BIOLOGICAL OVERVIEW

Neuroendocrine cells are specialized to produce, maintain and release large stores of secretory peptides. The Drosophila dimmed/Mist1 Atonal family bHLH gene confers just such a pro-secretory phenotype on neuroendocrine cells. dimmed is expressed selectively in central and peripheral neuroendocrine cells. In dimmed mutants, these cells survive, and adopt normal cell fates and morphology. However, they display greatly diminished levels of secretory peptide mRNAs, and of diverse peptides and proteins destined for regulated secretion. Secretory peptide levels are lowered even in the presence of artificially high secretory peptide mRNA levels. In addition, overexpression of dimmed in a wild-type background produces a complimentary phenotype: an increase in secretory peptide levels by neuroendocrine cells, and an increase in the number of cells displaying a neuroendocrine phenotype. It is proposed that dimmed encodes an integral component of a novel mechanism by which diverse neuroendocrine lineages differentiate and maintain the pro-secretory state (Hewes, 2003).

Dimm is the first example of a dedicated pro-secretory factor. Dimm is necessary to confer neuroendocrine features onto peptidergic neurons that, in its absence, survive with normal neuronal properties. In addition, Dimm overexpression produces supra-normal levels of neuropeptide expression in peptidergic neurons and the appearance of additional cells with neuroendocrine features. From this genetic analysis, it is suggested that neuroendocrine cell differentiation includes two interrelated, but separate sets of instructions. The first specifies the identity of the neuropeptide(s) or peptide hormone(s) to be expressed, while the second, which involves Dimm, specifies the level of regulated secretory activity (Hewes, 2003).

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, 2000). Interestingly, mouse Mist1 is present in many adult peripheral tissues, but within these tissues it is found only in serous exocrine cells (Pin, 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). Several mammalian Atonal family bHLH proteins have previously been implicated in earlier stages of endocrine cell development, including cell lineage commitment (Hewes, 2003 and references therein).

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, a characteristic and fundamental property of peptidergic secretory cells. How such cells acquire and maintain this capacity is largely unknown. This capacity 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 (Hewes, 2003).

A working model is proposed 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, dimm mutation was found to reduce 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. 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 (Hewes, 2003).

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. 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 the observation that secretory peptide levels are reduced in a dimm mutant background, despite the artificial elevation of the cognate secretory peptide mRNA (Hewes, 2003).

Does dimm also regulate the constitutive secretory pathway? Although constitutive secretion is quantitatively affected by loss of dimm function, mutant neurons maintain 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, the reduction in constitutive secretion may reflect an indirect effect of disruptions in the regulated pathway (Hewes, 2003 and references therein).

The view is favored that during development and maturity, dimm expression is a crucial determinant of high secretory protein expression in neuroendocrine cells. This hypothesis is supported by the gain-of-function analysis. Overexpression of dimm in a wild-type background produces higher levels of leucokinin (LK) expression in the normally LK-positive neuroendocrine neuron Br1. It also increases 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 (Hewes, 2003).

Accordingly, it is proposed that dimm promotes diverse neuroendocrine cell fates in different cellular locales, depending on local cellular context and identity. dimm expression is observed soon after cells cease dividing, and in its absence, most of these cells are 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 (Hewes, 2003).

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 apterous, Chip and dimm 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. 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. 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 (Hewes, 2003 and references therein). As a first step, it has been 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 (Hewes, 2003).

PROTEIN STRUCTURE

Amino Acids - 390

Structural Domains

Among Drosophila bHLH proteins CG5545 is closely related to the vertebrate Beta 3 protein, a repressor molecule (96% sequence identity in the bHLH domain), and the Olig proteins involved in oligodendritic precursor formation. CG8667 has closest sequence identity to the vertebrate Mist1 protein (Lemercier, 1997), a negative regulatory factor of MyoD activity (78% identical over the entire bHLH domain and 92% identical in the basic domain alone). It is proposed that this protein should be named Mistr (Mist 1-related protein) (Moore, 2000).

date revised: 20 July 2003

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