, and develop into the most anterior VUM neurons. At stage 10, Hedgehog activates the expression of Lethal of scute and Engrailed in midline cells posterior to the Gooseberry-distal domain. Lateral inhibition by Notch/Delta signalling selects one cell from the Lethal of scute cluster to become the MNB. The remaining cells become VUM neurons. At stage 10, the absence of Engrailed in the six midline cells anterior to the Gooseberry-distal domain defines a cell cluster that will give rise to midline glia and MP1 interneurons. Based on the expression of Odd, Delta mutants have an increased number of MP1 interneurons, up to six per segment. In Notch mutants, midline glial-specific markers are absent and the number of cells expressing a neuronal marker increases. Therefore, Notch/Delta signalling appears to determine midline glial versus MP1 interneuron cell fates in the anterior cluster. In the current model, midline cell determination takes place mainly after the division of the precursors. Although the initial determination of midline cells appears to be directed by a small number of genes, a far larger number is needed to control the differentiation of the various midline subsets. This work, and the recent identification of more than 200 genes expressed in midline cells, is the beginning of a comprehensive understanding of the differentiation of the ventral midline (Bossing, 2006).

REGULATION

Promoter Structure

Thirty nucleotides upstream from the start of transcription is an unconventional TATA box (TATTTAAA). Seven CANNTG motifs (E-boxes), putitive binding sites for bHLH transcription factors, are located in this upstream region 200 to 1000 bp above the start site. These local upstream elements could function in l'sc autoregulation or activation by Achaete or Scute (Martin-Bermudo, 1993).

Transcriptional Regulation

Snail represses l'sc transcription in the presumptive embryonic mesoderm (Kosman, 1991). Elements regulating l'sc are scattered throughout 75 kb between achaete and asense. These elements activate l'sc in specific proneural clusters and as a consequence, also in their corresponding neuroblasts (Martin-Bermudo, 1993).

Short gastrulation prevents Decapentaplegic from suppressing neurogenesis laterally in the blastoderm embryo. It is possible to exacerbate defects in sog mutants by increasing the level of DPP. The earliest neuroectodermal marker affected in sog mutants with a double dose of dpp is rhomboid, which is normally expressed in lateral stripes 8-10 cells wide in wild-type embryos but rapidly narrows to stripes 4-6 cells across in sog mutants with elevated DPP. Similarly l'sc expression is reduced in sog mutants with elevated DPP. A striking feature of the affects of DPP on neural suppression and dorsalization is that neuronal suppression is induced by a lower threshold of DPP activity than is dorsalization. Much less DPP is required to suppress expression of neuroectodermal genes than is required to activate dorsal markers. For example, brief submaximal heat induction of heat shock dpp in a wild type sog background leads to nearly maximal suppression of lethal of scute, scratch and snail expression during germ band extension, but there is no detectable ectopic expression of zerknüllt in the neuroectoderm (Biehs, 1996).

The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage that this expression produces. Little is known as to the control of neuroblast identity along the DV axis. The Drosophila Egfr receptor (Egfr) has been shown to promote the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Molecular markers identify particular neuroectodermal domains, composed of neuroblast clusters or single neuroblasts, and show that in Egfr mutant embryos (1) intermediate column neuroblasts do not form; (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Active Egfr signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. The concomitant loss of rhomboid and vein yields CNS phenotypes indistinguishable from Egfr mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that Egfr plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS (Skeath, 1998).

In a screen to identify mutations that disrupt embryonic CNS development, one P element mutation, l(2)03033, was identified that causes a loss of essentially all Eve-positive RP2/RP2 sib neurons. This P element maps to cytological position 57F1-2 in the right arm of the second chromosome and is known to be inserted within the Egfr locus. To verify that lesions in Egfr result in a nearly complete loss of RP2 motoneurons, three additional Egfr mutants were obtained, including the Egfr null allele, flb 1K35Egfr allele (Skeath, 1998).

The first phase of CNS development, as gastrulation commences, involves the activation of the Ac-S proneural genes in a precise pattern of proneural clusters. To investigate whether Egfr regulates As-C expression in the neuroectoderm, the expression patterns of the achaete (ac) and lethal of scute (lísc) genes were followed in Egfr mutant embryos. Loss of Egfr causes specific defects to the DV registration of ac and lísc gene expression in the neuroectoderm; however, no defects to the AP registration for either ac or lísc gene expression were found. In wild-type embryos during stages 8/9, ac is expressed in the medial and lateral, but not intermediate, clusters of rows 3 and 7; lísc is expressed in the medial and lateral, but not intermediate, clusters of row 7 and in the medial, intermediate and lateral clusters of rows 1 and 5. A single neuroblast subsequently forms from each proneural cluster. In Egfr mutant embryos, ac gene expression expands into the intermediate column in rows 3 and 7 and lísc expression expands into the intermediate column in row 7; lísc is expressed normally in rows 1 and 5. The lateral limits of ac and lísc gene expression in the neuroectoderm are unaltered in Egfr mutant embryos. The changes to the DV registration of ac and lísc gene expression in Egfr mutant embryos suggest that neuroectodermal cells in the intermediate column change their fate. Both ac and lísc are normally expressed in the medial and lateral columns in the affected rows, thus the phenotype is consistent with intermediate cells acquiring either a lateral or a medial fate. msh-1, which is expressed exclusively in the lateral column, expands into the intermediate column in Egfr mutant embryos. In this context, it appears that ac and lísc expression expand from the lateral column into the intermediate column in the absence of Egfr (Skeath, 1998).

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. This study investigates the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, Notch^IC, in transgenic embryos using the eve stripe2 enhancer. The Notch^IC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'naïve' embryos that contain low uniform levels of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch target genes and stimulation of Notch signaling -- help define precise lines of sim expression within the neurogenic ectoderm. It is proposed that Snail functions as a gradient repressor to restrict Notch signaling. In precellular embryos, the initial snail expression pattern is broad and extends into the future mesectoderm. During cellularization, the pattern is refined and snail expression is lost in the mesectoderm and restricted to the mesoderm. The early, broad snail pattern might create a broad domain of potential Notch signaling by repressing components of the Notch pathway, such as Delta and lethal of scute. After cellularization, Notch signaling is blocked in the presumptive mesoderm by sustained, high levels of the Snail repressor. However, Notch can be activated in the mesectoderm because of the loss of Notch inhibitors repressed by transient expression of the Snail repressor. According to this model, the dynamic snail expression pattern determines both the timing and limits of Notch signaling (Cowden, 2002).

Targets of Activity

L'SC activates Enhancer of split and HLH-M5 of the Enhancer of split complex, and possibly dorsal as well (Hinz, 1994).

Protein Interactions

Extramachrochaete forms inactivating heterodimers with L'SC as it does with AC and SC (Cabrera, 1994). Daughterless is required as a dimerization partner for L'SC function (Hinz, 1994).

Classical genetics indicates that the achaete-scute gene complex (AS-C) of Drosophila promotes development of neural progenitor cells. To further analyze the function of proneural genes, the effects of Gal4-mediated expression of lethal of scute, a member of the AS-C, were studied during embryogenesis. Expression of lethal of scute forces progenitor cells of larval internal sensory organs to take on features of external sensory organs. Normally, these cells are committed to this fate independent of AS-C activity. Surprisingly, overexpression of l'sc does not result in supernumerary neural cells. Supernumerary neural cells can be induced ectopically only if daughterless is overexpressed, either alone or together with lethal of scute: cells of the amnioserosa and the hindgut then express neuronal markers. Cells of the proctodeal anlage, which normally lack neural competence, acquire the ability to develop as neuroblasts following transplantation into the neuroectoderm. Activated Notch prevents the cells of the neuroectoderm from forming extra neural tissue when they express an excess of proneural proteins. Under the present conditions, lateral inhibition is thus dominant over the activity of proneural genes (Giebel, 1997).

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of l(sc) at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Like achaete and scute, l'sc is expressed in very specific subsets of cells in neuroblasts of the ventral neurectoderm (Martin-Bermudo, 1993). Gene expression in the ventral neuroectoderm is discussed at the achaete-scute complex site.

As with achaete and scute, l'sc is expressed in every neurogenic region of the fly, including the cephalic and gnathal regions. After stage 9, l'sc is expressed in the mesectodermal, central and peripheral nervous system anlage, as well as the stomatogastric nervous system and the optic lobes (Cabrera, 1990).

In head midline structures, in particular the optic lobe and stomatogastric nervous system, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express lísc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998).

The expression of the proneural gene lethal of scute is required for the development of the majority of the procephalic neuroblasts. lethal of scute expression patterns correspond to many of the identifiable 23 groups of neuroblasts in the developing brain. l'sc expression in the procephalic neurectoderm is controlled in partially overlapping domains of the neuroectoderm. Loss of function of a given head gap gene results in the absence of l'sc expression in its domain, followed by the absence of neuroblasts that would normally segregate from this domain (Younossi-Hartenstein, 1997).

Neuroblasts delaminate from the procephalic neurectoderm in a stereotyped spatiotemporal pattern that is tightly correlated with the expression of l'sc. The pattern of neuroblasts was reconstructed by using the marker asense; similar to its expression in the ventral neuroblasts, asense labels all brain neuroblasts. seven-up, expressed in specific subsets of neuroblasts making up approximately one-third of the total, is also used as a marker. For most, if not all, of these clusters the number of neuroblasts and the time of onset of svp expression are absolutely invariant (Younossi-Hartenstein, 1996).

Effects of Mutation or Deletion

lethal of scute mutants may reach adulthood (Martin-Bermudo, 1993). This attests to the many redundancies or biological fail safe mechanisms created by the duplication of function in proneural genes.

klumpfuss shows genetic interactions with achaete, scute, lethal of scute and asense. l'sc is able to activate klu expression, but apparently only in the wing disc. There appears to be only a weak influence of the AS-C genes on klu expression, restricted to the wing area of the wing disc. However, the overall expression pattern of klu is largely independent of proneural genes. The assumption that SOPs enter apoptosis in klu mutants is supported by the observation of abundant cell death in other developing organs of klu mutants, like the legs. At certain bristle positions, such as that of the anterior sternopleura, klu is required during early bristle development immediately after proneural gene function, in order to allow a particular epidermal cell to develop as a SOP. It is suggested that klu is required only for initiation of bristle development, being downregulated once specification takes place (Klein, 1997).

During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the formation of a single muscle (muscle DA3^[A] in the abdominal segments; DA4^[T] in the thoracic segments T2 and T3). Col expression is first observed in two promuscular clusters (in segments A1-A7), corresponding to two progenitors and then their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3^[A]. It is proposed that specification of the DA3^[A] muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles (Crozatier, 1999).

Following establishment of the promuscular clusters, specification of the progenitors is controlled by lateral inhibition, a cell-cell interaction process mediated by the neurogenic genes Notch (N) and Delta (Dl)). In both N and Dl mutant embryos, promuscular Col expression is initiated normally but fails to become restricted to a single cell per cluster, similar to observations previously made for the expression of lísc. As a consequence, a hyperplasic expression of Col is observed from stage 11. Since it is expressed in promuscular clusters and segregating muscle progenitors, lísc has been proposed to play a role in muscle progenitor selection similar to the role of achaete and scute in neuroblast specification. However, in embryos lacking lísc activity, selection of the Col-expressing progenitors occurs normally at stage 11 and muscle DA3^[A] forms as in wild type (Crozatier, 1999).

In the embryonic ventral neuroectoderm of Drosophila the proneural genes achaete, scute, and lethal of scute are expressed in clusters of cells from which the neuroblasts delaminate in a stereotyped orthogonal array. Analyses of the ventral neuroectoderm before and during delamination of the first two populations of neuroblasts show that cells in all regions of proneural gene activity change their form prior to delamination. Furthermore, the form changes in the neuroectodermal cells of embryos lacking the achaete-scute complex, of embryos mutant for the neurogenic gene Delta, and of embryos overexpressing l'sc, suggest that these genes are responsible for most of the morphological alterations observed (Stollewerk, 2000).

Almost all neuroectodermal cells are larger than the cells of the dorsal epidermal anlage (DEA). In comparison with the cells of the DEA in early stage 8 embryos the dorsoectodermal cells of mid-stage 8 embryos are clearly smaller. A comparison of the neurogenic region in early and mid-stage 8 embryos shows that the medial and intermediate regions of the ventral neuroectoderm (VNE) do not increase further in size whereas the lateral region enlarges considerably during this time. Due to these morphological changes the VNE can now be subdivided in relation to the cell sizes into three longitudinal regions on both sides of the midline: medial, intermediate, and lateral regions. In contrast to the cells of the medial and lateral regions, which now have approximately the same average values, the intermediate cells are smaller. Only 20% of all cells in the intermediate region are larger than the average, whereas 63% of the medial and 64% of the lateral cells exceed the average value. Most of the enlarged cells have a cuboidal shape. In every hemisegment the apical surfaces of two to four cells in the medial and lateral regions are very small (12-16 µm²) but expand basally to cover an area of 65-80 µm². One or two cells of this shape are also located in the intermediate regions but are smaller basally (48-58 µm²) than the medial and lateral cells. The number and position of these cells suggest that they correspond to the delaminating neuroblasts; this was confirmed by staining the embryos with anti-Hunchback antibody, an early marker for neuroblasts (Stollewerk, 2000).

During delamination of the SI neuroblasts the neuroectodermal cells gradually decrease in size, with the exception of a few cells located close to the midline. The cells that remain enlarged are either elongated perpendicularly to the midline or have a rounded appearance. Basally, between neuroectoderm and mesoderm, large round cells are located that lose contact with the apical surface at about 60% EL. On the basis of their position and arrangement, as well as the analysis of embryos stained for Hunchback, these cells can be identified as the SI neuroblasts. Before delamination of the SII neuroblasts, cells in the intermediate region of the neuroectoderm increase in size. Most of the SII neuroblasts delaminate from this region, whereas only a few neuroblasts arise from the medial region, where enlarged cells can also be detected. After delamination of the SII neuroblasts the enlarged cells shrink once again, as revealed by double staining with anti-Hunchback antibody and phalloidin. Cells in all regions of the VNE increase in size again prior to delamination of the SIII neuroblasts. Thus, the VNE of wild-type embryos becomes morphologically distinguishable from the DEA shortly before delamination of the SI neuroblasts. At this point the cells of the DEA have already divided, and about two-thirds of all cells in the medial and the lateral regions have become enlarged so that the DEA and the VNE are clearly distinguishable due to differences in cell size. In addition, almost all cells of the intermediate region increase in size prior to delamination of the SII neuroblasts. These data are at odds with claims that only the neuroblasts enlarge prior to delamination, both in grasshopper and Drosophila (Stollewerk, 2000).

Is there a correlation between the activity of the ASC genes and the observed morphological changes? The results presented indicate that the ASC genes are not the only ones responsible for the morphological changes that occur before delamination of the SI neuroblasts. Although the number of enlarged cells corresponds closely to the number of cells that express the ASC genes at this time point, the lack of the ASC does not result in all cells remaining the same size. Whereas in the medial region of the VNE of Df (1)260-1 embryos (that is, those lacking the ASC) only about 50% of the cells are smaller in size than in the wild type, and the lateral region is most strongly affected in comparison to the medial and intermediate regions. Therefore the enlargement of the neuroectodermal cells depends to a varying degree on the activity of the ASC genes and is additionally influenced by other factors. However, a clear correlation can be seen prior to delamination of the SII neuroblasts. At this time almost all cells of the intermediate region increase in size, which coincides with the expression of l'sc in this region. Furthermore, analysis of the VNE of embryos lacking the ASC reveals that the intermediate cells do not become enlarged prior to delamination of the SII neuroblasts, suggesting that the observed morphological changes are due to the activity of the ASC genes at this point. In addition, the shrinkage of the cells that had enlarged during delamination of the SI and SII neuroblasts is correlated with the decrease in ASC gene expression in the VNE at these time points (Stollewerk, 2000).

Analysis of wild-type and Delta mutant embryos also suggests that the ASC genes are important for the maintenance of the morphology of the neuroectodermal cells. Despite the fact that the total area of the intermediate region does not change significantly between early and mid-stage 8, cell size changes can be detected in this region shortly before delamination of the SI neuroblasts. While 20% of the intermediate cells remain larger than the average, the cells that had an average cell size in the VNE of early stage 8 embryos now split into groups of smaller cells. The fact that the number of cells that remain larger than the average corresponds to the number of cells that express the ASC genes in the intermediate region suggests that the proneural genes are required to keep these cells enlarged. This view is confirmed by analyses of the VNE of Delta mutant embryos. In Delta mutant embryos all cells of a proneural cluster continue to express the proneural genes and become neuroblasts. This altered gene expression causes all cells of a proneural cluster to remain enlarged until proneural gene expression is turned off (Stollewerk, 2000).

A correlation between increase in cell size and ASC gene expression has also been shown by the analysis of embryos labeled for ac protein and embryos overexpressing l'sc. Area measurements reveal that 85% of all cells that express ac are enlarged in these embryos. The fact that not all ac-expressing cells are larger than the average at the time point analyzed may be due to the rapidity of the morphological changes (enlargement and shrinkage) that occur immediately before and during delamination of the neuroblasts. A clear influence of a proneural gene on the cell sizes in the VNE can be seen in embryos overexpressing l'sc: 45% more cells become enlarged in the intermediate region in comparison to the wild type. Only a minor increase in the numbers of enlarged cells can be seen in the medial and lateral regions, because two-thirds of these cells already express proneural genes. In addition, the high proneural gene activity in the VNE of embryos overexpressing l'sc causes the future neuroblasts to change their morphologies: they expand not only their basal but also their apical surfaces. These data clearly show that the ASC genes have an influence on the morphologies of the neuroectodermal cells (Stollewerk, 2000).

A key role of Pox meso in somatic myogenesis of Drosophila: Poxm and l(1)sc exhibit partially redundant functions during muscle development

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 our 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. 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).

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, 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).

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date revised: 25 March 2008

 

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