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).
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 apGal4 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).
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 (Mist1KO), pancreatic exocrine cells display reduced intracellular organization. Moreover, the Mist1KO 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).
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).
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).
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).
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).
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 (dimmKG02598) located 111 bp upstream was detected. dimmKG02598 displays homozygous lethality, and represents a severe hypomorphic dimm allele, because CG8667 mRNA expression appears low or undetectable in dimmKG02598 homozygous mutant embryos. Hatchling dimmKG02598/Rev4 larvae display reduced immunostaining for PT2-positive neuropeptides. Normal PT2 immunostaining is restored after precise excision of the dimmKG02598 P element. Consistent with the conclusion that dimm and crc are separate genes, KG02598 was lethal when trans-heterozygous with Rev4, but not with crc1. The dimmKG02598 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).
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 apdsRNAi was not found to alter apGAL4 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).
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date revised: 20 April 2012
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