engrailed

DEVELOPMENTAL BIOLOGY

Larval

Engrailed functions to establish anterior-posterior polarity in various organs derived during the larval stage from imaginal discs, including leg, wing and eye.

engrailed is expressed in the posterior compartment of wing, leg, antennal, haltere and labial imaginal discs (Kornberg, 1985).

Unlike the thoracic discs, the anterior and posterior compartmental organization of the genital imaginal disc is compound, consisting of three primordia Ð the female genital, male genital, and anal primordia. Each primordium is divided into anterior and posterior compartments. Genes that are known to be expressed in a compartment-specific manner in other discs (engrailed, hedgehog, patched, decapentaplegic, wingless and cubitus interruptus) are expressed in analogous patterns in each primordium of the genital disc. Specifically, engrailed and cubitus interruptus are expressed in complementary domains, while patched, decapentaplegic and wingless are expressed along the border between the two domains. en and inv are required in the posterior comparment of the genital disc to repress dpp and activate hh. Mitotic clones induced at the beginning of the second larval instar do not cross the boundary between the engrailed-expressing and cubitus interruptus-expressing domains, indicating that these domains are true genetic compartments (Chen, 1997).

cAMP-dependent protein kinase A and engrailed-invected are genes known to play compartment-specific functions in other discs. The anterior/posterior patterning functions of these genes are conserved in the genital disc. en-inv mutant clones cause posterior to anterior transformations in adult terminalia. Pka is required to repress ptc, dpp and wg expression in the anterior compartment of the genital disc. Pka mutant clones result in pattern duplications in adult terminalia (Chen, 1997).

Comparison of the wg expression pattern with the fate maps of the genital discs suggests that the wg expression domains in both the female and male genital discs correspond to the internal, but not the external, genitalia. wg mutation results in deletion of all internal structures in both males and females. In addition, the male external genitalia show deletions in certain structures, such as clasper teeth, the lateral plate bristles and the penis apparatus. The female external genitalia are generally unaffected (Chen, 1997).

The adult clonal phenotypes of protein kinase A and engrailed-invected mutants provide a more detailed map of the adult genitalia and analia with respect to the anterior/posterior compartmental subdivision. A new model has been proposed to describe the anterior and posterior compartmental organization of the genital disc. Each of the three primordia (female, male and anal) is composed of its own anterior and posterior compartments. Each primordium has a larger anterior compartment and a smaller posterior compartment. Each genital disc is divided into anterior and posterior compartment (Chen, 1997).

The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs; the absence of engrailed and invected causes duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development (Casares, 1997).

Mutations in Abd-B transform female genitalia into abdomen, suggesting that the activity of Abd-B is a prerequisite for the specification of the terminalia by the sex-determing genes. abd-A is expressed only in female genital discs, in the region corresponding to the female genital primordium, particularly in the prospective internal female genitalia. abd-A expression is coincident with engrailed in the central region of the female genital primordium engrailed band. Abd-B transcripts are located in the genital disc. The Abd-B protein is present in the male and female primordia in both male and female discs, leaving unstained the region where the analia map. Abd-B expression is coincident with en bands 1 and 2. In female discs, Abd-B m transcript is present only in the female genital primordium: transcript levels are strong in the prospective external genitalia and faint in the prospective internal genitalia. In the male disc, only the repressed male primordium is labelled. Abd-B r transcript is expressed in the repressed male primordium of female discs and the male genetal primordium of male discs. caudal is located in the analia primordium of the genital disc, overlapping with the third engrailed band. However, caudal and enoverlap in only a few, dorsally located, epidermal nuclei of stage 14 embryos. This overlap is not seen in the ventrally located embryonic genital disc where caudal expression is observed in its posterior region. This suggests that en expression in anal primordium of mature genital discs appears during larval development. The perianal ring corresponds to the terminal band of en, and the co-expression of en and cad is maintained from the third instar disc until the adult stage (Casares, 1997)

Very little information is available about gene expression during the larval period, a developmental interval critical to the formation of the adult. To what extent does gene expression during this period resemble that in the embryonic stages, and how does gene expression during the larval period contribute to segment polarity in the adult? In fact, all the genes expressed during embryonic segment polarity also play a similar role in the formation of the adult. Cells destined to form the cuticle of the adult abdomen are present as clusters of small, non-dividing diploid cells (the anterior dorsal, posterior dorsal and ventral histoblast nests) located at stereotyped postions in the larval epidermis. These cells, just as do their embryonic counterparts, express engrailed, hedgehog, wingless, patched, cubitus interruptus and sloppy paired in a stereotyped manner dependent on their positions within each segment. Each segment is subdivided into an anterior (A) and posterior (P) compartment, distinguished by activity of the selector gene engrailed (en) in P but not A compartment cells. The ventral epidermis of each abdominal segment forms a flexible cuticle, the pleura, with a small plate of sclerotised cuticle, the sternite, centered on the ventral midline. The pleura is covered with a uniform lawn of hairs, all pointed posteriorly, whereas the sternite contains a stereotyped pattern of bristles. Posterior compartments are to a large degree devoid of hairs and bristles, while the sternite cuticle of the A compartment consists of an anterior-to posterior progression of six types of cuticle distinguished by ornamentation and pigmentation. Just anterior to the posterior compartment, A6 is unpigmented, with hairs and none of the larger ornaments called bristles. A5 is darkly pigmented with hairs and bristles of large size. A4 and A3 are darkly and lightly pigmented respectively with moderately sized hairs and bristles. A2 is lightly pigmented with hairs, and A1, adjacent to the next more anteriorly located "posterior" compartment is unpigmented without hairs (Struhl, 1997).

Hedgehog (Hh), a protein secreted by engrailed expressing P compartment cells, spreads into each A compartment across the anterior and the posterior boundaries to form opposing concentration gradients that organize cell pattern and polarity. Anteriorly and posteriorly situated cells within the A compartment respond in distinct ways to Hh: they express different combinations of genes and form different cell types. patched is expressed at both boundaries. patched is expressed in a graded fashion within each stripe, just anterior to each P compartment. ci peaks at high level in those cells abutting Hh- secreting cells of the P compartment and declines progressively in cells further away. wingless is also expressed in this domain and sloppy paired is expressed in the same region as wingless. decapentaplegic is expressed only in the ventral pleura in those A compartment cells neighboring P compartment cells within the same segment. dpp is not expressed immediately behind posterior compartments (Struhl, 1997a).

Each abdominal segment produces a large dorsal cuticular plate (the tergite) and a smaller ventral plate (the sternite). Each tergite can be divided into three regions: an acrotergite that contains undecorated sclerotized cuticle, a central region containing an array of microchaetes, and a posterior region that contains a dark pigment band as well as a row of large macrochaetes at the posterior edge of this posterior region. All of the tergite, except the acrotergite, is covered with trichomes. For convenience, the posterior boundary of the tergite is defined to be the posterior edge of the pigment band. The intertergal cuticle is unpigmented and composed of an anterior trichome-bearing region (the posterior hairy zone or PHZ) and a posterior region of naked cuticle (the intersegmental membrane or ISM). All trichomes and bristles in the abdomen are oriented from the anterior to the posterior. The tergite and anterior portion of the PHZ develop from the anterior dorsal histoblast nest; the rest of the PHZ and the ISM develop from the posterior dorsal nest (Kopp, 1997).

Hedgehog protein secreted by posterior compartment cells plays a key role in patterning the posterior portion of the anterior compartment in adult abdominal segments. Loss of function of hh in the hh(ts2) mutant causes the loss of posterior tergite characteristics in the anterior compartment, whereas ectopic expression driven by hs-hh or the gain-of-function allele hh(Mir) causes transformation of anterior structures toward the posterior. hh-expressing clones in the anterior compartment induce surrounding wild-type cells to produce posterior tergite structures, establishing that HH functions nonautonomously. The effects of pulses of ectopic expression driven by hs-hh indicate that bristle type and pigmentation are patterned by HH at widely different times in pupal development. The primary polarization of abdominal segments is symmetric. This symmetry is strikingly revealed by ectopic expression of engrailed. As expected, this transforms anterior compartment cells to a posterior compartment identity. However, ectopic en expression causes an autonomous reversal of polarity in the anterior portion of the anterior compartment, but not the posterior portion. By determining the position of polarity reversal within en-expressing clones, a cryptic line of symmetry can be defined that lies within the pigment band of the normal tergite. This line appears to be retained in hh(ts2) mutants raised at the restrictive temperature, suggesting it is not established by hh signaling. It is argued that the primary role of hh in controlling polarity is to cause anterior compartment cells to reverse their interpretation of an underlying symmetric polarization. Consistent with this, it is found that strong ectopic expression of hh causes mirror-symmetric double posterior patterning, whereas hh loss of function can cause mirror-symmetric double anterior patterning (Kopp, 1997).

The role of buttonhead and Sp1 in the development of the ventral imaginal discs: The role of Engrailed in subdividing the leg disc

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homolog of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. A novel function of btd and Sp1 is reported: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. It is proposed that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth (Estella, 2003).

In a search for genes with restricted expression in the adult cuticle, the MD808 Gal4 line was found to direct expression in the ventral derivatives of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and analia no clear expression was discerned. It was also noticed that the insertion was located in the first chromosome and associated with a lethal mutation. The mutant larvae showed a head phenotype resembling that described for mutants at the btd gene: loss of antennal organ and the ventral arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that reported for btd, suggesting that the Gal4 insertion was located at this gene. In addition, the imaginal expression of MD808 and of btd was largely coincident (Estella, 2003).

Further to the genetic analysis and the expression data, DNA fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the btd gene. The related gene Sp1 is immediately adjacent. It is likely that btd and Sp1 have originated by a tandem duplication of a primordial btd-like gene (Estella, 2003).

One particularly significant result about the mode of action of btd comes from the analysis of the ectopic leg patterns observed with ectopic btb expression in the wing and halteres. The clones of cells ectopically expressing btd tend to recapitulate the complete development of leg and antennal discs. For example, the whole genetic network necessary to make a leg appears to be activated. btd induces the activity of hth, dac and Dll, the domains of which account for the entire disc. Furthermore, hth, dac and Dll are activated in a spatially discriminated manner. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds. In one clone, for example hth is expressed only in the peripheral region, resembling the normal expression in the leg disc; in another clone the discriminate expressions of dac and Dll define three distinct regions. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds, but the hth domain is independent from Wg and Dpp (Estella, 2003).

The generation of distinct hth, dac and Dll domains within the clones suggested that btd-expressing cells in the wing and haltere generate their own signalling process. Indeed, within these clones there is local activation of en, the transcription factor that initiates Hh/Wg/Dpp signalling in imaginal discs. btd-expressing clones also acquire wg and dpp activity in subsets of cells. It is probably in the boundary of en-expressing with non expressing cells where the Wg and Dpp signals are generated de novo; subsequently, their diffusion initiates the same patterning mechanism which operates during normal leg development. The result of this process is that the hth, dac and Dll genes are expressed in different domains contributing to form leg patterns containing DV and PD axes. One question for which there is no clear answer is how the initial asymmetry is generated, so that a few cells within the group gain (or lose) en activity. The cells expressing en within the clones are those closer to the posterior compartment cells. It is conceivable that there might be an external signal, perhaps mediated by Hh, which triggers the initial asymmetry (Estella, 2003).

The ability of cells expressing btd to build their own patterning mechanism is also indicated by the observation that inducing btd activity in different parts of the wing disc results in the production of similar sets of leg pattern elements. For example, in MD743/UAS-btd and omb-Gal4/UAS-btd flies, btd is induced in different, non-overlapping wing regions, and yet all leg pattern elements are produced in both genotypes. Thus, the effect of btd is by and large independent of the position where it is induced, e.g., it does not depend on local positional signals (Estella, 2003).

A relevant issue is whether the ability of the Btd product to induce the formation of the full array of ventral structures has a functional significance in normal development. This property may be a faithful reflection of the original btd/Sp1 function: the activation of the developmental program to build the ventral adult patterns. btd/Sp1 function can be envisaged as follows. During the embryonic period, the conjunction of several regulatory factors (Wg, Dpp, EGF, Hox genes) allows activation of btd/Sp1 in a group of cells in each thoracic segment (it is assumed that a similar process takes place in the head). These cells become the precursors of the ventral imaginal discs and will eventually form the ventral thorax of the adult -- these include the trunk (the hth domain) and appendage (the Dll domain) regions. The activity of btd/Sp1 is instrumental in segregating these ventral discs precursors from those of the larval epidermis and determining their imaginal fate. It is involved in specifying their segment identity (in collaboration with the Hox genes) and in establishing their pattern and growth. To achieve the latter role btd/Sp1 induces the production of the growth signals Wg and Dpp, probably in response to localized activation of en and subsequent signalling by hedgehog (hh) (Estella, 2003).

A problem with this model is that in normal development all the genes involved, hth, en, hh, wg and dpp, are expressed in embryos prior to btd/Sp1. Why should a new round of activation be necessary? Although a totally satisfactory answer can not be provided, it is noted that clones of btd-expressing cells in wing or haltere lose their memory of en expression. Those that originated in the A compartment had no previous en expression, but gained it in some cells. Conversely, all cells in P compartment clones contained en activity but some lose it. The best explanation for this unexpected behavior is that btd provokes a 'naïve' cell state in which the previous commitment for en activity is lost. Later, en activity is re-established. This phenomenon may reflect the process that occurs in normal development. The initial btd/Sp1 domain may not inherit the previous developmental commitments and has to build a new developmental program. It is worth considering that the btd/Sp1function appears to determine ventral imaginal fate as different from larval fate. This is a major developmental decision, which may require de novo establishment of the genetic system responsible for pattern and growth. A key aspect of this would be the localized activation of en in part of the btd/Sp1 domain. It is speculated that there might be a short-range signal, perhaps Hh, emanating from nearby en-expressing embryonic cells, that acts as an inducer in the btd/Sp1 primordium. There is evidence that Hh can induce en activity (Estella, 2003).

Engrailed homeoprotein acts as a signaling molecule in the developing fly

Homeodomain transcription factors classically exert their morphogenetic activities through the cell-autonomous regulation of developmental programs. In vertebrates, several homeoproteins have also been shown to have direct non-cell-autonomous activities in the developing nervous system. This study presents the first in vivo evidence for homeoprotein signaling in Drosophila. Focusing on wing development as a model, it was first demonstrated that Engrailed (En) is secreted. Using single-chain anti-En antibodies expressed under the control of a variety of promoters, the wing territories in which secreted En acts were delineated. En is shown to be a short-range signaling molecule that participates in anterior crossvein (ACV) development, interacting with the Dpp signaling pathway. This report thus suggests that direct signaling with homeoproteins is an evolutionarily conserved phenomenon that is not restricted to neural tissues and involves interactions with bona fide signal transduction pathways (Layalle, 2011).

Direct, non-cell-autonomous homeoprotein activity has been previously reported in vivo for Pax6, Otx2 and En1/2. However, these reports only concern the developing vertebrate nervous system, raising the issue of the phylogenetic conservation and tissue specificity of this mode of signaling. This study shows that homeoprotein signaling occurs outside of the nervous system and in an invertebrate. Specifically, using a series of biochemical and genetic tools it has been demonstrated that En is secreted in Drosophila and participates in the morphogenesis of anterior structures of the wing, in particular in the formation of the ACV (Layalle, 2011).

Performing detergent-free immunostaining, an extracellular pool of En protein was first identified that is produced both within the P compartment (En/Hh domain) and in the anterior-most region of the En domain (Ptc/En domain). A tool (SP4F11 secreted single-chain antibody) was devised to interfere specifically with the extracellular En signal. Using this antibody, it was found that whereas the formation of both the PCV and the ACV depends on En expression, the formation of the ACV requires extracellular En. Further, using particular Gal4 drivers, it was shown that the pool of En protein secreted from the En/Hh P compartment is dispensable for ACV formation. By contrast, ACV formation requires an En signal that originates from the Ptc/En domain. It was also found that En secreted from this Ptc/En territory acts both cell-autonomously in the same Ptc/En domain and non-cell-autonomously in territories in which en is never expressed (the Dpp domain) (Layalle, 2011).

When expressed with the dpp driver, SP4F11 and enRNAi show different effects on ACV formation, ruling out an effect on En synthesis in this context. The fact that different effects are also observed on pMad levels strongly supports a role of extracellular En in ACV morphogenesis. Indeed, inhibition of Mad phosphorylation following SP4F11 expression does not correlate with a decrease in intracellular En levels. In view of all of these results, it is proposed that En should be considered not only as a nuclear transcription factor, but also as a short-range signaling molecule with morphogenetic activities (Layalle, 2011).

Although the results demonstrate a function for secreted En, they do not establish that the protein is transferred into recipient cells, even though this hypothesis is favored on the basis of the presence of the internalization sequence in the En homeodomain and by analogy with previous studies. Technical limitations might derive from the assertion that, if En secretion is similar in flies and chick, its secretion would be limited to 5% of the intracellular content (Layalle, 2011).

The results show that the extracellular and intracellular En pools have distinct activities. Indeed, based on genetic interactions between en and dpp, tkv or cv-2 and on analysis of Mad phosphorylation, it has been established that extracellular En activity enhances Dpp signaling. This positive action of extracellular En on Dpp signaling contrasts with the known repressive role that En has on dpp expression when it acts as a transcription factor. In other words, a reduction of nuclear En might lead on the one hand to dpp activation, but on the other hand to reduced Dpp signaling. Considering that the initiation of ACV formation in the presumptive six cell row anterior domain requires Dpp signaling, the action of nuclear En on dpp expression must be compensated by an independent action of the En signal, which together with Cv-2 and Tkv modulates Dpp signaling (Layalle, 2011).

How the En signal acts during wing morphogenesis remains an open question. A first observation is that the En signal does not cross the A/P boundary. Indeed, blocking the En signal from the posterior cells (in Hh>SP4F11 flies, for instance) does not modify the formation of the ACV, in sharp contrast with the absence of the ACV in Ptc>SP4F11 flies. This suggests that the extracellular En posterior pool, which is intact in this genetic background, is not able to compensate for this loss of En signal from the anterior cells. It was found that the En signal from the posterior cells might have a function in the formation of the posterior margin that is completely independent of its role in the anterior part. Previous work in an in vitro model for axonal guidance showed that extracellular En must be internalized in order to exert its guidance activity, and that it regulates local protein synthesis within the growth cone. A similar mechanism might be at work with En signaling in Drosophila. Indeed, an extracellular pool of En protein was identified in the embryonic ventral nerve cord. This suggests that En secretion might be a general feature in Drosophila. In addition, it was found that other homeoproteins, such as Ubx, are also potentially secreted, suggesting that this property of En might be extended to other homeoproteins in Drosophila (Layalle, 2011).

The observation that En signaling cooperates with the Dpp signaling pathway is reminiscent of recent observations made in the vertebrate nervous system that En, following internalization by growth cones, reduces the threshold of activation for the Ephrin signaling pathway. The fact that En cooperates with the Ephrin and Dpp signaling pathways, as well as the existence of homeoprotein signaling in plants, support the notion that homeoproteins are very ancient signaling entities and that distinct classical transduction mechanisms have been recruited more recently, possibly because they increase developmental and physiological robustness (Layalle, 2011).

Pupal

The cuticle of the adult abdomen of Drosophila is produced by nests of imaginal histoblasts, which proliferate and migrate during metamorphosis to replace the polyploid larval epidermal cells. In this report, a detailed description is presented of the expression of four key patterning genes, engrailed (en), hedgehog (hh), patched (ptc), and optomotor-blind (omb), in abdominal histoblasts during the first 42 h after pupariation, a period in which the adult pattern is established. In addition, the expression is described of the homeotic genes Ultrabithorax, abdominal-A, and Abdominal-B, which specify the fates of adult abdominal segments. The results indicate that abdominal segments develop in isolation from one another during early pupal stages, and that some patterning events are independent of hh, wg, and dpp signaling. Pattern and polarity in a large anterior portion of the segment are specified without input from Hh, and evidence is presented that abdominal tergites possess an underlying symmetric pattern upon which patterning by Hh is superimposed. The signals responsible for this underlying symmetry remain to be identified (Kopp, 2002).

The dorsal cuticle of a typical abdominal segment contains a stereotyped sequence of pattern elements. At the anterior edge of each segment is the acrotergite, a narrow strip of naked sclerotized cuticle (a1). The remainder of the tergite is covered by trichomes, and can be subdivided into four regions. From anterior to posterior these regions are: a lightly pigmented region with no bristles (a2 fate); a lightly pigmented region that contains two to three rows of microchaetes (a3); a darkly pigmented region with one to two rows of microchaetes (a4); and a darkly pigmented region with a single row of macrochaetes (a5). The tergite is followed by the unpigmented posterior hairy zone (PHZ), which is composed of both anterior (a6) and posterior (p3) compartment cells. All trichomes and bristles in the segment are oriented uniformly from anterior to posterior. Finally, at the posterior edge of the segment is a zone of thin, naked intersegmental membrane (ISM), which can be subdivided into anterior smooth (p2) and posterior crinkled (p1) regions (Kopp, 2002).

The adult abdominal pattern is established in the first 2 days of pupal development, concurrent with the proliferation and migration of histoblasts and the destruction of the larval epidermal cells (LECs.) The spatial and temporal evolution of en, hh, ptc, and omb expression is followed during this critical period. The cuticle of each abdominal hemisegment is formed by three major histoblast nests. The anterior dorsal nest (aDHN) is composed of anterior compartment histoblasts and produces the tergite and part of the PHZ (a1-a6), whereas the posterior dorsal nest (pDHN) is composed of posterior compartment cells and produces the intersegmental membrane and the remainder of the PHZ (p1-p3). The ventral histoblast nest, which produces the sternite and pleura, contains both anterior and posterior compartment cells. en, hh, ptc, and omb are expressed in similar patterns in dorsal and ventral histoblasts, and the description is limited to the dorsal abdomen (Kopp, 2002).

en-lacZ and hh-lacZ are expressed throughout the pDHN, but are not expressed in the aDHN. hh-lacZ is expressed in a gradient within the pDHN, with expression highest at the anterior edge. A similar gradient can be detected in understained preparations of en-lacZ. ptc-lacZ expression is present in only a few cells at the posterior edge of the aDHN. omb-GAL4 expression is seen in the posterior of the aDHN and the anterior of the pDHN. omb-GAL4 expression is highest near the compartment boundary and decreases symmetrically in both anterior and posterior directions. By 20-24 h APF, the aDHN and pDHN fuse to form a combined dorsal histoblast nest (DHN). The gradients of en-lacZ and hh-lacZ expression within the posterior compartment become more pronounced at this stage. ptc-lacZ is expressed in a narrow stripe in the middle of the DHN, which is presumably located just anterior to the compartment boundary. The posterior border of this stripe is sharply defined, whereas a short gradient forms in the anterior direction; no ptc-lacZ expression can be detected at the anterior edge of the DHN at this time. omb-GAL4 is expressed in a wide, double-sided gradient in the middle of the DHN. Double labeling for ß-galactosidase and En protein in omb-GAL4²/UAS-lacZ pupae shows that omb-GAL4 is expressed in both compartments (Kopp, 2002).

At ~30 h APF, the DHN of consecutive segments begin to merge. Contact occurs as the border cells, a specialized row of LECs located at the posterior edge of each segment, are lost. At this stage, expression of en-lacZ and hh-lacZ is still highest at the compartment boundary, and lowest at the posterior edge of the segment. At high magnification, a clear gradient of En protein can be seen at this stage on a cell-by-cell basis. The ptc-lacZ stripe in the middle of the segment widens somewhat, but retains a sharp posterior limit. As the border cells are eliminated and histoblasts of consecutive segments come into contact, cells at the anterior edge of each segment activate ptc-lacZ. Activation occurs only where border cells have been lost; no expression of ptc-lacZ is detected posterior to persisting border cells. This pattern strongly suggests that the border cells insulate anterior histoblasts from the Hh protein secreted by the posterior compartment cells of the preceding segment. Consistent with such a role, the border cells do not express hh transcript, although they do express En. omb-GAL4 continues to be expressed in a symmetric, double-sided gradient at this stage (Kopp, 2002).

By 40-42 h APF, the border cells, which are the last LECs to be replaced by histoblasts, have been eliminated and segmental fusion has been completed. en-lacZ and hh-lacZ are upregulated at the posterior edge of the segment at this time, and soon the expression of both genes becomes uniform within the posterior compartment. For a short time, En levels are highest in cells at both edges of the posterior compartment, and lower in the middle cells, suggesting that en expression is upregulated by contact of anterior and posterior compartment histoblasts. In addition to the main ptc-lacZ stripe, a weak second stripe develops at the anterior edge of the segment. omb-GAL4 expression becomes asymmetric, with a well-defined posterior and graded anterior boundaries; based on the positions of muscle insertion points, most or all of omb-GAL4 expression at this stage is in the anterior compartment (Kopp, 2002).

To test whether Hh signaling is required for ptc and omb expression, homozygous hh^ts2 individuals were grown at 29°C for 48 h prior to dissection. Under these conditions, ptc-lacZ expression was completely eliminated at all stages. However, the effect on omb-GAL4 expression was different, depending on the stage of development. In early pupae, the symmetric expression of omb-GAL4 about the compartment boundary was only slightly reduced, while expression in the LECs appeared normal. In contrast, the later asymmetric expression of omb-GAL4 in the anterior compartment was virtually eliminated. No change was seen in the expression of en-lacZ or En protein in hh^ts2 pupae raised at 29°C, suggesting that the gradients of en expression in the posterior compartment are established independently of Hh function (Kopp, 2002).

Ectopic expression of en causes transformation of anterior compartment structures to posterior compartment identity, and produces a mirror-symmetric double-posterior pattern (p1-p2-p3-p3-p2-p1). This phenotype is seen in the en gain-of-function en mutant, which causes near-ubiquitous expression of en in the pupal abdomen and in T155-GAL4/UAS-en heterozygotes. Examination of En-expressing clones in otherwise wild-type flies reveals that the line of symmetry lies within the anterior compartment. En-expressing cells located posterior to this line orient to the posterior, whereas En-expressing cells located anterior to it orient to the anterior. This effect of En on cell fate and polarity is strictly cell autonomous. Whether Hh signaling plays a role in the symmetric polarization of en-expressing cells has been tested. No activation of en-lacZ is seen in the anterior compartment of gain of function en heterozygotes, although sporadic activation of hh-lacZ and hh transcript is observed. However, it is difficult to see how such variable activation of hh could be responsible for the highly regular mirror-symmetric cuticular pattern produced. ptc-lacZ expression is reduced at both edges of the anterior compartment in gain of function en, consistent with repression of ptc by En. omb-GAL4 expression appears unchanged relative to wild type (Koop, 2002).

To ask directly whether en-expressing cells in gain of function en flies are patterned by Hh, smo³ clones were generated in gain of function en heterozygotes. These clones had no effect on cell fate or polarity: smo mutant cells located posterior to the line of symmetry retained posterior orientation, whereas cells located anterior to this line retained anterior orientation. The affinity of smo mutant cells in a gain of function en background also appeared unchanged, since all clones interdigitated freely with surrounding cells (Koop, 2002).

In a reciprocal experiment, it was asked whether the patterning of en-expressing cells is affected by ectopic Hh expression. Flip-out Hh-expressing clones were generated in en gain of function heterozygotes. Hh-expressing clones had no effect on cell fate or polarity. Thus, Hh signaling does not appear to play a role in the mirror-symmetric polarization of en-expressing tergites (Koop, 2002).

Mirror-symmetric patterning is also caused by ectopic expression of Hh itself. Ubiquitous Hh expression driven by hs-hh or UAS-hh;T155-GAL4 results in a mirror-symmetric double-posterior pattern (p2-p3-a6-a5-a5-a6-p3-p2). Interpretation of this phenotype has been complicated by the observation that ectopic Hh induces localized expression of en-lacZ in the anterior compartment. This induction leaves open the possibility that the mirror-symmetric patterning may be mediated by changes in endogenous hh expression (Koop, 2002).

To test this possibility, the expression of en-lacZ, hh-lacZ, and ptc-lacZ was examined in the abdomens of UAS-hh;T155-GAL4 pupae that were shifted from 17°C to 29°C at pupariation to enhance GAL4-induced ectopic expression. During the early pupal stages, ptc-lacZ expression was strongly and evenly expanded to the anterior, while the expression of hh-lacZ, en-lacZ, and En protein was unchanged. However, by 40-42 h APF some pupae showed weak ectopic expression of en-lacZ and hh-lacZ in a narrow stripe in the middle of the anterior compartment. ptc-lacZ expression was upregulated to each side of this stripe as well as at both edges of the anterior compartment (Koop, 2002).

In conclusion, abdominal tergites display mirror-symmetric patterning in several different genotypes. These genotypes include loss-of-function mutants of omb or hh, and genotypes in which omb, en, or hh are expressed ubiquitously. It is thought that these cases reveal an underlying symmetric patterning of the tergite. However, after the loss of the border cells, anterior compartments are exposed to Hh from both anterior and posterior edges, raising the possibility that these mirror-symmetric phenotypes result from symmetric Hh signaling. Indeed, it has been suggested that a U-shaped gradient of Hh produced by diffusion across the compartment and segment boundaries specifies polarity throughout the tergite. This report, tested the role of Hh in three separate cases of mirror-symmetric patterning. The results are uniformly negative, and provide compelling evidence that abdominal tergites possess an underlying mirror-symmetric patterning that is specified independently of Hh (Koop, 2002).

Lineage-specific cell death in postembryonic brain development of Drosophila

The Drosophila central brain is composed of thousands of neurons that derive from approximately 100 neuroblasts per hemisphere. Functional circuits in the brain require precise neuronal wiring and tight control of neuronal numbers. How this accurate control of neuronal numbers is achieved during neural development is largely unclear. Specifically, the role of programmed cell death in control of cell numbers has not been studied in the central brain neuroblast lineages. This study focusses on four postembryonic neuroblast lineages in the central brain identified on the basis that they express the homeobox gene engrailed (en). For each lineage, the total number of adult-specific neurons generated was determined, as well as number and pattern of en-expressing cells. Programmed cell death has a pronounced effect on the number of cells in the four lineages; approximately half of the immature adult-specific neurons in three of the four lineages are eliminated by cell death during postembryonic development. Moreover, programmed cell death selectively affects en-positive versus en-negative cells in a lineage-specific manner and, thus, controls the relative number of en-expressing neurons in each lineage. Furthermore, evidence is provided that Notch signaling is involved in the regulation of en expression. Based on these findings, it is concluded that lineage-specific programmed cell death plays a prominent role in the generation of neuronal number and lineage diversity in the Drosophila brain (Kumar, 2009).

In postembryonic CNS development of holometabolous insects such as flies, a combination of programmed cell death and neuronal process re-innervation allows the larval nervous system to reorganize and innervate new body structures. During metamorphosis many adult-specific neurons in the ventral ganglia are targeted by programmed cell death, particularly in abdominal segments. Furthermore, extensive cell death occurs during postembryonic development in the insect visual system, where cells are overproduced and those that do not make the appropriate targets are eliminated by apoptosis. By contrast, very little is currently known about the prevalence and functional roles of programmed cell death in development of the insect adult central brain (Kumar, 2009).

This report identifies four neuroblast lineages in the postembryonic central brain and finds that programmed cell death occurs in all four lineages, albeit to different extents. Whereas cell death plays only a minor role in the medial cluster MC1 lineage, it has dramatic effects in anterior cluster (AC), posterior cluster (PC) and medial cluster MC2 lineages, in which nearly half of the adult-specific neuronal progeny are programmed to die during larval development. It is noteworthy that the adult-specific neurons targeted by cell death are generated during larval development and are eliminated before their respective neuroblasts stop proliferating (12-24 hours after pupal formation). Because the cell death reported here occurs before neuronal differentiation, it is probably not involved in events of brain reorganization that take place during metamorphosis (Kumar, 2009).

Another central feature of the cell death events demonstrated here is that none of the four lineages is completely eliminated by cell death; all four neuroblasts and a significant number of their neuronal progeny survive at the end of larval development, and these neuronal progeny are largely present in the adult. In this sense, the programmed cell death reported here is likely to be functionally different from the cell death observed in the ventral ganglia, where the neuroblast itself undergoes apoptosis, regulating neuronal numbers in the abdominal segments (Kumar, 2009)

These experiments indicate that programmed cell death plays a prominent role in determining lineage-specific features; if cell death is blocked the total neuronal number increases in all four lineages and the number of en-expressing neurons increases in AC, PC and MC2. Furthermore, the axonal projection pattern of H99 mutant (deleting rpr, hid and grim) and Notch mutant en-expressing lineages was examined, comparing them to wild type. Both cell death defective H99 and Notch mutant PC lineages showed an additional projection that was not present in the wild type, whereas the other three H99 lineages did not appear to change drastically in their projection patterns. In conclusion, programmed cell death appears to contribute to the cellular diversity of neuronal lineages in the central brain (Kumar, 2009).

Studies on neuroblast lineages in the developing ventral ganglia indicate that proliferating neuroblasts generate a largely invariant clone of neural cells. In general, each neuroblast division produces a distinctly fated GMC, and each GMC division produces two sibling progeny of different fates. There is some evidence that the fate of these progeny is controlled by the parental GMC; the two siblings are restricted to a pair of different cell fates, with one sibling adopting an 'A' fate and the other adopting a 'B' fate. This, in turn, has led to a model in which a neuroblast lineage can be thought of as composed of two hemilineages, with one hemilineage comprising 'A'-fate cells and the other hemilineage comprising 'B'-fate cells. It is thought that an interaction between Notch and Numb is responsible for generating distinct neural fates of the two GMC daughter cells, with a loss of Notch or Numb resulting in reciprocal cell-fate duplication. However, Notch signaling does not appear to confer a particular fate; rather, it acts generically as a mechanism to enable two siblings to acquire different fates, and other developmental control genes that are inherited from the specific parental GMC are thought to be instrumental in determining the final identity of each progeny (Kumar, 2009).

Findings on lineage-specific cell death support a comparable model in which all four brain neuroblasts can generate one en-positive hemilineage and one en-negative hemilineage. In this model, programmed cell death is then targeted in a lineage-specific manner to either the en-negative hemilineage (AC, PC), or the en-positive hemilineage (MC2), or neither hemilineage (MC1). Alternatively, en-positive and en-negative neurons in the lineages could be generated in a temporal fashion and subsequently en-positive or en-negative neurons could be eliminated in a lineage-specific manner. However, the results suggest that this is unlikely. In particular, in the PC lineage, more than 80% of the two-cell clones examined were composed of one en-positive and one en-negative cell. If the above did occur, a significant number of two-cell en-positive clones should have been obtained along with two-cell clones comprising one en-positive and one en-negative neuron. Similar analysis of single and two cell clones in the other three en lineages is further required to confirm the occurrence of hemilineage-specific programmed cell death (Kumar, 2009).

Based on these experimental results, it was postulate that Notch signaling is an important generic mechanism underlying generation of the two different hemilineages, as in the absence of Notch signaling, cell-fate duplication of GMC siblings occurs. Indeed, analysis of Notch loss-of-function neuroblast clones suggests that in the absence of Notch signaling most of the neurons in the four lineages acquire an en-positive cell fate. Alternatively, in the four lineages examined, being positive for en may be the default state of the cells, and Notch induces secondary fate by repressing en in subsets of cells in each lineage. These en-positive neurons then appear to survive or undergo programmed cell death depending on the lineage-specific context. However, it remains to be seen whether Notch itself acts on the apoptotic machinery, independent of en (Kumar, 2009).

This study, used en as a molecular marker to identify four lineages in the postembryonic central brain. Might en itself be functionally involved in regulating programmed cell death in these lineages? For the PC lineage, there is some indication that the total clone size is reduced by approximately half in en loss-of-function mutants, compared with wild type. Although this suggests that en may be involved in promoting survival of en-positive neurons in PC (and probably AC), it does not explain the role of en in the MC2 lineage, where it would have to play an opposing role, as en-positive neurons die in this lineage. Thus, en could act either as a pro-apoptotic or an anti-apoptotic factor, depending on the lineage-specific context. Moreover, a direct genetic interaction between en and the apoptotic machinery remains to be investigated. As en is known to have multiple interactions with other proteins, a complex regulatory network involving target proteins of en may be responsible for regulating apoptosis in a lineage-specific manner. Further analysis of interactions with such target proteins is necessary to reveal the full regulatory network in more detail (Kumar, 2009).

The lineage-specific effects of cell death and of Notch signaling in AC and PC are distinctly different from those observed in MC1 or MC2 lineages. However, when compared with each other, many aspects of AC and PC lineages are similar. In wild type, both lineages consist of similar numbers of adult-specific neurons, and the majority (approximately 80%) of these neurons are positive for en, whereas neuroblasts and GMCs are negative for en in both lineages. Blocking cell death results in a substantial (approximately double) increase in total cell number in both lineages, and this increase is almost exclusively due to an increase in the number of surviving en-negative neurons in both lineages. Moreover, loss of Notch function causes a marked increase in the number of surviving en-positive neurons without affecting the number of en-negative neurons in both lineages. The only significant difference between AC and PC lineages observed in this study is that the AC lineage is located in the protocerebrum, whereas the PC lineage is located in the deutocerebrum (Kumar et al., 2009) (Kumar, 2009).

What might be responsible for these similarities in the AC and PC neuroblast lineages? There is some evidence for the existence of serially homologous neuroblasts in the fly brain and VNC. In the VNC, serially homologous neuroblasts, defined by comparable time of formation, similar positions in the neuromeric progenitor array and similar expression of developmental control genes, such as segment polarity genes, dorsoventral patterning genes and other molecular markers, can give rise to almost identical cell lineages. This suggests that similar regulatory interactions take place during development of serially homologous neuroblasts and their neural lineages. A comparison of molecular expression patterns in neuroblasts from different neuromeres of the brain and ventral ganglia suggests that several of them might be serial homologs of each other. For example, neuroblasts NB5-6 in the abdominal, thoracic and subesophageal ganglia have been proposed to be homologous to NBDd7 in the deutocerebrum and NBTd4 in the tritocerebrum (Kumar, 2009).

Given the remarkable similarities in AC and PC neuroblast lineages, it is possible that the protocerebral AC lineage and the deutocerebral PC lineages represent serial homologs. If this is the case, then investigations of the cellular and molecular mechanisms that control their lineage-specific development should be useful for understanding of how regionalized neural diversity in the brain evolves from a basic metameric ground state. However, as neither the combination of developmental control genes expressed in AC and PC neuroblasts nor the position of the two brain neuroblasts in their neuromeres of origin are currently known in sufficient detail, further experiments are needed before the issue of serial homology can be resolved for these brain neuroblast lineages (Kumar, 2009).

Adult

The spatial and temporal pattern of expression of enhancer trap lines reporting on the wingless and engrailed genes was characterized in the adult antenna of Drosophila. wg is expressed in a subset of cells in the third and fifth segments of the antenna. In the third segment expression is restricted to a crescent-shaped area in the ventral-most region of the segment. In the fifth segment, part of the base of the arista, a subset of cells express wg, while engrailed expression is restricted to subsets of cells in the second, third, and fifth segments. The time courses of expression seen for wg and en, although different from one another, reveal a complex well-controlled pattern of temporal expression, providing evidence that regulatory mechanisms are preserved throughout the life span of the adult fly. Upon eclosion (hatching) the level of wg expression is at its maximum and the levels decline so that between days 10 and 15, expression is barely detectable. Re-expression is seen and peaks between 20 and 50 days of adult life. The temporal pattern of en expression is different from that seen with wingless. Expression is at its maximum upon eclosion and declines slowly over the entire life span of the fly. Altering the life span demonstrates that the temporal patterns of expression of both wg and en are linked to life span. One correlation that has been noted is an inverse relationship between transcription from the wg gene and fertility. These studies suggest that the expression of wg and en in the adult antenna is controlled by age-dependent mechanisms (Rogina, 1997).

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