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First published online 8 February 2006
doi: 10.1242/dev.02288

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

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

View larger version (71K): [in a new window]   Fig. 1. The anteroposterior position of the five subsets of midline cells at stage 11. Midline precursors were labelled with the fluorescent dyes DiD or DiI (red). The position of the two daughter cells is shown in embryos 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 lineages at stage 17. Midline subsets are indicated on the right. (A,B) At stage 11, midline glia are located either in the middle of the segment (A) or anterior to the Engrailed domain (B). (H,I) At stage 11, ventral unpaired median neurons are positioned within the Engrailed domain (H), or posterior to it (I). The three other subsets of midline cells show a reproducible anteroposterior position. In panel A and K only one of the two midline siblings is in focus. Note that the absence of DiI from the nucleus enables cells to be counted. Ventral views, anterior to the left.

View larger version (92K): [in a new window]   Fig. 2. A molecular map of midline cells during germband elongation. (A) Engrailed is expressed in two phases at the midline. Embryos expressing tau-GFP driven by engrailed-GAL4 were stained with anti-GFP (GFP, brown) and anti-Engrailed (En, blue). Midline cells that express Engrailed from gastrulation (early Engrailed) can be stained with anti-GFP and anti-Engrailed (dark brown cells). Midline cells that initiate Engrailed expression at stage 10 (late Engrailed) can be stained with anti-Engrailed but not yet with anti-GFP (blue cells, arrow). (B) Gooseberry-distal (Gsb-D, dark purple) expression coincides with midline cells expressing early Engrailed (dark brown, arrow), but not late Engrailed (light brown). (C) At stage 10, midline cells located directly posterior to the Engrailed domain (brown) express Lethal of scute (black, arrow) but not Engrailed. (D) About one hour later, all Lethal of scute-positive cells also express Engrailed (dark brown, arrow). The two midline cells comprising the early phase of Engrailed expression (arrowhead) continue to express Engrailed only (light brown). (E) Lethal of scute-positive midline cells express the Hedgehog receptor Patched. Expression of ß-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 the anterior midline cells, labelled by the early expression of Odd (brown). Due to repression of Patched by Engrailed, the early Engrailed domain is visible as a white stripe. (G) Using Engrailed as a reference, the anteroposterior position of midline subsets (see Fig. 1) is superimposed on the expression map. Segmentation of the ectoderm is shown by Engrailed and Hedgehog expression. Engrailed and Odd have two distinct phases of midline expression. Parasegmental (arrowhead) and segmental borders (arrow) are indicated. Because MP1 interneurons and midline glia have a very similar anteroposterior position, it is also possible that MP1 interneurons are the most anterior cells in every segment. Ventral views, anterior to the left; brackets outline the midline.

View larger version (107K): [in a new window]   Fig. 3. Expression of Lethal of scute and late Engrailed in 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) In hedgehog mutants, expression of Engrailed weakens, and Lethal of scute expression in the midline is absent. (C) At stage 13, Engrailed-positive midline cells (black) form a dense cluster. (D) In hedgehog mutants, the number of Engrailed-expressing midline cells is severely reduced. (E) The expression of a 76 kDa truncation of the signal transducer Cubitus interruptus (Ci76) blocks the reception of the Hedgehog signal. Blocking Hedgehog reception in all midline cells leads to a severe reduction in Engrailed-expressing midline cells. (F) The ectopic expression of Hedgehog (Hh, brown) in midline cells of hedgehog mutants is sufficient to activate Engrailed (black). Expression of Engrailed in the midline is limited to Hedgehog-positive cells but not all midline cells express Hedgehog. (G) Engrailed (red) is still expressed 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 not extend axons and the number of midline cells is severely reduced. Only a few Engrailed-positive midline cells (red) survive. Arrows indicate cell fragments. (I) The loss of lethal of scute in the mutant Tp(1;2)sc19 results in a reduction in Engrailed-positive midline cells, which is comparable to hedgehog mutants. Ventral views, anterior to the left; brackets outline the midline.

View larger version (119K): [in a new window]   Fig. 4. Wingless signalling opposes Hedgehog signalling in the regulation of Lethal of scute. (A) In wild-type embryos, Lethal of scute (L'sc, black) is expressed in about six midline cells (arrows) in each segment. (B) In wingless mutants, the number of Lethal of scute cells in a cluster (arrows) increases, up to 10 cells per segment. By contrast, in hedgehog 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-type embryos. 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 not affected (arrows). The expression of Gooseberry-Distal (Gsb-d, brown), which serves as a segmental marker, does not change. All embryos are at stage 10. Ventral views, anterior to the left; brackets outline the midline.

View larger version (91K): [in a new window]   Fig. 5. Wingless and Hedgehog control different phases of Engrailed expression at the ventral midline. (A) In wild-type, at stage 9, two to four midline cells per segment express Engrailed (En, dark purple). (B) In wingless (wg) mutants, Engrailed expression in the ectoderm and at the ventral midline is lost. (C) In hedgehog (hh) mutants, Engrailed expression in the midline weakens but persists. (D) At stage 13, Engrailed-positive midline cells form a prominent cluster. (E) In wingless mutants, despite the loss of early Engrailed expression, the late phase of Engrailed expression in the midline is initiated. (F) In hedgehog mutants, the late phase of Engrailed expression is never induced. The number of Engrailed-positive midline 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 into the CNS, resulting in the separation of the two sides of the CNS. (I) Ectopic expression of Hedgehog (Hh, brown) in all midline cells in wingless mutants 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.

View larger version (91K): [in a new window]   Fig. 6. Ectopic expression of Hedgehog interferes with midline cell differentiation. (A) At stage 11, about six midline cells per segment express 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 activates Engrailed expression in all midline cells and prevents the crossing of axons through the anterior commissure. (E) Axons (BP102, brown) in the mature CNS form a ladder with the anterior (arrowhead) and posterior commissure as rungs. (F) Ectopic expression of Hedgehog deletes the anterior commissure (arrowhead). (G) At the end of embryogenesis, midline glia cells (star; Slit, dark purple) tightly enwrap the commissures. (H) In embryos 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 of Hedgehog eliminates Odd expression from most MP1 neurons (brown, arrow) but does not affect expression in the dMP2 neurons. (K) At the end of embryogenesis, midline cells (GFP, green) show an intricate axonal pattern and a subset of cells continues to express Engrailed (En, red). (L) The ectopic expression of Wingless in all midline cells does not interfere with the differentiation of the cells or the expression of Engrailed. (M) Ectopic expression of Hedgehog in all midline cells activates the ectopic expression of Engrailed in the midline. (N) Ectopic Hedgehog causes a severe reduction in the number of midline cells. Ventral views, anterior to the left; brackets outline the midline.

View larger version (130K): [in a new window]   Fig. 7. Engrailed expression prevents the differentiation of anterior midline cells. (A) When cells derived from donors that ubiquitously express Hedgehog (V2H/UAS-hh, brown and marked by a star) 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 the differentiation of the MP1 neurons. The axons of MP1 interneurons (thin arrow) are absent, causing a gap in the MP1 fascicle (thick arrow). The Hedgehog-expressing cells only interfere with the development of the MP1 neurons and midline glia. Midline cells are labelled by expression of tau-ß-galactosidase (brown; UAS-tau-lacZ). Ventral view of a stage 17 embryo. 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 anterior commissure (arrowhead) and a posterior commissure. (D) The expression of Engrailed (En, black) in all midline cells prevents the formation of the anterior commissure (arrowhead). (E) Midline glia (star) ensheath both commissures. (F) Midline glia expressing Engrailed do not enwrap the remaining commissure but lie on the dorsal surface of the CNS (star). Most midline glia die (arrows indicate cell fragments). (G) In every segment Odd (brown) is expressed in two lateral neurons, dMP2, and two midline neurons, MP1. (H) Ectopic Engrailed in all midline cells abolishes the expression of Odd in the MP1 neurons, but does not affect dMP2. Ventral views, anterior to the left.

View larger version (77K): [in a new window]   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 differentiation of the progeny, DiI was photoconverted to generate a permanent brown stain. The subset of lineages are indicated: MP1, midline precursor 1 neuron; UMI, unpaired median interneurons; VUM, ventral unpaired median neurons; MNB, median neuroblast neurons. Clones in F,J are also stained with anti-Engrailed (En, black). (A) Midline glia tightly enwrap the commissures. (E) Midline glia ectopically expressing Engrailed are found on the dorsal surface of the CNS. (B) The bifurcated axons of MP1 neurons project along the longitudinal tracts. We never obtained a clone resembling MP1 neurons in embryos expressing Engrailed in all midline cells. (C) The projection of one UMI (arrow) differs from the second UMI axon (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 of VUM 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 the VUM motoneurons (arrow) exit the CNS on the same side. (I) Interneuronal axons (arrowhead) can show the same phenotype. (E) The axons of MNB motoneurons (arrows) and interneurons (arrowhead) bifurcate in the anterior commissure. (J,K) Ectopic Engrailed in MNB neurons prevents or delays the bifurcation of their axons. Ectopic Engrailed expression on the midline causes all subsets to be mispositioned along the dorsal surface of the CNS. Ventral views, anterior to the left.

View larger version (47K): [in a new window]   Fig. 9. Opposing functions of Hedgehog and Wingless signalling separate midline siblings into an anterior and posterior compartment. (A) At stage 9, after the division of the midline precursors, Wingless signalling maintains Engrailed expression in two midline siblings. (B) At stage 10, repression by Wingless and activation by Hedgehog positions the proneural Lethal of scute cluster directly posterior to the early Engrailed-expressing cells. (C) Expression of Hedgehog and Lethal of scute are needed to induce and maintain late Engrailed expression in the midline siblings. (D) The induction of Engrailed by Hedgehog divides midline cells into two compartments. The absence of Engrailed in the anterior compartment allows Notch/Delta signalling to select between the MP1 interneuron and midline glial fate; Engrailed expression in the posterior compartment restricts the selection of cell fate by Notch/Delta signalling to MNB and VUM neurons. Midline cells at the parasegmental border, in the middle of the segment, may be determined as precursors.