Peptidergic neurosecretory cells in insects: Organization and control by the bHLH protein DIMMED

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Abstract

This review considers evidence that defines a role for the transcription factor DIMMED in the regulation of insect neurosecretory cells. Genetic anatomical and molecular data all suggest DIMMED is a dedicated controller of the regulated secretory pathway. DIMM is normally expressed within diverse neuropeptide-expressing cells and appears highly correlated with a neurosecretory cell fate. Loss of DIMM is associated with deficits in display of neuropeptides and neuropeptide-associated enzymes. Gain of DIMM promotes such display in peptidergic cells and can confer such neurosecretory properties onto conventional neurons. We review models proposed to explain how DIMMED regulates these essential cellular properties.

Introduction

Neurosecretory (NS)1 cells are dedicated to make, store and release large amounts of specific peptides and peptide hormones via the regulated secretory pathway (RSP). Release often occurs quickly in response to precise and appropriate stimuli. Such cells are found at all levels of the central (CNS) and peripheral (PNS) nervous systems and release their products within the brain or into the circulation. NS cells play important roles in regulating developmental changes (e.g., McBrayer et al., 2007), physiological adaptations (e.g., Kim and Rulifson, 2004) and behavioral sequence (e.g., Renn et al., 1999). How are the complex properties of these professional secretory cells coordinated and maintained? This review considers the molecular mechanisms that distinguish insect NS cells from other neurons and permit them to make very high use of the RSP. We speculate that a better understanding of such mechanisms will help explain how different secretory cell populations arise, and how they are functionally-organized to support development, physiology and behavior.

Neuropeptides and peptide hormones derive from larger precursors and gain final forms due to processing by dedicated sets of enzymes. The precursors are targeted to the RSP via the trans-Golgi, then are packaged into and released from large dense-core vesicles (DCVs-70–200 nm in diameter—reviewed by Crivellato et al. (2006)). DCVs are distinguishable from the smaller synaptic vesicles (30–40 nm) that contain fast-acting transmitters. Although the release machinery that mediates DCV exocytosis is similar to that of synaptic vesicles, the timing of DCV release is typically much slower (Martin, 2003), and thus the regulation of DCV release appears to involve distinct molecules (e.g., Fukuda et al., 2004).

Many cell-intrinsic regulatory mechanisms that help establish specific transmitter phenotypes have been discovered (e.g., Ding et al., 2003, Cheng et al., 2004). However, the relevant developmental mechanisms that underlie peptidergic cell properties, especially those of peptidergic NS cells, remain poorly understood. Transcription factors such as Mash1, Otp, Brn2, Sim1 and Sim2 are known to regulate the early differentiation of hypothalamic neuroendocrine centers by their expression in neuronal progenitors and in pre-migratory neurons (Acampora et al., 1999, Wang and Lufkin, 2000, Schonemann et al., 1995, Nakai et al., 1995, Michaud et al., 1998, Goshu et al., 2004, Hosoya et al., 2001). However, where they have been examined in detail, these genes appear to function very early in the proliferation, migration or other initial steps in the determination of different neuroendocrine cell precursors. Hence, very little is known about the intrinsic regulatory factors that directly organize the maturation of peptidergic cellular properties. Such properties involve proper scaling and modulation of the RSP by which peptides and peptides hormones are packaged and released.

While there have been many mechanistic studies of proteins that utilize the RSP (e.g., Arvan and Castle, 1998, Beuret et al., 2004, Kim et al., 2001), only a handful of studies have addressed the genetic basis underlying its display. That there is a dedicated genetic basis is underscored by, for example, the identification of variants of well-known cell lines (AtT-20 and PC12) which are deficient in the maintenance of RSP molecular and sub-cellular features (Day et al., 1995, Malosio et al., 1999). Such variants display few if any DCVs and lack molecular expression profiles that are normally associated with dedicated secretory cells. This phenotype is reversible in response to stimulation of the cAMP pathway (Day et al., 1995), suggesting it may be normally regulated as a consequence of cellular interactions. Furthermore, several independent studies have implicated the transcription factor REST as a negative regulator of the differentiated secretory state in the PC12 cell variants (e.g., Pance et al., 2006). Interestingly, inhibition of REST permits transcription of genes associated with the regulated secretory pathway, but typically not the accumulation of their encoded proteins. It follows that regulation of the RSP in these PC12 variants must involve both transcriptional and post-transcriptional control mechanisms. Here we review studies in Drosophila that suggest there is a genetic basis underlying development and maintenance of the NS cell fate in insects. In particular, we consider evidence that a specific basic helix–loop–helix protein called DIMMED (DIMM) has a special role in this mechanism in Drosophila as a dedicated pro-secretory factor.

Section snippets

Molecular structure of the dimmed gene

The dimmed (CG8667, mist-r) gene consists of three exons spanning a ∼9 kb fragment of cytogenetic region 39CD (on the 2nd chromosome) (Hewes et al., 2003; D. Park and P. Taghert, unpublished data). The predicted DIMM protein consists of a single 390 amino acid (AA) open-reading frame with a centrally-positioned basic helix–loop–helix (bHLH) domain (AA 157–209). The specific sequence of DIMM’s bHLH domain signals its membership in the Atonal/NeuroD superfamily of bHLH proteins and it shows

How many DIMM cells are peptidergic?

To survey DIMM expression within peptidergic systems systematically, Park et al. (2008a) used a panel of markers representing 26 different peptide-encoding genes. As mentioned above, there are 306 DIMM-positive cells identifiable at the 3rd instar larval brain (Park et al., 2008a) and these correspond to the vast majority of larval neurons that display high levels of PHM immunostaining (Hewes et al., 2003). 64% of DIMM cells within the larval CNS can be identified by one or more of the 24 CNS

DIMM cells are LEAP cells

If DIMM-expressing peptidergic cells represent a minority of all peptidergic cells, do they have characteristics similar enough to suggest a unifying theme? The available evidence suggests they do. Among peptidergic cells, several attributes set DIMM cells apart. These were especially evident from inspection of peptidergic neuronal cohorts that were mixed for DIMM expression (e.g., some corazonin cells are consistently DIMM-positive and others are not). Three essential properties distinguish

DIMM controls NS differentiation but not survival

Strong dimm loss-of-function alleles display a large-scale reduction in the staining intensity for neuropeptides, and for neuropeptide processing enzymes, in dimm-positive neurons within the larval CNS and PNS. Importantly, loss of dimm function does not appear to affect cell survival or cell morphogenesis (Hewes et al., 2003). When DIMM is mis-expressed or over-expressed, it can cause cellular disruption; wide-scale mis-expression (with a pan-neuronal gal4 driver like elav-gal4) causes

DIMM forms homodimers to activate PHM

DIMM is a Class 2 bHLH transcription factor, related to the ATONAL superfamily, many members of which are involved in neurogenesis (Moore et al., 2000). Most Class 2 bHLH proteins form heterodimers with a Class 1 (ubiquitous) bHLH protein, E12/E47 in mammals, and daughterless in Drosophila, to activate downstream target genes (Massari and Murre, 2000). In the case of DIMM, homodimers appear to be preferred over heterodimeric combination with DAUGHTERLESS (Allan et al., 2005) and are essential

The DIMM ortholog Mist1 is expressed in serous exocrine cells

It is reasonable to assume that NS cells in different animals operate within the same cellular constraints. If so, then regulatory programs that are organized by DIMM in insect NS cells may help define similar programs in vertebrate NS cells. DIMM is an ortholog of the vertebrate Mist1 (Moore et al., 2000), based on significant similarity (78%) that is limited to the bHLH domain. In spite of this restricted sequence similarity, it is remarkable that both proteins display restricted expression

Summary

DIMM appears to control packaging, storage and/or secretion of NS peptides within DCVs, but its action mechanisms remain obscure. Conceptually, there are three principal means by which DIMM could achieve this result (Fig. 3): (i) by driving the synthesis of rate-limiting secretory components (e.g., critical DCV constituent proteins); (ii) by diminishing the rate of turnover of secretory components, or (iii) by diminishing the rate of release of peptidergic DCVs. Having identified the DIMM

Acknowledgments

We thank Tarik Hadzic for comments on a draft of this review. Work in the laboratory is supported by NIH Grants NS21749, MH067122, and GM85788, and by the Imaging Core Facility of the P30 NIH Neuroscience Blueprint Core Grant (#NS057105) to Washington University.

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