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First published online 10 November 2004
doi: 10.1242/dev.01521

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Segment-specific prevention of pioneer neuron apoptosis by cell-autonomous, postmitotic Hox gene activity

Irene Miguel-Aliaga^* and Stefan Thor^*,

Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA

View larger version (75K): [in a new window]   Fig. 1. Anterior dMP2 motor neurons degenerate in late embryogenesis. dMP2-GAL4/UAS-nls-myc-EGFP expression in the VNC. (A) At stage 16, a pair of dMP2 neurons is present in every segment. (B) At stage 17, most anterior dMP2s undergo apoptosis, as apparent from their pyknotic cell bodies (C,D, arrow) and fragmented axons (D, arrowhead, E). (F) In first instar larvae, only three pairs of dMP2s (A6 to A8) remain. These posterior dMP2s persist throughout larval stages (not shown). (G,H) dMP2 neurons express the transcription factor Odd-skipped (arrowheads). Expression of dMP2-GAL4 is not always robust in A8 dMP2. However, Odd staining confirms the presence of two dMP2 neurons in this segment (asterisk). The smaller, more dorsal MP1 neuron (arrows) also expresses Odd. (I) At late embryonic stages, dMP2s mature into peptidergic neurons that express the neuropeptide Proctolin. (J) dMP2 neurons project their axons posteriorly, fasciculating with their homologues in the next segment to form a discrete axonal tract that runs the length of the nerve cord (dMP2-GAL4/UAS-myc-EGFPF). This tract exits in the posterior nerve (arrows) and innervates the hindgut (K, arrow). (L) Lipophilic dye (DiI) injections from the hindgut backfills dMP2 neurons in segments A6-8, but not in more anterior segments (n=11 VNCs). (M) dMP2 pioneer neurons are generated at embryonic stage 10 in segments S3 to A8. They extend their axons at stage 12, mature and then degenerate anteriorly at stage 17. Posterior dMP2 become peptidergic motor neurons that express Proctolin and pMad (Allan et al., 2003). dMP2-GAL4 expression in dMP2 neurons is not apparent until stage 14 (not shown) and is never detected in the vMP2 neuron, the dMP2 sibling. dMP2-GAL4 is, therefore, a postmitotic driver.

View larger version (17K): [in a new window]   Fig. 7. RHG-motif genes involved in the apoptosis of pioneer neurons. (A) Organization of the genomic region containing four of the five known RHG-motif cell death activators in Drosophila. The H99 deletion removes hid, grim and rpr. skl resides proximal to rpr, just outside the H99 region. XR38 removes rpr and, perhaps, skl. X14 removes hid only, while X25 removes hid and grim (based upon Peterson et al., 2002). (B) Effect of different mutant combinations, removing one or more of the RHG-motif genes, on the survival of anterior dMP2 (black bars) and MP1 (grey bars) neurons. The vertical axis shows the number of surviving anterior pioneer neurons in late embryos (air-filled trachea stage) for each genotype (n>7 VNCs). See text for details.

View larger version (72K): [in a new window]   Fig. 2. Anterior dMP2 neurons survive when apoptosis is prevented. (A) Wild-type expression of dMP2-GAL4/+;UAS-nls-myc-EGFP/+ in late embryos (air-filled trachea stage). Only the three posterior pairs of dMP2 neurons are apparent. (B) In late embryos homozygous for the H99 deficiency, which removes hid, grim and rpr, anterior dMP2s survive. (C) Anterior dMP2s also survive in XR38/H99 mutant embryos (dMP2-GAL4,H99/XR38; UAS-nls-myc-EGFP), indicating that rpr mediates dMP2 death. (D) Postmitotic expression of anti-apoptotic baculoviral P35 in the dMP2 neurons (UAS-p35/+; dMP2-GAL4/dMP2-GAL4, UAS-nls-myc-EGFP) efficiently suppresses cell death. Surviving anterior dMP2s always express Odd in both XR38/H99 embryos (E) and when apoptosis is blocked with UAS-p35 (F). For each genotype in A to D n>10 VNCs (see Fig. 7B for additional numbers). (G,H) In larvae, anterior dMP2s express Proctolin in 30% and 21% of cases, respectively (n>37 rescued cells). (I) rprGAL4 expression in a stage 16 embryo (rprGAL4/+; UAS-nls-myc-EGFP). Expression is dynamic, but double labeling with Odd (J) reveals that rprGAL4 expression is often observed in 45% of anterior dMP2 neurons. It is, however, always absent from posterior dMP2 neurons (n>40 VNCs).

View larger version (95K): [in a new window]   Fig. 3. The expression of Hox proteins in the VNC is mainly neuronal, and only Abd-B parallels the segmental survival of dMP2 neurons. (A-D) Hox gene expression at embryonic stage 16 in a subset of embryonic postmitotic neurons (UAS-nls-myc-EGFP/+; HB9-GAL4/UAS-nls-myc-EGFP). Within a Hox expression domain, most neurons express the relevant Hox gene (arrows). However, a few do not (arrowheads). (E-L) Hox proteins are largely absent from midline (E-H) and lateral (I-L) glial cells at the same stage, with a few exceptions (L, arrow). (M-P) Only the Abd-B expression profile fits the dMP2 survival/death boundary: neither Antp (M) nor Ubx (N) is present in A5 or A6, Abd-A is expressed in both (O) and only Abd-B is expressed in A6, but not in A5 (P). (Q) Diagram summarizing the expression of Hox genes in the VNC (left) and in dMP2 neurons (circles). Hox gene expression within a specific cell type, in this case the dMP2 neurons, cannot necessarily be inferred from the general Hox expression domain boundaries in the VNC. At stage 16, Abd-B is specifically expressed in dMP2 neurons that will survive into larval stages.

View larger version (55K): [in a new window]   Fig. 4. Abd-B, but not other Hox genes, can fully prevent apoptosis of anterior dMP2 neurons. Postmitotic ectopic expression of specific Hox genes using dMP2-GAL4. dMP2 neurons are visualized by simultaneous expression of UAS-nls-myc-EGFP. No anterior dMP2 neurons survive in late embryos (air-filled stage) expressing Antp (A) or Abd-A (C). Ubx expression results in occasional rescue (B, arrow), and only Abd-B expression can prevent anterior death robustly (D). White numbers in A-D indicate the percentage of anterior dMP2 neurons rescued using dMP2-GAL4 (n>8 VNCs for each genotype). Percentages in yellow were obtained using elavGAL4 and staining dMP2 neurons with Odd (not shown; n>8 VNCs for each genotype). (I,J) Anterior dMP2 neurons rescued by ectopic Abd-B expression express Odd in late embryos (I, 100%, n>100 neurons) and, in larvae, Proctolin (J, 70%, n=38 neurons). (E-H) All four transgenes are robustly expressed at stage 16, just before anterior dMP2 neurons begin to die. For purposes of clarity, segments with no endogenous expression of the relevant Hox gene are displayed.

View larger version (70K): [in a new window]   Fig. 6. Abd-B prevents grim-mediated apoptosis of MP1 pioneer neurons. (A) Odd expression in late embryos (air-filled trachea stage). In addition to the larger, more lateral dMP2 neurons in A6-A8, Odd is expressed in two MP1 neurons (smaller, more medial and more dorsal) up to A5. Odd is also expressed in an unidentified posterior cluster of cells in the posterior tip of the VNC. (G) At early stage 17 (when anterior dMP2 neurons have already died), rprGAL4 is occasionally observed in MP1 neurons (arrowhead). (B) In XR38/H99 mutant embryos (dMP2-GAL4, H99/XR38; UAS-nls-myc-EGFP) both anterior dMP2 and MP1 neurons survive, and four Odd-positive cells are typically observed per segment. Two of these cells are dMP2 neurons, as revealed by dMP2-GAL4 expression (H, arrows), and the other two are surviving MP1 neurons (H, arrowheads). (C) In X25/X25 mutant embryos, loss of grim results in the survival of most anterior MP1 neurons. However, no extra dMP2 neurons are rescued, and only A6-A8 dMP2s are present and express Odd. (D) In Abd-Bm mutants (Abd-BM5/Abd-BM2), both posterior dMP2 and MP1 neurons are lost, and only the posterior cluster of Odd-expressing cells remains. For each genotype in A to D, n>9 VNCs (see Fig. 7B for numbers). (E) Postmitotic rescue of anterior dMP2 and MP1 neurons (elavGAL4/+; UAS-Abd-B/+). Both cell types are rescued in every anterior segment (100% rescue, n=9 VNCs). Hence, four Odd-positive cells are invariably observed per segment (I): two dMP2 neurons (arrow) and two MP1 neurons (arrowhead). (F) Expression of Abd-B in A5 to A7 dMP2 (arrows) and MP1 (arrowheads) pioneer neurons at stage 16. In segments A6 and A7, both dMP2 and MP1 neurons express Abd-B. However, only MP1 neurons express Abd-B in segment A5.

View larger version (64K): [in a new window]   Fig. 5. Abd-B is critical for the survival of posterior dMP2 neurons. In wild-type embryos, all dMP2 neurons are present at stage 16 as visualized by dMP2-GAL4/+; UAS-nls-myc-EGFP (A), while only posterior ones survive at a later stage (C). In Abd-Bm mutants (dMP2-GAL4, Abd-BM5/Abd-BM2; UAS-nls-myc-EGFP), no Abd-Bm protein is detected and Abd-Br expression is confined to the posterior tip of the VNC (B). Posterior dMP2 and MP1 neurons are still normally generated and always express Odd (F, arrowhead and asterisk, respectively). However, at stage 17 they also undergo apoptosis and no dMP2 neurons survive in late Abd-Bm mutant embryos (D). In A to D, n>7 VNCs. At the onset of their degeneration, Abd-Bm is always absent from posterior dMP2 neurons (E, arrowhead). As a result, 55% of posterior dMP2 neurons express rprGAL4 in Abd-Bm mutants (G, arrowheads; rprGAL4, Abd-BM5/Abd-BM2; UAS-nls-myc-EGFP; n>90 cells). (H-J) Cell-autonomous, postmitotic rescue of posterior dMP2 neurons in Abd-Bm mutants with dMP2-GAL4/UAS-Abd-Bm (in red; genotype is UAS-Abd-Bm; dMP2-GAL4, Abd-BM5/Abd-BM2). At stage 16, all posterior dMP2 neurons express Abd-B (H) and Odd (J) while no other cells express Abd-B in segments A6-A8. (I) As a result, posterior dMP2 death is rescued in late embryos (76%, n=10 VNCs).

View larger version (43K): [in a new window]   Fig. 8. Abd-B prevents apoptosis by repressing rpr and grim. (A) In H99, Abd-Bm double-mutant embryos (dMP2-GAL4, Df(3L)H99, Abd-BM5/dMP2-GAL4, Df(3L)H99, Abd-BM5; UAS-nls-myc-EGFP) posterior dMP2 and MP1 neurons are generated normally; at stage 16, two dMP2 neurons express Odd and dMP2-GAL4, whereas two MP1 neurons express Odd only. Abd-B expression is absent from both neuronal types, and only the posterior tip of the VNC expresses Abd-Br. (B) In late embryos, neither anterior nor posterior dMP2 and MP1 neurons undergo apoptosis (100% survival, n=6 VNCs), indicating that rpr and grim are epistatic to Abd-B. Insets show two dMP2 and two MP1 neurons in A6, both at stage 16 (left) and at air-filled trachea stages (right). (C) Proposed mechanism of apoptosis regulation in dMP2 and MP1 neurons. See text for details.

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