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doi: 10.1242/10.1242/dev.00475

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Multiple roles for u-turn/ventral veinless in the development of Drosophila PNS

¹ Department of Genetics and the Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel
² Unit of Electron Microscopy, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel

View larger version (104K): [in a new window]   Fig. 3. VVL expression and loss-of-function effects in embryonic es organs. Anterior is towards the left and dorsal is upwards in all panels. (A-C) A wild-type embryo, stained with anti-VVL (A) and anti-CT (B). (C) A merge of A and B. Colocalization of CT and VVL is evident in many es cells (yellow). (D) The sensory organs of a single abdominal segment (A1-A7) in the embryonic PNS [adapted, with permission, from Bodmer et al. (Bodmer et al., 1989)]. es, ch and multiple dendritic neurons are depicted in red, green and gray, respectively. The nomenclature is according to Ghysen et al. and Bodmer et al. (Ghysen et al., 1986; Bodmer et al., 1989). (E,F) A normal embryo (E) and a vvlGA3 homozygous embryo (F) stained with anti-ELAV to demonstrate the nuclei of all neurons. In the lateral cluster of a normal embryo there are 13 neuronal nuclei (arrow and inset in E). In a lateral cluster from a vvl mutant embryo a reduced number of neurons, in this case only six, is evident (arrow and inset in F). The five nuclei of lch5 neurons can be detected near the dorsal cluster (arrowhead). (G,H) es organs of abdominal dorsal PNS clusters, demonstrated by anti-CT staining. (G) Normal embryo and (H) utH599 homozygous embryo. Braces mark the position of desC and desD cells; square brackets mark the position of des2 and desB cells. A marked decrease in the number of cells in desC and desD is evident in the mutant embryo, whereas des2 and desB seem largely unaffected. (I-K) TUNEL analysis in normal (I) and vvl mutant embryos (J,K). Neuronal nuclei are demonstrated by ELAV staining (green) and apoptotic nuclei are labeled in red. The embryo in K carries also the A101 enhancer trap and is stained with anti-ß-GAL (blue). Asterisks mark the location of lch5. D and L, dorsal and lateral PNS clusters, respectively. (I) In the lateral cluster of a normal embryo only one or two apoptotic nuclei are typically observed. Many more apoptotic nuclei are evident in this cluster in utH599 (J) and vvlGA3 (K) homozygous embryos. (K) Colocalization of the red (TUNEL) and blue (PNS) signals indicates that at least some of the apoptotic nuclei belong to PNS cells (arrowheads).

View larger version (131K): [in a new window]   Fig. 1. Localization of lch5 cells in normal and vvl mutant embryos. (A-C) Dorsal and lateral PNS clusters visualized by mAb 22C10. Anterior is towards the left and dorsal is upwards in all panels. (A) The lch5 neurons (arrows) of a normal embryo are located in the lateral PNS cluster of abdominal segments A1-A7, and their dendrites point dorsally. The v'ch1 neurons can also be observed in these clusters (arrowheads). In utH599 (B) and vvlGA3 (C) homozygous embryos, the lch5 neurons are often aberrantly positioned (arrows). When associated with the dorsal cluster, the dendrites of lch5 neurons point posteriorly or ventrally (compare A1 segments in A-C). The v'ch1 neuron remains in its normal position (arrowheads). (D-G) lch5 organs in normal (D) and utH599 embryos (E-G). Neurons in D and E are labeled with mAb 22C10 (red); ligament, cap and attachment cells are labeled with anti-85E (green). a, attachment cells; c, cap cells; n, neurons; l, ligament cells. Yellow labeling of the ligament cells (D,E) is due to the projection of several focal sections. (D) In a normal embryo, the organ is stretched with the ligament and attachment cells at its ventral and dorsal edges respectively. (E) White arrows indicate misplaced lch5 organs, associated with the ventral part of the dorsal cluster. The organs are collapsed. Note the correct orientation of the dendrites in A2, where the ligament cells (yellow arrow) have descended below the level of the dbd neuron (white line). (F) a85E staining of the segments shown in E. All support cells of the misplaced organs can be detected. The attachment cells are in their normal positions (white arrows). The ligament cells (yellow arrow) and cap cells (green arrow) fail to localize to their correct positions. (G) Occasionally the affected lch5 organs are oriented as thoracic dch3 (compare with T3 in D and F). The arrow indicates the five nuclei of the ligament cells, labeled with anti-REPO (red). (H-J) ato-lacZ embryos stained with anti-ß-GAL (red) and anti-REPO (green), revealing the relative position of the ligament cells in developing lch5 organs. (H) Arrows and arrowhead indicate abdominal and thoracic ligament cells, respectively. Square bracket marks the lch5 cells. In A2 the ligament cells (yellow) are at the dorsal edge of the organ as in T3. In A1 and A3, these cells have already started descending (arrows). (I) An early stage 13 embryo. The ligament cells are assuming a more ventral position relative to the other cells of the organ (arrows). (J) At stage 16, the ligament cells are at their final position at the ventral edge of the lch5 organs (arrows).

View larger version (121K): [in a new window]   Fig. 2. VVL expression pattern in lch5 organs and rescue experiments. Anterior is towards the left and dorsal is upwards in all panels. (A-F) ato-lacZ embryos stained with anti-VVL (green) and anti-ß-GAL (red). (A) An early stage 11 embryo showing VVL expression in the tracheal placodes (t). VVL is not expressed in the ch precursors. (B,C) Abdominal segments of a stage 12 embryo. The box in B marks VVL-positive cells that belong to the lch5 lineage (arrow) and the oenocytes (arrowheads). (C) A merge of anti-VVL (B) and anti-ß-GAL staining. VVL-positive nuclei boxed in B colocalize with the ato-lacZ staining (arrows). The oenocytes are marked with arrowheads. (D-F) A stage 15 embryo. VVL expression is clearly detected in the nuclei of lch5 neurons (white box in D and arrows in F). (G-J) ß-GAL expression under the regulation of arm-Gal4 (G,H), ato-Gal4 (I) and elav-Gal4 (J). (G,H) Two focal planes of the same stage 14 embryo. arm-Gal4 drives strong ß-GAL expression (red) in the ectoderm (G), and much weaker expression in the PNS (H). Ectodermal and PNS focal planes were determined by staining with anti-85E (green). The arrow in G indicates lch5 ectodermal attachment cells. The arrow in H indicates the position of neurons and sheath cells. c, cap cells; l, ligament cells. (I) ato-Gal4 drives expression in the ch lineage and in a group of ectodermal cells that remains confined to the posterior compartment of each segment. lch5 in the A1 segment are boxed. White arrows in the T3 segment indicate the edges of dch3 organs, and black arrows indicate the cluster of the ß-GAL-expressing ectodermal cells. (J) lacZ expression induced by elav-Gal4 can be observed in the neurons (n) and sheath cells (s) of lch5 organs. (K-M) Rescue experiments with elav-Gal4 and arm-Gal4. (K,L) Embryos are labeled with mAb 22C10 (red) and anti-VVL (green). (K) A normal embryo carrying both the elav-Gal4 and UAS-vvl transgenes. The lch5 organs are positioned correctly in the lateral clusters in all abdominal segments (arrow). VVL expression is seen in the ectoderm and trachea (endogenous protein), and in neurons and sheath cells (induced protein). (L) A utH599 homozygous embryo, carrying both transgenes as the embryo in K. VVL expression can be observed only in neurons and sheath cells. This expression is clearly insufficient for rescuing the abnormal localization of lch5 organs. Abnormally positioned lch5 neurons are marked with arrowheads. (M) Abdominal segments of a utH599 homozygous embryo, carrying the arm-Gal4 and UAS-vvl transgenes. Note the correct positioning of lch5 neurons. D, dorsal PNS cluster; L, lateral PNS cluster.

View larger version (87K): [in a new window]   Fig. 4. VVL expression and loss-of-function effects in developing adult bristles. (A-D) Nuclear expression of VVL in developing bristles in pupal nota. (A) At 16 hours APF VVL expression is observed both in the precursors that have not divided yet and in the two daughter cells. (B) At 24 hours APF, VVL is evenly expressed in the nuclei of all four cells of the developing bristles. (C) By 37-40 hours APF, the expression of VVL decreases in three of the four cells of the organ. (D) At 42 hours APF strong VVL expression is evident in only one cell per organ; this is the socket cell, as indicated by colocalization of VVL (green) and Su(H) (red) (arrow in inset). Weaker expression is occasionally seen in the adjacent shaft cell nucleus (arrowhead). (E-G) Expression in a pupal retina (30 hours APF) of VVL (E), CT (F) and a merge panel (G). Colocalization of the two proteins can be observed in all four cells of the developing interommatidial bristles. Note the lack of VVL expression from the cone cells (arrow in G). (H-N) Cuticle preparations and scanning electron micrographs of wild type (H,M) and vvl mutant clones (I-L,N). (H,I) a2 antennal segments. Note shaft duplications accompanied by two fused sockets in the mutant (arrows). (J-L) Other bristle defects include reduced or missing shafts (black arrowheads in J), distorted shafts (white arrowhead in J), supernumerary sockets (arrows in K) and splitting of shafts (L). The supernumerary sockets can be abnormally flat (arrow in J). (M,N) In the mutant eye, the shafts are dramatically reduced, but the sockets and the distribution of the bristles appear normal. (O,P) Anti-CT staining of developing bristles in wild type (O) and vvl mutant heads (P) at 42 hours APF. Enlarged clusters, containing five or more cells are observed in the mutant, as opposed to only four cells normally (arrows in O and P).

View larger version (80K): [in a new window]   Fig. 5. A model for the lateral localization of lch5 organs. Anterior is towards the left and dorsal is upwards in all panels. lch5 organs in embryonic stages 12-16. The neurons are depicted in red, ligament cells in light green, scolopale cells in light blue, cap cells in dark green and the attachment cells in dark blue. In early stage 12 the ligament cells are located most dorsally within the developing organ. During stage 12 and early stage 13, they migrate ventrally, pulling with them the neuron, scolopale and cap cells. This leads to the rotation of the whole organ. The attachment cells do not change their position. Once the organs acquire the right orientation the ligament cells stretch ventrally and attach to the ectoderm in the lateral region. For simplicity, the lch5 cells in stages 12-13 are drawn next to each other, whereas in reality they are more tightly packed. (B) An embryo overexpressing abd-A under the regulation of da-Gal4, stained with anti 85E. dch3 organs in T2 and T3 segments have descended and reversed their orientation (compare with dch3 in figure 1F). Arrows and arrowheads indicate ligament and cap cells, respectively. (C) A stage 12 wild-type embryo stained with anti-VVL. Note the stronger VVL expression in the dorsal half of the embryo.