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First published online 25 August 2004
doi: 10.1242/dev.01366

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Dynactin is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons

Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue 68-230B, Cambridge, MA 02139, USA

View larger version (88K): [in a new window]   Fig. 2. Nuclei are mispositioned within Glued mutant photoreceptors. (A-K) All neuronal nuclei in a third instar eye–brain complex are labeled with anti-Elav (blue in C-H,K, white in B,J). Individual cells are labeled with GFP (green in C-H,K, white in A,I) using a heat-shock FLP Act-FRT-FRT-Gal4:UAS-GFP chromosome in a wild-type background (A-D) or a Glued1 mutant animal (E-K). An individual wild-type photoreceptor is marked with GFP in A, and all neuronal nuclei labeled with anti-Elav in B. A and B are merged in C, with a higher magnification view of the marked photoreceptor containing a nucleus in the inset. (D) An individual photoreceptor in another sample is imaged from a side view, with apical at the top. In Glued1 mutants, individual cells in the optic stalk in E (marked with arrowheads) are shown at higher magnification in F. Examples of trailing processes are marked with arrows in F. Cells in F marked with both an arrowhead and a letter are shown at right, with their corresponding letter, at higher magnification. An individual labeled cell in another animal is shown in I, with trailing process indicated by the arrow. I and J are merged in K.

View larger version (112K): [in a new window]   Fig. 1. Dynactin is required to position photoreceptor cell bodies and nuclei in the developing third instar eye disc. Photoreceptor cell membranes are stained with anti-Chaoptin in A-G. (A) In wild type, photoreceptor cell bodies (as defined in this figure by the position of the nucleus) are precisely arranged in clusters in the apical region of the eye disc and project axons through the optic stalk (os) into the brain's optic lobe. (B) In Glued1 mutants, many photoreceptor neuron cell bodies leave the apical region of the eye disc (arrowheads) and travel into the optic stalk (bracket) and brain (arrow). (C) Heterozygous cpbM143 animals have a wild-type photoreceptor axon projection pattern, with photoreceptor cell bodies positioned in the eye disc. (D) In heterozygous cpbM143 animals with homozygous cpbM143/cpbM143 patches in the visual system, many photoreceptor cell bodies leave the eye disc and enter the brain (arrows). (E) An independently generated cpb allele, cpbF44, also causes photoreceptor cell bodies to enter the brain (arrows) in eye clones. (F) Photoreceptor mispositioning (arrows) is also observed in cpbM143/Df(2L)E.2 animals rescued from early lethality by expression of Cpb from a genomic transgene [pYES-ß] (see text for details). (G) cpbM143/Df(2L)E.2 animals rescued by ubiquitous expression of a wild-type Cpb cDNA have normal photoreceptor positioning. Photoreceptor nuclei are stained with anti-Elav in H-J. (H) In wild type, photoreceptor nuclei remain in the eye disc and do not enter the optic stalk. (I) Photoreceptor nuclei are mispositioned in cpbM143 mosaic eye discs, with patches of eye tissue missing nuclei (arrows) and Elav-staining nuclei found in the optic stalk (arrowheads). (J) cpbM143/Df(2L)E.2 animals rescued by ubiquitous expression of a Cpb cDNA have normal photoreceptor nuclear positioning.

View larger version (52K): [in a new window]   Fig. 3. Dynactin is required postmitotically to maintain photoreceptor nuclear positioning. Third instar eye–brain complexes are stained with anti-Chaoptin (red) and anti-Elav (green). No photoreceptor nuclei (arrowheads) are seen in the optic stalk in wild type (A). Expression of dominant-negative Glued or overexpression of Dynamitin in differentiated photoreceptors under the control of Glass38-1 causes nuclei to leave the eye disc and enter the optic stalk (B,C, arrowheads).

View larger version (63K): [in a new window]   Fig. 4. Apical markers are not disrupted, but Nod:LacZ is mislocalized, in Glued mutants. Third instar eye discs are stained with anti-Elav (blue), anti-Armadillo (red), and anti-PATJ (green) (A-D). Views from the apical surface (A,B) show evenly spaced apical markers in wild type (A), with each ommatidial cluster (dashed outline) centered under a concentration of Armadillo (arrowhead). Armadillo staining is largely normal in Glued1 (B), despite the presence of ommatidia devoid of nuclei (arrowhead). Side views show that in wild type (C), each ommatidium (dashed line) has a distinct apical clustering of Armadillo and PATJ (arrowhead). Glued animals appear to retain apical markers (arrowheads) even when photoreceptor nuclei are mispositioned (D). Nod:LacZ (yellow) expressed in postmitotic photoreceptor neurons under the control of the Glass38-1 promotor localizes apical to photoreceptor nuclei (anti-Elav, blue) in the most mature photoreceptor neurons (E, apical surface, and F, side view) and is not found in axons or in the optic stalk (G). When dominant-negative Glued is expressed in photoreceptor neurons using the Glass38-1 promoter, Nod:LacZ staining is distributed throughout the axons of the most mature cells (H, arrowhead). In (I), consequences of Dynactin disruption are summarized, synthesizing the data obtained in Figs 2, 3, 4. Inhibition of Dynactin function in the postmitotic neuron causes the photoreceptor nucleus (blue) to be displaced toward the axon terminal. Despite nuclear movement, a trailing process remains and apical markers (PATJ in green, Armadillo in red) are retained. Nod: LacZ (yellow), however, becomes mislocalized from its wild-type apical position and enters the axon.

View larger version (109K): [in a new window]   Fig. 5. Glued nuclear mispositioning is enhanced by dynein intermediate chain reduction. Third instar eye discs were stained with anti-Elav. (A,B) Apical surface of eye disc. Glued1 mutants show small areas devoid of apical photoreceptor nuclei (A), while dic2/+; Glued1/+ animals (B) have much larger areas devoid of nuclei (arrowheads). (C,D) Basal surface of eye disc and optic stalk. The greater absence of photoreceptor neuron nuclei in apical regions of the eye disc in dic2/+; Glued1/+ animals is not simply due to an absence of photoreceptor neurons, as large numbers of photoreceptor nuclei are clustered at the base of the optic stalk in both Glued1 (C) and dic2/+; Glued1/+ animals (D).

View larger version (109K): [in a new window]   Fig. 6. Glued nuclear mispositioning is suppressed by kinesin heavy chain reduction. Scanning electron micrographs of adult eyes (A-D). (A) Wild type. (B) Gl1/+ animals have smaller eyes with disorganized ommatidia. (C,D) Reduction of kinesin heavy chain gene dosage partially suppresses Gl1/+ eye defect. Apical regions of third instar eye discs in which photoreceptor nuclei are stained with anti-Elav (E-H). (I) Suppression of the Glass-GluedDN phenotype quantified by counting the number of Elav-positive nuclei in the optic stalks of animals with 15 to 22 rows of photoreceptor development. The average for each genotype was 19 rows of development. Error bars are s.e.m. and asterisks denote P-value of unpaired t-test (**P<0.01, ***P<0.001).

View larger version (65K): [in a new window]   Fig. 7. Glued function is required to position Bolwig organ nuclei, where it is antagonized by kinesin heavy chain. Wild-type second instar eye-antennal discs stained with anti-Elav show no neuronal nuclei in the eye-antennal disc along the path of the Bolwig nerve (A), while animals expressing dominant-negative Glued in postmitotic Bolwig photoreceptor neurons, using either the Elav promoter (B) or Glass38-1 (C), have neuronal nuclei (arrowheads in B,C) along the path of the Bolwig nerve (in C, anti-Chaoptin in red, anti-Elav in blue, arrow indicates Bolwig nerve). Misplaced Bolwig nuclei in animals that express dominant-negative Glued under Glass38-1 control were quantified in D. The number of misplaced Bolwig neuron nuclei was reduced when animals were heterozygous for loss-of-function mutations in khc. *P<0.05.

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