development 133, 1001-1012 doi:10.1242/dev.02288 ... · determination of cell fate along the...

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INTRODUCTIONThe ventral midline in Drosophila and the floor plate in vertebratesare specialised tissues that serve as central organisers during CNSdevelopment. During early embryogenesis, the floor plate and theventral midline secrete signals that determine cell fates in adjacenttissues (reviewed by Arendt and Nubler-Jung, 1999). In vertebrates,Sonic hedgehog is secreted by floor plate cells, and controls cell fatein the adjacent somites and motoneuron cell fate in the neural tube(Echelard et al., 1993; Ericson et al., 1996; Hynes et al., 1995;Roelink et al., 1994; Teillet et al., 1998). In Drosophila, a TGF�homologue called Spitz is secreted by the ventral midline and directsventral cell fates in the ectoderm, mesoderm and developing CNS(Buescher et al., 1998; Gabay et al., 1996; Golembo et al., 1996;Luer et al., 1997; Schweitzer et al., 1995; Skeath, 1998; Yagi et al.,1998). In later embryogenesis, the floor plate and the ventral midlineare essential for establishing a functional axon network. The twotissues secrete proteins of the widely conserved Netrin and Slitfamilies to organise extending axons into commissures andlongitudinal tracts (reviewed by Araujo and Tear, 2003; Strahle etal., 2004).

Owing to its functional similarity to the vertebrate floor plate andto the amenability of Drosophila embryos to genetic and physicalmanipulations, the ventral midline has attracted the interest ofdevelopmental neurobiologists for more than a decade. Severalaspects of the development of the ventral midline have been wellcharacterised (reviewed by Jacobs, 2000). During gastrulation,midline precursors from each side of the embryo merge into twoparallel rows on the ventral side of the embryo (Bossing andTechnau, 1994). About 20 minutes later, the eight midline precursorsin each segment undergo synchronous equal divisions to give rise to16 midline cells. These midline cells, with the exception of themedian neuroblast (MNB), differentiate without further division togenerate three to four midline glia, 13-15 interneurons and four

motoneurons in each neuromere (Bossing and Technau, 1994). Oneprecursor gives rise to the two MP1 interneurons. A secondgenerates two unpaired median interneurons (UMI) interneurons.Three precursors each generate one ventral unpaired median (VUM)interneuron and one VUM motoneuron. One midline precursor, themedian neuroblast (MNB), divides in a stem cell-like manner to givefive to eight interneurons and at least one motoneuron.

In spite of our detailed knowledge of the morphology of midlinecells, our understanding of cell fate determination at the midline isstill limited. The master regulator of midline development, single-minded (sim), is activated and maintained by genes that specify thedorsoventral embryonic axis and by Notch dependent cell-cellsignalling (Menne and Klambt, 1994; Morel et al., 2003; Nambu etal., 1993). Sim and its dimerisation partner Tango (Tgo) areexpressed in all midline cells and control the expression of amultitude of midline specific genes (reviewed by Crews, 1998).Expression of the first subset-specific gene on the midline is onlydetected at stage 10, about one hour after the midline precursors firstdivide. At this stage, the pair-rule gene odd skipped (odd) becomesrestricted to the MP1 interneurons (Ward and Coulter, 2000).However, the mechanism by which subsets of midline cells acquiretheir different fates is still unknown.

Because all midline cells have the same dorsoventral positionalinformation, it is likely that genes conferring anteroposteriorinformation control midline cell fate. Using Engrailed expression asa reference, we identified the anteroposterior origin and the geneexpression patterns of midline subsets at stage 10. In the midline,Engrailed is expressed in two distinct periods. Early Engrailedexpression in the midline starts at the blastoderm stage and itsmaintenance in midline daughter cells depends on Winglesssignalling. At stage 10, repression by Wingless and activation byHedgehog restricts Lethal of scute expression to the most anteriormidline daughter cells of the neighbouring posterior segment.Subsequently, Hedgehog induces Engrailed expression in all cellsof the Lethal of scute cluster. In spite of their anterior origin, lateEngrailed-expressing midline cells join the adjacent anteriorsegment and develop into posterior midline cells, VUM and MNBneurons. Ectopic expression of Hedgehog is sufficient to induceectopic Engrailed expression in anterior midline cells, and to

Determination of cell fate along the anteroposterior axis ofthe Drosophila ventral midlineTorsten Bossing and Andrea H. Brand*

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers duringneurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in thelateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is stillnot well understood. Here, we show 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 theproneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog andLethal 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.

KEY WORDS: Cell fate determination, Ventral midline, Embryonic CNS, Drosophila, Engrailed, Hedgehog, Wingless

Development 133, 1001-1012 doi:10.1242/dev.02288

The Gurdon Institute and Department of Physiology, Development and Neuroscience,University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

*Author for correspondence (e-mail: [email protected])

Accepted 18 January 2006

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suppress the differentiation of MP1 interneurons and midline glia.Our data indicate that an early step in midline cell determination isthe separation of midline siblings into non-Engrailed- andEngrailed-expressing compartments by the opposing functions ofWingless and Hedgehog signalling.

MATERIALS AND METHODSDrosophila stocksThe following Drosophila lines were used: Oregon R (for wild type),wg1-17/CyO (Baker, 1988), wgt s (wg1-12) (Bejsovec and Martinez Arias,1991), hh3/TM3sb (Mohler, 1988) and hhts2 (Ma et al., 1993). hhts2 is a strongtemperature-sensitive allele. Homozygous flies were kept at the permissivetemperature (16°C) and embryos were shifted to the restrictive temperature(29°C) at different stages of development. The developmental stage of eachembryo was determined by inspection prior to each temperature shift.

We used the following GAL4 lines: sca-GAL4 (Klaes et al., 1994), whichdrives expression in the neuroectoderm, CNS and, transiently, in the midline(stage 10-13), with expression gradually decreasing from stage 13; V2H-GAL4 (kindly provided by D. St Johnston) (Haecker and Perrimon, 1998),which drives expression of Hedgehog and Wingless ubiquitously fromcellular blastoderm until early stage 11, and then in scattered cell clusters inthe epidermis; sim-GAL4/CyO; sim-GAL4/sim-GAL4 (Scholz et al., 1997),which drives expression in all midline cells from stage 10 to stage 13 and isgradually restricted to midline glia, although tau-GFP and tau-�-galactosidase perdure in all midline cells throughout embryogenesis; and en-GAL4 (A.H.B., K. B. Yoffe and N. Perrimon, unpublished) (Fietz et al.,1995), to drive UAS-tau-GFP expression (Brand, 1998).

The UAS lines are: UAS-en (Yoffe et al., 1995), UAS-hh M4 (Fietz et al.,1995) and UAS-wgts. Embryos expressing UAS-wgts were raised at thepermissive temperature, 18°C. Hedgehog signalling was blocked byexpression of UAS-Ci76 (Aza-Blanc et al., 1997). To follow cellmorphology, we expressed UAS-tau-lacZ (Hidalgo et al., 1995) and UAS-tau-GFP (Brand, 1998).

The following crosses were used to study the differentiation of midlinecells after loss, or ectopic expression, of Hedgehog and Wingless: UAS-hh/sim-GAL4;UAS-tau-GFP/sim-GAL4, sim-GAL4/UAS-tau-GFP;hh3/hh3,sim-GAL4/UAS-hh;hh3/hh3, UAS-tau-GFP/sim-GAL4;UAS-wgts/sim-GAL4,wg1-17/wg1-17;sim-GAL4/UAS-tau-GFP and wg1-12,ts/wg1-17,UAS-hh;sim-GAL4/sim-GAL4. Mutant embryos were selected by the loss of Hedgehogexpression, of Wingless expression, or, in the case of wgts embryos, ofEngrailed expression.

Cell transplantation, DiI-labelling and immunohistochemistryCell transplantation was performed as described previously (Technau andCampos-Ortega, 1986). yw; V2H-GAL4/UAS-hh and yw; V2H-GAL4/+;UAS-wgts/+ embryos were used as donors. Donors were labelled by injectionof 1% lysine-conjugated Texas Red dextran (70,000 Mr; Molecular Probes)and 5% HRP in 0.2 M KCl. Cells were removed along the ventral midlinefrom four to five segments: the donor cells come from differentanteroposterior positions but the same dorsoventral position. Up to six donorcells were implanted into the same host segment and placed in a dorsoventralposition comparable to their origin. Donors and hosts were both at stage 8.Midline precursors were labelled with DiI about 10 minutes aftergastrulation. The anteroposterior position of the two midline siblings wasdocumented about two hours (stage 10) and three hours (stage 11) afterlabelling. DiI labelling, photoconversion and immunohistochemistry wereperformed as previously described (Bossing and Technau, 1994; Bossing etal., 1996).

Antibodies were diluted in PBT (0.3% Triton in PBS) and newborn calfserum (20%) as follows: rabbit anti-�-galactosidase, 1:1000 (Cappel);mAbBP102, 1:50 (kindly provided by N. Patel) (Seeger et al., 1993); rabbitanti-Ci, 1:50 (M. Fietz, unpublished; kindly provided by M. van den Heuveland P. Ingham); mouse anti-Futsch (mAb22C10), 1:3 (kindly provided byM. Bate and S. Benzer) (Fujita et al., 1982); mouse anti-En, 1:2, and rat anti-Gsbd, 1:3 (kindly provided by R. Holmgren); rabbit anti-Hh, 1:2000 (A.Taylor, unpublished; kindly provided by M. van den Heuvel and P. Ingham);mouse anti-Inv, 1:10 (kindly provided by N. Patel) (Patel et al., 1989); rabbitanti-Odd, 1:1000 (Ward and Coulter, 2000); mouse anti-Ptc, 1:250 (kindly

provided by I. Guerrero) (Capdevilla et al., 1994); mouse anti-Slit, 1:10(kindly provided by D. Hartley) (Rothberg et al., 1988); mouse anti-Wg,1:10 (Strigini and Cohen, 2000).

Secondary antibodies conjugated to alkaline phosphatase, biotin, HRP(Jackson Laboratories), Alexa488 or Alexa568 (Molecular Probes) wereused at a dilution of 1:250 in PBT (0.3% Triton in PBS) and newborn calfserum (20%). Biotin-coupled antibody reactions were enhanced using theVectastain ABC Kit (Vector Laboratories).

All embryos were mounted as flat preparations in 90% glycerol in PBS.Images were taken using a Zeiss axiophot with DIC optics or a BioRad MRC1024 confocal scan head on a Nikon E800 microscope. Images wereassembled in Adobe Photoshop.

RESULTSThe anteroposterior position of midline lineagesat stage 10The determination of midline cells appears to take place duringgermband elongation, as by germband retraction most midlinesubsets can be identified by the expression of unique molecules(Jacobs, 2000). We decided, therefore, to identify the anteroposteriorposition of midline siblings during germband elongation. Welabelled midline precursors with the lipophilic dye DiD or DiI(Molecular Probes) in embryos expressing GFP in the Engraileddomain (en-GAL4/ UAS-tauGFP). After division of the precursors,we followed the daughter cells throughout development, recordingtheir segmental position at stage 10 and stage 11 (Fig. 1). MP1interneurons (n=4, Fig. 1D,E), UMI (n=5, Fig. 1F,G) and MNBneurons (n=9, Fig. 1K,L) each arise from one precursor, and theirdaughter cells occupy fixed anteroposterior positions duringgermband elongation. The four daughter cells of the two glialprecursors can be located either in the middle of the segment (n=2,Fig. 1A) or just anterior to the Engrailed domain (n=3, Fig. 1B).VUM neurons (Fig. 1J) arise from three midline precursors, and thesix daughter cells of these precursors are located inside the Engraileddomain (n=3, Fig. 1H) and immediately posterior to the domain, inthe anterior of the next segment (n=9, Fig. 1I).

In summary, we show that the midline glia and MP1 interneuronsare the most anterior midline subsets, followed by a second pair ofmidline glia and a pair of UMIs, and, finally, the VUM and MNBneurons. DiI labelling cannot resolve whether the MP1 interneuronsor the midline glia are the most anterior cells. As determination ofthe MP1 interneurons depends on Notch/Delta signalling (Spana andDoe, 1996), it is possible that the anteroposterior position of the mostanterior midline cells, the midline glia and MP1 interneurons, israndom. Interestingly, four VUM neurons and the MNB neuronsseem to arise from the anterior compartment of the next posteriorsegment. These cells initiate Engrailed expression half-way throughgermband elongation, and, during germband retraction, they join theadjacent anterior segment to become the most posterior midlinesubsets.

A molecular map of midline cells at stage 10To identify molecules involved in the determination of midline cells,we combined the results from our in vivo studies with an expressionanalysis of midline cells throughout development. This approachallowed us to deduce a subset-specific expression for the differentmidline cell types (Fig. 2G). Surprisingly, Engrailed is expressed inmidline cells in two phases (Fig. 2A). The early phase of Engrailedexpression (early Engrailed) starts with two midline precursors.After the division of the two precursors, the number of Engrailed-positive midline siblings is reduced to about two cells at stage 10(Fig. 2A-D). Early Engrailed-positive cells, also express thesegmentation gene Gooseberry distal (Fig. 2B). During mid stage

RESEARCH ARTICLE Development 133 (6)

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10, the late phase of Engrailed expression (late Engrailed) starts insix midline cells positioned immediately posterior to the earlyEngrailed cells (Fig. 2A-D). Engrailed continues to be expressed inVUM interneurons (Siegler and Jia, 1999) and MNB neurons (Patelet al., 1989). The onset of late Engrailed expression is preceded bythe expression of the proneural gene lethal of scute (Fig. 2C,D), andfor about one hour expression of Engrailed and Lethal of scutecoincide (until early stage 11). Interestingly, Lethal of scute-expressing cells (Fig. 2E) and cells expressing late Engrailed (datanot shown) show strong expression of the Hedgehog receptor

Patched, as revealed by the expression of the lacZ gene controlledby the patched enhancer element (Fig. 2E), or by anti-Patched (datanot shown). The expression of Patched in Lethal of scute- andEngrailed-positive midline cells suggests that these midline cellsreceive the Hedgehog signal (see below).

Our in vivo analysis indicated that midline cells expressing lateEngrailed appear to be part of the anterior half of the adjacentposterior segment. To confirm their anterior identity, we examinedthe expression of odd skipped (odd), a segmentation gene expressedin stripes in the anterior of each segment (Ward and Coulter, 2000).Unfortunately, Odd expression disappears from the midline with theonset of lethal of scute expression. Yet, the six Odd-positive midlinecells, like the six Lethal of scute-expressing cells, also expressPatched (Fig. 2F). In summary, a late phase of Engrailedexpression is initiated in midline cells of the anterior half of thenext posterior segment.

Hedgehog induces the expression of Lethal ofscute and late Engrailed in midline cellsCells receiving the Hedgehog signal activate Patched, the Hedgehogreceptor (Chen and Struhl, 1996; Goodrich et al., 1996). Patched isupregulated in midline cells expressing Lethal of scute and lateEngrailed, suggesting that Hedgehog may control the expression ofboth genes. In hedgehog mutants, Lethal of scute expression is lostin midline cells, but not in the adjacent CNS (Fig. 3A,B); earlyEngrailed expression is unaffected but late Engrailed is neverinduced (Fig. 3C,D). In wild-type embryos at stage 10, the numberof Engrailed-positive cells increases from two to eight per segment,but hedgehog mutants have zero to five, with most segments havingonly two Engrailed midline cells. At the end of embryogenesis, wild-type embryos have six to nine Engrailed-positive midline cells persegment, whereas hedgehog mutants have between zero and three.

In the adjacent ectoderm, hedgehog is essential for themaintenance of wingless expression, and Wingless signallingactivates Engrailed (Heemskerk et al., 1991). To establish thatHedgehog, and not Wingless, controls the expression of lateEngrailed at the midline, we blocked Hedgehog signalling in midlinecells by expressing a truncated version of the Hedgehog signaltransducer Cubitus interruptus (Ci76). The Ci76 truncation mimicsthe short form of Cubitus interruptus, which is able to repressHedgehog, but not Wingless, target genes (Aza-Blanc et al., 1997).Midline targeted expression of Ci76 from stage 10 leads to areduction in Engrailed expression during germband retraction (Fig.3E). Furthermore, restoring Hedgehog expression from stage 10 inmidline cells of hedgehog mutants (sim-GAL4/UAS-hh; hh3) issufficient to activate Engrailed (Fig. 3F). Surprisingly, not allmidline cells express UAS-hedgehog in a hedgehog mutantbackground. To activate expression in early midline cells, we used afly strain carrying a fusion between the single-minded (sim)promoter and GAL4 (Brand and Perrimon, 1993; Scholz et al.,1997). In hedgehog mutants, Sim expression disappears from mostmidline cells during stage 10 (see Fig. S1 in the supplementarymaterial). The downregulation of Sim in hedgehog mutants may alsoaffect the sim promoter element driving GAL4, and thereby reducethe number of cells producing the GAL4 activator.

Our experiments suggest that Hedgehog, and not Wingless,controls late Engrailed expression in midline cells. Hedgehog isexpressed in midline cells until mid stage 11 (see Fig. S2A in thesupplementary material), after which time Hedgehog continues tobe expressed in the adjacent neuroectoderm and CNS (see Fig. S2Bin the supplementary material). To determine the time period duringwhich Hedgehog is required to induce Engrailed expression, we

1003RESEARCH ARTICLEMidline cell fate determination in the Drosophila CNS

Fig. 1. The anteroposterior position of the five subsets of midlinecells at stage 11. Midline precursors were labelled with the fluorescentdyes DiD or DiI (red). The position of the two daughter cells is shown inembryos expressing tau-GFP (green) in the Engrailed domain.(A,B,D,F,H,I,K) Midline siblings at stage 11; (C,E,G,J,L) midline lineagesat stage 17. Midline subsets are indicated on the right. (A,B) At stage11, midline glia are located either in the middle of the segment (A) oranterior to the Engrailed domain (B). (H,I) At stage 11, ventral unpairedmedian neurons are positioned within the Engrailed domain (H), orposterior to it (I). The three other subsets of midline cells show areproducible anteroposterior position. In panel A and K only one of thetwo midline siblings is in focus. Note that the absence of DiI from thenucleus enables cells to be counted. Ventral views, anterior to the left.

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inactivated Hedgehog at different time points during embryogenesisusing a temperature-sensitive hedgehog allele. When Hedgehog isinactivated during stage 10 and stage 11, the number of midline cellsexpressing Engrailed at stage 17 is reduced to zero to three cells, thesame number as in hedgehog mutants (see Fig. S2C in thesupplementary material). Even when Hedgehog is inactivated at latestage 11, about 30 minutes after the end of midline expression ofHedgehog, the number of Engrailed-expressing cells is reduced toabout four cells in most segments. Later inactivation has no effecton Engrailed expression in the midline (Fig. S1C). The temperatureshifts indicate that Hedgehog expressed in midline cells, as wellas neuroectodermal Hedgehog, controls the expression of lateEngrailed in the midline.

We labelled all midline cells with GFP to examine theirdifferentiation in hedgehog mutant embryos (Fig. 3G,H). Midlinecells are not incorporated into the CNS, but are positioned alongthe dorsal surface of the nerve cord. The number of midline cellsper segment is severely reduced and the remaining cells stayundifferentiated. The reduction of midline cells may be partially dueto the loss of Sim expression in hedgehog mutants. Yet, the increasednumber of GFP-labelled cell fragments indicates that many midlinecells die in hedgehog mutants (Fig. 3H).

Because the expression of Lethal of scute precedes the expressionof late Engrailed, we also examined whether Engrailed expressiondepends on lethal of scute. We followed Engrailed expression in thedeficiency Tp(1;2)sc19, which removes the proneural genes achaete,scute and lethal of scute. This deficiency can be used as a lethal of

scute mutant, because achaete and scute are not expressed in midlinecells (Skeath and Carroll, 1992). Consistent with earlier reports(Martin-Bermudo et al., 1995), we confirm that the loss of lethal ofscute causes a severe reduction in midline Engrailed expression (Fig.3I). The expression of early Engrailed is not affected. At the end ofstage 10, instead of about the eight cells in wild type, only four to sixmidline cells in every segment express Engrailed. Engrailedexpression in the midline of Tp(1;2)sc19 embryos declines furtherduring germband retraction. Ectopic expression of Lethal of scute inall midline cells is not sufficient to activate Engrailed (data notshown).

In summary, Hedgehog is needed to activate Lethal of scuteexpression in midline cells. From stage 10 to stage 12, Hedgehogand Lethal of scute are essential to induce and maintain lateEngrailed expression in the midline. In hedgehog mutants, manymidline cells die and surviving midline cells are undifferentiated.

Wingless represses lethal of scute expressionBecause lethal of scute is essential for the expression of lateEngrailed in midline cells, we assayed whether Wingless signallingcontrols Lethal of scute expression. In wild-type embryos, about sixmidline cells per segment form the Lethal of scute cluster (Fig. 4A).In wingless mutants, the number of Lethal of scute-positive midlinecells per segment increases to eight to ten cells per segment. Thisincrease suggested that in wild type, Wingless signalling repressesLethal of scute at the midline. The earliest time we can expressWingless in all midline cells is stage 10. At this stage, ectopic


Fig. 2. A molecular map of midline cells duringgermband elongation. (A) Engrailed is expressed intwo phases at the midline. Embryos expressing tau-GFP driven by engrailed-GAL4 were stained withanti-GFP (GFP, brown) and anti-Engrailed (En, blue).Midline cells that express Engrailed from gastrulation(early Engrailed) can be stained with anti-GFP andanti-Engrailed (dark brown cells). Midline cells thatinitiate Engrailed expression at stage 10 (lateEngrailed) can be stained with anti-Engrailed but notyet with anti-GFP (blue cells, arrow). (B) Gooseberry-distal (Gsb-D, dark purple) expression coincides withmidline cells expressing early Engrailed (dark brown,arrow), but not late Engrailed (light brown). (C) Atstage 10, midline cells located directly posterior tothe Engrailed domain (brown) express Lethal of scute(black, arrow) but not Engrailed. (D) About one hourlater, all Lethal of scute-positive cells also expressEngrailed (dark brown, arrow). The two midline cellscomprising the early phase of Engrailed expression(arrowhead) continue to express Engrailed only (lightbrown). (E) Lethal of scute-positive midline cellsexpress the Hedgehog receptor Patched. Expressionof �-galactosidase (red) from the patched promoter(ptc-lacZ) is very high in Lethal of scute (green)-positive midline cells (yellow cells, arrow). (F) Patched(Ptc) expression (purple) is upregulated in theanterior midline cells, labelled by the early expressionof Odd (brown). Due to repression of Patched byEngrailed, the early Engrailed domain is visible as awhite stripe. (G) Using Engrailed as a reference, theanteroposterior position of midline subsets (see Fig.1) is superimposed on the expression map.Segmentation of the ectoderm is shown byEngrailed and Hedgehog expression. Engrailed and Odd have two distinct phases of midline expression. Parasegmental (arrowhead) andsegmental borders (arrow) are indicated. Because MP1 interneurons and midline glia have a very similar anteroposterior position, it is alsopossible that MP1 interneurons are the most anterior cells in every segment. Ventral views, anterior to the left; brackets outline the midline.

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Wingless no longer interferes with midline cell differentiation (Fig.6L). Therefore, we decided to test whether Wingless repressesLethal of scute by removing neuroectodermal cells from embryosubiquitously expressing Wingless (V2H-GAL4/+; UASwgts/+) andimplanting them next to the midline of wild-type embryos. Hostembryos were examined for Lethal of scute expression at stage 10,using the expression of Gooseberry-distal or Wingless as a

segmental marker. All Wingless-expressing cells that integrateoutside the endogenous Wingless domain (n=3) repress theexpression of Lethal of scute at the midline (Fig. 4C). Most of thetransplanted cells integrate into the Wingless domain (n=8) andtherefore do not become sources of ectopic Wingless. These cells donot affect Lethal of scute expression. Cells transplanted as a controlbetween wild-type embryos never interfere with the expression of


Fig. 3. Expression of Lethal of scute and late Engrailedin midline cells depends on Hedgehog. (A) At stage 10,midline cells posterior to the Engrailed domain (En, brown)initiate Lethal of scute (L’sc, black) expression. (B) Inhedgehog mutants, expression of Engrailed weakens, andLethal of scute expression in the midline is absent. (C) Atstage 13, Engrailed-positive midline cells (black) form adense cluster. (D) In hedgehog mutants, the number ofEngrailed-expressing midline cells is severely reduced. (E) Theexpression of a 76 kDa truncation of the signal transducerCubitus interruptus (Ci76) blocks the reception of theHedgehog signal. Blocking Hedgehog reception in all midlinecells leads to a severe reduction in Engrailed-expressingmidline cells. (F) The ectopic expression of Hedgehog (Hh,brown) in midline cells of hedgehog mutants is sufficient toactivate Engrailed (black). Expression of Engrailed in themidline is limited to Hedgehog-positive cells but not allmidline cells express Hedgehog. (G) Engrailed (red) is stillexpressed in a subset of midline cells in a stage 17 embryo.The axons of midline cells are labelled by expression of tau-GFP (green). (H) In hedgehog mutants, midline cells (yellow)are not incorporated into the CNS. Midline cells do notextend axons and the number of midline cells is severelyreduced. Only a few Engrailed-positive midline cells (red)survive. Arrows indicate cell fragments. (I) The loss of lethalof scute in the mutant Tp(1;2)sc19 results in a reduction inEngrailed-positive midline cells, which is comparable tohedgehog mutants. Ventral views, anterior to the left;brackets outline the midline.

Fig. 4. Wingless signalling opposes Hedgehog signalling in theregulation of Lethal of scute. (A) In wild-type embryos, Lethal ofscute (L’sc, black) is expressed in about six midline cells (arrows) in eachsegment. (B) In wingless mutants, the number of Lethal of scute cells ina cluster (arrows) increases, up to 10 cells per segment. By contrast, inhedgehog mutants, Lethal of scute expression is lost (see Fig. 3B).(C) Cells taken from donors that express Wingless ubiquitously (V2H-GAL4/+; UAS-wg/+) were transplanted next to the midline of wild-typeembryos. Wingless-expressing cells (asterisk) repress Lethal of scute(dark purple) in the segment into which they integrate (arrowhead),whereas the expression of Lethal of scute in adjacent segments is notaffected (arrows). The expression of Gooseberry-Distal (Gsb-d, brown),which serves as a segmental marker, does not change. All embryos areat stage 10. Ventral views, anterior to the left; brackets outline themidline.

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Lethal of scute (data not shown, n=10). Hence, in contrast toHedgehog, which activates Lethal of scute in midline cells, Winglesssignalling represses Lethal of scute.

Wingless maintains early Engrailed expression atthe midlineIn hedgehog mutants and lethal of scute mutants, the earlyexpression of Engrailed that is maintained from the blastoderm stageis never affected. We investigated whether this early expression ofEngrailed depends on Wingless. At stage 9 in wild-type embryos,two to three midline cells are Engrailed positive (Fig. 5A). Inwingless mutants, the expression of Engrailed after the division ofmidline precursors is not maintained, resulting in a completeabsence of midline Engrailed at stage 9 (Fig. 5B). In hedgehogmutants, the normal number of midline cells express Engrailed,albeit at a slightly lower level than in wild type (Fig. 5C). In winglessmutants, the late phase of Engrailed is initiated at the correct time(stage 10), and shortly after germband retraction there are still threeto five Engrailed-positive midline cells (Fig. 5E), about half of thenumber present in wild type (Fig. 5D). hedgehog mutants fail toinitiate late Engrailed expression and, after germband retraction,only about two midline cells are still Engrailed positive (Fig. 5F).

Because Wingless counteracts Hedgehog signalling at the ventralmidline by repressing Lethal of scute, we examined whether midlinetargeted expression of Hedgehog could increase the number of lateEngrailed-expressing midline cells in wingless mutants. At stage 16,wild-type embryos have six to nine Engrailed midline cells per

segment (Fig. 5G), whereas in wingless mutants only one to threeEngrailed-positive midline cells per segment persist (Fig. 5H).Midline expression of Hedgehog in wingless mutants only slightlyincreases the number of Engrailed-positive cells (Fig. 5I). Ectopicexpression of Hedgehog results in cell death, as indicated by theincreased number of cell fragments (Fig. 5I), and the survivingmidline cells are not integrated into the CNS.

Finally, we expressed tau-GFP in wingless mutants to analysemidline cell differentiation. In wild-type embryos, the differentiationof midline cells is far advanced at stage 14 (Fig. 5J). In winglessmutants, we detect no morphological differentiation and all midlinecells are positioned at the dorsal CNS surface (Fig. 5K). After stage14, cell death reduces the number of midline cells to a numbercomparable to that in hedgehog mutants (data not shown). Weconclude that Wingless is essential to maintain early Engrailedexpression after the division of midline precursors, whereasHedgehog activates and induces late Engrailed at the ventralmidline. In wingless mutants, as in hedgehog mutants, midline cellsdo not differentiate, and they die during late embryogenesis.

Ectopic Hedgehog induces Lethal of scute andEngrailed in all midline cells and interferes withmidline cell differentiationBecause Hedgehog is needed to activate and maintain the expressionof Lethal of scute and late Engrailed, we investigated whetherectopic expression of Hedgehog in the neuroectoderm anddeveloping CNS (sca-GAL4) is sufficient to expand the expression


Fig. 5. Wingless and Hedgehog control different phases of Engrailed expression at the ventral midline. (A) In wild-type, at stage 9, twoto four midline cells per segment express Engrailed (En, dark purple). (B) In wingless (wg) mutants, Engrailed expression in the ectoderm and at theventral midline is lost. (C) In hedgehog (hh) mutants, Engrailed expression in the midline weakens but persists. (D) At stage 13, Engrailed-positivemidline cells form a prominent cluster. (E) In wingless mutants, despite the loss of early Engrailed expression, the late phase of Engrailed expressionin the midline is initiated. (F) In hedgehog mutants, the late phase of Engrailed expression is never induced. The number of Engrailed-positivemidline cells does not increase after stage 9. (G) In wild-type, Engrailed continues to be expressed in the midline until stage 17. (H) At stage 16,wingless mutants show a severe reduction in Engrailed-expressing midline cells. Most midline cells die. Surviving midline cells do not integrate intothe CNS, resulting in the separation of the two sides of the CNS. (I) Ectopic expression of Hedgehog (Hh, brown) in all midline cells in winglessmutants only partially rescues the loss of Engrailed (black). Midline cells are excluded from the CNS. Cell fragments can be found in macrophages(arrows). (J) At stage 14, the expression of tau-GFP (GFP, brown) in all midline cells reveals an elaborate axon pattern. (K) In wingless mutants,midline cells are not integrated into the CNS and show no differentiation. Ventral views, anterior to the left; brackets outline the midline.

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of these two genes. At the onset of stage 11, ectopic expression ofHedgehog induces Lethal of scute in all midline cells (compare Fig.6A with 6B). At the end of stage 11, all midline cells also start toexpress Engrailed (compare Fig. 6C with 6D). For both Lethal ofscute and Engrailed, the expression in the endogenous cluster isstronger than in cells in which ectopic expression is induced byHedgehog (Fig. 6B,D). This is most likely due to continuedrepression by Wingless.

Midline cells are essential for the formation of commissures –axon bundles crossing the midline (reviewed by Jacobs, 2000). Thefirst axons to cross the midline can be detected in the anteriorcommissure by staining for Futsch, the Drosophila orthologue of thevertebrate microtubule associated protein 1B (MAP1B; Fig. 6C).The Futsch-positive commissural axons are absent in embryos inwhich ectopic Hedgehog activates Engrailed in all midline cells (Fig.6D). Also, the staining of all axons with the monoclonal antibodyBP102 reveals that Hedgehog-induced Engrailed expressionabolishes the formation of the anterior commissure, but not of the

posterior commissure (compare Fig. 6E with 6F). Ectopic Hedgehogin the neuroectoderm and CNS also interferes with thedifferentiation of midline glia (compare Fig. 6G with 6H) and MP1interneurons (compare Fig. 6I with 6J), midline subsets that in wild-type embryos do not express Engrailed.

We examined whether induction of Engrailed by ectopicHedgehog is able to transform non-Engrailed-expressing midlinesubsets (midline glia, MP1 interneurons, UMI) into Engrailed-expressing midline subsets (VUM, MNB). In wild-type embryos,the expression of tau-GFP in midline cells outlines neurons and glialcells (Fig. 6K). Wingless expression in all midline cells from latestage 9 does not interfere with differentiation (Fig. 6L). Hedgehogexpression in all midline cells activates Engrailed in all cells (Fig.6M), and results in a severe reduction in cell number (Fig. 6N). Thefew remaining axons of the surviving midline cells suggest that theUMI and VUM interneurons are still able to differentiate. Inconclusion, ectopic Hedgehog induces Lethal of scute and lateEngrailed in all midline cells. Ectopic Hedgehog interferes with the


Fig. 6. Ectopic expression of Hedgehog interferes with midline cell differentiation. (A) At stage 11, about six midline cells per segmentexpress the proneural gene lethal of scute (L’sc, black). (B) Ectopic expression of Hedgehog in the neuroectoderm and developing CNS (sca-GAL4)induces Lethal of scute expression in all midline cells. (C) At stage 13, the first axons cross through the anterior commissure (arrow; Futsch, brown).Engrailed-positive midline cells (En, dark purple) cluster at the developing posterior commissure. (D) Ectopic expression of Hedgehog activatesEngrailed expression in all midline cells and prevents the crossing of axons through the anterior commissure. (E) Axons (BP102, brown) in themature CNS form a ladder with the anterior (arrowhead) and posterior commissure as rungs. (F) Ectopic expression of Hedgehog deletes theanterior commissure (arrowhead). (G) At the end of embryogenesis, midline glia cells (star; Slit, dark purple) tightly enwrap the commissures. (H) Inembryos expressing Hedgehog ectopically, midline glia cells do not enwrap the remaining commissure. Often midline glia cells become apoptotic(arrow). (I) In each segment Odd (brown) is expressed in two midline-derived MP1 neurons and two dMP2 neurons. (J) Ectopic expression ofHedgehog eliminates Odd expression from most MP1 neurons (brown, arrow) but does not affect expression in the dMP2 neurons. (K) At the endof embryogenesis, midline cells (GFP, green) show an intricate axonal pattern and a subset of cells continues to express Engrailed (En, red). (L) Theectopic expression of Wingless in all midline cells does not interfere with the differentiation of the cells or the expression of Engrailed. (M) Ectopicexpression of Hedgehog in all midline cells activates the ectopic expression of Engrailed in the midline. (N) Ectopic Hedgehog causes a severereduction in the number of midline cells. Ventral views, anterior to the left; brackets outline the midline.

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differentiation of most midline subsets but cannot transform non-Engrailed-expressing midline subsets into Engrailed-expressingsubsets.

Ectopic Engrailed expression in midline cellsprevents the differentiation of midline glia andMP1 interneuronsHedgehog acts as a morphogen: different concentrations ofHedgehog can instruct different cell fates (reviewed by Hooper andScott, 2005). Ectopic expression of Hedgehog in the neuroectodermand the developing CNS, or in all midline cells, equalises themorphogen gradient and not only exposes midline cells to ectopicHedgehog but also to concentrations that may be too high to allowtheir differentiation.

We created single sources of ectopic Hedgehog by transplantingHedgehog-expressing cells (from V2H-GAL4/UAS-hh embryos) intoembryos in which all midline cells are outlined by the expression oftau-�-galactosidase (Callahan and Thomas, 1994; Hidalgo et al.,1995). Transplantation of Hedgehog-expressing cells activatesEngrailed expression in midline cells (n=6, Fig. 7A). The majorityof transplanted cells (n=14) do not serve as an ectopic source ofHedgehog because they integrate into the endogenous Hedgehogdomain. These cells fail to induce Engrailed. Cells transplanted as acontrol between wild-type embryos never induce Engrailed (n=8,data not shown).

Ectopic Hedgehog interferes with the differentiation of the MP1interneurons (11/17 embryos). MP1 axons are missing (n=8),causing a gap in the MP1 axon fascicle (Fig. 7B), or the axonsproject randomly across the longitudinal tracts (n=3). The numberof midline cells in the affected neuromeres is not reduced, althoughthe midline glia are occasionally absent or pushed out of the CNS(2/17; data not shown). The axons of the VUM interneuronsdefasciculate slightly (2/17; data not shown). All other midline celllineages develop normally. Transplantation of wild-type cells doesnot interfere with midline cell differentiation (n=5, data not shown).

We investigated whether the ectopic expression of Engrailed inmidline cells is sufficient to explain the changes in midline celldifferentiation caused by ectopic Hedgehog. Midline targetedexpression of Engrailed from stage 10 mimics the phenotypescaused by the ectopic expression of Hedgehog in the neuroectodermor in all midline cells. Ectopic Engrailed in midline cells preventsthe formation of the anterior commissure (Fig. 7C,D), interferes withthe differentiation of midline glia (Fig. 7E,F) and abolishes Oddexpression in MP1 interneurons (Fig. 7G,H).

Finally, we labelled single midline precursors in embryosexpressing Engrailed in all midline cells. Most unusually, nearly halfof the labelled precursors (15/32) either generated only twoundifferentiated cells (n=4) or the progeny died duringembryogenesis (n=11). All the surviving progeny of the labelledprecursors are abnormally positioned at the dorsal surface of theCNS. Midline cells expressing ectopic Engrailed rarely develop intomidline glia (2/32 precursors) and the glial cells fail to enwrap theremaining, posterior, commissure (compare Fig. 8A with 8F). Wenever obtained MP1 interneurons (Fig. 8B), UMI neurons (compareFig. 8C with 8G) showed only slight axonal pathfinding defects(3/32), and axons of VUM motoneurons were more severelyaffected than interneuronal axons (8/32; Fig. 8D,H,I). In wild-typeembryos, VUM motoneuron axons bifurcate in the anteriorcommissure and the branches extend to both sides of the embryo(Fig. 8D). Ectopic Engrailed at the midline prevents the formationof the anterior commissure, and VUM motoneuron axons now turnrandomly to one side of the CNS, where the axons bifurcate (Fig.

8H,I). Normally VUM interneuronal axons bifurcate in the posteriorcommissure (Fig. 8D). In spite of the presence of a posteriorcommissure, most of the VUM interneurons (5/8) also project toonly one side (Fig. 8H). MNB progeny (4/32) show severelyretarded axonal growth (Fig. 8E,J,K). The frequency with which thedifferent subsets of neurons and glial cells are found in our clonalanalysis suggests that the non-Engrailed-expressing subsets have nottaken on the identity of the Engrailed-expressing subsets. Instead,ectopic expression of Engrailed in midline cells prevents thedifferentiation of midline glia and MP1 interneurons, and results inincreased cell death. It is possible that the observed axonal defectsin the other lineages are not cell autonomous but result from the lossof the anterior commissure or the loss of anterior midline subsets.


Fig. 7. Engrailed expression prevents the differentiation ofanterior midline cells. (A) When cells derived from donors thatubiquitously express Hedgehog (V2H/UAS-hh, brown and marked by astar) are implanted close to the midline, they induce Engrailed expression(black) in midline cells (arrowhead). Ventral view of a stage 12 embryo;anterior to the left. (B) Transplanted cells (star) interfere with thedifferentiation of the MP1 neurons. The axons of MP1 interneurons (thinarrow) are absent, causing a gap in the MP1 fascicle (thick arrow). TheHedgehog-expressing cells only interfere with the development of theMP1 neurons and midline glia. Midline cells are labelled by expression oftau-�-galactosidase (brown; UAS-tau-lacZ). Ventral view of a stage 17embryo. Only the MP1 axons are in focus. (C-H) Left column, wild type;right column, sim-GAL4/UAS-en. Embryos are at stage 17. (C) Axons(BP102, brown) in the mature CNS can cross through an anteriorcommissure (arrowhead) and a posterior commissure. (D) The expressionof Engrailed (En, black) in all midline cells prevents the formation of theanterior commissure (arrowhead). (E) Midline glia (star) ensheath bothcommissures. (F) Midline glia expressing Engrailed do not enwrap theremaining commissure but lie on the dorsal surface of the CNS (star).Most midline glia die (arrows indicate cell fragments). (G) In everysegment Odd (brown) is expressed in two lateral neurons, dMP2, andtwo midline neurons, MP1. (H) Ectopic Engrailed in all midline cellsabolishes the expression of Odd in the MP1 neurons, but does not affectdMP2. Ventral views, anterior to the left.

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DISCUSSIONThe ventral midline provides an ideal system for a comprehensivegenomic approach to the development of a CNS organiser. This isbecause of the limited number of midline precursors, which giverise to only a few neuronal and glial progeny, and because of ourcomplete knowledge of each of the lineages. Here, we show thatthe separation of midline cells into two compartments is an earlyand crucial step in midline cell determination. During germbandelongation, a second phase of Engrailed expression is initiated atthe midline in the anterior cells of the next posterior segment.During germband retraction, these cells join the anterior segmentwhere they develop into posterior midline cells. Expression oflate Engrailed depends on Hedgehog signalling and theproneural gene lethal of scute. Lethal of scute precedes Engrailedexpression and is also activated by Hedgehog. Hedgehog andWingless signalling counteract each other to define the positionof the Lethal of scute cluster, and to divide the 16 midlinedaughter cells into eight non-Engrailed- and eight Engrailed-expressing cells.

Posterior midline cells are determined after thedivision of the precursorsIt has generally been believed that the determination of thedifferent subsets of midline cells occurs before the precursorsundergo their simultaneous division at stage 8 (reviewed byJacobs, 2000). This view is challenged by our observation that

expression of the proneural gene lethal of scute, and thesubsequent expression of Engrailed, is initiated in midlinedaughter cells at stage 10, about one hour after the precursorsdivide. In the neuroectoderm, proneural genes confer neuralcompetence to a cluster of ectodermal cells (reviewed by Skeathand Thor, 2003). Lateral inhibition by Notch/Delta signalling thenlimits the expression of proneural genes to a single cell, whichdelaminates from the ectoderm and becomes a neural precursor(neuroblast). Because we have shown that the only neuroblast atthe ventral midline (median neuroblast, MNB) originates from theproneural Lethal of scute cluster, it seems likely that the MNB isselected by lateral inhibition from a cluster of midline daughtercells. However, the process of lateral inhibition in the midlinediffers from that in the adjacent neuroectoderm. In theneuroectoderm, a single cell delaminates and the remaining cellsof the cluster cease proneural expression and give rise to theepidermis (reviewed by Skeath and Thor, 2003). The proneuralcluster in the midline consists of three pairs of siblings generatedby the division of three separate precursors. Labelling of singleprecursors shows that, during the selection of the MNB, only oneof the two, labelled siblings enlarges but both delaminate from theembryo (data not shown). In contrast to the neuroectoderm, theremaining cells of the midline cluster continue to express Lethalof scute after delamination of the MNB. This extended proneuralexpression might be necessary to maintain neural competence inthe non-delaminating cells that develop into VUM neurons.


Fig. 8. Ectopic Engrailed expression in all midline cells affects the differentiation of all midline lineages. Midline precursors in wild type(A-E) and embryos expressing Engrailed in all midline cells (sim-GAL4/ UAS-En, F-K) were labelled with the fluorescent dye, DiI. After differentiationof the progeny, DiI was photoconverted to generate a permanent brown stain. The subset of lineages are indicated: MP1, midline precursor 1neuron; UMI, unpaired median interneurons; VUM, ventral unpaired median neurons; MNB, median neuroblast neurons. Clones in F,J are alsostained with anti-Engrailed (En, black). (A) Midline glia tightly enwrap the commissures. (E) Midline glia ectopically expressing Engrailed are foundon the dorsal surface of the CNS. (B) The bifurcated axons of MP1 neurons project along the longitudinal tracts. We never obtained a cloneresembling MP1 neurons in embryos expressing Engrailed in all midline cells. (C) The projection of one UMI (arrow) differs from the second UMIaxon (arrowhead). (G) Expression of ectopic Engrailed in UMI only slightly affects their projections. (D) The axons of VUM motoneurons (arrow)bifurcate in the anterior commissure, whereas the VUM interneuronal axons (arrowhead) bifurcate in the posterior commissure. The cell bodies ofVUM neurons are out of focus, at the ventral surface of the CNS. (H) When Engrailed is expressed ectopically in midline cells, all of the axons of theVUM motoneurons (arrow) exit the CNS on the same side. (I) Interneuronal axons (arrowhead) can show the same phenotype. (E) The axons ofMNB motoneurons (arrows) and interneurons (arrowhead) bifurcate in the anterior commissure. (J,K) Ectopic Engrailed in MNB neurons prevents ordelays the bifurcation of their axons. Ectopic Engrailed expression on the midline causes all subsets to be mispositioned along the dorsal surface ofthe CNS. Ventral views, anterior to the left.

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Our results cannot exclude the possibility that some of the midlinesubsets are determined as precursors, but we show that at least twoof the five midline subsets, the VUM neurons and the MNB, aredetermined after precursor cell division. There are strikingsimilarities between the development of the ventral midline ofDrosophila and grasshopper embryos (Bastiani et al., 1985; Bossingand Technau, 1994). In grasshopper, Engrailed expression can bedetected in the MNB, its progeny and the midline precursors MP4to MP6, which each give rise to two neurons with projection patternscomparable to the Drosophila VUM neurons (Jia and Siegler, 2002).Hence, the same types of midline cells express Engrailed ingrasshopper and Drosophila, but in grasshopper Engrailedexpression is initiated in all midline precursors prior to division (Jiaand Siegler, 2002).

The role of Hedgehog and Wingless in midline celldeterminationIn the ectoderm from stage 10 onwards, Wingless, Engrailed andHedgehog maintain the expression of one another by a feedbackloop: Wingless maintains Engrailed expression, Engrailed is neededfor the expression of Hedgehog and Hedgehog maintains Winglessexpression (Bejsovec and Martinez Arias, 1991; Ingham andHidalgo, 1993). In the developing CNS, Wingless and Hedgehogexpression seem to be independent of each other (Bhat, 1999). Atthe ventral midline there are two separate stages of Engrailedexpression (Fig. 9): the early phase is maintained by Wingless (Fig.9A); the late phase does not require Wingless and is instead activatedat stage 10 by Hedgehog signalling and Lethal of scute (Fig. 9B,C).In the ectoderm, Wingless and Hedgehog act in concert to maintainEngrailed expression (Bejsovec and Martinez Arias, 1991), but atthe midline Wingless and Hedgehog act in opposition: Winglessrepresses and Hedgehog activates Lethal of scute expression (Fig.9B).

Wingless may repress Lethal of scute expression indirectly, viaits maintenance of early Engrailed. As in the ectoderm, midlineEngrailed represses expression of the Hedgehog receptor Patchedand the Hedgehog signal transducer Cubitus interruptus (reviewedby St Johnston and Nusslein-Volhard, 1992). It is possible that earlyEngrailed-expressing midline cells are not able to receive theHedgehog signal. However, ectopic expression of Hedgehog is ableto induce Lethal of scute in all midline cells, suggesting thatWingless 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 byattenuating the expression of proneural and neurogenic genes (Kuboet al., 2005).

We examined the differentiation of midline cells in winglessand hedgehog mutants. Consistent with earlier reports (Hummelet al., 1999), many midline cells become apoptotic in bothmutants. The surviving midline cells are not integrated into theCNS and show no morphological differentiation. The reduction inthe number of Engrailed-positive midline cells in hedgehogmutant embryos may be mainly due to the loss of midlinecell identity. In hedgehog mutants, midline cells lose theexpression of Sim, the master regulator of midline development(reviewed by Crews, 1998). As described for sim mutants, the lossof midline identity results in increased cell death and mis-specification of the surviving midline cells as ectoderm (Xiao etal., 1996).

Ectopic Hedgehog induces the expression ofLethal of scute and late Engrailed in all midlinecellsEctopic expression of Hedgehog in the neuroectoderm and thedeveloping CNS induces the expression of Lethal of scute and,approximately 40 minutes later, the expression of late Engrailed inall midline cells. It seems likely that Lethal of scute is an early targetof Hedgehog signalling, and its activation may only require releasefrom repression by the short form of Cubitus interruptus (Methot andBasler, 1999; Muller and Basler, 2000). By contrast, the delay ininduction of late Engrailed in the same midline cells indicates thatEngrailed activation may not only require release from repression,but also activation by the long form of Cubitus interruptus (reviewedby Hooper and Scott, 2005).

Uniformly high levels of ectopic Hedgehog prevent thedifferentiation of most midline subsets and cause increased celldeath. A single source of ectopic Hedgehog, achieved by celltransplantation, does not result in midline cell death, but reveals thatthe differentiation of the MP1 interneurons is more sensitive toHedgehog levels than is the differentiation of midline glia. No othermidline subsets are affected. It seems likely that Hedgehog not onlyactivates Lethal of scute and late Engrailed, but also acts as amorphogen to control the differentiation of the MP1 neurons andmidline glia.


Fig. 9. Opposing functions of Hedgehog andWingless signalling separate midline siblings into ananterior and posterior compartment. (A) At stage 9,after the division of the midline precursors, Winglesssignalling maintains Engrailed expression in two midlinesiblings. (B) At stage 10, repression by Wingless andactivation by Hedgehog positions the proneural Lethal ofscute cluster directly posterior to the early Engrailed-expressing cells. (C) Expression of Hedgehog and Lethal ofscute are needed to induce and maintain late Engrailedexpression in the midline siblings. (D) The induction ofEngrailed by Hedgehog divides midline cells into twocompartments. The absence of Engrailed in the anteriorcompartment allows Notch/Delta signalling to selectbetween the MP1 interneuron and midline glial fate;Engrailed expression in the posterior compartmentrestricts the selection of cell fate by Notch/Delta signallingto MNB and VUM neurons. Midline cells at theparasegmental border, in the middle of the segment, maybe determined as precursors.

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Engrailed prevents the differentiation of midlineglia and MP1 interneuronsThe phenotypes caused by ectopic Hedgehog are due to theinduction of Engrailed in all midline cells. Expression of ectopicHedgehog and ectopic Engrailed blocks the differentiation ofmidline glia and MP1 interneurons, and also prevents the formationof the anterior commissure. Labelling single midline precursorsenabled us to examine cell fates in embryos expressing ectopicEngrailed in the midline. The frequency of clones obtainedindicates that ectopic Engrailed expression does not transform non-Engrailed-expressing midline subsets (MP1 interneurons, midlineglia and UMI) into Engrailed-expressing subsets (VUM and MNB).Instead, embryos expressing midline Engrailed show increased celldeath. In particular, the MP1 interneurons seem to be affected andwere never obtained during our analysis. The low frequency ofmidline glia also points to apoptosis caused by expression ofEngrailed. Surviving midline glia are not able to differentiateproperly and cannot enwrap the remaining, posterior, commissure.All other midline subsets, including the UMIs, are able todifferentiate, but they show a variety of axonal pathfinding defectsthat may result from the loss of anterior midline subsets and theabsence of the anterior commissure.

A model for midline cell determinationIt is likely that genes other than hedgehog and wingless are crucialfor midline cell determination. In our experiments, non-Engrailed-expressing midline subsets are never transformed into Engrailed-expressing subsets, or vice versa. gooseberry-distal may be one ofthese genes. From the blastoderm stage, Gooseberry-distal isexpressed by two midline precursors and their four daughter cells.During early embryogenesis Gooseberry-distal expression at themidline does not depend on Wingless and Hedgehog (Bhat andSchedl, 1997). The anterior Gooseberry-distal cells also expressWingless and most likely give rise to the UMIs. The posteriorGooseberry-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 Engrailedin midline cells posterior to the Gooseberry-distal domain. Lateralinhibition by Notch/Delta signalling selects one cell from the Lethalof scute cluster to become the MNB. The remaining cells becomeVUM neurons. At stage 10, the absence of Engrailed in the sixmidline cells anterior to the Gooseberry-distal domain defines a cellcluster that will give rise to midline glia and MP1 interneurons.Based on the expression of Odd, Delta mutants have an increasednumber of MP1 interneurons, up to six per segment (Spana andDoe, 1996). In Notch mutants, midline glial-specific markers areabsent and the number of cells expressing a neuronal markerincreases (Menne and Klambt, 1994). Therefore, Notch/Deltasignalling appears to determine midline glial versus MP1interneuron cell fates in the anterior cluster. In our model, midlinecell determination takes place mainly after the division of theprecursors. Although the initial determination of midline cellsappears to be directed by a small number of genes, a far largernumber is needed to control the differentiation of the variousmidline subsets. Our work, and the recent identification of morethan 200 genes expressed in midline cells (Kearney et al., 2004), isthe beginning of a comprehensive understanding of thedifferentiation of the ventral midline.

For generously providing fly lines, antibodies and constructs, we thank M.Bate, S. Benzer, K. Bhat, S. Bray, I. Guerrero, D. Hartley, R. Holmgren, P.Ingham, A. Jacinto, F. Jimenez, K. Kaiser, C. Klambt, T. Kornberg, L. Martin-Bermudo, K. Mitchell, J. Nambu, N. Patel, M. Peifer, D. St Johnston and M. van

den Heuvel. We thank R. Jacobs, T. Southall and G. Technau for comments onthe manuscript. This work was funded by a Wellcome Trust Senior Fellowshipand a Wellcome Trust Programme Grant to A.H.B.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/133/6/1001/DC1

ReferencesAraujo, S. J. and Tear, G. (2003). Axon guidance mechanisms and molecules:

lessons from invertebrates. Nat. Rev. Neurosci. 4, 910-922.Arendt, D. and Nubler-Jung, K. (1999). Comparison of early nerve cord

development in insects and vertebrates. Development 126, 2309-2325.Aza-Blanc, P., Ramirez-Weber, F.-A., Laget, M.-P., Schwartz, C. and Kornberg,

T. B. (1997). Proteolysis that is inhibited by hedgehog targets cubitus interruptusprotein to the nucleus and converts it to a repressor. Cell 89, 1043-1053.

Baker, N. E. (1988). Embryonic and imaginal requirements for wingless, asegment-polarity gene in Drosophila. Dev. Biol. 125, 96-108.

Bastiani, M. J., Doe, C. Q., Helfand, S. L. and Goodman, C. S. (1985). Neuronalspecificity and growth cone guidance in grasshopper and Drosophila embryos.Trends Neurosci. 8, 257-266.

Bejsovec, A. and Martinez Arias, A. (1991). Roles of wingless in patterning thelarval epidermis of Drosophila. Development 113, 471-485.

Bhat, K. M. (1999). Segment polarity genes in neuroblast formation and identityspecification during Drosophila neurogenesis. Bioessays 21, 472-485.

Bhat, K. M. and Schedl, P. (1997). Requirement for engrailed and invected genesreveals novel regulatory interactions between engrailed/invected, patched,gooseberry and wingless during Drosophila neurogenesis. Development 124,1675-1688.

Bossing, T. and Technau, G. M. (1994). The fate of the CNS midline progenitorsin Drosophila as revealed by a new method for single cell labelling. Development120, 1895-1906.

Bossing, T., Udolph, G., Doe, C. Q. and Technau, G. M. (1996). The embryoniccentral nervous system lineages of Drosophila melanogaster. I. Neuroblastlineages derived from the ventral half of the neuroectoderm. Dev. Biol. 179, 41-64.

Brand, A. (1998). GFP as a cell and developmental marker in the Drosophilanervous system. In Green Fluorescent Proteins, vol. 58 (ed. K. F. Sullivan and S.A. Kay), pp. 165-181. La Jolla: Academic Press.

Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means ofaltering cell fates and generating dominant phenotypes. Development 118, 401-415.

Buescher, M., Yeo, S. L., Udolph, G., Zavortink, M., Yang, X., Tear, G. andChia, W. (1998). Binary sibling neuronal cell fate decisions in the Drosophilaembryonic central nervous system are nonstochastic and require inscuteable-mediated asymmetry of ganglion mother cells. Genes Dev. 12, 1858-1870.

Callahan, C. A. and Thomas, J. B. (1994). Tau-�-galactosidase, an axon-targetedfusion protein. Proc. Natl. Acad. Sci. USA 91, 5972-5976.

Capdevilla, J., Estrada, M. P., Sanchez-Herero, E. and Guerrero, I. (1994). TheDrosophila segment polarity gene patched interacts with decapentaplegic inwing development. EMBO J. 13, 71-82.

Chen, Y. and Struhl, G. (1996). Dual roles for patched in sequestering andtransducing hedgehog. Cell 87, 553-563.

Crews, S. T. (1998). Control of cell lineage-specific development and transcriptionby bHLH-PAS proteins. Genes Dev. 12, 607-620.

Echelard, Y., Epstein, D. G., St-Jacques, B., Shen, L., Mohler, J., McMahon, J.A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family ofputative signaling molecules, is implicated in the regulation of CNS polarity. Cell75, 1417-1430.

Ericson, J., Morton, S., Kawakami, A., Roelink, H. and Jessell, T. M. (1996).Two critical periods of Sonic hedgehog signaling required for the specification ofmotorneuron identity. Cell 87, 661-673.

Fietz, M. J., Jacinto, A., Taylor, A. M., Alexandre, C. and Ingham, P. W. (1995).Secretion of the amino-terminal fragment of the hedgehog protein is necessaryand sufficient for hedgehog signalling in Drosophila. Curr. Biol. 5, 643-650.

Fujita, S. C., Zipursky, S., Benzer, S., Ferrus, A. and Shotwell, S. L. (1982).Monoclonal antibodies against the Drosophila nervous system. Proc. Natl. Acad.Sci. USA 79, 7929-7933.

Gabay, L., Scholz, H., Golembo, M., Klaes, A., Klambt, C. and Shilo, B.-Z.(1996). EGF receptor signaling induces pointed P1 transcription and inactivatesYan protein in the Drosophila embryonic ventral ectoderm. Development 122,3355-3362.

Golembo, M., Raz, E. and Shilo, B.-Z. (1996). The Drosophila embryonic midlineis the site of Spitz processing and induces activation of the EGF receptor in theventral ectoderm. Development 122, 3363-3370.

Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. and Scott, M.(1996). Conservation of the hedgehog/patched signaling pathway from flies tomice: induction of a mouse patched gene by Hedgehog. Genes Dev. 10, 301-312.


DEVELO

PMENT

DEVELO

PMENT

1012

Haecker, U. and Perrimon, N. (1998). DRhoGEF2 encodes a member of the Dblfamily of oncogenes and controls cell shape changes during gastrulation inDrosophila. Genes Dev. 12, 274-284.

Heemskerk, J., diNardo, S., Kostriken, R. and O’Farrell, P. H. (1991). Multiple-modes of engrailed regulation in the progression towards cell fatedetermination. Nature 352, 404-410.

Hidalgo, A., Urban, J. and Brand, A. H. (1995). Targeted ablation of gliadisrupts axon tract formatiom in the Drosophila CNS. Development 121, 3703-3712.

Hooper, J. E. and Scott, M. P. (2005). Communicating with Hedgehogs. Nat. Rev.Mol. Cell Biol. 6, 306-317.

Hummel, T., Schimmelpfeng, K. and Klambt, C. (1999). Commissure formationin the embryonic CNS of Drosophila. Dev. Biol. 209, 381-398.

Hynes, M., Porter, J. A., Chiang, C., Chang, D., Tessier-Lavigne, M., Beachy, P.A. and Rosenthal, A. (1995). Induction of midbrain dopaminergic neurons bySonic hedgehog. Neuron 15, 35-44.

Ingham, P. W. and Hidalgo, A. (1993). Regulation of wingless transcription in theDrosophila embryo. Development 117, 283-291.

Jacobs, J. R. (2000). The midline glia of Drosophila: a molecular genetic model forthe developmental functions of glia. Prog. Neurobiol. 62, 475-508.

Jia, X. X. and Siegler, M. V. (2002). Midline lineages in grasshopper produceneuronal siblings with asymmetric expression of Engrailed. Development 129,5181-5193.

Kearney, J. B., Wheeler, S. R., Estes, P., Parente, B. and Crews, S. T. (2004).Gene expression profiling of the developing Drosophila CNS midline cells. Dev.Biol. 275, 473-492.

Klaes, A., Menne, T., Stollewerk, A., Scholz, H. and Klambt, C. (1994). The Etstranscription factors encoded by the Drosophila gene pointed direct glial celldifferentiation in the embryonic CNS. Cell 78, 149-160.

Kubo, F., Takeichi, M. and Nakagawa, S. (2005). Wnt2b inhibits differentiationof retinal progenitor cells in the absence of Notch activity by downregulating theexpression of proneural genes. Development 132, 2759-2770.

Luer, K., Urban, J., Klambt, C. and Technau, G. M. (1997). Induction ofidentified mesodermal cells by CNS midline progenitors in Drosophila.Development 124, 2681-2690.

Ma, C., Zhou, Y., Beachy, P. A. and Moses, K. (1993). The segment polarity genehedgehog is required for progression of the morphogenetic furrow in thedeveloping Drosophila eye. Cell 75, 927-938.

Martin-Bermudo, M. D., Carmena, A. and Jimenez, F. (1995). Neurogenicgenes control gene expression at the transcriptional level in early neurogenesisand in mesectoderm specification. Development 121, 219-224.

Menne, T. V. and Klambt, C. (1994). The formation of commissures in theDrosophila CNS depends on the midline cells and on the Notch gene.Development 120, 123-133.

Methot, N. and Basler, K. (1999). Hedgehog controls limb development byregulating the activities of distinct transcriptional activator and repressor formsof Cubitus Interruptus. Cell 96, 819-831.

Mohler, J. (1988). Requirements for hedgehod, a segmental polarity gene, inpatterning larval and adult cuticle of Drosophila. Genetics 120, 1061-1072.

Morel, V., Le Borgne, R. and Schweisguth, F. (2003). Snail is required for Deltaendocytosis and Notch-dependent activation of single-minded expression. Dev.Genes Evol. 213, 65-72.

Muller, B. and Basler, K. (2000). The repressor and activator forms of Cubitusinterruptus control Hedgehog target genes through common generic gli-bindingsites. Development 127, 2999-3007.

Nambu, J. R., Lewis, J. O. and Crews, S. T. (1993). The development and

function of the Drosophila CNS midline cells. Comp. Biochem. Physiol. Comp.Physiol. 104, 399-409.

Patel, N. H., Schafer, B., Goodman, C. S. and Holmgren, R. (1989). The role ofsegment polarity genes during Drosophila neurogenesis. Genes Dev. 3, 890-904.

Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Altaba, A.R., Tanabe, Y., Placzek, M., Edlund, T. and Jessell, T. M. (1994). Floor plateand motor neuron by vhh-1 a vertebrate homolog of hedgehog expressed bythe notochord. Cell 76, 761-775.

Rothberg, J. M., Hartley, D. A., Walther, Z. and Artavanis-Tsakonas, S.(1988). slit: an EGF-homologous locus of D. melanogaster involved in thedevelopment of the embryonic central nervous system. Cell 55, 1047-1059.

Scholz, H., Sadlowski, E., Klaes, A. and Klambt, C. (1997). Control of midlineglia development in the embryonic Drosophila CNS. Mech. Dev. 62, 79-91.

Schweitzer, R., Shaharabany, M., Seger, R. and Shilo, B.-Z. (1995). SecretedSpitz triggers the DER signaling pathway and is a limiting component inembryonic ventral ectoderm determination. Genes Dev. 9, 1518-1529.

Seeger, M., Tear, G., Ferres-Marco, D. and Goodman, C. S. (1993). Mutationsaffecting growth cone guidance in Drosophila: genes necessary for guidancetoward or away from the midline. Neuron 10, 409-426.

Siegler, M. V. and Jia, X. X. (1999). Engrailed negatively regulates the expressionof cell adhesion molecules connectin and neuroglian in embryonic Drosophilanervous system. Neuron 22, 265-276.

Skeath, J. B. (1998). The Drosophila EGF receptor controls the formation andspecification of neuroblasts along the dorsal-ventral axis of the Drosophilaembryo. Development 125, 3301-3312.

Skeath, J. B. and Carroll, S. B. (1992). Regulation of proneural gene expressionand cell fate during neuroblast segregation in the Drosophila embryo.Development 114, 939-946.

Skeath, J. B. and Thor, S. (2003). Genetic control of Drosophila nerve corddevelopment. Curr. Opin. Neurobiol. 13, 8-15.

Spana, E. P. and Doe, C. Q. (1996). Numb antagonizes Notch signaling to specifysibling neuron cell fates. Neuron 17, 21-26.

St Johnston, D. and Nusslein-Volhard, C. (1992). The origin of pattern andpolarity in the Drosophila embryo. Cell 68, 201-219.

Strahle, U., Lam, C. S., Ertzer, R. and Rastegar, S. (2004). Vertebrate floor-platespecification: variations on common themes. Trends Genet. 20, 155-162.

Strigini, M. and Cohen, S. M. (2000). Wingless gradient formation in theDrosophila wing. Curr. Biol. 10, 293-300.

Technau, G. and Campos-Ortega, J. A. (1986). Lineage analysis of transplantedindividual cells in embryos of Drosophila melanogaster. II. Commitment andproliferative capabilities of neural and epidermal progenitors. Rouxs Arch. Dev.Biol. 195, 445-454.

Teillet, M. A., Watanabe, Y., Jeffs, P., Dupres, D., Lapointe, F. and LeDouarin,N. M. (1998). Sonic Hedgehog is required for survival of both myogenic andchondrogenic somitic lineages. Development 125, 2019-2030.

Ward, E. J. and Coulter, D. E. (2000). odd-skipped is expressed in multiple tissuesduring Drosophila embryogenesis. Mech. Dev. 96, 233-236.

Xiao, H., Hrdlicka, L. A. and Nambu, J. R. (1996). Alternate functions of thesingle-minded and rhomboid genes in development of the Drosophila ventralneuroectoderm. Mech. Dev. 58, 65-74.

Yagi, Y., Suzuki, T. and Hayashi, S. (1998). Interaction between Drosophila EGFreceptor and vnd determines three dorsoventral domains of the neuroectderm.Development 125, 3625-3633.

Yoffe, K. B., Manoukian, A. S., Wilder, E. L., Brand, A. H. and Perrimon, N.(1995). Evidence for engrailed-independent wingless autoregulation inDrosophila. Dev. Biol. 170, 636-650.


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