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
Author for correspondence (e-mail:
taghertp{at}pcg.wustl.edu)
Accepted 9 January 2003
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SUMMARY |
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Key words: Neuroendocrine, Drosophila, bHLH, Neuropeptide, Differentiation, Regulated secretion
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INTRODUCTION |
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Neuroendocrine cell differentiation also involves the integrated assembly
of cellular machinery needed to produce large amounts of secretory peptides.
Such mechanisms coordinate several events associated with the regulated
secretory pathway (Arvan and Castle,
1998): the ability to synthesize, process, sort, traffic and
accumulate dense-core secretory granules and their contents. Neurons differ
greatly and reproducibly in the amount of secretory peptides that they
produce, and in their elaboration of the secretory pathway. For example,
mammalian motoneurons produce low levels of neuropeptides such CGRP or galanin
(Streit et al., 1989
), and at
the ultrastructural level, their terminals contain many small, clear vesicles,
but very few large, dense-core (peptide-containing) granules
(Hall and Sanes, 1993
). By
contrast, hypothalamic neurosecretory neurons produce large amounts of
vasopressin, oxytocin, or corticotrophin-releasing factor, and they contain
correspondingly large numbers of dense-core secretory granules
(Burbach et al., 2001
). Cells
also transiently modify their levels of secretory activity following injury
(e.g. Blake-Bruzzini et al.,
1997
) or stimulation (e.g.
Herman et al., 1991
). The
mechanisms underlying these differences in levels of secretory activity are
unknown.
Because the amplified expression of the secretory pathway is a stable and cell-specific feature of neuroendocrine cells, we hypothesize the existence of genetic factors that control this phenotype. Identifying such factors will facilitate a detailed, mechanistic analysis of neuroendocrine cell organization and physiology. Such an analysis will be crucial to a general understanding of neuroendocrine cell biology and will support efforts to produce a program of neuroendocrine differentiation from stem cells in vitro. We describe a Drosophila bHLH gene, dimmed (dimm), with an expression pattern that corresponds precisely to the neuronal and endocrine cells that accumulate large amounts of secretory peptides. We present both loss-of-function and gain-of-function analyses to argue that dimm confers a pro-secretory phenotype within these diverse cells, and that its actions appear confined to that aspect of cellular differentiation. Thus, we propose a novel and general mechanism, of which dimm is an essential component, for the amplification of the regulated secretory pathway by dedicated secretory cells.
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MATERIALS AND METHODS |
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Scoring of dimm larvae
Eggs were collected on apple juice-agar plates supplemented with yeast
paste. Larvae were collected from the plates, and heterozygotes (y*
w* and balanced over CyO-y+) were distinguished
by mouthpart color. After scoring, size-matched pairs of
y- and y+ larvae were dissected and
stained in parallel.
Immunostaining
Immunostaining was performed as described previously
(Benveniste et al., 1998).
Briefly, tissues were fixed in 4% paraformaldehyde (PFA), Bouin's, or 4%
paraformaldehyde/7% picric acid (PFA-PA). Polyclonal or monoclonal primary
antisera were used (overnight at 4°C) to detect the following proteins:
ß-gal (1:1000, PFA-PA; Promega); PHM (1:750 pre-absorbed to
PHM-/- larvae, Bouin's) (Jiang
et al., 2000
); -RFa (`PT2')(1:2000, PFA-PA)
(Taghert, 1999
); FMRF (1:2000,
PFA-PA) (Chin et al., 1990
);
corazonin (1:500, Texas Red-conjugated; PFA-PA)
(Veenstra, 1994
); LK (1:500,
PFA-PA) (Nässel and Lundquist,
1991
); CCAP (1:400, PFA-PA)
(Ewer and Truman, 1996
); PDH
and PAP (each 1:2000, PFA-PA) (Renn et
al., 1999
); MM (1:800, PFA-PA)
(O'Brien and Taghert, 1998
);
dopa decarboxylase (affinity purified 1:100, PFA)
(Scholnick et al., 1991
);
Furin-1 (1:1000, Bouin's) (Jiang et al.,
2000
); and Myc (1:500, PFA-PA; a gift from Y.-N. Jan; Sigma).
Secondaries used were goat Cy3, FITC, Texas Red or ALEXA 488 conjugates
(Jackson ImmunoResearch) at a 1:500 dilution. Confocal z-series
projections were obtained using an Olympus Fluoview microscope.
RACE
A cDNA library was made from RNA of y w adult heads using
commercial reagents (Clontech). 5' RACE was performed according to
manufacturer's recommendations using CG8667-specific primers.
dimm RNAi
RNAi was performed based on the methods of Kennerdell and Carthew
(Kennerdell and Carthew, 1998)
and Clemens et al. (Clemens et al.,
2000
). The template for RNA synthesis was generated by PCR, using
primers containing a T7 promoter sequence
(5'-GAATTAATACGACTCACTATAGGGAGA-3') at the 5' ends and P1
DNA (DS00532) as the PCR template. The gene specific primers
5'-CAGATTCCAGTTCGCAAAGCGAT-3' and
5'-GGGCTCGTCGAAATTATCATTGATA-3' amplified a 951 bp segment of open
reading frame in exon 3, including the entire bHLH domain. Transcription and
analysis of the double-stranded RNA (dsRNA) were performed as described
(Clemens et al., 2000
). dsRNA
(3 µM) was injected into syncytial blastoderm embryos (Canton-S)
75%
along the anteroposterior axis. For the mock controls, all steps were
performed in parallel, except that the P1 DNA was omitted from the PCR
reaction. Mock- and RNA-injected larvae were dissected during or within 6
hours after hatching.
UAS-dimm transgene
The predicted coding region of CG8667 was amplified by PCR using
cDNA generated from y w adult head RNA (Clontech). The primers
5'-CAGATCTCGACGATTTTTGTTCAGCCAT-3' (5' UTR) and
5'-TGCGGCCGCAGAAACTCTCGAAAGGGCT-3' (end of the ORF) were used to
construct a 1236 bp fragment that was cloned into pBSK+ and then
transferred to pP{UAST-Myc} at the BglII and NotI
sites. Transgenic lines containing P{UAS-dimm::Myc} insertions were
created using standard techniques
(Benveniste and Taghert, 1999).
UAS-dimm::Myc2-A-3, Rev8/CyO, Act-GFP flies were crossed
to c127-Gal4, UAS-GFP; Rev4/CyO, Act-GFP, and first instar larvae
were scored for Act-GFP- and c127-specific GFP labeling
patterns.
mRNA in situ hybridization
Whole-mount in situ hybridization
(Tautz and Pfeifle, 1989) was
performed using single-stranded, digoxigenin-labeled RNA or DNA probes
(Patel, 1996
) prepared from P1
or cDNA templates.
Staining quantification
Cells were imaged on a Zeiss Axioplan fitted with a SPOT CCD camera and
software (Diagnostic Instruments) or in confocal z-series scans.
Exposure settings were adjusted to optimize detection without saturating the
signal. For a given neuron, identical settings were used for all preparations
and genotypes. Mean pixel luminosity for the area covering the soma (S) was
measured for each neuron using Adobe Photoshop. An adjacent area was sampled
to measure the background signal (B). The intensity index=(S-B)/B. Cells not
visible were scored 0. Cells that were obscured or lost due to tissue damage
were not analyzed. Brightfield images were inverted before quantification. CNS
size was measured as an additional control in each case, mean brain
lobe diameters were not significantly different between genotypes (data not
shown). Statistics were performed using the NCSS-2000 Statistical Analysis
System or StatView (MANOVAs; Games-Howell). Variances are reported as
±s.e.m.
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RESULTS |
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Conversely, most neurons displaying strong PHM immunostaining were also
c929 positive, while most weakly PHM-positive neurons were not
c929 positive (data not shown). In addition, PHM was expressed in all
three c929-positive endocrine cell types and in the LBD peripheral
neurons (O'Brien and Taghert,
1998) (data not shown). Thus, in the larval CNS and in several
peripheral tissues, c929 primarily labels neuroendocrine cells as its
expression was highly correlated with the production of large amounts of
amidating enzyme, amidated neuropeptides and peptide hormones.
To assess the degree of heterogeneity among c929-positive cells,
we compared the expression pattern of c929 with a variety of other
peptidergic cell markers. This population of cells was chemically diverse. For
example, seven bilateral pairs of c929-positive neurons were
double-labeled with the PT2 antiserum (Fig.
1C,E). PT2 is a marker for -RFamide containing neuropeptides,
which include the products of at least three Drosophila genes
(Taghert, 1999). Additional
subsets of c929-positive neurons were immunostained with antisera
directed against a variety of neuropeptides. These included the
Drosophila FMRF propeptide (n=8 specimens), cockroach
corazonin (n=5), cricket leucokinin-1 (LK), crustacean cardioactive
peptide (CCAP; n=4), crustacean beta-PDH (n=4) and
Aplysia myomodulin (MM; Fig.
1D,E). Finally, a distinct subset of 34 c929-positive
neurons (see below) was immunopositive for an additional, putative
Drosophila peptide biosynthetic enzyme (n=10; P.H.T. and M.
Han, unpublished) Furin 1 (De Bie et al.,
1995
). Based on their positions, cellular morphologies, and
immunostaining with the above markers, the cells within the c929
pattern represent more than 26 distinct classes of peptidergic neurons and
endocrine cells. No c929-positive neurons were stained with an
antiserum to dopa decarboxylase (n=8), an enzyme required for
synthesis of the biogenic amines, serotonin and dopamine
(Hirsh, 1989
).
No single transmitter system we tested was entirely c929 positive.
For example, among the 17 Fmrf cell types
(Benveniste and Taghert, 1999),
only the Tv neurons were c929-positive. However, in third instar
larvae there were some c929-negative neurons, such as the peptidergic
MP1s and VAs, which displayed weak and/or transient c929 reporter
expression during other stages of development (e.g.
Fig. 5G). Thus, our
identification of c929-positive peptidergic neurons is likely to be
an underestimate of the total population of peptidergic cells that express the
reporter gene.
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We used six additional neurosecretory markers in dimm mutant larvae, and found that all six displayed moderate to severe reductions in immunostaining in spatial patterns corresponding to the c929 reporter pattern. The affected proteins included several known or presumed neuropeptides MM (Fig. 2C), LK (n>25), the FMRF propeptide (n>12) and several PT2 positive neuropeptides (n>50) and the putative neuropeptide biosynthetic enzyme Furin 1 (see below). All c929-positive neurons displayed the mutant phenotype for at least one marker, PHM (Fig. 2B); many showed reduced immunostaining with multiple markers. For example, the Tv neurons had reduced levels of PHM, the FMRF propeptide, RFamide peptides and Furin 1 (Fig. 2B, see Fig. 3B, see Fig. 6B). Thus, in a large and diverse population of CNS peptidergic neurons, dimm regulates levels of a broad array of secretory proteins.
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dimm encodes a basic helix-loop-helix protein
Using chromosomal deletions, we genetically mapped the dimm gene.
We performed peptide immunostaining on Rev8 homozygotes
(n=15) and on hemizygotes (n>50) bearing one copy of
R6 (or Rev8) over one of several independently derived
deficiencies of the entire 39C4-D1 region of chromosome 2L (e.g.
Rev4). In each case, the effects on peptide immunostaining were
comparable, although the reduction in MM staining in larvae homozygous for
Rev4, a null allele (Fig.
2A), was more pronounced than in R6/Rev8
trans-heterozygotes (n=12; data not shown). Thus, R6 and
Rev8 are hypomorphic alleles. Normal peptide immunostaining was
restored in male Rev8 homozygotes (n=6) bearing a
duplication of chromosome bands 35A-40 [Tp(2;Y)J54], consistent with
the location of dimm in 39C4-D1.
In contrast to R6/Rev8 mutants, larvae with disruptions in the crc gene (c929 homozygotes, n=6; crc1/R6 trans-heterozygotes, n>40; R2 homozygotes, n=5), or deletions of DNA extending up to 200-300 kb towards the telomere (TW1/Rev18 trans-heterozygotes, n=7) displayed wild-type neuropeptide levels (see Table S1 at http://dev.biologists.org/supplemental/). Thus, dimm is not crc, nor is it any other gene located distal to the site of the c929 insertion.
The closest gene proximal to c929 is CG8667
(Mistr), found within 25 kb (Fig.
2A). It encodes a basic helix-loop-helix (bHLH) protein that is a
member of the Atonal subfamily of transcription factors
(Moore et al., 2000). Its bHLH
domain displays 79% identity with the mouse Mist1 protein
(Pin et al., 1999
). In
Rev8 homozygous embryos, CG8667 mRNA expression was markedly
reduced, but not eliminated (n>50; data not shown), consistent
with the identification of Rev8 as a hypomorphic dimm
allele. After 5' RACE identification of the 5' end of
CG8667, we identified a P-element insertion
(dimmKG02598) located 111 bp upstream
(Fig. 2A).
dimmKG02598 displays homozygous lethality (see Table S2 at
http://dev.biologists.org/supplemental/),
and represents a severe hypomorphic dimm allele, because
CG8667 mRNA expression appeared low or undetectable in
dimmKG02598 homozygous mutant embryos
(Fig. 3A). Hatchling
dimmKG02598/Rev4 larvae displayed reduced immunostaining
for PT2-positive neuropeptides (n>15;
Fig. 3B). Normal PT2
immunostaining was restored (n>15;
Fig. 3B) after precise excision
of the dimmKG02598 P element. Consistent with the
conclusion that dimm and crc are separate genes,
KG02598 was lethal when trans-heterozygous with Rev4, but
not with crc1 (see Table S2 at
http://dev.biologists.org/supplemental/).
The dimmKG02598 mutation also reduces levels of secretory
peptide mRNAs in the Tv neuroendocrine cells, which display high levels of
Fmrf mRNA expression (Schneider
et al., 1993
): when assessed using in situ hybridization, the mean
number of Fmrf-positive Tv neurons per CNS was 5.57 in dimm
heterozygotes (n=7; Fig.
3C) and 2.33 in dimm hemizygotes (n=9;
Fig. 3C; P<0.01).
These combined data indicate that in the absence of dimm, there is a
reduction in levels of both secretory peptide mRNAs and secretory
peptides.
To examine further the effect of disruptions in CG8667 expression, we performed RNAi analysis and observed reduced levels of MM immunostaining in hatchling stage larvae (Fig. 2D). The reduction in MM immunostaining was comparable with the phenotype in null dimm-/- mutants (Fig. 2C). We obtained the same results using two additional antisera, PT2 and anti-LK (n=5 and n=6; data not shown). We also tested the ability of a UAS-dimm::Myc transgene to restore neuropeptide levels in dimm-/- animals. We used the c127-Gal4 line to drive dimm::Myc expression in a small set of ventral CNS neurons, which included the 14 LK-positive cells in abdominal neuromeres (Fig. 4A). Expression of dimm::Myc selectively restored normal levels of LK immunostaining in Rev8/Rev4 animals (n=19; Fig. 4C), but not in the absence of the Gal4 driver (n=17; Fig. 4B). The rescue displayed cell specificity: the FMRF-positive MP2 neurons did not express UAS-GFP by c127-Gal4, and they were not rescued (n=10; data not shown). Together, these results support the hypothesis that dimm is the Drosophila Mist1 ortholog, CG8667.
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CG8667 is specifically expressed in peptidergic neurons and
endocrine cells
CG8667 mRNAs were ubiquitous in pre-cellular blastoderm embryos
(Moore et al., 2000; data not
shown) and later were expressed in the developing nervous system
(Moore et al., 2000
). Presumed
zygotic CG8667 expression was first visible as nascent transcripts
scattered throughout the CNS in stage 12 embryos. Cytoplasmic CG8667
hybridization was visible in many of these cells beginning around stage 14
(Fig. 5A), was strong by stage
16 (Fig. 5B) and persisted in
stage 17 embryos (Fig. 5C) and
in hatchling larvae less than 24 hour old
(Fig. 5G).
The pattern of CNS CG8667 in situ hybridization resembled the c929 reporter pattern (Fig. 5A-C,G). Based on their positions and morphologies, more than 12 separate types of CG8667-expressing neurons were putatively identified as c929 positive. These included dorsal chain neurons (e.g. d3-d5), T1-3v, LP1, MP1, MP2, SP1, T1-3vb and VA, as well as several bilateral clusters of neurons: large, midline protocerebral brain cells (MC), lateral protocerebral brain cells (LC), ventral subesophageal neurons (SE) and lateral abdominal neurons (neuromeres N1, N4 and N5).
We also observed expression of the c929 reporter and CG8667 in strikingly similar patterns in peripheral tissues (Fig. 5). These sites included the LBD neurons and several endocrine tissues: intrinsic cells of the corpora cardiaca, Inka cells and a few midgut cells. Numerically, all peripheral cell types were equally represented, except that there were fewer CG8667-expressing gut cells in embryos than c929-positive gut cells of larvae. CG8667 was not expressed in any other location, except for a few unidentified non-CNS cells scattered throughout the anterior and lateral regions (stages 12-15). Thus, in CNS, PNS and endocrine tissues, expression of the c929 reporter closely mirrored CG8667 expression. These expression analyses support the genetic mapping, genetic identification and RNAi data. Thus, from this point onwards we refer to CG8667 as dimmed.
dimm mutant cells survive and arborize normally
We next determined whether secretory cells survived and differentiated in
dimm-/- mutant animals. In larvae homozygous for the null
allele, Rev4, some of the affected cells displayed low residual
immunostaining for secretory proteins (e.g.
Fig. 2B,C). Thus, some
dimm-expressing cells survived in mutant larvae and were at least
partially differentiated. Others displayed a complete loss of peptide
immunostaining, and their status was unclear.
In order to determine the fates of the latter cells, we used Gal4/UAS mosaics to express ectopic, non-secretory proteins in dimm mutant neurons. We studied 34 CNS neurons that co-expressed three different markers: the c929 reporter, the putative peptide biosynthetic enzyme Furin 1, and ap-Gal4 (Fig. 6A; P.H.T. and M. Han, unpublished). In dimm-/- larval CNS, all 34 neurons displayed strongly reduced, and often undetectable, immunostaining for Furin 1 (Fig. 6B). Using ap-Gal4 to drive heterologous expression of a tau::Myc fusion protein, we found that all 34 of these neurons were present and displayed normal morphology in the dimm-/- larvae (Fig. 6C). In addition, the intensity of anti-Myc immunostaining was not affected (Fig. 6D). We obtained identical results using green fluorescent protein (GFP) to mark the cells (n=6; data not shown). Thus, dimm mutant neurons displayed multiple differentiated features and synthesized non-secretory proteins at normal levels throughout larval development.
We also examined the effects of dimm on the terminal arbor of the LK-positive neurons. These cells displayed reduced soma LK immunostaining in dimm-/- CNS (Fig. 4B). Each neuron had a single efferent axon that projected across the posterior muscle 8 surface and terminated dorsally near a tracheal branch. In third instar dimm-/- larvae, these axons also displayed reduced LK immunostaining. However, a sufficient number of immunoreactive boutons remained to indicate a normal axonal expanse (see Fig. S1 at http://dev.biologists.org/supplemental/). Thus, the effects of dimm on this LK neuron appear limited to expression of the transmitter phenotype.
dimm affects levels of proteins destined for both regulated
and constitutive secretion
Our earlier measures of the dimmed phenotype were restricted to
analysis of proteins abundant in the regulated secretory pathway. We also
tested for an effect of dimm on constitutively secreted proteins.
With ap-Gal4, we directed expression of a CD8::GFP fusion protein
(UAS-CD8::GFP) to a subset of dimm-expressing neurons. CD8
is an integral membrane protein that is targeted to the plasma membrane in
Drosophila cells (Zito et al.,
1997). In dimm-/- mutant larvae, all 34
ap-Gal4 (Furin-1) neurons expressed CD8::GFP and displayed normal
neuritic projections. However, CD8::GFP levels were significantly lower in
c929-positive neurons in the dimm-/- background
(see Fig. S2 at
http://dev.biologists.org/supplemental/).
This effect was more subtle than the effects on levels of regulated secretory
proteins. However, it suggests that dimm influences both regulated
and constitutive secretory activity in neuroendocrine cells.
dimm regulates multiple elements of the secretory
pathway
Because ap-dependent expression of transgenes was unaffected by
dimm (Fig. 6C), we
were able to uncouple neuropeptide transcription from potential effects of
dimm on secretory activity. Thus, when ap-Gal4 drove ectopic
expression of the pdf neuropeptide gene, ectopic pdf mRNA
levels were unaffected in dimm-/- larvae
(Fig. 7A,B). By contrast,
ectopic PDF protein levels were severely reduced. We performed immunostaining
for two peptide epitopes of the proPDF precursor
(Renn et al., 1999): PAP
(Fig. 7C,F) and PDF
(n=20; data not shown). All 34 (c929-positive) neurons
displayed severely reduced immunostaining for both PDF-related epitopes.
Additional ventral abdominal neurons served as internal controls. These
included 44 neurons that also displayed ectopic pdf expression driven
by ap-Gal4, and a set of approximately eight native pdf
neurons (not ap-positive). All of the internal control cells were
c929 negative, and PAP/PDF immunostaining in these neurons was
unaffected in dimm-/- larvae
(Fig. 7C-F). Thus,
dimm was required within c929-positive neurons for the
maintenance of ectopic PDF neuropeptide levels, but not of ectopic
pdf mRNA.
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DISCUSSION |
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The bHLH domain of the predicted Dimm protein showed the highest degree of
sequence identity with the mouse, rat and human Mist1 proteins. These proteins
may be orthologs (Moore et al.,
2000). Interestingly, mouse Mist1 is present in many adult
peripheral tissues, but within these tissues it is found only in serous
exocrine cells (Pin et al.,
2000
). The restriction of mouse Mist1 expression to dedicated
secretory cells suggests that dimm and mouse Mist1 may both control
levels of secretory activity, and so may perform evolutionarily conserved
functions. Other members of the Atonal family are expressed in both
differentiating and terminally differentiated cells (e.g. NeuroD)
(Morrow et al., 1999
). Several
mammalian Atonal family bHLH proteins have previously been implicated in
earlier stages of endocrine cell development, including cell lineage
commitment (e.g. Yang et al.,
2001
) and endocrine cell differentiation
(Sheng and Westphal,
1999
).
In Drosophila, dimm performs a novel, pro-secretory function in a
diverse population of peptidergic CNS and PNS neurons and endocrine cells. In
its absence, peptidergic cells complete many aspects of their differentiation
some express low levels of appropriate peptide transmitters. However,
they uniformly fail to display normal amplified levels of secretory activity,
which is a characteristic and fundamental property of peptidergic secretory
cells (Arvan and Castle, 1998).
How such cells acquire and maintain this capacity is largely unknown. We have
shown that it is under the control of specific genetic mechanisms, as revealed
by animals deficient in expression of the dimm gene. These
experiments indicate that dimm plays a fundamental role in the
differentiation of neuroendocrine lineages.
We propose a working model in which Dimm directly regulates transcription
of genes required for production of a neuroendocrine phenotype genes
encoding neuropeptides, peptide hormones and peptide biosynthetic enzymes.
Consistent with this model, we found that dimm reduces the normally
high levels of Fmrf neuropeptide mRNA in specific neuroendocrine
cells. In addition, Dimm also may regulate expression of proteins (e.g.
transcription factors, or structural or regulative proteins of dense core
granules) that are important for the function and amplification of the
secretory pathway [e.g., as suggested by Kim et al.
(Kim et al., 2001)]. Dimm
functions after cell fate determination and during the early differentiation
of these neurons in dimm mutants, affected peptidergic
neurons are present, arborize normally and often express low levels of
appropriate neuropeptides.
Some secretory proteins form dense aggregations (`progranules') in the
trans-Golgi network prior to their uptake into immature secretory granules.
Similarly, condensation of secretory proteins during subsequent granule
maturation may be required for their retention in maturing granules
(Arvan and Castle, 1998).
Therefore, direct reductions in the levels of a small number of target
secretory proteins in dimm mutant cells may lead to a secondary
disruption in aggregation or condensation of other proteins. In turn, these
effects could lead to loss of most secretory proteins by mis-routing and
degradation. This may account for our observation that secretory peptide
levels could be reduced in a dimm mutant background, despite the
artificial elevation of the cognate secretory peptide mRNA
(Fig. 7).
Does dimm also regulate the constitutive secretory pathway?
Although constitutive secretion was quantitatively affected by loss of
dimm function, mutant neurons maintained their normal cellular
morphology. These observations suggest that Dimm has only moderate effects on
the constitutive secretory pathway. Given the physical interactions between
cargoes destined for the regulated and constitutive pathways
(Arvan and Castle, 1998), the
reduction in constitutive secretion may reflect an indirect effect of
disruptions in the regulated pathway.
We favor the view that during development and maturity, dimm
expression is a crucial determinant of high secretory protein expression in
neuroendocrine cells. This hypothesis was supported by the gain-of-function
analysis. Overexpression of dimm in a wild-type background produced
higher levels of LK expression in the normally LK-positive Br1 neuroendocrine
neuron. It also increased the number of cells that display the specific LK
neuroendocrine phenotype, but only within the immediate proximity of Br1. In
this case, dimm overexpression was driven by a promoter
(ap-GAL4) that is only expressed in postmitotic neurons. Therefore,
it appears likely that the additional LK immunoreactive neurons represent
cells that normally express LK but at levels that are too low to be detected.
In addition, the limited number of ectopic leukokinin cells is likely a
function of the specific GAL4 driver used (ap is only
expressed in a subset of cells), and the marker assayed (LK is only expressed
in 20 out of 10,000 neurons). Although the complete extent of the effects
of dimm, when overexpressed, is not yet known it is likely to be
large, as UAS-dimm produces large-scale embryonic lethality when
driven by the pan-neuronal elav-GAL4 (D. P., unpublished).
Accordingly, we propose that dimm promotes diverse neuroendocrine
cell fates in different cellular locales, depending on local cellular context
and identity. We observed dimm expression soon after cells cease
dividing, and in its absence, most of these cells were deficient in
`transmitter expression'. Thus, Dimm appears to function like NeuroD proteins,
which are also members of the Atonal family and which act as cell
differentiation factors (Hassan and
Bellen, 2000).
Analysis within the identified, neuroendocrine Tv neurons may be especially
informative to reveal further details of the mechanisms of dimm
action. Four regulatory factors have now been defined that affect FMRF
neuropeptide levels in Tv neurons. Loss-of-function ap
(Benveniste et al., 1998),
Chip (Van Meyel et al.,
2000
) and dimm (this report) alleles all decrease
Tv-specific FMRF expression, but do not influence Tv survival or morphology.
Likewise, the squeeze (sqz) gene helps regulate Tv-specific
FMRF levels (S. Thor, personal communication). Within Tv neurons, ap,
Chip, dimm and sqz may function in a linear pathway to regulate
Fmrf gene expression, akin to the sequential actions of the bHLH
protein MASH1 and the Phox2 homeoproteins in neurons of the locus coeruleus
(Pattyn et al., 2000
).
Alternatively, they may work in parallel fashion, akin to the synergistic
interactions between the bHLH NeuroD1 and the LIM homeoproteins Lmx1.1 and
Lmx1.2 to control insulin expression
(Ohneda et al., 2000
). As a
first step, we have shown that ap promoter function is independent of
dimm. Further work will permit description of the molecular pathways
controlling qualitative and quantitative aspects of neuroendocrine cell
differentiation in vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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