QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00404

This Article Summary Full Text Full Text (PDF) Supplemental Data Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hewes, R. S. Articles by Taghert, P. H. Search for Related Content PubMed PubMed Citation Articles by Hewes, R. S. Articles by Taghert, P. H.

The bHLH protein Dimmed controls neuroendocrine cell differentiation in Drosophila

Randall S. Hewes^1,2,*, Dongkook Park¹, Sebastien A. Gauthier², Anneliese M. Schaefer¹ and Paul H. Taghert^1,

¹ Department of Anatomy and Neurobiology, Washington University School of Medicine, Saint Louis, MO 63110, USA
² Department of Zoology, University of Oklahoma, Norman, OK 73019, USA
^* Present address: Department of Zoology, University of Oklahoma, 730 Van Vleet Oval, Norman, OK 73019, USA

View larger version (76K): [in a new window]   Fig. 1. The c929 reporter gene is expressed in 200 peptidergic CNS neurons. (A) Confocal z-series of c929 reporter (c929-Gal4, P{UAS-lacZ}Bg4-1-2) expression in the larval CNS. Expression is detected in 200 neurons, in neuroendocrine release sites (asterisks) and in intrinsic cells of the endocrine corpora cardiaca (CC) (n>50). (B,B') Double-labeling for c929 reporter (B) and the peptide biosynthetic enzyme, PHM (B'). Yellow cells in the overlay (B'') were positive for both markers (n=8). (C) Double-labeling (C'') for c929 reporter (C; P{UAS-GFP}D1) (Yeh et al., 1995) and neuropeptides ending in the epitope, RF-amide (PT2 antiserum; RFa, C') (n=10). (D) Double-labeling (D'') for c929 reporter (D) and neuropeptides related to MM (D') (n=10). (E) Schematic illustration of the expression patterns for the c929 reporter, the PHM enzyme, and MM- or RFa-immunoreactive neuropeptides. Although generally c929 negative at this stage (mid-third larval instar), the MP1 and VA neurons express the c929 reporter gene for one or more brief periods during development. Scale bars: 50 µm.

View larger version (106K): [in a new window]   Fig. 5. dimm mRNA is expressed in a c929-like pattern of differentiating and post-embryonic peptidergic cells. (A-C) Photomontages of dimm in situ hybridization in embryonic CNS (A, stage 14; B, stage 16; C, stage 17). (D-F) Photomontages of dimm in situ hybridization in the Inka cells (D, stage 17), the peptidergic LBD neurons (LBD), which are adjacent to muscle 8 (m8) (E, stage 15), and a gut cell (mg) at the base of the anterior region of the midgut (F, stage 17). The ventral midline (D-E) is towards the left and anterior is upwards. dv, dorsal vessel; tr, tracheae. (G) Matching patterns of dimm expression revealed by in situ hybridization (G, photomontage) and the c929 reporter (G', confocal image) in the brain lobes of hatchling (first instar) larvae. The LC cells are present but out of focus in G (compare with C). The intrinsic cells of the corpora cardiaca (CC) were removed during dissection of the brain in G'. Scale bars: 50 µm in A-G; 20 µm in insets in A-C.

View larger version (79K): [in a new window]   Fig. 2. dimm is required for expression of normal levels of peptide transmitters. (A) The 39C4-D1 region of chromosome 2L showing the locations of the c929 and KG02598 (`2598') P element insertions (triangles), the CG18362, cryptocephal (crc) and dimm (CG8667, Mistr) genes, and local deletions (wavy lines; bars indicate breakpoint uncertainties). TW1 (not shown) deletes 38A7-B1 to 39C3, ending 17 kb distal to the c929 insertion (Hewes et al., 2000). Rev18 deletes chromosomal bands 39A3-7 to 39D3-5 (A. Carpenter, personal communication). Rev4 deletes 39C1-4 (Hewes et al., 2000) and is a molecular null for the dimm gene (D. Eberl, personal communication; data not shown). Introns smaller than 70 bp are not shown. A segment of DNA extending toward the telomere (wild type) was deleted without affecting neuropeptide levels in the CNS. A second region (dimm 5') was required for the maintenance of normal neuropeptide levels and probably contains dimm 5' enhancer elements. (B) The dimm mutant phenotype for a peptide biosynthetic enzyme, PHM. Reduction in PHM immunostaining in the CNS of a third instar dimm-/- larva (B'), compared with a dimm+/- sibling control (B) (n=28). (C) The dimm mutant phenotype for neuropeptides related to MM. Reduction in MM immunostaining in the CNS of a first instar dimm-/- larva (C'), compared with a dimm+/- sibling control (C) (n>50). (D) Phenocopy of the dimm-/- phenotype (MM immunostaining) by injection of double-stranded CG8667 RNA into wild-type embryos (D'), compared with a mock (saline-injected) control (D) (n=7). Asterisk, weak signal in midline S2 and/or S1a neurons. (E,E') The dimm mutant phenotype in the endocrine Inka cells. Anti-MM immunostaining of an Inka cell in a third instar dimm-/+ larva Inka cell immunostaining [E; compare with O'Brien and Taghert (O'Brien and Taghert, 1998)] was no longer detectable in a dimm-/- larva (E'). The apparent increase in background in E' is due to the incidence of air pockets in the tracheae of this specimen. The precise location of the Inka cell is variable and not certain in (E'); however, it is normally within this image field. Scale bars: 50 µm.

View larger version (84K): [in a new window]   Fig. 3. The KG02598 P-element insertion reduces CG8667 expression and is a strong dimm mutant allele. (A) Photomontages of CG8667 in situ hybridization in homozygous stage 16 and 17 embryonic CNS: normal staining levels were observed in 7/15 specimens from the stock dimmKG02598/CyO-y+ (left); reduced staining levels were observed in the remaining eight specimens (right). In the presumed homozygotes, residual staining was seen only in unpaired cell bodies in caudal abdominal neuromeres that normally stain strongly (arrow). Normal staining levels were also found in 15 out of 15 comparable specimens from the cross dimmKG02598/CyO-y+ x Canton S (not shown). (B) Reduction in immunostaining with the PT2 antiserum in the CNS of a first instar dimmKG02598/Rev4 larva (B'), compared with a dimm+/- sibling control (B) and a dimmS2a/Rev4 control (B''). dimmS2a is a precise excision of the KG02598 P-element. (C). Photomontages of reduced Fmrf in situ hybridization in the CNS of a first instar dimmKG02598/Rev4 larva (C') compared with a dimm+/- sibling control (C). Scale bars: 50 µm.

View larger version (43K): [in a new window]   Fig. 6. dimm-/- mutant neurons differentiate, survive, and express non-secretory proteins at normal levels. (A) Thirty-four neurons (black circles) in the ventral nerve cord that co-express the c929 reporter, Furin 1 and ap-Gal4 (apGal4). Additional ventral neurons (white circles) express ap-Gal4 but do not express the other two markers. (B) The dimm mutant phenotype for the 34 Furin 1 neurons. Reduction in Furin 1 immunostaining in the CNS of a third instar dimm-/- (R6/Rev8) larva (B'), compared with a dimm+/- (R6 or Rev8/+) sibling control (B) (n>50). Arrowheads indicate Tv and Tvb neurons. (C) Ectopic expression of the non-secretory fusion protein tau::Myc in the 34 Furin 1-positive neurons. In dimm-/- mutant CNS (third instar; R6, ap-Gal4/Rev8; UAS-tau::Myc/+), all 34 Furin 1 neurons displayed anti-Myc immunostaining (C') that was comparable with that in the dimm+/- sibling control (C; R6, ap-Gal4/+; UAS-tau::Myc/+). In both genotypes, each of the dorsal chain neurons (d1-d11) displayed projections that extended toward the midline (vertical arrows) and then ran longitudinally (horizontal arrows). We also observed normal immunostained projections in each abdominal nerve (arrowheads). (D) Mean pixel intensity (intensity index) for soma Furin 1 immunostaining in five pairs of dorsal chain neurons in seven dimm+/- and 13 dimm-/- CNSs. *P<0.05. Scale bars: 50 µm.

View larger version (41K): [in a new window]   Fig. 4. Expression of wild-type CG8667 restores neuropeptide levels in dimm mutants (left) and increases normal neuropeptide levels in wild-type animals (right). (A) LK immunostaining of the A1-A7 neurons in the ventral nerve cord in animals heterozygous for the Rev8 or Rev4 deficiencies. The P{UAS-dimm::Myc} transgene, which contains the entire predicted CG8667 ORF, was present on the Rev8 chromosome. (B) Markedly reduced LK immunostaining in dimm-/- (Rev8/Rev4) animals. (C) Staining levels in A1-A7 were restored to normal in Rev8/Rev4 animals by inclusion of the c127-Gal4 element. The A1-7 LK-positive neurons were all GFP positive at this stage (C'). (D) Mean pixel intensity (intensity index) for four pairs of LK-positive neurons in the three genotypes (NS, heterozygous versus rescue; P<0.01, homozygous versus rescue). (E) LK immunostaining of the Br1 neuron in the lateral brain of an animal wild type for dimm and containing one copy of the UAS-dimm::myc transgene. (F) LK-immunostaining in Br1 (soma and arbor) is markedly increased, and a neighboring neuron Br2 becomes LK positive, when UAS-dimm-Myc is driven by ap-GAL4. Br1 was identified based on the retained shape of its axonal arbor. (F') Anti-Myc immunostaining of the specimen in F reveals that Br1 and Br2 neuron are both ap-GAL4 positive. Scale bar, 50 µm in C; 20 µm in F. (G) Mean pixel intensity (intensity index) for the Br1 LK-positive neuron in the two genotypes (P<0.001).

View larger version (74K): [in a new window]   Fig. 7. dimm controls levels of ectopic neuropeptide (proPDF) but not levels of ectopic pro-pdf mRNA. (A) Brightfield photomontages of pdf mRNA in situ hybridization in the dorsal chain neurons of dimm+/- (A; R6, ap-Gal4/+; UAS-pdf/+) and dimm-/- (A'; R6, ap-Gal4/Rev8; UAS-pdf/+) third instar stage CNS. Heterologous expression of the pdf neuropeptide gene, which encodes the PAP neuropeptide, was directed to these neurons using UAS-pdf (Renn et al., 1999) and ap-GAL4. (B) Mean pixel intensity (intensity index) for pdf mRNA in situ hybridization in the somata of selected pairs of c929-positive dorsal neurons (dimm+/-, n=11-12; dimm-/-, n=8-9). (C) Confocal z-series images of dorsal PAP (proPDF) immunostaining in the CNS from dimm-/+ (C) and dimm-/- (C') third instar larvae. Reduced staining (C') was observed in the dorsal chain neurons (d1-11) and the dorsal neurohemal organs (asterisks), which contain the neuroendocrine terminals of the Tv neuron (Benveniste and Taghert, 1999). Note that staining levels in c929-negative cells were unaffected: these included PN (natively pdf-positive), and v7 and v8 (ectopic, ap-Gal4-dependent expression). (D) Mean pixel intensity (intensity index) for PAP immunostaining in the somata of selected pairs of dorsal neurons and in the PN neurons (dimm+/-, n=6; dimm-/-, n=5). (E) Confocal z-series images of ventral PAP immunostaining in T2v (arrows) and in the ventral chain neurons, including v2a and v8, of a dimm-/+ third instar stage CNS (E; same CNS as C) and a dimm-/- CNS (E'; same CNS as C'). The Tv neurons (arrows) express the c929 reporter gene (Fig. 1C,E) and display the dimm mutant phenotype. By contrast, the ventral chain neurons do not express the reporter gene (data not shown), and they do not display the dimm mutant phenotype. (F) Mean pixel intensity (intensity index) for PAP immunostaining in the somata of selected pairs of ventral neurons (dimm+/-, n=6; dimm-/-, n=5). The brightest cell in each Tv cluster was assumed to be Tvb (P.H.T. and M. Han, unpublished). *P<0.05, **P<0.01, ***P<0.001. Scale bars: 50 µm.