InteractiveFly: GeneBrief

dimmed: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

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).

REGULATION

Transcriptional Regulation

Ap-let neurons--a peptidergic circuit potentially controlling ecdysial behavior in Drosophila

A set of peptidergic neurons is conserved throughout all developmental stages in the Drosophila central nervous system. A small complement of 28 apterous-expressing cells (Ap-let neurons) in the ventral nerve cord (VNC) of Drosophila larvae co-express numerous gene products. The products include the neuroendocrine-specific bHLH regulator called Dimmed (Dimm), four neuropeptide biosynthetic enzymes (PC2, Fur1, PAL2, and PHM), and a specific dopamine receptor subtype (dDA1). For the PC2, Fur1, and PAL2 enzymes, and for the dDA1 receptor, this neuronal pattern represents the vast majority of their total expression in the VNC. In addition, while Dimm and PHM are present in the peritracheal Inka cells in larvae, pupae, and adults, Ap, PC2, Fur1, PAL2, and dDA1 are not. PC2, PAL2, and DA1 receptor expression are all controlled by both dimm and ap. Previous genetic analysis of animals deficient in PC2 revealed an abnormal larval ecdysis phenotype. Together, these data support the hypothesis that the small cohort of Ap-let interneurons regulates larval ecdysis behavior by secretion of an unidentified amidated peptide(s). This hypothesis further predicts that the production of the Ap-let neuropeptide(s) is dependent on each of four specific enzymes, and that a certain aspect(s) of its production and/or release is regulated by dopamine input (Park, 2004).

Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development

Drosophila embryonic neuroblasts generate different cell types at different time points. This is controlled by a temporal cascade of Hb->Kr->Pdm->Cas->Grh, which acts to dictate distinct competence windows sequentially. In addition, Seven up (Svp), a member of the nuclear hormone receptor family, acts early in the temporal cascade, to ensure the transition from Hb to Kr, and has been referred to as a 'switching factor'. However, Svp is also expressed in a second wave within the developing CNS, but here, the possible role of Svp has not been previously addressed. In a genetic screen for mutants affecting the last-born cell in the embryonic NB5-6T lineage, the Ap4/FMRFamide neuron, a novel allele of svp was isolated. Expression analysis shows that Svp is expressed in two distinct pulses in NB5-6T, and mutant analysis reveals that svp plays two distinct roles. In the first pulse, svp acts to ensure proper downregulation of Hb. In the second pulse, which occurs in a Cas/Grh double-positive window, svp acts to ensure proper sub-division of this window. These studies show that a temporal factor may play dual roles, acting at two different stages during the development of one neural lineage (Benito-Sipos, 2011).

This study has found that Svp is expressed in two pulses and plays two different roles in the NB5-6T lineage. Initially, Svp is expressed briefly in the early part of this lineage, where it acts to control the downregulation of the first temporal factor, Hb. Subsequently, Svp is expressed in the late part of this lineage, in the Ap window, in a highly dynamic fashion: initiated in all four Ap neurons, to be downregulated in the first- and last-born Ap cells. In the second expression phase, Svp acts to suppress Col and Dimm, thereby preventing the first-born Ap neuron fate, Ap1/Nplp1, from being established in the subsequently born Ap2 and Ap3 neurons. Misexpression studies further indicate that Svp also suppresses the last-born Ap neuron fate, Ap4/FMRFa, from being established in Ap2/3 (Benito-Sipos, 2011).

Previous studies of Svp demonstrated that it is expressed in a brief pulse in the majority of early embryonic neuroblasts, where it acts to suppress Hb, thereby allowing for the switch to the next stage of temporal competence. Recently, studies have identified additional factors involved in the downregulation of Hb: the pipsqueak-domain proteins Distal antenna and Distal antenna-related (herein referred to collectively as 'Dan'). Dan is expressed somewhat earlier than Svp, and is also maintained in a longer pulse. svp and dan do not regulate each other, and although they can be activated by ectopic hb expression, neither Svp nor Dan expression is lost in hb mutants. This raises the intriguing questions of how Svp and Dan are activated during early stages of lineage progression, and how they become downregulated at the appropriate stage (Benito-Sipos, 2011).

Another interesting complexity with respect to Svp expression and function pertains to the fact that the Hb window is of different size in different lineages. For example, in NB6-4T and NB7-3, Hb is downregulated in the neuroblast immediately after the first division, whereas in NB5-6T, Hb expression is evident during three divisions. In line with this, no Svp expression is observed in NB5-6T until stage 10, when the neuroblast has already gone through two rounds of division. How the on- and offset of Svp, and perhaps Dan, expression is matched to the specific lineage progression of each unique neuroblast lineage, to thereby allow for differing Hb window sizes, is an interesting topic for future studies (Benito-Sipos, 2011).

Svp is re-expressed in the NB5-6T lineage in a second pulse. In contrast to the early pulse of Svp expression, where there is no evidence for temporal genes controlling Svp, it was found that the second pulse of Svp expression is regulated by the temporal genes cas and grh. However, it was not found that svp is important for the expression of Cas or Grh. Instead, svp participates in the sub-division of the Cas/Grh temporal window, i.e. the Ap window. Based upon the idea that Svp is regulated by temporal genes, and acts to sub-divide a broader temporal window, it could be referred to as a 'sub-temporal' factor in the latter part of the NB5-6T lineage (Benito-Sipos, 2011).

The expression of Svp is dynamic also in the second pulse of expression, commencing in the neuroblast at stage 14 -- after the three first Ap neurons are born -- and being maintained in the neuroblast until it exits the cell cycle at stage 15. At late stage 14 and 15, Svp expression becomes evident in all four Ap neurons, but it is rapidly downregulated from Ap1 and Ap4 during stages 16 and 17. Svp is, however, maintained in the Ap2 and Ap3 neurons into late embryogenesis. The role of svp in the Ap window appears to be to ensure proper specification of the Ap2/3 interneurons, generated in the middle of the Ap window. This is achieved by svp suppressing the first- and last-born Ap neuron fates: the Ap1/Nplp1 and Ap4/FMRFa fates. With regard to the suppression of the Ap1 fate, one important role for svp is to suppress Col expression in Ap2/3. Importantly, the temporal delay in Svp expression when compared to Col -- commencing two stages after Col in the Ap neurons -- allows for col to play its critical early role in Ap neuron specification: activating ap and eya. The timely suppression of Col in Ap2/3 is mediated also by sqz and nab, and the loss of Nab expression in svp mutants may be a contributing factor to the failure of Col downregulation in svp. However, the potent function of svp in suppressing Ap1/Nplp1 fate when misexpressed postmitotically from ap^Gal4 does not appear to require Nab, as Nab is not ectopically expressed in these experiments. Thus, svp may act via several routes to prevent Ap1/Nplp1 fate from being established in the Ap2/3 cells: by suppressing Col and by activating Nab (Benito-Sipos, 2011).

Regarding the second role of svp in the Ap window -- the suppression of the Ap4/FMRFa fate -- it is less clear what the target(s) may be. However, a common denominator for both the Ap1/Nplp1 and the Ap4/FMRFa neurons is the expression of Dimm. Dimm, a basic-helix-loop-helix protein, is a critical determinant of the neuropeptidergic cell fate, and also controls high-level neuropeptide expression in many neuropeptide neurons. Both svp loss and gain of function results in robust effects upon Dimm expression in the NB5-6T lineage, indicating that Dimm is an important target for svp. However, dimm mutants show only reduced levels of FMRFa expression, and thus svp is likely to regulate additional targets to prevent the Ap4/FMRFa cell fate in the Ap2/3 neurons (Benito-Sipos, 2011).

Another interesting phenotype in svp mutants, pertaining to the second pulse of Svp expression in the NB5-6T lineage, is the finding of one to two extra Ap neurons. This indicates that the NB5-6T neuroblast undergoes one to two extra rounds of division, and that the expression of Svp in the neuroblast during stage age 14-16 is important for precise cell cycle exit. Interestingly, the other temporal (cas and grh) and sub-temporal (sqz and nab) genes acting in the latter part of the NB5-6T lineage also play roles in controlling cell cycle exit. Moreover, studies of neuroblast cell cycle exit in other neuroblasts, both embryonic and postembryonic, have also shown roles for grh and svp in these decisions. Thus, a picture is emerging in which late temporal and sub-temporal genes may be broadly involved in controlling timely cell cycle exit of many neuroblasts (Benito-Sipos, 2011).

The early role of svp, in its first expression pulse, is to suppress Hb expression. Svp is expressed transiently by most if not all neuroblasts, and the regulation of Hb also appears to be a global event. Similarly, the second pulse of Svp expression has been observed in many lineages, although the role for svp in this later pulse was hitherto unknown. The findings of a role for svp as a sub-temporal gene in the latter part of the NB5-6T lineage indicates that svp may play such roles in many lineages. However, it should be noted that global changes in Col, Dimm and Eya expression in the embryonic central nervous system (CNS) are not seen. Thus, unlike the more universal role of svp in regulating Hb during the first pulse, the putative sub-temporal function of the second pulse of svp expression in other lineages must be highly context-dependent and involve other targets (Benito-Sipos, 2011).

In mammals, the svp orthologues COUP-TFI and -II are expressed dynamically in the developing CNS. Functional studies reveal a number of important roles for COUP-TFI/II during nervous system development, and mutant mice display aberrant neuro- and gliogenesis, accompanied by axon pathfinding defects. Intriguingly, recent studies have revealed that COUP-TFI/II acts in a temporal manner to control the timing of generation of sub-classes of neurons and glia in the developing mouse brain. Given that the other genes described in this study are also conserved, it is tempting to speculate that temporal and sub-temporal cascades similar to those outlined in this study are also used in the mammalian CNS during development (Benito-Sipos, 2011).

Targets of Activity

Transcriptional regulation of neuropeptide and peptide hormone expression by the Drosophila dimmed and cryptocephal genes

The regulation of neuropeptide and peptide hormone gene expression is essential for the development and function of neuroendocrine cells in integrated physiological networks. In insects, a decline in circulating ecdysteroids triggers the activation of a neuroendocrine system to stimulate ecdysis, the behaviors used to shed the old cuticle at the culmination of each molt. Two evolutionarily conserved transcription factor genes, the basic helix-loop-helix (bHLH) gene dimmed (dimm) and the basic-leucine zipper (bZIP) gene cryptocephal (crc), control expression of diverse neuropeptides and peptide hormones in Drosophila. Central nervous system expression of three neuropeptide genes (Dromyosuppressin, FMRFamide-related and Leucokinin) is activated by dimm. Expression of Ecdysis triggering hormone (ETH) in the endocrine Inka cells requires crc; homozygous crc mutant larvae display markedly reduced ETH levels and corresponding defects in ecdysis. crc activates ETH expression though a 382 bp enhancer, which completely recapitulates the ETH expression pattern. The enhancer contains two evolutionarily conserved regions, and both are imperfect matches to recognition elements for activating transcription factor-4 (ATF-4), the vertebrate ortholog of the CRC protein and an important intermediate in cellular responses to endoplasmic reticulum stress. These regions also contain a putative ecdysteroid response element and a predicted binding site for the products of the E74 ecdysone response gene. These results suggest that convergence between ATF-related signaling and an important intracellular steroid response pathway may contribute to the neuroendocrine regulation of insect molting (Gauthier, 2006).

DIMM has been proposed as a direct regulator of neuroendocrine gene expression in most neuropeptidergic cells. Quantitative RTPCR results, supplemented by in situ hybridization, show that DIMM upregulates the levels of mRNAs derived from at least three neuropeptide genes, Fmrf, Lk and Dms. These findings provide strong support for DIMM as a key regulator of multiple neuroendocrine genes. The LIM-homeodomain gene apterous (ap) also controls Fmrf and Lk gene expression. ap acts cell-autonomously to stimulate dimm expression, but the AP and DIMM proteins can also physically interact, and they may function together in regulating Fmrf. Several other factors, including the transcriptional co-factors encoded by dachshund and eyes absent, the zinc-finger gene squeeze, and the retrograde bone morphogenetic protein (BMP) pathway, act in combinatorial fashion with dimm and ap to control Fmrf expression. However, other neuropeptidergic cells appear to use only portions of this code. For example, ap and dimm appear to contribute to the expression of Lk in Fmrf-negative cells (the segmental cells A1–A7 and possibly the brain lobe cells Br1). Even within the population of Lk cells, loss of dimm results in very different effects in different neurons, with a reduction in Lk transcript levels in cells A1–A7, and an increase (or no change) in Lk levels in the Br1 and the subesophageal SE neurons. How do these relatively widely expressed factors interact with other regulatory proteins to produce cell type-specific patterns of neuropeptide gene expression? It will be of interest to determine which other elements of the combinatorial pro-Fmrf code are used to control Lk and Dms expression, and to identify additional factors that interact with dimm to control expression of these neuropeptide genes (Gauthier, 2006).

Does dimm control neuropeptide levels through an additional indirect mechanism? No changes were detected in levels of three neuropeptide biosynthetic enzyme mRNAs, Phm, Fur1 and amon, in the qRTPCR analysis. This is in contrast to earlier immunocytochemical studies, in which a marked reduction was observed in the protein products of these genes in dimm mutant CNS. In some cases, these differences may reflect the spatial insensitivity of the qRTPCR methods, as was confirmed by in situ hybridization analysis of Lk expression. Phm, in particular, may belong in this category. Although levels of PHM and DIMM expression are strongly correlated, PHM is also highly expressed in many other tissues that do not express dimm. Any dimm-dependent change in Phm expression may have been obscured by the much larger pool of dimm-independent Phm mRNA in whole-animal qRTPCR analysis (Gauthier, 2006).

DIMM may regulate levels of other neuroendocrine proteins through a route that does not involve interactions between DIMM and cis-regulatory elements in the respective genes. Evidence was obtained in support of this hypothesis in an earlier analysis of an ectopically expressed neuropeptide in dimm mutant cells; levels of ectopic PDF protein were strongly reduced while dimm had no effect on levels of the cognate Pdf mRNA. This study showed that larvae homozygous for a specific loss-of-function mutation in dimm displayed reduced levels of endogenous ETH-like protein(s), but not ETH mRNA, in the endocrine Inka cells, a site of dimm gene expression. This may occur simply through a dimm-dependent change in levels of one secreted protein, such as PHM, that may disrupt the formation of multi-protein aggregates required for neuropeptide sorting into secretory granules. Alternatively, recent studies on the mouse ortholog of dimm, Mist1, suggest that dimm may play a more direct role in the management of secretory granule budding from the trans-Golgi network. In Mist1 knockout mice (Mist1^KO), pancreatic exocrine cells display reduced intracellular organization. Moreover, the Mist1^KO phenotype is partially phenocopied in animals mutant for the Rab3D gene, a small GTPase involved in secretory granule trafficking. Further studies on the regulation of ETH, PHM and Rab3-like proteins, and on the biochemical interactions among them, may shed light on the cellular mechanisms underlying the indirect actions of DIMM (Gauthier, 2006).

Mutations in the crc gene result in pleiotropic defects in ecdysone-regulated events during molting and metamorphosis. Many of the morphological defects are associated with a failure of the insect to properly complete ecdysis, a stereotyped set of behaviors required for shedding of the old cuticle at the culmination of each molt. Several neuropeptides and peptide hormones, including ETH, play critical roles in organizing and triggering ecdysis behavior (Gauthier, 2006).

This study provides four independent lines of evidence that demonstrate a crucial role for crc in the upregulation of ETH mRNA levels. First, a marked reduction by qRTPCR is observed in levels of ETH transcripts [but not in mRNAs encoding CCAP or EH, two other neuropeptides involved in the neuropeptide hierarchy controlling ecdysis in crc mutant larvae. Second, in situ hybridization revealed a strong reduction in ETH mRNA levels in the endocrine Inka cells in crc mutant larvae. Third, the intensity of anti-PETH immunoreactivity was markedly reduced in crc¹/crc¹ homozygotes. Fourth, EGFP fluorescence driven by an ETH-EGFP reporter gene was reduced in crc mutant larvae. Therefore, CRC is a strong activator of ETH gene expression, and loss of CRC results in a corresponding reduction in levels of the ETH protein (Gauthier, 2006).

Despite the molecular identification of the crc locus, almost six decades after the original description of the first crc allele, the causes of the molting and metamorphosis defects in crc mutants remained unclear. The current results suggest a simple model to explain the crc mutant phenotype. Strong hypomorphic or null mutations in crc and ETH both severely disrupt ecdysis. These defects include weak, irregular and slower ecdysis contractions and a failure to shed old cuticular structures, leading to retention of two and sometimes three sets of mouthparts into the next larval stage. These similarities in molting defects, taken together with the observation that crc is required for normal expression of ETH mRNA and ETH protein, point to the loss of ETH signaling as the likely proximate cause of the ecdysis defects observed in crc mutants (Gauthier, 2006).

Despite the specific effects of crc on ETH transcription in the Inka cells, crc is widely expressed, suggesting a cellular housekeeping function. The vertebrate ATF-4 protein is also ubiquitously expressed. In addition, the upregulation of ATF-4 constitutes a milestone of many cellular stress response pathways including oxidative stress, amino acid deprivation, and hypoxia. In the tobacco hornworm, Manduca sexta, levels of ETH fluctuate during the molts and are regulated by circulating ecdysteroids. It is hypothesized that CRC contributes to the regulation of ETH gene expression during this period, perhaps in response to signals from the tracheae (Gauthier, 2006).

Peaks in circulating levels of the ecdysteroid hormone, 20-hydroxyecdysone (20HE), initiate and coordinate each molt. A subsequent decline in 20HE levels is required for ecdysis, and the activation of these behaviors involves a hierarchical cascade of peptide hormone and neuropeptide signals that is triggered by ETH. Is CRC required in order to maintain ETH expression, or is CRC involved in regulating transcription of the ETH gene during the molts? While it is not known whether ETH mRNA levels fluctuate during Drosophila post-embryonic development, the regulation of ETH levels by ecdysteroids in molting Manduca sexta, and the analysis of the conserved region sequences CR1 and CR2 (located 91-171 bp upstream of the ETH translational start site), provides tantalizing clues to possible coordinate regulation of ETH gene expression by CRC and ecdysone response genes. There is substantial overlap between the predicted CRC binding site in CR1 and a putative ecdysteroid response element (EcRE). In addition, a potential binding site in CR2 for products of the E74 early ecdysone-inducible gene. E74 expression is induced directly by 20HE, and it encodes transcription factors that regulate other ecdysone response genes. Mutations that specifically disrupt E74B, which likely binds the same consensus as E74A, display defects associated with pupal ecdysis that closely phenocopy crc. In future, studies will focus on whether ETH expression is regulated by elements in both CR1 and CR2 in an ecdysteroid-dependent manner, and whether CRC, E74B and other factors in the ecdysone-response pathway interact competitively or cooperatively at these sites (Gauthier, 2006).

Neuronal cell fate specification by the molecular convergence of different spatio-temporal cues on a common initiator terminal selector gene

The extensive genetic regulatory flows underlying specification of different neuronal subtypes are not well understood at the molecular level. The Nplp1 neuropeptide neurons in the developing Drosophila nerve cord belong to two sub-classes; Tv1 and dAp neurons, generated by two distinct progenitors. Nplp1 neurons are specified by spatial cues; the Hox homeotic network and GATA factor grn, and temporal cues; the hb -> Kr -> Pdm -> cas -> grh temporal cascade. These spatio-temporal cues combine into two distinct codes; one for Tv1 and one for dAp neurons that activate a common terminal selector feedforward cascade of col -> ap/eya -> dimm -> Nplp1. This study molecularly decodes the specification of Nplp1 neurons, and finds that the cis-regulatory organization of col functions as an integratory node for the different spatio-temporal combinatorial codes. These findings may provide a logical framework for addressing spatio-temporal control of neuronal sub-type specification in other systems (Stratmann, 2017).

The Drosophila ventral nerve cord (VNC; defined here as thoracic segments T1-T3 and abdominal A1-A10) contains ~10,000 cells at the end of embryogenesis, which are generated by a defined set of ~800 neuroblasts (NBs). The Apterous neurons constitute a small sub-group of interneurons, identifiable by the selective expression of the Apterous (Ap) LIM-homeodomain factor, as well as the Eyes absent (Eya) transcriptional co-factor and nuclear phosphatase. A subset of Ap neurons express the Nplp1 neuropeptide, but can be sub-divided into the lateral thoracic Tv1 neurons, part of the thoracic Ap cluster of four cells, and the dorsal medial row of dAp neurons. In line with the distinct location of the Tv1 and dAp neurons, studies have revealed that they are generated by distinct NBs; NB5-6T and NB4-3, respectively. A number of studies have addressed the genetic mechanisms underlying the specification of the Tv1 and dAp neurons, and the regulation of the Nplp1 neuropeptide. These have revealed that two distinct spatio-temporal combinatorial transcription factor codes, one acting in NB5-6T and the other in NB4-3, converge on a common initiator terminal selector gene; collier, encoding a COE/EBF transcription factor. Col in turn is necessary and sufficient to trigger a feed forward loop (FFL) consisting of Ap, Eya and the Dimmed (Dimm) bHLH transcription factor, which ultimately activates the Nplp1 gene. Strikingly, the combinatorial coding selectivity of the spatio-temporal cues combined with the information-coding capacity of the FFL results in the selective activation of Nplp1 in only 28 out of the ~10,000 cells within the VNC. While these genetic studies have helped resolve the regulatory logic of this cell specification event, they have not addressed the molecular mechanisms by which the two different spatio-temporal combinatorial codes intersect upon the col initiator terminal selector, to trigger a common terminal FFL, or the molecular nature of the FFL (Stratmann, 2017).

To address this issue, this study has identified enhancers for Tv and dAp neuron expression for the genes in the common Tv1/dAp FFL: col, ap, eya, dimm and Nplp1. Transgenic reporters were generated for these enhancers, both wildtype and mutant for specific transcription factor binding sites, to test their regulation in mutant and misexpression backgrounds. CRISPR/Cas9 technology was used to delete these enhancers in their normal genomic location to test their necessity for gene regulation. Strikingly, this study found that the distinct upstream spatio-temporal combinatorial codes, which trigger col expression in Tv1 versus dAp neurons, converge onto different enhancer elements in the col gene. Hence, the col Tv1 neuron enhancer is triggered by Antp, hth, exd, lbe and cas, while the dAp enhancer is triggered by Kr, pdm and grn. In contrast to this subset-specific enhancer set-up for col activation, the subsequent, col-driven Nplp1 FFL feeds onto common enhancers in each downstream gene. These findings reveal that distinct spatio-temporal cues, acting in different neural progenitors, can trigger the same FFL by converging on discrete enhancer elements in an initiator terminal selector, to thereby dictate the same ultimate neuronal subtype cell fate (Stratmann, 2017).

This study has been able to molecularly decode the Tv1/dAp genetic FFL cascades, bolstering evidence for a complex molecular FFL, based upon sequential transcription factor binding to the downstream genes. The NB4-3 and NB5-6T neuroblasts are born in different regions of the VNC, and express different spatial determinants i.e., Antp, Lbe, Hth, Exd and Gr. As lineage progression commences, they undergo a programmed cascade of transcription factor expression; the temporal cascade. Early temporal factors Kr and Pdm integrate with Grn in NB4-3, while the late temporal factor Cas integrates with Antp, Lbe, Hth and Exd in NB5-6T, to create two distinct combinatorial spatio-temporal codes. These two codes converge on two different enhancers in the col gene, triggering Col expression, and hence the Nplp1 FFL. The FFL, in this case a so-called coherent FFL, where regulators act positively at one or several steps of a cascade, was first identified in E.coli and yeast regulatory networks, but have also been identified in C.elegans and Drosophila. Coherent FFLs can act as regulatory timing devices, exemplified by the action of col in NB5-6T: The initial expression of col in Ap cluster cells triggers a generic Ap/Eya interneuron fate in all four cells, while its downregulation in Tv2-4 and maintenance in Tv1 helps propagate the FFL leading to Nplp1 expression (Stratmann, 2017).

This study has found that the two different spatio-temporal programs converge on col, but on different enhancer elements. However, neither enhancer element gave complete null effects when deleted. Specifically, the 6.3kb col-Tv-CRM shows robust reporter expression, overlaps with endogenous col expression, responds to the upstream mutants, and is affected by TFBS mutations. However, when deleted (generating the col^ΔTv-CRM mutant), it had weak effects upon endogenous col expression in NB5-6T, and no effect upon Eya and Nplp1 expression. Deletion of the col-dAp-CRM (generating the col^ΔdAp-CRM mutant), gave more robust effects with reduction of Col, Eya and Nplp1 in dAp cells, although the expression was not lost completely (Stratmann, 2017).

Early developmental genes, which often are dynamically expressed, may be controlled by multiple enhancer modules, to thereby ensure robust onset of gene expression. This has been reported previously in studies of early mesodermal and neuro-ectodermal development, in which several genes i.e., twist, sog, snail are controlled by multiple distal enhancer fragments, so called 'shadow enhancers', in order to ensure reliable onset of gene expression. The shadow enhancer principle is also supported by recent findings on the Kr gene. Moreover, extensive CRM transgenic analysis, scoring thousands of fragments in transgenic flies, has also supported the shadow enhancer idea, revealing that a number of early regulators, several of which encode for transcription factors, indeed have shadow enhancers. The dichotomy between the col transgenic reporter results and the partial impact on col expression upon deletion of its Tv1 and dAp enhancers, gives reason to speculate that col may be under control of additional enhancers, some of which may be referred to as shadow enhancers (Stratmann, 2017).

The results on the eya, ap, dimm and Nplp1 enhancer mutants stand in stark contrast to the col CRMs findings. For these four genes, the enhancer deletion resulted in robust, near null effects, on expression. It is tempting to speculate that the current findings, combined with previous studies, points to a different logic for early regulators, with highly dynamic patterns, requiring several functionally overlapping enhancers for fidelity, and late regulators and terminal differentiation genes, which may operate with one enhancer that is inactive until the pertinent combinatorial TF codes have been established (Stratmann, 2017).

Analysis of the ap and eya enhancers indicates that Col directly interacts with these enhancers. Both of these enhancer-reporter transgenes are affected in col mutants, and can be activated by ectopic col. Moreover, mutation of one Col binding site in the ap enhancer and two sites in the eya enhancer, was enough to dramatically reduce enhancer activity. Direct action of Col on ap and eya is furthermore supported by recent data on Col genome-wide binding, using ChIP, which demonstrated direct binding of Col to these regions of ap and eya in the embryo. The regulation of ap is an excellent example of the complexity of gene regulation, and studies have identified additional enhancers controlling ap expression in the wing, muscle and brain (Stratmann, 2017).

In contrast to regulation of ap and eya, a direct action of Col on dimm and Nplp1 is less clear. Analysis of the dimm and Nplp1 enhancers did not reveal perfectly conserved Col binding sites. Mutation of multiple non-perfect Col binding sites in the dimm enhancer did not affect reporter expression in the Ap cluster, but did however reduce levels in the dorsal Ap cells. Mutation of non-perfect Col binding sites in the Nplp1 enhancer had no impact on enhancer activity, neither in Tv1 nor dAp. These findings support a model where Col is crucial for directly activating ap and eya, which in turn directly activate dimm and Nplp1, with some involvement of Col on dimm. However, support for a direct role for Col on Nplp1 comes from RNAi studies in larvae or adult flies, showing that knockdown of col resulted in loss of Nplp1, while Ap, Eya and Dimm expression was unaffected (Stratmann, 2017).

It is tempting to speculate that Col regulates Nplp1 not via direct interaction with its enhancer, but rather as a chromatin state modulator, keeping the chromatin around the Nplp1 locus in an accessible state, in order for Dimm, Ap and Eya to be able to access the Nplp1 gene. Support for this notion comes from studies on the mammalian Col orthologue EBF, which is connected to the chromatin remodeling complex SWI/SNF during EBF-mediated gene regulation in lymphocytes (Gao, 2009). Moreover, the central SWI/SNF component Brahma was recently identified in a genetic screen for Ap cluster neurons, and found to affect FMRFa neuropeptide expression in Tv4 without affecting Eya expression, indicating a late role in Ap cluster differentiation. Alternatively, Col may activate Nplp1 via unidentified, low affinity sites, similar to the mechanism by which Ubx regulates some of its embryonic target genes (Stratmann, 2017).

ap encodes a LIM-HD protein, a family of transcription factors well known to control multiple aspects of terminal neuronal subtype fate, including neurotransmitter identity, axon pathfinding and ion channel expression. The current results indicate that Ap in turn acts upon dimm, and subsequently with Dimm on Nplp1. eya encodes an evolutionary well-conserved phosphatase and does not bind DNA directly, instead acting as a transcriptional co-factor. Eya (and its orthologues) have been found to interact with several transcription factors in different systems, but whether it forms complexes with Col and Ap is not known (Stratmann, 2017).

The final transcription factor in the FFL is Dimm, a bHLH protein. Dimm is selectively expressed by the majority of neuropeptide neurons in Drosophila, and is important for expression of many neuropeptides. Intriguingly, Dimm is also both necessary and sufficient to establish the dense-core secretory machinery, found in neuropeptide neurons. Based upon these findings Dimm has been viewed as a cell type selector gene, acting to up-regulate the secretory machinery. This study found evidence for that Dimm acts directly on the Nplp1 enhancer, and this raises the possibility that Dimm is both a selector gene for the dense-core secretory machinery, and can act in some neuropeptide neurons to directly regulate specific neuropeptide gene expression (Stratmann, 2017).

Regulators acting in combinatorial codes also act independently in single differentiating neurons

In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, how each of these factors contributes to ap neuron diversity has been resolved. These factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, it was also found that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits (Allan, 2005).

Within every VNC hemisegment, ap is expressed by one dorsal neuron (dAp) and two ventral neurons (vAp). Additionally, in thoracic VNC hemisegments, ap is expressed by a lateral cluster of four neurons (the ap cluster), termed the Tv, Tvb, Tva, and Tvc neurons. These ap neurons are phenotypically diverse. The axons of most ap neurons project within an ipsilateral fascicle (ap fascicle) that projects to the brain, whereas the axons of the Tv cell exit the VNC at the midline to innervate the dorsal neurohemal organs (DNH). A subset of ap neurons is peptidergic (the Tv, Tvb, and dAp neurons). As is characteristic for the vast majority of Drosophila peptidergic neurons, these cells express high levels of the peptide biosynthetic enzyme peptidylglycine alpha-hydroxylating monooxygenase (PHM). However, this peptidergic subset is also diverse: Tv cells selectively express the dFMRFa neuropeptide, whereas Tvb and dAp cells selectively coexpress three peptide biosynthetic enzymes -- PC2, Furin1, and PAL2 -- although the identity of their secreted neuropeptide(s) remains unknown. This coexpression in Tvb and dAp cells suggested a functional grouping and a common name, 'Ap-let' cells. For clarity, the ap neurons will be considered as three classes: (1) Tv cells express dFMRFa and PHM and innervate the DNH; (2) Ap-let (Tvb and dAp) cells express PHM, PC2, Furin1, and PAL2; (3) the vAp, Tva, and Tvc cells are nonpeptidergic (Allan, 2005).

ap, sqz, dimm, and the BMP pathway act in a combinatorial code to regulate dFMRFa in the Tv cell (ap, sqz, dimm, and the BMP pathway) and furin1 (ap, dimm) in Ap-let cells. Importantly, however, each regulator also plays critical roles within these ap neurons independent of the other regulators. Ap independently acts to regulate axon pathfinding by all ap cells except the Tv. Dimm independently controls PHM in the Tv and Ap-let cells. Moreover, Sqz independently acts via the N pathway to regulate cell identity within the ap cluster, upstream of both Ap and Dimm, apparently by suppressing the Tvb cell fate to favor the Tv fate. The Ap-let cells do not express Sqz, nor do they have an activated BMP pathway. In these neurons, Ap activates the expression of Dimm, and both act together to activate the expression of the peptide-processing enzyme Fur1. The Tva and Tvc cells of the ap cluster do not express Dimm and do not have an activated BMP pathway. Remarkably, the differences inferred between regulatory circuits for the two classes of peptidergic cells are highly reminiscent of differences in regulatory circuits that operate during the differentiation of distinct noradrenergic neurons. Together, these sets of studies support the proposition that epistatic relations between regulators underlying the production of a common phenotype may differ according to cell type (Allan, 2005).

The loss-of-function and gain-of-function phenotypes presented for ap, sqz, dimm, and the BMP pathway, suggest that they act in a combinatorial fashion to regulate dFMRFa expression in the Tv neuron. Likewise, the results indicate that ap and dimm, in the absence of sqz and the BMP pathway, combine to activate Fur1 in the Ap-let neurons, Tvb and dAp. In order to determine whether these regulators act simultaneously on dFMRFa and Fur1, rather than in a genetic hierarchy, the epistatic and biochemical relationship between these regulators were studied. Only one clear epistatic relationship was found; Ap activates the expression of Dimm in the majority of ap neurons. Therefore, it was important to determine whether Dimm acted downstream of Ap to independently and more directly regulate dFMRFa and Fur1 expression. This hypothesis was tested in two complementary tests. (1) Rescuing Dimm function in ap neurons that were absent for Ap function, yielded a nearly complete rescue of dFMRFa in Tv neurons, but only relatively weak rescue of Fur1 in Ap-let neurons. (2) Panneuronal co-misexpression of both ap and dimm triggers ectopic dFMRFa expression in a much greater number of neurons than does either regulator alone. These data indicate that Dimm functions together with Ap to achieve wild-type levels of dFMRFa and, more notably, Fur1. Thus, ap and dimm appear to display both hierarchical and combinatorial interactions. This hypothesis has precedent in studies of the developing pancreas, in which Foxa2 is required for pdx-1 transcription in β cells and later interacts directly with PDX-1 protein to regulate target gene expression, including maintained pdx-1 expression. Biochemical data are also consistent with the possibility that a combinatorial Ap, Dimm, and Sqz code that activates dFMRFa and dFur1 involves direct protein interactions. These may exist within larger complexes bridged by Chip, since Dimm can interact directly with both Ap and Chip, and this in turn may explain why Dimm partially rescues both the ap mutant dFMRFa and Fur1 phenotypes. These multiple interactions are reminiscent of synergistic interactions suggested between mammalian bHLH proteins, LIM-HD proteins, and the Chip homolog, LDB1/NLI. The simplest explanation for restricted patterns of neuropeptides and certain neuropeptide biosynthetic enzymes features a combinatorial hypothesis. More specifically, it is proposed that different combinatorial codes of transcription factors act cell specifically to effect differing patterns of neuropeptides and associated processing enzymes (Allan, 2005).

Ap expression is an early marker of ap cell differentiation, and it is required for proper axonal pathfinding by most ap neurons, although not by the Tv cell. In contrast, neither Sqz nor Dimm appear to control ap cell morphogenesis. An independent role for Sqz occurs early in ap cell differentiation, at a time when postmitotic cell fates are being determined. It is surprising that such cell fate changes can be rescued by UAS-Dl. Why would the frequently used N pathway signaling system depend upon a much more restricted regulator like sqz for proper activity? Increasing evidence points to major mechanistic differences between N signaling during neuroblast specification and during asymmetric division, where asymmetric divisions specifically require neuralized, numb, and sanpodo. No expression of sqz is found in neuroblasts, but expression is evident in many VNC cells. Therefore, it is proposed that factors like Sqz coordinate late N signaling with cell specification and/or cell cycle genes (Allan, 2005).

Dimm acts independently of Ap, Sqz, and the BMP pathway to activate expression of the neuropeptide-processing enzyme PHM. The evidence regarding the independent role of Dimm suggests that it is a master regulator of neuroendocrine cell fate. dimm expression is highly correlated with a neuroendocrine/peptidergic cellular identity, where it regulates the expression of almost all neuropeptides and their processing enzymes examined to date, especially within those neurons that express peptides that are processed to include an α-amidated C terminus. This is a significant cellular pattern, because more than 90% of Drosophila neuropeptides are amidated. Furthermore, high-level expression of the PHM enzyme is absolutely required for amidation and serves as an excellent marker for most peptidergic neurons in Drosophila. Finally, PHM expression appears to be dedicated to neuroendocrine peptide biosynthesis; it is exclusively found within the luminal domain of secretory vesicles. Thus, PHM expression provides a faithful marker for the peptidergic/neuroendocrine cell fate. This study has shown that PHM is dominantly induced by dimm overexpression throughout most or all of the CNS. This evidence, together with the loss-of-function data argues strongly that dimm is a neuroendocrine master regulator, with properties akin to those of other bHLH proteins in regulating cell fate (Allan, 2005).

As anticipated, more restricted peptidergic traits such as dFMRFa and Fur1 expression are dependent upon combinatorial codes. Importantly, however, the selection of cell-specific peptidergic markers arises from a deterministic interaction between a peptidergic master regulator and a cell-specific combinatorial code. There exists a clear analogy between the action of dimm in developing neurons and results regarding the glial cells missing (gcm) gene. Studies have shown that gcm is both necessary and sufficient for glial cell specification within the DrosophilaVNC. gcm is able to ectopically activate generic glial genes, such as reversed polarity, and also activates subclass-specific glial genes, but only in certain prescribed subsets of cells. Thus, similar to gcm, it is predicted that dimm is a master regulator of core neuroendocrine genes in most peptidergic/neuroendocrine cells. It will be of interest to determine which genes beyond PHM are under dimm control. In parallel, dimm combines with local-acting factors to help activate subclass-specific genes (e.g., neuropeptide-encoding genes) within peptidergic cell subsets (Allan, 2005).

The genes studied here combine to regulate dFMRFa and Fur1 but also have independent roles within the same cells. This raises the issue of how Dimm, for instance, can complex with Ap/Sqz on dFMRFa and also act independently on PHM within the same nucleus. Surprisingly, no clear evidence of an antagonistic relationship between the individual roles of Ap, Sqz, and Dimm was found. For example, co-misexpression of ap with dimm does not obviously suppress the ectopic PHM expression observed when dimm alone is misexpressed. Likewise, misexpression of sqz in the Fur1-expressing dAp/Tvb cells does not suppress Fur1. Thus, it appears that the independent mechanisms of regulator action are robust and can coexist with combinatorial functions. Therefore, it is proposed that these regulators operate within a bistable organizational mechanism. With respect to independent roles, it is proposed that Dimm operates independently of Ap and Sqz to dominantly induce specific target genes (e.g., PHM) within all neuronal lineages by forming heterodimers with a class A bHLH like Da, or by forming homodimers. The Drosophila bHLH Twist protein has distinct regulatory roles in vivo, acting either as a heterodimer with Da, or as a homodimer. Notably, the mammalian ortholog of Dimm, Mist1, forms functional homodimers to promote the differentiation of pancreatic secretory cells (Allan, 2005).

The TGFβ/BMP signal transduction pathway plays critical roles during a number of developmental events, and mutants affecting the Drosophila BMP pathway show dramatic defects in embryonic development. In contrast, in the Tv neuron, BMP signaling plays a much more subtle role, and although it is critical for dFMRFa expression, no effects were found upon the expression of sqz, ap, or dimm or on the general peptidergic marker PHM in wit mutants. Although these studies cannot rule out other roles for the BMP pathway in Tv neurons, it is tempting to speculate that target-derived BMP signaling in neurons may have quite a limited set of nuclear readouts in each specific neuronal subclass (Allan, 2005).

Regulation of secretory protein expression in mature cells by DIMM, a basic helix-loop-helix neuroendocrine differentiation factor

During differentiation, neuroendocrine cells acquire highly amplified capacities to synthesize neuropeptides to overcome dilution of these signals in the general circulation. Once mature, the normal functioning of integrated physiological systems requires that neuroendocrine cells remain plastic to dramatically alter neuropeptide expression for long periods in response to hormonal and electrical cues. The mechanisms underlying the long-term regulation of neuroendocrine systems are poorly understood. This study shows that the Drosophila basic helix-loop-helix protein DIMM, a critical regulator of neuroendocrine cell differentiation, controls secretory capacity in mature neurons. DIMM expression begins embryonically but persists in adults. Through spatial and temporal manipulation of transgene expression in vivo, two phases of prosecretory DIMM activity have been defined. During an embryonic critical window, DIMM controls the differentiation of amplified expression of the neuropeptide leucokinin. At the onset of metamorphosis, levels of DIMM decreases in the insulin-producing cells (IPCs) in parallel with a marked reduction in levels of Drosophila insulin-like peptide 2 and a key neuropeptide biosynthetic enzyme peptidylglycine α-monooxygenase (PHM). Overexpression of DIMM in the IPCs prevented the decrease in PHM levels at this stage. In addition, transient overexpression of DIMM in adults produces a dramatic increase in PHM levels in numerous neurons located throughout the brain. These findings provide insights into the mechanisms controlling the maintenance of differentiated cell states, and they suggest an effective means for dynamically adjusting the strength of hormonal signals in diverse homeostatic systems (Hewes, 2006).

DIMM is required for the embryonic development of secretory peptide expression in diverse neuronal and endocrine cell lineages. In the current study, these findings were extended through spatiotemporal manipulation of dimm transgene expression. A critical window was found, that closed at the end of embryogenesis, during which DIMM must be present to induce full expression of the neuropeptide leucokinin. Thus, under some conditions, DIMM is a differentiation factor (Hewes, 2006).

If DIMM also regulates mature neuronal cell phenotypes, then it should satisfy five conditions. First, it must be present in terminally differentiated cells. Through analysis of dimm reporter gene expression, dimm in situ hybridization, and anti-DIMM immunocytochemistry, it was shown that DIMM is expressed in mature neurons. Second, levels of DIMM must be positively correlated with levels of secretory proteins, and this should occur without significant changes in the cell fates of the affected neurons. Consistent with this prediction, reduced dimm expression results in lower secretory protein levels, elevated expression of dimm results in higher secretory protein levels, and neither effect was accompanied by gross changes in cell morphology or transmitter identity. Third, acute changes in DIMM expression in mature cells should produce corresponding changes in the abundance of secretory proteins: PHM levels were markedly increased after transient DIMM overexpression in the adult brain. Fourth, levels of both DIMM and secretory proteins should fluctuate in tandem in some cells under native conditions. Positively correlated changes have been observed in cellular expression of secretory proteins and DIMM in the context of normal physiological regulation or postembryonic developmental transitions. Fifth, these natural changes in neuropeptide levels should be sensitive to experimental manipulation of DIMM. Overexpression of DIMM in the IPCs prevented the decrease in PHM levels that normally occurs in these cells at the onset of metamorphosis. Together, these results provide the first direct evidence for the postembryonic regulation of differentiated cell properties by an Atonal family protein in living animals (Hewes, 2006).

The induction of PHM and FMRFamide-related neuropeptide expression in the Tva neurons during Drosophila metamorphosis is accompanied by increased expression of a dimm reporter gene. These results are consistent with a role for DIMM in the postembryonic regulation of both Phm and FMRFamide-related (Fmrf) expression in these neurons, because both genes are regulated embryonically by dimm. Early expression of DIMM in the Tva neurons produced early PHM expression, although Fmrf expression was not affected. However, because the Tva neurons are born in the embryo, and their larval function (if any) is unknown, it is not clear whether the correlated changes in DIMM and PHM in these cells reflect cell regulation or delayed differentiation. In contrast, the IPCs are fully functional neurons that control growth rates and circulating carbohydrate levels in larvae. Thus, the regulation of PHM in the IPCs is a clear example of DIMM activity in terminally differentiated cells (Hewes, 2006).

The control of PHM expression by DIMM may serve to regulate the capacity of neurons to produce amidated neuropeptides. In Drosophila, PHM is essential for neuropeptide amidation. Most insect neuropeptides (>90%) are amidated at the C terminus, and amidation is often required for neuropeptide signaling. In addition, many secretory proteins, including the vertebrate ortholog of PHM, peptidylglycine α-amidating monooxygenase, may play indirect roles in the sorting of coexpressed neuropeptides into secretory granules. Because Drosophila insulin-like peptide 2 (dILP2) is not an amidated peptide, the role of the PHM in the insulin-producing cells (IPCs) is unclear. It will be interesting to determine whether PHM is required for dILP packaging and sorting into secretory granules or whether other amidated neuropeptides contribute to signaling by the IPCs. Nevertheless, because DIMM regulates PHM levels pan-neuronally, it likely effects dynamic changes in levels of bioactive, secretion-competent neuropeptides in diverse neurons in addition to the IPCs. In turn, these changes may alter the gain of neuropeptide signaling in the context of homeostatic and developmental regulation of neuroendocrine systems (Hewes, 2006).

The differential effects of dimm on expression of Phm versus Leucokinin (Lk) and Fmrf may reflect differences in the combinatorial transcription factor codes that control the expression of these secretory genes. PHM can be induced (or elevated) in most if not all neurons by expression of a wild-type dimm transgene, although other factors likely contribute secondarily to the fine-tuning of PHM expression, because the responses to dimm overexpression were not linear. Therefore, the code for Phm expression is primarily binary and depends on whether or not DIMM is expressed and generally not on developmental stage. In contrast, the overexpression of dimm alone is not sufficient in most cells to drive Lk and Fmrf expression, and other factors, such as the LIM homeodomain gene apterous and the zinc finger gene squeeze, are also required. Thus, if some elements of these combinatorial codes are only present in differentiating cells, then the induction of Lk, Fmrf, and other similarly regulated genes may only be possible during differentiation. After this stage, other unknown mechanisms would be needed to maintain Lk and Fmrf expression (Hewes, 2006).

Atonal-related proteins operate in transcriptional hierarchies, with proteins such as the neurogenins involved in selection of cell precursors, and later acting factors such as the NeuroD proteins regulating terminal differentiation. NeuroD1/BETA2, for example, is a member of the latter class, and it is expressed in endocrine cells of the pancreas, intestine, and pituitary and in several classes of neurons. It is essential for the complete differentiation of several neuronal and endocrine cell types. Moreover, NeuroD1/BETA2 has been shown to control neurite outgrowth, cell excitability, and the expression of several peptide hormone genes, including insulin, secretin, and proopiomelanocortin (Hewes, 2006).

Interestingly, hypothalamic NeuroD mRNA levels are reduced in obese ob/ob and food-deprived mice, suggesting a functional relationship in mature neurons between NeuroD and the neuroendocrine/endocrine signaling pathways that control energy balance. NeuroD is also required for activity-dependent granule neuron dendritic growth in the intact rat cerebellar cortex, and Gal4-NeuroD chimeras can activate insulin promoter elements in response to glucose stimulation of cultured pancreatic beta cells. In addition, mutations in NeuroD are associated with the development of certain forms of type 2 diabetes mellitus in young people. Together, these findings provide strong, albeit indirect, support for roles of other Atonal family proteins in the regulation or maintenance of neuropeptide and peptide hormone levels in fully differentiated cells. The current results on the dual functionality of DIMM provide additional indirect evidence for this model and suggest that regulation by Atonal proteins is a conserved and important feature of many neuroendocrine systems (Hewes, 2006).

In summary, this study demonstrates that DIMM controls neuropeptide expression in developing and mature neurons. This is the first direct evidence, in situ, for continued function of an Atonal family transcription factor in differentiated cells. The findings provide insights into the general mechanisms for maintenance of terminally differentiated cells after the induction signals are gone. In addition, they suggest an effective means for the regulation of the gain of neuropeptide signaling in mature animals (Hewes, 2006).

Molecular organization of Drosophila neuroendocrine cells by Dimmed.

In Drosophila, the basic-helix-loop-helix protein Dimm coordinates the molecular and cellular properties of all major neuroendocrine cells, irrespective of the secretory peptides they produce. When expressed by nonneuroendocrine neurons, Dimm confers the major properties of the regulated secretory pathway and converts such cells away from fast neurotransmission and toward a neuroendocrine state. 134 transcripts were identified that were upregulated by Dimm in embryos, and they were evaluated systematically using diverse assays (including embryo in situ hybridization, in vivo chromatin immunoprecipitation, and cell-based transactivation assays). It is concluded that of eleven strong candidates, six are strongly and directly controlled by Dimm in vivo. The six targets include several large dense-core vesicle (LDCV) proteins, but also proteins in non-LDCV compartments such as the RNA-associated protein Maelstrom. In addition, a functional in vivo assay, combining transgenic RNA interference with MS-based peptidomics, revealed that three Dimm targets are especially critical for its action. These include two well-established LDCV proteins, the amidation enzyme PHM and the ascorbate-regenerating electron transporter cytochrome b(561-1). The third key Dimm target, CAT-4 (CG13248), has not previously been associated with peptide neurosecretion. It encodes a putative cationic amino acid transporter, closely related to the Slimfast arginine transporter. Finally, Transcripts upregulated by Dimm were compared with those normally enriched in Dimm neurons of the adult brain, and an intersection of 18 Dimm-regulated genes was found that included all six direct Dimm targets. The results provide a rigorous molecular framework with which to describe the fundamental regulatory organization of diverse neuroendocrine cells (Park, 2011).

These experiments address the mechanisms underlying Dimm's regulatory functions within peptidergic neuroendocrine cells in Drosophila. The results from a genome-wide screening revealed a diverse array of potential Dimm targets and illustrated that the scope of Dimm actions is likely broad (see Molecular Targes of Dimm's control of neuroendocrine cell physiology). The actions of its direct targets appear to extend from the nucleus (CG17293) to regulation of mRNAs (CG11254) to the endoplasmic reticulum and Golgi (CG13248) to peptide-containing LDCVs (Phm and CG1254). No neuropeptide-encoding genes were found on any of the lists, even the larger 134-gene list of transcripts exhibiting upregulation with Dimm overexpression. Previously work has shown that Dimm is inefficient on its own at driving ectopic neuropeptide gene expression. Together, these findings are consistent with previous speculation that in Drosophila, specific neuropeptide expression is controlled by differing sets of transcription factors working within complex combinatorial codes. In contrast, Dimm provides parallel instructions for the cell biological machinery within which neuropeptides can be made, stored, and trafficked (Park, 2011).

Because an overexpression screen was used to generate a primary list of candidate targets, it was important to authenticate those results by reference to genes enriched in “normal” Dimm cells (i.e., cells in which Dimm levels were not artificially manipulated). Access to such information is available from the recently published gene array study of Kula-Eversole (2010), from which it was found that 13% of the 134 gene candidates were in fact highly enriched in Dimm-positive neurons (versus Dimm-negative peptidergic neurons). Although several candidates performed well in many of these tests and exhibit properties of direct Dimm targets, most did not score positive in all tests employed (only Phm, CG1275, CG13248, CG11254, and CG17293 did so). The results emphasize the importance of employing multiple tests to fully evaluate and properly interpret lists of regulated transcripts. Of the 11 genes passing the first test, diverse experimental criteria were then used to divide them into sets of six direct targets, two likely direct targets, and three indirect targets. It is emphasized that the categorization of direct targets is based on highly stringent criteria (Park, 2011).

The inclusion of Phm and Cyt b_561-1 genes in the original list of 11 candidates increased confidence in the list's authenticity because both play well-established roles in LDCVs. Furthermore, it has been demonstrated previously that Phm is a true transcriptional Dimm target both in heterologous cells and in vivo. Likewise, the subsequent strong performance of Phm and Cyt b_561-1 in all four downstream assays provided further support for the validity of the experimental design to identify authentic Dimm targets (Park, 2011).

In addition to Phm and Cyt b_561-1, these studies show that a third bona fide Dimm target gene, CG13248, is critical to normal regulation of neuroendocrine cell properties. Notably, in the results reported by Kula-Eversole (2010), Phm, Cyt b_561-1, and CG13248 all ranked near the top for absolute transcript abundance in Dimm-positive neurons. The identification of CG13248 as an integral component of neuroendocrine physiology is a significant new finding, but its specific contribution is a mystery because its precise molecular functions are not known. It is the clear sequence ortholog to mammalian cationic amino acid transporter 4 (CAT-4) and is therefore a candidate member of the system y+ (Na⁺- and pH-independent) cationic amino acid-preferring transport activities (Park, 2011).

CAT proteins form a branch of the solute carrier family 7 (SLC7). Murine CAT-1, -2, and -3 all display arginine transporter activity when heterologously expressed, but to date, CAT-4 does not. Notably, the Drosophila ortholog of the CAT-1 protein is the transporter Slimfast, which mediates arginine transport in fat body and acts as a nutrient sensor. In murine pancreatic acinar cells (which are regulated by the Dimm ortholog MIST1, CAT-4 is a membrane-associated protein of secretory granules. Future pursuit of the exact mechanisms by and pathways in which CAT-4 operates in Dimm-expressing neurons will help to illuminate fundamental neuroendocrine cell physiology (Park, 2011).

Regarding the other direct Dimm targets, a few are mentioned for potential novel insights into mechanisms of neuroendocrine cell regulation. By transcript profiling, Kula-Eversole (2010) reported that CG11254 (maelstrom [mael]) is highly enriched in the Dimm-positive l-LNvs. In germ cells, Mael localizes components of the microRNA pathway and contributes to cellular polarization. CG17293 encodes a protein highly related to mammalian WDR82, and CG7785 encodes a protein highly related to CCLD6—both of which suggest a connection of Dimm mechanisms to chromatin-modifying properties (Park, 2011).

There were two genes that were concluded to be likely directly targeted. CG6522 encodes a member of the Testin/Prickle family of proteins. Notably, the Prickle-like protein RILP interacts with REST and acts as a nuclear translocation factor. The significance of potential Prickle-REST interactions is that REST displays a suppressive effect on neurosecretory properties of PC12 cells. In addition, CG32850 encodes a protein orthologous to RING finger protein 11, which is a membrane-associated E3 ligase expressed widely in brain. Finally, in the larger list of 18 genes representing the intersection of the embryonic and adult Dimm-regulated transcripts, there was sizable representation of genes encoding proteins previously implicated in regulated neuropeptide secretion (Rph) and probable elements of the secretory pathway (PPADC1, RCN2, and Rabx4) (Park, 2011).

These results define principal elements of what is anticipated to be a core program for neuroendocrine cell organization. Among mammalian basic-helix-loop-helix proteins, Dimm is most similar to MIST1. It is noted that five of the six Dimm targets that responded to Dimm in the transactivation assay contained E boxes in their first exon-intron regions. The importance of first-intron E boxes has already been established for the case of Phm and is also true for MIST1 target genes identified thus far. Furthermore, studies of Phm and CG13248 suggest that they can define a consensus Dimm binding profile: they both contain three boxes within the first intron, two of which have the sequence CATATG, all of which contribute synergistically, and one of which appears to have the strongest contribution to Dimm transactivation. It is predicted that many other Dimm targets will display a similar E box profile. Furthermore, how individual target gene products contribute to the Dimm program and how many more genes are involved are now pertinent questions that will require additional studies. It is anticipated that further analysis of this core Dimm program will help explain the regulatory organization of neuroendocrine cells and their evolution in different phyla. Because Dimm protein persists for the life of neuroendocrine cells in Drosophila, this work may also inform studies of neuroendocrine cell physiology and plasticity (Park, 2011).

In the case of neurons that utilize fast conventional neurotransmitters, transcriptional regulatory systems typically exert direct control over genes that encode biosynthetic enzymes, as well as ones for key transporter proteins that retrieve and recycle transmitters back into the lumen of synaptic vesicles. For example, PET-1 supports serotonergic differentiation and directly targets genes that encode the critical biosynthetic enzyme TBH-1 and the serotonin transporter SERT1. It is striking, therefore, that the limited but highly validated list of Dimm targets similarly includes genes essential for neuropeptide biosynthesis (Phm and Cyt b_561-1) as well as a transporter that is specifically expressed by neuroendocrine cells (CAT-4). It is proposed that there may exist an unexpected but essential parallelism in the developmental regulation of secretory systems for small transmitters and small amidated peptides. This hypothesis can help design experiments to further illuminate the mechanisms that underlie the developmental generation of peptidergic phenotypes (Park, 2011).

Peptidergic cell-specific Synaptotagmins in Drosophila: Localization to dense-core granules and regulation by the bHLH protein DimmED

Bioactive peptides are packaged in large dense-core secretory vesicles, which mediate regulated secretion by exocytosis. In a variety of tissues, the regulated release of neurotransmitters and hormones is dependent on calcium levels and controlled by vesicle-associated synaptotagmin (SYT) proteins. Drosophila express seven SYT isoforms, of which two (SYT-α and SYT-β) were previously found to be enriched in neuroendocrine cells. This study shows that SYT-α and SYT-β tissue expression patterns are similar, though not identical. Furthermore, both display significant overlap with the bHLH transcription factor Dimm, a known neuroendocrine (NE) regulator. RNAi-mediated knockdown indicates that both SYT-α and SYT-β functions are essential in identified NE cells as these manipulations phenocopy loss-of-function states for the indicated peptide hormones. In Drosophila cell culture, both SYT-α and neuropeptide cargo form Dimm-dependent fluorescent puncta that are coassociated by super-resolution microscopy. Dimm is required to maintain SYT-α and SYT-β protein levels in Dimm-expressing cells in vivo. In neurons normally lacking all three proteins (Dimm^-/SYT-α^-/SYT-β^-), Dimm misexpression conferred accumulation of endogenous SYT-α and SYT-β proteins. Furthermore transgenic SYT-α does not appreciably accumulate in nonpeptidergic neurons in vivo but does so if Dimm is comisexpressed. Among Drosophila syt genes, only syt-α and syt-β RNA levels are upregulated by Dimm overexpression. Together, these data suggest that SYT-α and SYT-β are important for NE cell physiology, that one or both are integral membrane components of the large dense-core vesicles, and that they are closely regulated by Dimm at a post-transcriptional level (Park 2014).

Bioactive peptides are packaged in large dense-core secretory vesicles, which mediate regulated secretion by exocytosis. In a variety of tissues, the regulated release of neurotransmitters and hormones is dependent on calcium levels and controlled by vesicle-associated synaptotagmin (Syt) proteins. Drosophila express seven Syt isoforms, of which two (Syt-α and Syt-β) were previously found to be enriched in neuroendocrine cells. This study shows that Syt-α and Syt-β tissue expression patterns are similar, though not identical. Furthermore, both display significant overlap with the bHLH transcription factor Dimm, a known neuroendocrine (NE) regulator. RNAi-mediated knockdown indicates that both Syt-α and Syt-β functions are essential in identified NE cells as these manipulations phenocopy loss-of-function states for the indicated peptide hormones. In Drosophila cell culture, both Syt-α and neuropeptide cargo form Dimm-dependent fluorescent puncta that are coassociated by super-resolution microscopy. Dimm is required to maintain Syt-α and Syt-β protein levels in Dimm-expressing cells in vivo. In neurons normally lacking all three proteins (Dimm^-/Syt-α^-/Syt-β^-), Dimm misexpression conferred accumulation of endogenous Syt-α and Syt-β proteins. Furthermore transgenic Syt-α does not appreciably accumulate in nonpeptidergic neurons in vivo but does so if Dimm is comisexpressed. Among Drosophila syt genes, only syt-α and syt-β RNA levels are upregulated by Dimm overexpression. Together, these data suggest that Syt-α and Syt-β are important for NE cell physiology, that one or both are integral membrane components of the large dense-core vesicles, and that they are closely regulated by Dimm at a post-transcriptional level (Park 2014).

Large dense-core vesicles (LDCVs) are the critical subcellular organelles for peptidergic neurotransmission: to understand peptide cell biology in detail, it is critical to provide an in-depth understanding of how LDCVs are made, trafficked, and released. In pursuit of this goal, a study was carried out of the two 'peptidergic-specific' Drosophila synaptotagmin isoforms, syt-α and syt-β. The results provide a foundation for future genetic and cell biological studies of LDCV synthesis and regulation (Park 2014).

The subcellular distribution of Syt-α in Dimm-transformed Drosophila neurons in vitro displayed extensive overlap with puncta of a fluorescently labeled neuropeptide. The major protein components of LDCV have been identified in a variety of systems. This study used super-resolution microscopy to reveal that Syt-α is strongly associated with LDCV peptide cargo in Drosophila neuronal cell culture and is detected unevenly at LDCVs with two different super-resolution microsopy. That result is novel in that it identifies Syt-α as an integral membrane protein of the LDCV and supports the hypothesis that it may serve as a calcium sensor for LDCV exocytosis. Different Syt isoforms have been associated with diverse LDCVs in a variety of different cell types, both functionally and by expression. Future studies on the functional roles of different Syt isoforms in LDCV will benefit greatly from the genetic repertoire available for Drosophila studies. These experiments are further significant because they indicate that the nanoscale arrangement of numerous LDCV proteins and their structure–function relationships could be dissected in subsequent studies using super-resolution light microscopy (Park 2014).

The effects were tested of knocking down syt-α and syt-β RNA levels specifically in two small peptidergic NE populations: the peripheral ETH-secreting Inka cells and the CCAP-secreting cells of the CNS. Peptides released from either population normally trigger and/or shape characteristic innate patterns of behavior, ecdysis and wing-spreading, respectively. The loss of either peptide signal, and also the loss of the peptide-secreting cell population, produces severe disruptions in the regulated behaviors. syt-α and syt-β RNAi produced strong phenotypes in Inka cells, that mimic the genetic loss of the Inka bioactive peptide (ETH/PETH) and the effects of Inka cell ablation. Because Inka cells were present and appeared healthy and because this study used a conditional RNAi transgene design, it is presumed the Inka cell phenotypes were due to altered function of mature Inka cells. Hence, it is concluded that Syt-α and Syt-β play critical roles in the physiology of Inka cells, presumably by contributing to the episodic release of the ETH and PETH peptides to drive ecdysial behaviors (Park 2014).

Notably, the RNAi construct directed to Inka cells for syt-α was as effective in creating lethality as was the one for syt-β. Adolfsen (2004) reported that embryonic Inka cells contain syt-β but not syt-α. To explain these results, it is speculated that the α isoform may appear later in postembryonic stages of Inka cell function and hence be vulnerable to RNAi-mediated attack during larval stages. Alternatively, the syt-α RNAi construct may have produced a nonspecific effect on syt-β. The possibility of nonspecific RNAi effects are considered to be remote because they were not observed in parallel experiments in the case of CCAP neurons. RNAi for syt-β, but not for syt-α, show the clear phenocopy of the CCAP loss of function syndrome, affecting a fixed wing spreading routine. On this basis, it is argued that the different syt RNAi constructs displayed overall specificity of effects. Together, the genetic results support the hypothesis that the Syt-α and Syt-β isoforms play critical roles for neuropeptide release in Dimm⁺ NE cells, most likely acting as calcium sensors (Park 2014).

Although expression of Syt-α and Syt-β proteins was not exclusively associated with Dimm, their strongest expression sites are well-known peripheral and central Dimm⁺ NE cells. These observations strongly support the prediction by Adolfsen (2004) that Syt-α and Syt-β would be closely aligned with peptidergic cell physiology. Based on this conclusion, the regulation of Syt-α and Syt-β expression levels and their precise subcellular localizations become significant issues. dimm loss of function alleles produced marked declines in Syt-α and Syt-β expression. Additionally, Dimm contributed to endogenous and transgenic Syt protein stability. Dimm therefore appears both necessary and sufficient to control Syt-α and -β expression. Overexpressing dimm upregulated syt-α and syt-β RNAs selectively among seven syt genes tested; paradoxically, however, syt-α and syt-β RNA levels did not decline in a loss-of-dimm background. It is proposed that dimm contributes to syt RNA stability, but that it is not normally an important activator of syt-α or syt-β gene expression (other regulators must control their transcription) (Park 2014).

These general effects mirror previous findings on how Dimm controls levels of secretory peptides, such as PDF. In that case, Dimm indirectly dictates peptide accumulation in Dimm neurons, without directly activating neuropeptide gene expression. In the present example, it was found that accumulation of ectopic Syt-α and Syt-β transgenic proteins is largely dependent on whether or not Dimm is also misexpressed. Likewise, average Drosophila neurons cannot accumulate neuropeptides ectopically, suggesting that only peptidergic neurons have an enhanced ability to accumulate and/or release neuropeptides compared with neurons that primarily release classical neurotransmitters. At the molecular level, Dimm cannot transactivate syt-α promoter fragments in an in vitro assay, in which regulation of true Dimm direct targets is readily measured. Also, in vivo ChIP-chip genomic surveys indicate that Dimm protein is not normally resident at either the syt-α or syt-β loci. Thus, both cellular and molecular analyses suggest that Dimm critically regulates syt-α or syt-β levels post-transcriptionally, not transcriptionally (Park 2014).

There are at least 17 synaptotagmin isoforms in mammals: as yet, none appears to be a clear molecular ortholog of Syt-α and Syt-β, although Syt-IX and Syt-XVII are the most closely related. In mammals, several Syt isoforms (especially synaptotagmins 1, 4, 7, 9, and 10) are implicated in regulating exocytosis of peptide-containing LDCVs. The detailed roles of Syt isoforms in regulating the trafficking and exocytosis of peptidergic LDCVs appear complex. It has been reported that different Syt isoforms are associated with different size DCVs. Syt-1 regulates an early phase of calcium-dependent exocytosis from chromaffin cells vesicles, whereas Syt-7 regulates a second, slower one. Likewise, Syt-4, Syt-7, Syt-9, Syt-10, and Syt-17 have all been implicated by expression and/or by functional studies to be associated with release of bioactive peptides. Further analysis of these issues in a genetic model system, such as Drosophila, will help clarify some of these essential contributions (Park 2014).

The results suggest a model whereby Dimm organizes the regulated secretory pathway in NE cells by both direct and indirect mechanisms. Direct mechanisms refer to Dimm's transcriptional activation of a defined set of target genes. Using several independent methods, six direct targets have been identified, and these include neuropeptide biosynthetic enzymes, putative transporters, and chromatin remodeling factors. There are many additional direct Dimm targets that are identified by genome-wide methods, such as ChiP-CHIP. A significant outcome of these studies when summed is that Dimm does not target genes that encode neuropeptides or peptide hormones (Park 2014).

Indirect Dimm mechanisms could influence protein sorting by directing certain specific seed proteins (e.g., PHM and/ or Cyt_b-561) to exceed threshold concentrations. Seed proteins could permit accumulation and stabilization of 'indirect targets' (e.g., Syt-α and Syt-β) that are regulated by transcription factors other than Dimm. Importantly, the possibility that interactions between proteins in the LDCV membrane and those in the LDCV lumen may contribute to protein sorting has already been proposed. Thus, direct Dimm targets may represent rate-limiting components that can affect the protein composition of LDCV. Regardless of the precise molecular mechanism, this study has shown that this general explanation holds for Syt-α and Syt-β, and it is therefore proposed that a significant fraction of LDCV-associated components may be likewise dependent indirectly on Dimm actions. Cell-specific neuronal gene expression is most often ascribed to direct gene control by specific transcription factors. This study now extends that general hypothesis to include what is termed 'indirect transcription factor effects' as major contributors to cellular differentiation. Thus, patterns of cell-specific protein expression reflect in part underlying indirect support by critical transcription factors (Park 2014)

DEVELOPMENTAL BIOLOGY

Embryonic

There is a strong maternal contribution of CG8667/mistr mRNA (dimmed). Zygotic transcription is initiated at stage 14. It is expressed in bilateral domains in the cephalic region, which, as development proceeds, fuse into a U shape forming part of the ring gland. Concomitant expression of CG8667/mistr also begins in the CNS. By stage 17, CG8667/mistr is in clusters of cells at the anterior and posterior of the VNC and bilaterally in two lateral cells per hemisegment in the VNC (Moore, 2000).

The c929 P-element insertion into the dimmed gene was isolated in a P{Gal4} enhancer detection screen for genes expressed in the Tv neuroendocrine neurons (O'Brien, 1998), ventral cord neurons known to express the neuropeptide FMRFamide (Benveniste, 1998). In addition to the Tv neurons, c929 drives reporter gene expression (GFP or ß-galactosidase) in ~200 neurons scattered throughout the larval CNS and in neuroendocrine projections to the ring gland, the dorsal neurohemal organs and the transverse nerves. Outside the CNS, this pattern includes at least three classes of endocrine cells: intrinsic cells of the corpora cardiaca, 10-20 midgut cells and the peritracheal myomodulin-immunoreactive cells. The latter appear homologous to the endocrine Inka cells (O'Brien, 1998). c929 reporter expression also appeares in several other tissues, including peptidergic PNS neurons (LBD neurons; D. Allan and S. Thor, personal communication to Hewes, 2003), fat body, epithelial cells and salivary glands (Hewes, 2003).

To determine whether c929-positive neurons express neuropeptides, double-label experiments were performed for the c929 reporter and for the peptide biosynthetic enzyme, peptidylglycine-alpha-hydroxylating mono-oxygenase (PHM). In Drosophila, PHM is a marker for most peptidergic cells. It is required for neuropeptide amidation, which is a highly specific modification of secretory peptides; greater than 90% of all known or predicted Drosophila peptide transmitters are amidated. Most if not all c929-positive CNS neurons are immunostained very strongly by PHM antibodies (Hewes, 2003).

Conversely, most neurons displaying strong PHM immunostaining are also c929 positive, while most weakly PHM-positive neurons are not c929 positive. In addition, PHM is expressed in all three c929-positive endocrine cell types and in the LBD peripheral neurons (O'Brien, 1998). Thus, in the larval CNS and in several peripheral tissues, c929 primarily labels neuroendocrine cells as its expression is highly correlated with the production of large amounts of amidating enzyme, amidated neuropeptides and peptide hormones (Hewes, 2003).

To assess the degree of heterogeneity among c929-positive cells, the expression pattern of c929 was compared with a variety of other peptidergic cell markers. This population of cells is chemically diverse. For example, seven bilateral pairs of c929-positive neurons were double-labeled with the PT2 antiserum. 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, cockroach corazonin, cricket leucokinin-1 (LK), crustacean cardioactive peptide (CCAP; see Drosophila Ccap), crustacean beta-PDH and Aplysia myomodulin (MM). Finally, a distinct subset of 34 c929-positive neurons was immunopositive for an additional, putative Drosophila peptide biosynthetic enzyme Furin 1. 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, an enzyme required for synthesis of the biogenic amines, serotonin and dopamine (Hewes, 2003).

No single transmitter system tested was entirely c929 positive. For example, among the 17 Fmrf cell types (Benveniste, 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. Thus, the identification of c929-positive peptidergic neurons is likely to be an underestimate of the total population of peptidergic cells that express the reporter gene (Hewes, 2003).

Dimm is specifically expressed in peptidergic neurons and endocrine cells. CG8667 mRNAs are ubiquitous in pre-cellular blastoderm embryos (Moore, 2000) and later are expressed in the developing nervous system (Moore, 2000). Presumed zygotic CG8667 expression is first visible as nascent transcripts scattered throughout the CNS in stage 12 embryos. Cytoplasmic CG8667 hybridization is visible in many of these cells beginning around stage 14, is strong by stage 16 and persists in stage 17 embryos and in hatchling larvae less than 24 hour old (Hewes, 2003).

The pattern of CNS CG8667 in situ hybridization resembled the c929 reporter pattern. 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) (Hewes, 2003).

Expression of the c929 reporter and CG8667 was also observed in strikingly similar patterns in peripheral tissues. 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 is not expressed in any other location, except for a few unidentified non-CNS cells scattered throughout the anterior and lateral regions. Thus, in CNS, PNS and endocrine tissues, expression of the c929 reporter closely mirrors CG8667 expression. These expression analyses support the genetic mapping, genetic identification and RNAi data (Hewes, 2003).

Postmitotic specification of Drosophila insulinergic neurons from pioneer neurons

Insulin and related peptides play important and conserved functions in growth and metabolism. Although Drosophila has proved useful for the genetic analysis of insulin functions, little is known about the transcription factors and cell lineages involved in insulin production. Within the embryonic central nervous system, the MP2 neuroblast divides once to generate a dMP2 neuron that initially functions as a pioneer, guiding the axons of other later-born embryonic neurons. Later during development, dMP2 neurons in anterior segments undergo apoptosis but their posterior counterparts persist. Surviving posterior dMP2 neurons no longer function in axonal scaffolding but differentiate into neuroendocrine cells that express insulin-like peptide 7 (Ilp7) and innervate the hindgut. The find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Forkhead, and Dimmed. These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation. Ectopic reconstitution of this code is sufficient to activate Ilp7 expression in other postmitotic neurons. These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic beta-cells (Miguel-Aliaga, 2008).

The observed death of some Drosophila pioneer neurons has been used to argue that their function is transient, but persistence in other cases suggested that, either they continue to play an axonal-scaffolding role, or that they adopt some other identity. The current findings resolve this long-standing issue by clearly demonstrating that, for dMP2 neurons, the axonal scaffolding function is only transient. After this role is no longer required, surviving dMP2 neurons become insulinergic and innervate the hindgut. The other known innervation of the Drosophila gut occurs much more anteriorly, in the foregut and anterior midgut, from neuronal cell bodies located in the peripheral ganglia of the stomatogastric nervous system. Unlike dMP2 neurons, however, the individual identities of the stomatogastric neurons and their cell lineages remain to be clearly defined. Thus, dMP2 neurons may provide a simple and well-characterised system for studies of the guidance cues involved in enteric innervation. Future studies, however, will be needed to determine the functions of Ilp7 in dMP2 neurons. It will be important to distinguish if this posterior neural source of insulin acts humorally to promote growth, like the more anterior brain mNSCs, or if it has more local effects in abdominal tissues. In this regard, the presence of Ilp7-expressing neurites in close proximity to the Ilp2-producing mNSCs is intriguing (Miguel-Aliaga, 2008).

The transition from pioneer to neuroendocrine neuron is not unique to dMP2 neurons, as Drosophila MP1 pioneer neurons also become neuropeptidergic at larval stages (Wheeler, 2006). In the grasshopper, segment-specific survival of pioneer neurons has also been reported, raising the possibility that they too may become neuroendocrine. Studies in other species, including vertebrates, will be needed to reveal the extent to which the linkage between pioneer and neuroendocrine functions is conserved. Identifying pioneer neurons with an 'ancestral' neuroendocrine identity in other phyla would lend further support to the proposal that pioneer neurons are highly conserved in evolution (Miguel-Aliaga, 2008).

Apoptosis of postmitotic neurons is a widespread feature of normal VNC development, but few developmental regulators of core pro-apoptotic genes such as grim, hid, and rpr have been identified. This study uncovers roles for Fkh and Hb9. Hb9, at least, appears linked to cell death in neurons other than dMP2: in Df(3L)H99 mutant embryos, where apoptosis is blocked, ectopic Hb9-positive RP motor neurons are observed in segments A7-A8. Hb9 is an important regulator of motor neuron identity in both Drosophila and vertebrates. Finding of a pro-apoptotic function for Hb9 in Drosophila, together with the neurotrophic requirement for motor neuron survival in vertebrates, raises the possibility that the same genetic programs specifying the identities of motor neurons also sensitize them for postmitotic editing via apoptosis (Miguel-Aliaga, 2008).

Fkh function in CNS development has not been characterized. Fkh is expressed in segmentally repeated clusters of midline neurons, including dMP2, vMP2, MP1 neurons, and the VUM interneurons. Within the MP2 lineage, Fkh is first expressed in the MP2 neuroblast at stage 9-10 and continues to be expressed in both the dMP2 and vMP2 daughters throughout embryonic and larval stages. In fkh mutants, 95% of anterior dMP2 neurons fail to undergo apoptosis, and 95.3% of posterior dMP2 neurons (and 100% of ectopic anterior counterparts) fail to express Ilp7. Both of these dramatic phenotypes could be rescued to near wild-type levels by reintroducing Fkh under odd-GAL4 regulation, indicating a cell-autonomous requirement for promoting dMP2 apoptosis and Ilp7 expression (Miguel-Aliaga, 2008).

Hb9 and Fkh expression in many neurons that do not die suggests a combinatorial mechanism for the control of developmental apoptosis. One possibility is that several transcription factors function in combination to activate the core pro-apoptotic genes. Given the proposed role for Foxa proteins in chromatin accessibility, Fkh expression in dMP2 neurons may render the promoters of core pro-apoptotic genes responsive to activation by Hb9. An alternative but not mutually exclusive mechanism involves individual transcription factors activating different pro-apoptotic genes such that a combination of these would then be required to trigger neuronal death. For example, Hb9 could be required for rpr/skl but not grim expression. Some support for this idea comes from the observation that loss of hb9 activity blocks rpr/skl-mediated death of dMP2 neurons but not the largely grim-dependent apoptosis of anterior MP1 neurons (Miguel-Aliaga, 2008).

An important conclusion from this study is that the combinatorial transcription factor code controlling apoptosis partially overlaps with that regulating insulinergic identity. Thus, Fkh and Hb9 are both essential components of the codes for anterior apoptosis and also Ilp7 expression, illustrating that these transcription factors play surprising dual roles as pro-apoptotic and pro-differentiation factors within the same neuronal subtype. Importantly, the results also show that the segment-specific Hox protein Abd-B acts as a postmitotic switch, converting the pro-apoptotic Fkh⁺ Hb9⁺ code into an insulinergic Fkh⁺ Hb9⁺ Abd-B⁺ code (Miguel-Aliaga, 2008).

Three Ilp7 regulators (Hb9, Abd-B, and Fkh) are expressed at least 12 h before Ilp7 is first activated: from the time when the MP2 neuroblast exits the cell cycle. In the case of Hb9, it was not possible to uncouple two temporally separable functions. Early postmitotic expression of Hb9 is important for its death-activating function, whereas later expression suffices for activating Ilp7. Similarly, the Hox protein Abd-B generates a segment-specific neuropeptide pattern via postmitotic regulation of posterior dMP2 survival and also Ilp7 activation. As vertebrate neuropeptides are also expressed in restricted neuronal populations within specific rostrocaudal domains, they may be similarly regulated by Hox survival/neuroendocrine inputs. In the case of Fkh, it is required for many different aspects of the progression from the early to the late postmitotic dMP2 fate. Fkh expression is restricted to VNC midline neurons and its vertebrate orthologue Foxa2 functions in the differentiation of the floor plate and ventral dopaminergic and serotonergic neurons (Ferri, 2007; Jacob, 2007; Norton, 2005). Thus, in both the Drosophila midline and its vertebrate counterpart, the floor plate, Fkh proteins play a conserved role in the differentiation of ventral neuronal subtypes (Miguel-Aliaga, 2008).

The other two dMP2 regulators identified in this study, Dimm and the BMP pathway, are switched on shortly before the onset of Ilp7 expression. The timing of onset of these two broad neuroendocrine regulators is likely to specify when Ilp7 is first activated, whereas the earlier factors Fkh, Hb9, and Abd-B may contribute more specifically to insulinergic identity. Together, the genetic and expression analyses in this study demonstrate that the combinatorial code of genetic inputs required for Ilp7 expression is assembled in a step-wise manner during postmitotic maturation. Importantly, this allows a subset of the components to be shared (such as Fkh and Hb9) between sequential neuronal programmes (survival and Ilp7 expression) without losing output specificity (Miguel-Aliaga, 2008).

Two observations from this study indicate that insulinergic combinatorial codes can vary from cell-to-cell and also from one Ilp to another. (1) None of the regulators of Ilp7 in dMP2 neurons appear to regulate it in DP neurons. (2) The dMP2 insulinergic code is sufficient to trigger ectopic expression of Ilp7 but not Ilp2 or other neuropeptides such as FMRFa. These findings suggest the existence of additional, as yet unidentified, insulinergic factors in DP neurons and also in the brain mNSCs where Ilp2 is expressed. Identification of the neural progenitor for these mNSCs (Wang, 2007) should facilitate characterization of the Ilp1/Ilp2/Ilp3/Ilp5 combinatorial codes and thus clarify the extent to which different insulinergic transcriptional programmes overlap (Miguel-Aliaga, 2008).

The finding that an Ilp7-expressing neuron derives from the MP2 lineage reveals that at least some insulinergic regulators are similar in insects and mammals. Three apparent similarities may not be very insulin-specific but reflect more general processes shared by neural and endocrine programmes in many species. (1) Notch signalling singles out the MP2 neuroblast and distinguishes its two progeny neurons, while in mammals, it limits pancreatic expression of the 'proneural' gene Ngn3 to prospective endocrine cells. (2) The survival and pro-Ilp7 functions mediated by Abd-B in the dMP2 neuron could also have their postmitotic counterparts in ß-cells, either mediated by related Hox genes or via another homeobox gene, Pdx-1, following its early input into pancreatic induction. (3) Nerfin-1 is required for dMP2 pioneer function (Kuzin, 2005), while its mammalian orthologue Insm1/IA1 is important for pancreatic ß-cell specification (Miguel-Aliaga, 2008).

Several more specific regulatory similarities exist between the insulinergic differentiation factors active in postmitotic dMP2 neurons. For example, the role of fkh in dMP2 neurosecretory differentiation described in this study is similar to the functions of HNF3b/Foxa2 in islet maturation and insulin secretion (Sund, 2001). In addition, mammalian Nkx2.2 is important for pancreatic ß-cell specification and is known to activate transcription of the insulin regulator Nkx6.1: an important late event in ß-cell differentiation. Intriguingly, the Drosophila orthologue of Nkx2.2, Vnd, is required for dMP2 formation. Drosophila Nkx6.1, the orthologue of mammalian Nkx6 (FlyBase name HGTX), is expressed by postmitotic dMP2 neurons, and it will be interesting to determine whether it too functions downstream of Vnd during Ilp7 regulation. Most strikingly, mammalian equivalents of two of the insulinergic inputs identified in this study, Hb9 and BMP signalling, are also required for several aspects of late ß-cell differentiation including the expression of Nkx6.1 and insulin. Together, these insect-mammalian comparisons provide evidence that, although the cell types involved look very different, some of the genetic circuitry regulating insulin is conserved between arthropods and chordates. This suggests that the power of fly genetics can now be harnessed to identify additional mammalian regulators of neuroendocrine cell fates and insulin expression (Miguel-Aliaga, 2008).

Origin and specification of the brain leucokinergic neurons of Drosophila: Similarities to and differences from abdominal leucokinergic neurons

The Drosophila central nervous system contains many types of neurons that are derived from a limited number of progenitors as evidenced in the ventral ganglion. The situation is much more complex in the developing brain. The main neuronal structures in the adult brain are generated in the larval neurogenesis, although the basic neuropil structures are already laid down during embryogenesis. The embryonic factors involved in adult neuron origin are largely unknown. To shed light on how brain cell diversity is achieved, a study was carried out of the early temporal and spatial cues involved in the specification of lateral horn leucokinin peptidergic neurons (LHLKs). The analysis revealed that these neurons have an embryonic origin. Their progenitor neuroblast were identified as Pcd6 in the Technau and Urbach terminology. Evidence was obtained that a temporal series involving the transcription factors Kr, Pdm, and Cas participates in the genesis of the LHLK lineage, the Castor window being the one in which the LHLKs neurons are generated. It was also shown that Notch signalling and Dimmed are involved in the specification of the LHLKs. It is concluded that serial homologies with the origin and factors involved in specification of the abdominal leucokinergic neurons (ABLKs) have been detected (Herrero, 2013).

Studies on neuroblast lineages in the developing ventral ganglia are numerous, but investigations of which lineages are present in cerebral ganglia and which are not have only just begun. Drosophila neurogenesis takes place at two stages: an embryonic stage, in which larval functions are established, and a larval stage, in which neurons involved in adult functions are added. Temporal genes regulating the postembryonic neuroblast lineages in the central brain and in the optic lobes have been identified, but little is known of brain neuroblast embryonic lineages. LHLK neurons offer the possibility of studying the embryonic origins of brain neurons and comparing them to the origins of other lineages including LK-expressing progeny. This study shows that LK-expressing neurons from different segments of brain and abdomen not only share neuropeptide expression but also cell number per hemisegment and neuronal cell appearance, characterized by long axons full of varicosities, large superficially located somas, but lack of coexpression of any small neurotransmitters. The results obtained above provide clues for defining the serial homology between neuroblasts from the protocerebrum and from the ventral ganglia, and for analyzing differences between the complex combinatorial code that defines the fates of LK-expressing neurons (Herrero, 2013).

The results suggest that the canonical temporal gene cascade Hb-Kr-Pdm-Cas-Grh is active in protocerebral neuroblasts as it is in thoracic and abdominal neuroblasts. Consequently, as in the VNC, temporal factors in the brain also activate the next gene and repress the 'next plus one' or the previous one. These factors, except for Hb and Kr, are weakly expressed in LHLK neurons at the early first instar larva, but the most important clues concerning their temporal implications are the effects of their loss and gain of function: LHLK specification is partially inhibited in kr and pdm mutants, and completely blocked in cas mutant. Only the grh mutant has no phenotypic effect on LHLKs, although its overexpression does have a phenotype, indicating that the Cas window is negatively regulated by Grh. On the other hand, svp is also involved in LHLK specification, probably not via its relation to hb but because it is expressed in another phase after the Cas window, as in many embryonic abdominal neuroblast lineages. Although the temporal factors implicated in the origin of LHLKs fit the model accepted for other NB lineages in the embryonic CNS, more studies are required to provide precise information about the timing of temporal factor expression and about the specification of the other progeny in the lineage and in other embryonic brain lineages (Herrero, 2013).

The results obtained in dimm overexpression experiments demonstrate the existence of other neurons with potential LK fates in the Drosophila brain. In this situation it seems that expression of the neuroendocrine differentiation gene dimm forces the 'almost' leucokinergic neurons to complete their differentiation. There are analogies with the results obtained for FMRFamide, where ectopic FMRFamide expression in Tv neurons is only observed when dimm is misexpressed. dimm is essential for transforming the synaptic vesicles of neurons into functional peptidergic vesicles. This study demonstrates that other neurons in the brain have the LK fate determinants but not the ability to adopt the neuropeptidergic cell fate. Interestingly, the ectopic LK neurons found in dimm overexpression correspond to different brain segments, namely deutocerebrum, tritocerebrum and protocerebrum. This could be pointing to serial homology in some brain lineages. Further analysis is needed to probe the LK fate in these segments (Herrero, 2013).

The two LK-expressing cell types share two main characteristics: the ventral-lateral location within their segments and their embryonic origin. LHLK neurons arise from a lineage located dorsally and near to the optic primordium, which corresponds to the protocerebral dorsal central lineage in Urbach (2003) terminology, or the basolateral dorsal lineage in Pereanu (2004) terminology. ABLKs arise from abdominal NB5-5, which is laterally located in the VNC, both are lateral in their respective segments, arise during embryonic neurogenesis and start expressing LK at the end of stage 17 (Herrero, 2013).

There are some differences in terms of temporal genes between LHLK and ABLK lineages. The analysis suggests that Cas is the temporal factor window specifying LHLK fate, whilea Cas/Grh temporal window has been proposed for ABLKs. There is evidence that the Cas window is long in some NBs of the trunk, and Cas has also been identified in postembryonic brain development. In the light of these findings it is proposed that, as in trunk neuroblasts, the Cas time window in the neuroblast Pcd6 lineage is extensive and the Cas inhibitory effect of Grh is delayed with respect to the abdominal segments. As a result, the LHLKs can be generated before grh expression; so that this factor is dispensable for the appearance of LHLKs. Hence, Grh effects on LHLKs are only observed when grh is overexpressed (Herrero, 2013).

Of the 27 genes, 7 were not expressed in either of the two types of LK neurons and their loss of function had different effects on their phenotypes. Three of these genes expressed in the ABLKs were hkb, gsb and ind, whose NB expression is weak in the protocerebrum. The expression of other two genes, also expressed in the ABLKs: unpg and runt, is sustained until the end of embryogenesis in the postmitotic cells. However ABLKs are controlled by the pair rule gene runt and the homeodomain gene unpg. It has been reported that runt regulates the expression boundaries of segment polarity genes in the VNC but not in the procephalon, while unpg, together with otd, is involved in the protocerebrum/deutocerebrum interface in the procephalic neuroctoderm. Hence these different functions could explain the different expression (Herrero, 2013).

Finally, ap and klu show extended brain expression in neuroblasts (klu) and in postmitotic neurons (ap) in the brain; however their effects are not the same in LHLKs and ABLKs: Ap regulates LK expression in LHLKs, while Klu does it in ABLKs. Xiao (2012) has shown that Klu is necessary in the brain for the renewal maintenance of type II neuroblasts, whereas VNC type I neuroblasts are probably not affected because other factors provide this function. Thus Klu has different functions in the brain and the VNC. In spite of these differences, ABLKs and LHLKs do share the presence or absence of expression of 19 genes, among which are not only the aforementioned temporal genes and postmitotic cofactors nab and sqz, but the segment polarity gene wg. Just as engrailed (en) marks the posterior border segment, wg marks the anterior one, as in the trunk segment, although less obviously. Four cephalic segments have been describe: intercalary, antennal, ocular and labral, the last two being part of the protocerebrum. The wg, en, gsb-d and hh segment polarity genes and the ind, msh, vnd columnar genes mark some of their boundary. The ocular segment contains the largest number of neuroblasts (60), and it is the most difficult to study because of its complexity. However it is clear that the anterior region of this neuromere is extended the most, with more than 25% of the wg-expressing neuroblasts at stage 11. On the other hand, the en expressing region is very much smaller (only 10 NBs). The LHLKs, like the ABLKs, belong to an anterior segment lineage. ABLK-progenitor neuroblast expresses ind but LHLKs cannot be assigned to a particular columnar neuroblast because the ocular segment has almost no ind identity. It may be concluded that the neuroblasts Pcd6 and NB5-5, from which the LK-expressing neurons arise in an equivalent temporal embryonic window, are serially homologous, although several individual characteristics distinguish their development. In some of the serially homologous neuroblast lineages of the VNC, there are differences between thoracic and abdominal neuromeres, and it is expected that such segment-specific differences would be more pronounced between the brain and the VNC where the genetic backgrounds are different, and the canonical orthogonal expression genes described in the VNC are mainly not conserved in the protocerebral neuromeres. Clarification of the progression of the Leucokinin-progenitor neuroblasts during brain development and comparison with the situation in the trunk could help in an understanding of what makes the brain different from the VNC (Herrero, 2013).

Insulin/IGF-Regulated Size Scaling of Neuroendocrine Cells Expressing the bHLH Transcription Factor Dimmed in Drosophila

Neurons and other cells display a large variation in size in an organism. Thus, a fundamental question is how growth of individual cells and their organelles is regulated. Is size scaling of individual neurons regulated post-mitotically, independent of growth of the entire CNS? Although the role of insulin/IGF-signaling (IIS) in growth of tissues and whole organisms is well established, it is not known whether it regulates the size of individual neurons. The role of IIS in the size scaling of neurons in the Drosophila CNS was studied. By targeted genetic manipulations of insulin receptor (dInR) expression in a variety of neuron types it was demonstrated that the cell size is affected only in neuroendocrine cells specified by the bHLH transcription factor DimmedD (Dimm). Several populations of Dimm-positive neurons tested displayed enlarged cell bodies after overexpression of the dInR, as well as PI3 kinase and Akt1 (protein kinase B), whereas Dimm-negative neurons did not respond to dInR manipulations. Knockdown of these components produce the opposite phenotype. Increased growth can also be induced by targeted overexpression of nutrient-dependent TOR (target of rapamycin) signaling components, such as Rheb (small GTPase), Tor and S6K (S6 kinase). After Dimm-knockdown in neuroendocrine cells manipulations of dInR expression have significantly less effects on cell size. It was also shown that dInR expression in neuroendocrine cells can be altered by up or down-regulation of Dimm. This novel dInR-regulated size scaling is seen during postembryonic development, continues in the aging adult and is diet dependent. The increase in cell size includes cell body, axon terminations, nucleus and Golgi apparatus. It is suggested that the dInR-mediated scaling of neuroendocrine cells is part of a plasticity that adapts the secretory capacity to changing physiological conditions and nutrient-dependent organismal growth (Luo, 2013).

EFFECTS OF MUTATION

To test for roles of a putative 'c929' gene in the development and/or function of peptidergic cells, deletions flanking the c929 insertion site were generated. These deletions cause recessive lethality, owing to disruption of at least one essential gene, cryptocephal (crc). However, many homozygous mutant animals survived into the larval stages, when the fates of CNS peptidergic neurons could be examined (Hewes, 2003).

By immunostaining the mutant animals for a peptide biosynthetic enzyme (peptidylglycine-alpha-hydroxylating mono-oxygenase: PHM), a novel phenotype was detected: R6/Rev8 trans-heterozygous animals contain small deficiencies around the c929 insertion site that are ~12 and ~35 kb respectively. Transheterozygous larvae display marked reductions in PHM protein levels in all strongly c929-positive CNS neurons. c929-negative neurons are unaffected in R6/Rev8 larvae, and weakly or transiently c929-positive neurons, such as the peptidergic VA neurons, show smaller reductions in PHM immunostaining. The mutant phenotype is detectable at the time of larval hatching and throughout all larval stages. By contrast, heterozygous R6 or Rev8/+ larvae are essentially wild type, although these alleles display mild haploinsufficiency with other markers. These results demonstrate a requirement for ~10 kb of DNA flanking the c929 insertion site for the normal expression and/or maintenance of PHM in c929-positive CNS neurons. The affected gene was named dimmed to reflect the diminished staining (Hewes, 2003).

Six additional neurosecretory markers were used in dimm mutant larvae; all six display 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, LK, the FMRF propeptide and several PT2 positive neuropeptides — and the putative neuropeptide biosynthetic enzyme Furin 1. All c929-positive neurons display the mutant phenotype for at least one marker, PHM; many show reduced immunostaining with multiple markers. For example, the Tv neurons have reduced levels of four markers: PHM, the FMRF propeptide, --RFamide peptides and Furin 1. Thus, in a large and diverse population of CNS peptidergic neurons, dimm regulates levels of a broad array of secretory proteins (Hewes, 2003).

Since the three classes of c929-positive endocrine cells also likely secrete peptide hormones, they were also tested for effects of the dimm mutation. The ring gland and tracheal endocrine cells display severe reductions in peptide immunostaining for PHM and/or MM in dimm^-/- mutants; the gut endocrine cells were not tested. Taken together, these results suggest a crucial role for dimm in controlling bioactive peptide levels in diverse neuronal and endocrine secretory cells (Hewes, 2003).

Using chromosomal deletions, the dimm gene was genetically mapped. Peptide immunostaining was performed on deficiency (Rev8) homozygotes and on hemizygotes bearing one copy of R6 (or Rev8) over one of several independently derived deficiencies of the entire 39C4-D1 region of chromosome 2L. In each case, the effects on peptide immunostaining were comparable, although the reduction in myomodulin (MM) staining in larvae homozygous for Rev4, a null allele, was more pronounced than in R6/Rev8 trans-heterozygotes. Thus, R6 and Rev8 are hypomorphic alleles. Normal peptide immunostaining was restored in male Rev8 homozygotes bearing a duplication of chromosome bands 35A-40, consistent with the location of dimm in 39C4-D1 (Hewes, 2003).

In contrast to R6/Rev8 mutants, larvae with disruptions in the crc gene, or deletions of DNA extending up to 200-300 kb towards the telomere display wild-type neuropeptide levels. Thus, dimm is not crc, nor is it any other gene located distal to the site of the c929 insertion (Hewes, 2003).

The closest gene proximal to c929 is CG8667 (Mistr), found within 25 kb. It encodes a basic helix-loop-helix (bHLH) protein that is a member of the Atonal subfamily of transcription factors (Moore, 2000). Its bHLH domain displays 79% identity with the mouse Mist1 protein (Pin, 1999). In Rev8 homozygous embryos, CG8667 mRNA expression is markedly reduced, but not eliminated, consistent with the identification of Rev8 as a hypomorphic dimm allele. After 5' RACE identification of the 5' end of CG8667, a P-element insertion (dimm^KG02598) located 111 bp upstream was detected. dimm^KG02598 displays homozygous lethality, and represents a severe hypomorphic dimm allele, because CG8667 mRNA expression appears low or undetectable in dimm^KG02598 homozygous mutant embryos. Hatchling dimm^KG02598/Rev4 larvae display reduced immunostaining for PT2-positive neuropeptides. Normal PT2 immunostaining is restored after precise excision of the dimm^KG02598 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. The dimm^KG02598 mutation also reduces levels of secretory peptide mRNAs in the Tv neuroendocrine cells, which display high levels of Fmrf mRNA expression: when assessed using in situ hybridization, the mean number of Fmrf-positive Tv neurons per CNS was 5.57 in dimm heterozygotes and 2.33 in dimm hemizygotes. These combined data indicate that in the absence of dimm, there is a reduction in levels of both secretory peptide mRNAs and secretory peptides (Hewes, 2003).

To examine further the effect of disruptions in CG8667 expression, RNAi analysis was performed and reduced levels of MM immunostaining was observed in hatchling stage larvae. The reduction in MM immunostaining is comparable with the phenotype in null dimm^-/- mutants. The same results were obtained using two additional antisera, PT2 and anti-LK. The ability of a UAS-dimm::Myc transgene to restore neuropeptide levels in dimm^-/- animals was also tested. The c127-Gal4 line was used to drive dimm::Myc expression in a small set of ventral CNS neurons, which included the 14 LK-positive cells in abdominal neuromeres. Expression of dimm::Myc selectively restores normal levels of LK immunostaining in Rev8/Rev4 animals, but not in the absence of the Gal4 driver. The rescue displays cell specificity: the FMRF-positive MP2 neurons do not express UAS-GFP by c127-Gal4, and they are not rescued. Together, these results support the hypothesis that dimm is the Drosophila Mist1 ortholog, CG8667 (Hewes, 2003).

A gain-of-function analysis was performed by driving UAS-dimm::myc in an otherwise wild-type background. When misexpressed using a pan-neuronal elav-GAL4 driver, most embryos die. This suggests that the effects of dimm on shaping neuronal properties can be widespread. To permit a more restricted analysis, ap-Gal4, a P{Gal4} reporter inserted in the apterous (ap) gene was used. When overexpressed in a subset of brain neurons, dimm increases the brightness of LK immunostaining in the cell body and processes of the LK-positive Br1 neuron. dimm overexpression does not produce widespread LK misexpression, but it reproducibly increases the number of LK-positive neurons from one (in animals lacking the ap-Gal4 element) to two. The additional LK-positive neuron is always adjacent to the normal one. Thus, dimm can alter the properties of normal neuroendocrine cells, and it can affect the number of cells displaying a neuroendocrine phenotype (Hewes, 2003).

Whether secretory cells survive and differentiate in dimm^-/- mutant animals was determined. In larvae homozygous for the null allele, Rev4, some of the affected cells displayed low residual immunostaining for secretory proteins. Thus, some dimm-expressing cells survive in mutant larvae and are at least partially differentiated. Others display a complete loss of peptide immunostaining, and their status is unclear (Hewes, 2003).

In order to determine the fates of the latter cells, Gal4/UAS mosaics were used to express ectopic, non-secretory proteins in dimm mutant neurons. Thirty four CNS neurons were used that co-expressed three different markers: the c929 reporter, the putative peptide biosynthetic enzyme Furin 1, and ap-Gal4. In dimm^-/- larval CNS, all 34 neurons display strongly reduced, and often undetectable, immunostaining for Furin 1. Using ap-Gal4 to drive heterologous expression of a tau::Myc fusion protein, it was found that all 34 of these neurons are present and display normal morphology in the dimm^-/- larvae. In addition, the intensity of anti-Myc immunostaining was not affected. Identical results were obtained using green fluorescent protein (GFP) to mark the cells. Thus, dimm mutant neurons display multiple differentiated features and synthesize non-secretory proteins at normal levels throughout larval development (Hewes, 2003).

The effects of dimm were also examined on the terminal arbor of the LK-positive neurons. These cells display reduced soma LK immunostaining in dimm^-/- CNS. Each neuron has a single efferent axon that projected across the posterior muscle 8 surface and terminates dorsally near a tracheal branch. In third instar dimm^-/- larvae, these axons also display reduced LK immunostaining. However, a sufficient number of immunoreactive boutons remain to indicate a normal axonal expanse. Thus, the effects of dimm on this LK neuron appear limited to expression of the transmitter phenotype (Hewes, 2003).

Earlier measures of the dimmed phenotype were restricted to analysis of proteins abundant in the regulated secretory pathway. Whether there is an effect of dimm on constitutively secreted proteins was also tested. With ap-Gal4, expression of a CD8::GFP fusion protein (UAS-CD8::GFP) was directed to a subset of dimm-expressing neurons. CD8 is an integral membrane protein that is targeted to the plasma membrane in Drosophila cells. In dimm^-/- mutant larvae, all 34 ap-Gal4 (Furin-1) neurons express CD8::GFP and display normal neuritic projections. However, CD8::GFP levels are significantly lower in c929-positive neurons in the dimm^-/- background. This effect is 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 (Hewes, 2003).

Because ap-dependent expression of transgenes is unaffected by dimm, it was not possible to uncouple neuropeptide transcription from potential effects of dimm on secretory activity. Thus, when ap-Gal4 drives ectopic expression of the pdf neuropeptide gene, ectopic pdf mRNA levels are unaffected in dimm^-/- larvae. By contrast, ectopic PDF protein levels are severely reduced. Immunostaining was performed for two peptide epitopes of the proPDF precursor: PAP and PDF. 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. Thus, dimm is required within c929-positive neurons for the maintenance of ectopic PDF neuropeptide levels, but not of ectopic pdf mRNA (Hewes, 2003).

Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), tests were performed to see whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, it was found that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply 'locked-in,' but must be actively maintained by persistent developmental transcription factor networks. The cross-regulatory relationships were examined between all transcription factors that persisted in adult Tv1/Tv4 neurons. Certain critical cross-regulatory relationships that had existed between these transcription factors during development are no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, these results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain (Eade, 2012).

The data provide novel insight supporting the view of Blau and Baltimore (1991) that cellular differentiation is a persistent process that requires active maintenance, rather than being passively 'locked-in' or unalterable. Two primary findings are made in this study regarding the long-term maintenance of neuronal identity. First, all known developmental transcription factors acting in postmitotic Tv1 and Tv4 neurons to initiate the expression of subtype terminal differentiation genes are then persistently required to maintain their expression. Second, it was found that key developmental cross-regulatory relationships that initiated the expression of certain transcription factors were no longer required for their maintained expression in adults. Notably, this was found to be the case even between transcription factors whose expression persists in adults (Eade, 2012).

In this study, all transcription factors implicated in the initiation of subtype-specific neuropeptide expression in Tv1 and Tv4 neurons were found to maintain subtype terminal differentiation gene expression in adults (see Summary of changes in subtype transcription network configuration between initiation and maintenance of subtype identity). In Tv1, col, eya, ap and dimm are required for Nplp1 initiation during development. In this study, knockdown of each transcription factor in adult Tv1 neurons was shown to dramatically downregulate Nplp1. In Tv4 neurons, FMRFa initiation during development requires eya, ap, sqz, dac, dimm and retrograde BMP signaling. Together with previous work showing that BMP signaling maintains FMRFa expression in adults (Eade, 2009), this study now demonstrates that all six regulatory inputs are required for FMRFa maintenance. Most transcription factors, except for dac, also retained their relative regulatory input for FMRFa and Nplp1 expression. In addition, individual transcription factors also retained their developmental subroutines. For example, as found during development, dimm was required in adults to maintain PHM (independently of other regulators) and FMRFa/Nplp1 expression (combinatorially with other regulators) (Eade, 2012).

The few genetic studies that test a persistent role for developmental transcription factors support their role in initiating and maintaining terminal differentiation gene expression. In C. elegans, where just one or two transcription factors initiate most neuronal subtype-specific terminal differentiation genes, they then also appear to maintain their target terminal differentiation genes. In ASE and dopaminergic neurons respectively, CHE-1 and AST-1 initiate and maintain expression of pertinent subtype-specific terminal differentiation genes. In vertebrate neurons, where there is increased complexity in the combinatorial activity of transcription factors in subtype-specific gene expression, certain transcription factors have been demonstrated to be required for maintenance of subtype identity. These are Hand2 that initiates and maintains tyrosine hydroxylase and dopa ß-hydroxylase expression in mouse sympathetic neurons, Pet-1, Gata3 and Lmx1b for serotonergic marker expression in mouse serotonergic neurons, and Nurr1 for dopaminergic marker expression in murine dopaminergic neurons (Eade, 2012).

However, while these studies confirm a role for certain developmental transcription factors in subtype maintenance, it had remained unclear whether the elaborate developmental subtype transcription networks, that mediate neuronal differentiation in Drosophila and vertebrates, are retained in their entirety for maintenance, or whether they become greatly simplified. This analysis of all known subtype transcription network factors in Tv1 and Tv4 neurons now indicates that the majority of a developmental subtype transcription network is indeed retained and required for maintenance. Why would an entire network of transcription factors be required to maintain subtype-specific gene expression? The combinatorial nature of subtype-specific gene expression entails cooperative transcription factor binding at clustered cognate DNA sequences and/or synergism in their activation of transcription. In such cases, the data would indicate that this is not dispensed with for maintaining terminal differentiation gene expression in mature neurons (Eade, 2012).

How the transcription factors of the subtype transcription networks are maintained is less well understood. An elegant model has emerged from studies in C. elegans, wherein transcription factors stably auto-maintain their own expression and can then maintain the expression of subtype terminal differentiation genes. The transcription factor CHE-1 is a key transcription factor that initiates and maintains subtype identity in ASE neurons. CHE-1 binds to a cognate DNA sequence motif (the ASE motif) in most terminal differentiation genes expressed in ASE neurons, as well as in its own cis-regulatory region. Notably, a promoter fusion of the che-1 transcription factor failed to express in che-1 mutants, indicative of CHE-1 autoregulation, and for the cooperatively-acting TTX-3 and CEH-10 transcription factors in AIY neurons. Thus, subtype maintenance in C. elegans is anchored by auto-maintenance of the transcription factors that initiate and maintain terminal differentiation gene expression (Eade, 2012).

In contrast, all available evidence in Tv1 and Tv4 neurons fails to support such an autoregulatory mechanism. An ap reporter (apC-t-lacZ) is expressed normally in ap mutants, and in this study ap^dsRNAi was not found to alter ap^GAL4 reporter activity. Moreover, col transcription was unaffected in col mutants that express a non-functional Col protein. This leaves unresolved the question of how the majority of the transcription factors are stably maintained. For transcription factors that are initiated by transiently expressed inputs, a shift to distinct maintenance mechanisms have been invoked and in certain cases shown. In this study, this was found for the loss of cas expression in Tv1 (required for col initiation) and the loss of cas, col and grh in Tv4 (required for eya, ap, dimm, sqz, dac initiation). However, it was surprising to find that the cross-regulatory relationships between persistently-expressed transcription factors were also significantly altered in adults. Notably, eya initiated but did not maintain dimm in Tv4. In Tv1, col initiated but did not maintain eya, ap or dimm. This was particularly unexpected as eya remained critical for FMRFa maintenance and col remained critical for Nplp1 maintenance. Indeed, although tests were performed for cross-regulatory interactions between all transcription factors in both the Tv1 and Tv4 subtype transcription networks, only Dimm was found to remain dependent upon its developmental input; Eya and Ap in Tv1 as well as Ap in Tv4. However, even in this case, the regulation of Dimm was changed; it no longer required eya in Tv4, and in Tv1 it no longer required col, in spite of the fact that both col and eya are retained in these neurons. It is anticipated that such changes in transcription factor cross-regulatory relationships will be found in other Drosophila and vertebrate neurons, which exhibit high complexity in their subtype transcription networks. Indeed, recent evidence has found that in murine serotonergic neurons, the initiation of Pet-1 requires Lmx-1b, but ablation of Lmx-1b in adults did not perturb the maintenance of Pet-1 expression (Eade, 2012).

The potential role of autoregulation for the other factors in the Tv1/Tv4 subtype transcription networks is being pursued. However, there are three additional, potentially overlapping, models for subtype transcription network maintenance. First, regulators may act increasingly redundantly upon one another. Second, unknown regulators may become increasingly sufficient for transcription factor maintenance. Third, transcription factors may be maintained by dedicated maintenance mechanisms, as has been shown for the role of trithorax group genes in the maintenance of Hox genes and Engrailed. Moreover, chromatin modification is undoubtedly involved and likely required to maintain high-level transcription of Tv transcription factors as well as FMRFa, Nplp1 and PHM. However, the extent to which these are instructive as opposed to permissive has yet to be established. In this light, it is intriguing that MYST-HAT complexes, in addition to the subtype transcription factors Che-1 and Die-1, are required for maintenance of ASE-Left subtype identity in C. elegans (Eade, 2012).

Taken together, these studies have identified two apparent types of maintenance mechanism that are operational in adult neurons. On one hand, there are sets of genes that are maintained by their initiating set of transcription factors. These include the terminal differentiation genes and the transcription factor dimm. On the other, most transcription factors appear to no longer require regulatory input from their initiating transcription factor(s). Further work will be required to better understand whether these differences represent truly distinct modes of gene maintenance or reflect the existence of yet unidentified regulatory inputs onto these transcription factors. One issue to consider here is that the expression of certain terminal differentiation genes in neurons, but perhaps not subtype transcription factors, can be plastic throughout life, with changes commonly occurring in response to a developmental switch or physiological stimulus. Thus, terminal differentiation genes may retain complex transcriptional control in order to remain responsive to change. It is notable, however, that FMRFa, Nplp1 and PHM appear to be stably expressed at high levels in Tv1/4 neurons, and no conditions were found that alter their expression throughout life. Thus, these are considered to be stable terminal differentiation genes akin to serotonergic or dopaminergic markers in their respective neurons that define those cells' functional identity and, where tested, are actively maintained by their developmental inputs. Tv1/4 neurons undoubtedly express a battery of terminal differentiation genes, and sets of unknown transcription factors are likely required for their subtype-specific expression. Subtype transcription networks are considered to encompass all regulators required for differentiating the expression of all subtype-specific terminal differentiation genes. Further, differentiation of subtype identity is viewed as the completion of a multitude of distinct gene regulatory events in which each gene is regulated by a subset of the overall subtype transcription network. As highly restricted terminal differentiation genes expressed in Tv1 and Tv4 neurons, it is believed that Nplp1, FMRFa and PHM provide a suitable model for the maintenance of overall identity, with the understanding that other unknown terminal differentiation genes expressed in Tv1 and Tv4 may not be perturbed by knockdown of the transcription factors tested in this study. In the future, it will be important to incorporate a more comprehensive list of regulators and terminal differentiation genes for each neuronal subtype. However, it is believed that the principles uncovered in this study for FMRFa, Nplp1 and PHM maintenance will hold for other terminal differentiation genes (Eade, 2012).

Finally, it is proposed that the active mechanisms utilized for maintenance of subtype differentiation represent an Achilles heel that renders long-lived neurons susceptible to degenerative disorders. Nurr1 ablation in adult mDA neurons reduced dopaminergic markers and promoted cell death. Notably, Nurr1 mutation is associated with Parkinson's disease, and its downregulation is observed in Parkinson's disease mDA neurons. Adult mDA are also susceptible to degeneration in foxa2 heterozygotes, another regulator of mDA neuron differentiation that is maintained in adult mDA neurons. Studies in other long-lived cell types draw similar conclusions. Adult conditional knockout of Pdx1 reduced insulin and ß-cell mass and, importantly, heterozygosity for Pdx1 leads to a rare monogenic form of non-immune diabetes, MODY4. Similarly, NeuroD1 haploinsufficiency is linked to MODY6 and adult ablation of NeuroD in β-islet cells results in β-cell dysfunction and diabetes. These data, together with current results, underscore the need to further explore the transcriptional networks that actively maintain subtype identity, and hence the function, of adult and aging cells (Eade, 2012).

EVOLUTIONARY HOMOLOGS

Mist1 is a basic helix-loop-helix (bHLH) transcription factor that is highly expressed in the adult pancreas. The mouse Mist1 gene has been sequenced and its genomic structure determined. Fluorescence in situ hybridization mapping located the Mist1 gene to the telomere of mouse chromosome 5 at position 5G2-5G3, an area that is syntenic to human chromosome 13q and which contains several additional pancreatic regulatory genes including IPF1 and CDX (Pin, 1999).

Basic helix-loop-helix (bHLH) proteins often belong to a family of transcription factors that bind to the DNA target sequence -CANNTG- (E-box) that is present in the promoter or enhancer regions of numerous developmentally regulated genes. A novel bHLH factor, termed Mist1, was identified by virtue of its ability to interact with E-box regulatory elements in a yeast 'one-hybrid' screening procedure. Northern analysis revealed that Mist1 transcripts are expressed in several adult tissues, including stomach, liver, lung, and spleen but no expression is detected in the heart, brain, kidney, or testis. During mouse embryogenesis, Mist1 mRNA is first observed at E10.5 in the primitive gut and in the developing lung bud. Expression persists through E16.5 and remains restricted primarily to the epithelial lining. Mist1 is also detected in skeletal muscle tissues beginning at E12.5, persisting throughout all embryonic stages examined although in older embryos and in the adult expression becomes severely reduced. At later developmental times, Mist1 transcripts also are found in the pancreas, submandibular gland, and adult spleen. As predicted, the Mist1 protein is nuclear and binds efficiently to E-box sites as a homodimer. Mist1 also is capable of binding to E-box elements when complexed as a heterodimer with the widely expressed E-proteins, E12 and E47. Surprisingly, although Mist1 binds to E-boxes in vivo, the Mist1 protein lacks a functional transcription activation domain. These observations suggest that Mist1 may function as a unique regulator of gene expression in several different embryonic and postnatal cell lineages (Lemercier, 1997).

A good model system to examine aspects of positive and negative transcriptional regulation is the muscle-specific regulatory factor, MyoD, which is a basic helix-loop-helix (bHLH) transcription factor. Although MyoD has the ability to induce skeletal muscle terminal differentiation in a variety of non-muscle cell types, MyoD activity itself is highly regulated through protein-protein interactions involving several different co-factors. A novel bHLH protein, Mist1, influences MyoD function. Mist1 accumulates in myogenic stem cells (myoblasts) and then decreases as myoblasts differentiate into myotubes. Mist1 functions as a negative regulator of MyoD activity, preventing muscle differentiation and the concomitant expression of muscle-specific genes. Mist1-induced inhibition occurs through a combination of mechanisms, including the formation of inactive MyoD-Mist1 heterodimers and occupancy of specific E-box target sites by Mist1 homodimers. Mist1 lacks a classic transcription activation domain and instead possesses an N-terminal repressor region capable of inhibiting heterologous activators. Thus, Mist1 may represent a new class of repressor molecules that play a role in controlling the transcriptional activity of MyoD, ensuring that expanding myoblast populations remain undifferentiated during early embryonic muscle formation (Lemercier, 1998).

Transcription factors of the basic Helix-Loop-Helix (bHLH) protein family play key roles in several developmental processes. Mist1 belongs to this group of proteins and shares several properties with the other family members. For example, Mist1 is capable of dimerization with the ubiquitously expressed E2A bHLH proteins and exhibits a strong DNA-binding activity to the core E-box sequence. Using in-situ hybridization and Northern blot hybridization, Mist1 mRNA has been detected in a variety of embryonic and adult rodent tissues. To understand the molecular mechanisms involved in the expression of the gene, the rat Mist1 gene has been cloned and 2.5 kb of its 5' flanking region have been analyzed. The Mist1 gene spans over 5 kilobases and is composed of two exons separated by a unique intron. The entire coding region is localized in the second exon. Sequence analysis of the promoter region indicates an absence of TATA-box or CAAT-box sequence, but several consensus Sp1-binding sites are present near the transcription start site. Deletion analysis of the promoter region identified a 272 bp proximal fragment to be sufficient to drive expression of a reporter gene in NIH3T3 fibroblasts. Subsequent deletion of potential Sp1 sites results in a marked decrease in promoter activity. Electrophoretic mobility shift assays reveal that Sp1 binds to two different regions in the proximal promoter, a typical Sp1 site located at (-38; -33) and a G/C-rich region between (-67; -62). These data suggest that the basal expression of this TATA-less gene might be driven by general transcription factors, such as Sp1 (Lemercier, 2000).

Mist1 is a basic helix-loop-helix transcription factor that represses E-box-mediated transcription. Previous studies have suggested that the Mist1 gene is expressed in a wide range of tissues, although a complete characterization of Mist1 protein accumulation in the adult organism has not been described. In an effort to identify specific cell types that contain the Mist1 protein, antibodies specific for Mist1 were generated and used in Western blot and immunohistochemical assays. These studies show that the Mist1 protein is present in many different tissues but that it is restricted to cell types that are exclusively secretory in nature. Pancreatic acinar cells, serous or seromucous cells of the salivary glands, chief cells of the stomach, and secretory cells of the prostate and seminal vesicle show high levels of Mist1 protein, whereas nonserous exocrine cells, including the mucus-producing cells of the salivary glands, remain Mist1 negative. These results identify Mist1 as the first transcription factor that exhibits this unique serous-specific expression pattern and suggest that Mist1 may have a key role in establishing and maintaining a pathway responsible for the exocytosis of serous secretions (Pin, 2000).

The pancreas is a complex organ that consists of separate endocrine and exocrine cell compartments. Although great strides have been made in identifying regulatory factors responsible for endocrine pancreas formation, the molecular regulatory circuits that control exocrine pancreas properties are just beginning to be elucidated. In an effort to identify genes involved in exocrine pancreas function, Mist1, a basic helix-loop-helix transcription factor expressed in pancreatic acinar cells, has been identified. Mist1-null [Mist1(KO)] mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation. The exocrine disorganization is accompanied by increases in p8, RegI/PSP, and PAP1/RegIII gene expression, mimicking the molecular changes observed in pancreatic injury. By 12 m, Mist1(KO) mice develop lesions that contain cells coexpressing acinar and duct cell markers. Analysis of the factors involved in cholecystokinin (CCK) signaling reveal inappropriate levels of the CCK receptor A and the inositol-1,4,5-trisphosphate receptor 3, suggesting that a functional defect exists in the regulated exocytosis pathway of Mist1(KO) mice. Based on these observations, it is proposed that Mist1(KO) mice represent a new genetic model for chronic pancreas injury and that the Mist1 protein serves as a key regulator of acinar cell function, stability, and identity (Pin, 2001).

Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26 (Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, Cx26 and Cx32 gene activities were examined in mice lacking the basic helix-loop-helix transcription factor Mist1 (Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice reveals a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription is greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remains unaffected. However, in the absence of Cx32 protein, Cx26 does not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirm that Mist1 transcriptionally regulates expression of the Cx32 gene. It is concluded that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted (Rukstalis, 2003).

The pancreas consists of three main cell lineages (endocrine, exocrine, and duct) that develop from common primitive foregut precursors. The transcriptional network responsible for endocrine cell development has been studied extensively, but much less is known about the transcription factors that maintain the exocrine and duct cell lineages. One transcription factor that may be important to exocrine cell function is Mist1, a basic helix-loop-helix (bHLH) factor that is expressed in acinar cells. In order to perform a molecular characterization of this protein, coimmunoprecipitation and bimolecular fluorescence complementation assays, coupled with electrophoretic mobility shift assay studies, were used to show that Mist1 exists in vivo as a homodimer complex. Analysis of transgenic mice expressing a dominant-negative Mist1 transgene [Mist1(mutant basic)] [Mist1(MB)]) revealed the cell autonomous effect of inhibiting endogenous Mist1. Mist1(MB) cells become disorganized, exhibit a severe depletion of intercellular gap junctions, and express high levels of the glycoprotein clusterin, which has been shown to demarcate immature acinar cells. Inhibition of Mist1 transcriptional activity also leads to activation of duct-specific genes, such as cytokeratin 19 and cytokeratin 20, suggesting that alterations in the bHLH network produce a direct acinar-to-ductal phenotypic switch in mature cells. It is proposed that Mist1 is a key transcriptional regulator of exocrine pancreatic cells and that in the absence of functional Mist1, acinar cells do not maintain their normal identity (Zhu, 2004).

Ca2+ signaling and exocytosis are highly polarized functions of pancreatic acinar cells. The role of cellular architecture in these activities and the capacity of animals to tolerate aberrant acinar cell function are not known. A key regulator of acinar cell polarity is Mist1, a basic helix-loop-helix transcription factor. Ca2+ signaling and amylase release were examined in pancreatic acini of wild type and Mist1 null mice to gain insight into the importance of cellular architecture for Ca2+ signaling and regulated exocytosis. Mist1^-/- acinar cells exhibit dramatically altered Ca2+ signaling with up-regulation of the cholecystokinin receptor but minimal effect upon expression of the M3 receptor. However, stimulation of inositol 1,4,5-trisphosphate production by cholecystokinin and carbachol is inefficient in Mist1^-/- cells. Although agonist stimulation of Mist1^-/- cells evokes a Ca2+ signal, often the Ca2+ increase is not in the form of typical Ca2+ oscillations but rather in the form of a peak/plateau-type response. Mist1^-/- cells also display distorted apical-to-basal Ca2+ waves. The aberrant Ca2+ signaling is associated with mislocalization and reduced Ca2+ uptake by the mitochondria of stimulated Mist1^-/- cells. Deletion of Mist1 also leads to mislocalization of the Golgi apparatus and markedly reduced digestive enzyme content. The combination of aberrant Ca2+ signaling and reduced digestive enzyme content results in poor secretion of digestive enzymes. Yet, food consumption and growth of Mist1^-/- mice are normal for at least 32 weeks. These findings reveal that Mist1 is critical to normal organelle localization in exocrine cells and highlight the critical importance of maintaining cellular architecture and polarized localization of cellular organelles in generating a propagating apical-to-basal Ca2+ wave. The studies also reveal the spare capacity of the exocrine pancreas that allows normal growth and development in the face of compromised exocrine pancreatic function (Luo, 2005).

A single transcription factor is sufficient to induce and maintain secretory cell architecture

This study hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) (see Drosophila dimm) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. That targeted deletion of MIST1 causes dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs cause them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induces a cohort of genes regulated by MIST1 in multiple organs but does not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. These results indicate that cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints" (Lo, 2017).

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date revised: 22 December 2017

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