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First published online 3 November 2004
doi: 10.1242/dev.01447

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Independent roles of the dachshund and eyes absent genes in BMP signaling, axon pathfinding and neuronal specification

Irene Miguel-Aliaga^*, Douglas W. Allan and Stefan Thor^*,

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

View larger version (31K): [in a new window]   Fig. 1. In the developing Drosophila VNC, a small group of 90 neurons express the ap gene. Most ap-neurons extend axons in a common fascicle running the length of the VNC. The Tv neurons are unique; they innervate the DNH and express the Fmrf neuropeptide. Specification of Tv neurons is dependent upon a combinatorial code of ap, sqz and a target-derived BMP signal mediated by the Gbb ligand and the Wit receptor (Allan et al., 2003; Marques et al., 2003).

View larger version (52K): [in a new window]   Fig. 2. The BMP pathway is active in many peptidergic cells, but only a subset is responsive to the ap/sqz Fmrf code. (A) The Drosophila VNC contains 150 peptidergic neurons, as revealed by c929-GAL4/UAS-nls-myc-EGFP expression. (B,C) pMad immunoreactivity in c929-GAL4+ neurons reveals that some peptidergic cells, such as Corazonin neurons, do not have active BMP signaling (arrowhead in B), whereas others, such as the peptidergic lateral cluster (Plc) do (arrow in C). (D) Based upon their responsiveness to sqz/ap co-misexpression (Allan et al., 2003), the pMad+, peptidergic cells of the VNC can be subdivided into a responsive and a non-responsive `compartment'.

View larger version (84K): [in a new window]   Fig. 3. Dachshund and Eyes Absent are expressed in subsets of peptidergic neurons. (A) Expression of dac (dacGAL4/UAS-myc-EGFPF) is observed in subsets of neurons in the embryonic VNC, including a lateral cluster of cells in the three thoracic segments (arrows). (B-L) Expression of Dac and Eya in late-stage-17 embryos. (B,B',B'') Expression of Dac and the glia-specific marker repo (repoGAL4/UAS-nls-myc-EGFP) reveals that Dac is not expressed in glia. (C,C',C'') Expression of Dac and pMad reveals that Dac is not expressed in motoneurons. Expression of Dac is evident in the pCC interneuron (arrow in C) and in the Va peptidergic cells (arrowheads in C''). (D) From embryonic stage 16, expression of Eya is observed in a highly restricted set of cells in the VNC. (E) Expression of Dac (red), Fmrf (blue) and ap (apGAL4/UAS-nls-myc-EGFP; green) shows that Dac is expressed in three of the four ap-cluster cells, including the Tv cell (arrow), but is absent from the dAp cell (arrowhead). (F) Expression of Eya (red), Fmrf (blue) and ap (apGAL4/UAS-nls-myc-EGFP; green) shows that Eya is expressed in all four ap-cluster cells, including the Tv cell (arrow) and in the dAp cell (arrowhead). (G,H) c929-GAL4 and Fmrf-lacZ co-labeling discriminates the Tv cell (c929-GAL4+, Fmrf-lacZ+) and the Tvb and the dAp cells (c929-GAL4+, Fmrf-lacZ-negative). (G) Expression of Dac (red), Fmrf (blue) and c929-GAL4/UAS-nls-myc-EGFP (green) shows that Dac is not expressed in the Tvb neuron (arrow) or the dAp cell (arrowhead). (H) Expression of Eya (red), Fmrf (blue) and c929-GAL4/UAS-nls-myc-EGFP (green) shows that Eya is expressed in both the Tvb neuron (arrow) and dAp cell (arrowhead). (I-L) Expression of Dac and c929-GAL4/UAS-nls-myc-EGFP shows that Dac is expressed in Va neurons (arrow in I) and posterior dMP2 neurons (arrow in J), is absent from corazonin neurons (arrowhead in K), present in the Vi neurons (arrow in L) and absent from the Plc neurons (arrowhead in L). The dMP2 neurons were previously described as Vap neurons (Allan et al., 2003). However, subsequent work has revealed that Vap neurons are, in fact, the well-characterized dMP2 neurons (I.M-A. and S.T., unpublished). (M) Summary of the expression of Ap, Dac, Eya and pMad within peptidergic neurons of the stage-17 embryonic and larval Drosophila VNC. Note that either Dac or Eya are expressed in all peptidergic neurons that express Fmrf in response to the ap/sqz/BMP code.

View larger version (109K): [in a new window]   Fig. 4. dachshund and eyes absent are both important for Fmrf expression but play different roles. Expression of proFmrf (A-D,M,N), morphology of ap-axons at the thoracic midline (E-H), innervation of the DNH using apGAL4; UAS-myc-EGFPF (green) and anti-Glutactin to visualize the DNH (red) (E'-H'), and pMad expression in the ap-cluster (I-L,R,S) in late-stage-17 embryos. In the wild type (A), proFmrf expression is readily observed in the six lateral Tv neurons in thoracic segments T1 to T3 and in the two anterior, medial SE2 neurons. (A,E,E',I) In controls (apGAL4/+; UAS-myc-EGFPF/+), ap-neurons project close to the midline (E) and innervate the DNH (E'), and pMad staining is evident in the Tv cell of the ap-cluster (I). (B,F,F',J) In dac mutants (dac3/dac4), the expression of proFmrf is weak and partly lost in Tv cells (B). However, in dac mutants (apGAL4, dac4/dac3; UAS-myc-EGFPF/+), there is entirely wild-type midline and DNH innervation (F,F'), and pMad staining of Tv cells (J). (C,G,G',K) In eya mutants (eyaCli-IID/eya10) proFmrf expression is detected in only 32% of Tv neurons, and this is reduced to 6% by removing one copy of ap (C). In eya mutants (apGAL4, eyaCli-IID/eya10; UAS-myc-EGFPF/+), TV axonal projections reach the midline (G) but fail to innervate the DNH (G', only 19% of DNH). Only 26% of Tv neurons express pMad (K). (D,H,H',L) Cell-autonomous reintroduction of eya (apGAL4, eyaCli-IID/eya10; UAS-myc-EGFPF/UAS-eya) rescues proFmrf (D), DNH innervation (H') and Tv pMad expression (L). (M,R) Direct activation of the BMP pathway in eya mutants (apGAL4, eyaCli-IID/eya10, UAS-tkvA, UAS-saxA; UAS-myc-EGFPF/+) only partly restores proFmrf (M), although pMad is expressed in most ap-cluster neurons and is rescued to 100% in Tv cells (R). (N,S) Providing gbb cell-autonomously in eya mutants (apGAL4, eyaCli-IID/eya10, UAS-gbb; UAS-myc-EGFPF/+) fails to restore either proFmrf (N) or pMad (S). (O,P,Q) Expression of -lacZ reveals abdominal ap-axon projections in the stage 16-17 embryo. In the control (apC--lacZ), dAp and vAp neurons project axons within the ipsilateral ap-fascicle and do not cross the midline (O). In two different eya mutant VNCs (eyaCli-IID/eya10; apC--lacZ), the dAp axons frequently (96%) cross the midline (P, arrow, Q). However, they join the contralateral ap fascicle and appear to project anteriorly, like wild-type dAp axons (Q, arrowhead). (T) Relative proFmrf staining intensity in wild type, dac mutant (dac3/dac4) and dac rescue (apGAL4, dac4/dac3; UAS-dac/+) late-stage-17 Tv neurons. dac mutants have reduced proFmrf expression and the dac rescue shows increased intensity, probably due to overexpression of dac. Percentages presented in white were obtained in a wild-type ap background, whereas those presented in green correspond to an ap heterozygous (apGAL4/+) background.

View larger version (33K): [in a new window]   Fig. 5. Expression of apterous, Dachshund and Eyes Absent in mutant backgrounds. Expression of ap (A-L) Dac (A-F) and Eya (G-L) in control and mutant late-stage-17 ap-clusters. In controls, expression of Dac is evident in three ap-cluster cells (A), being absent from the Tvb cell (see Fig. 3E,G). In ap mutants (apGAL4/apP44; UAS-nls-myc-EGFPF/+), Dac is often de-repressed in the Tvb cell (B). In eya mutants (apGAL4, eyaCli-IID/eya10; UAS-myc-EGFPF/+), Dac is often lost from one cell (D), but not from the Tv cell (identified as the highest EGFP-expressing cell). In wit mutants (apGAL4/+; witA12, UAS-myc-EGFPF/witB11), Dac expression is normal (E). In sqz mutants (apGAL4/+; sqzDf, UAS-myc-EGFPF/sqzie), expression of Dac is typically lost from one additional cell per ap-cluster (F), but not from the Tv cell (again, identified as the highest EGFP-expressing cell). Eya expression is not affected in any of the mutant backgrounds (G-L). As expected, expression of Dac and Eya is absent in dac (C; dac4, apGAL4/dac3; UAS-myc-EGFPF/+) and eya (J; apGAL4, eyaCli-IID/eya10; UAS-myc-EGFPF/+) mutants, respectively.

View larger version (126K): [in a new window]   Fig. 6. dachshund acts strongly to activate Fmrf expression in combination with ap and BMP signaling. Small inset panels show close-ups of Fmrf expression in the ap-cluster. (A-D) Misexpression within the peptidergic compartment using c929-GAL4. Misexpression of dac alone does not trigger ectopic Fmrf-lacZ (A), but co-misexpression of both dac and ap triggers ectopic Fmrf-lacZ in the Plc cells (B). Both endogenous and ectopic Fmrf-lacZ expression is dependent upon BMP signaling, as only SE2 cells express Fmrf in wit mutants (C; c929-GAL4, Fmrf-lacZ/UAS-ap; witA12, UAS-dac/witB11). Misexpression of dac and ap together with BMP activation triggers extensive ectopic Fmrf-lacZ expression (D; c929-GAL4, Fmrf-lacZ/UAS-tkvA, UAS-saxA; UAS-ap, UAS-dac/+). dAp, Crz and Tvb cells (inset) all express Fmrf (D). (E-G) Misexpression within all postmitotic neurons using elavGAL4. Misexpression of dac alone triggers Fmrf-lacZ expression in a small subset of posterior cells (E), but co-misexpression of both dac and ap triggers extensive ectopic Fmrf-lacZ expression (F). Staining for Fmrf-lacZ (green) and pMad (magenta) reveals that ectopic Fmrf-lacZ cells are all pMad-positive (G). Misexpression of dac and ap in RP motor neurons using HB9-GAL4 triggers ectopic proFmrf expression (H). (I-L) Misexpression in ap-neurons. Misexpression of dac alone does not trigger ectopic Fmrf-lacZ (I), but together with BMP activation (J) and ap (K), all ap-neurons, except the vAp neurons, are triggered to express Fmrf-lacZ. (L) Both ectopic and endogenous Fmrf expression is dependent upon eya (L; apGAL4, eyaCli-IID/eya10, UAS-tkvA, UAS-saxA; UAS-ap, UAS-dac/+).

View larger version (36K): [in a new window]   Fig. 7. Summary of Tv cell specification. (A) At stage 16, when ap-neurons appear, dac, eya and sqz are expressed in different subsets of ap-neurons. At stage 17, BMP activation via the Gbb BMP ligand and the Wit BMP receptor leads to nuclear translocation of pMad and subsequent Fmrf activation. (B) In the Tv neuron, our genetic analyses support a differential role for eya and dac. eya plays multiple roles, regulating axon pathfinding, competence to respond to Gbb, and Fmrf expression in response to activated BMP signaling. dac, by contrast, regulates only Fmrf. Although the exact target of eya action downstream of pMad is not known, the dashed arrows suggest two possibilities: eya may regulate the pMad/Medea (Med) complex and/or the expression of Fmrf directly.

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