FMRFamide


DEVELOPMENTAL BIOLOGY

In situ hybridization techniques were used to describe the cellular distribution of transcripts from a Drosophila gene that encodes multiple FMRFamide-related neuropeptides. Exon-specific oligonucleotide probes were used to assay transcription in both embryonic and larval stages. A pattern of hybridization signals is found that is restricted to the central nervous system and, within that tissue, is cell-specific. The pattern included 36 distinct signals distributed throughout both the brain and segmental nerve cord (ventral ganglion). These observations suggest that the cell-specific pattern of FMRFamide-like neuropeptide expression in the Drosophila CNS is due to the restricted expression of specific gene transcripts. With few exceptions, all previously identified FMRFamide-immunoreactive neurons in Drosophila larvae express FMRFamide gene transcripts. The 36 hybridization regions of the CNS could be divided into three categories, based on their signal intensities (strong, moderate, and weak). The differences in intensity are reproducible and suggest that steady-state levels of specific neuropeptide RNA differ among individual neurons. The two exon-specific probes produce patterns that are indistinguishable both in pattern and in intensity. This supports the previous conclusion that the one detectable FMRFamide transcript contains both exons. A single identifiable signal is detected during embryogenesis (beginning at stage 16), but the mature complement of signals is not fully established until the final larval stages (Schneider, 1991).

Protein expression of the FMRFamide neuropeptide gene in Drosophila has been mapped with polyclonal antisera against three small peptides whose sequences were derived from the Drosophila proFMRFamide precursor. One antiserum is affinity-purified and extensively characterized. The enriched antibodies label 15-21 bilaterally symmetric pairs of neurons in a pattern that correspond very closely to the pattern of in situ hybridization that was determined previously. The other antisera produce complementary results. These findings suggest that the antisera specifically label cells that express the FMRFamide gene. Strong staining was observed in identified larval interneurons and neuroendocrine cells, and moderate to weak staining in neurons of unknown function. The adult pattern of expression includes both larval neurons whose immunoreactivity persists through metamorphosis and adult-specific neurons. During metamorphosis, transient staining is observed in a small number of neurons and in specific neuropil regions that included the central body, the protocerebral bridge, and the optic ganglia. Based on these morphological features, it is suggested that the FMRFamide-like neuropeptides in Drosophila play a number of functional roles, perhaps affecting both physiological and developmental phenomena. Such roles include general modulation throughout all post-embryonic stages, via the blood, and also more stage- and region-specific modulation within the CNS (Schneider, 1993a).

DPKQDFMRFamide is one of five different FMRFamide-containing peptides encoded in the Drosophila FMRFamide gene. To study the cellular expression of DPKQDFMRFamide, antisera was generated to DPKQD, the N-terminal sequence of the peptide, to avoid crossreactivity with other -FMRFamide-containing peptides. The antisera were purified and the specificity characterized. DPKQDFMRFamide immunoreactive material is first observed in the embryonic central nervous system (CNS) in one cell of the subesophageal ganglion and one cell in each of the three thoracic ganglia. This pattern of expression is observed in larval, pupal, and adult neural tissue, albeit with increased signal intensity. In larva, pupa, and adult, additional cells in the superior protocerebrum (a thoracic ganglion) and an abdominal ganglion express DPKQDFMRFamide immunoreactive material. Immunoreactivity is observed in a cell in the lateral protocerebrum of pupa and adult and cells in the optic lobe of adult. No immunoreactive material is observed in gut tissue. DPKQDFMRFamide antisera stain a subset of cells previously identified by in situ hybridization and immunocytochemistry to express the FMRFamide transcript and polypeptide precursor. These data suggest that the Drosophila FMRFamide polypeptide precursor undergoes differential processing to produce DPKQDFMRFamide immunoreactive material in a limited number of cells expressing the FMRFamide precursor (Nichols, 1995a).

The expression of SDNFMRFamide, one of five different FMRFamide-containing peptides encoded by the Drosophila melanogaster FMRFamide gene, has been determined. To study expression, antisera to the N-terminus of SDNFMRFamide was generated to avoid crossreactivity with FMRFamide-containing peptides. The antisera were purified and the specificity characterized. SDNFMRFamide immunoreactive material is present in the central nervous system throughout development. Immunoreactivity is first observed in embryonic neural tissue in a cluster of cells in the subesophageal ganglion and immunoreactive fibers projecting from these cells to the brain and ventral ganglion. This pattern of expression is also observed in neural tissue dissected from larva, pupa, and adult. Double-labelling experiments indicate that cells recognized by SDNFM-antisera are also stained with FMRFamide antisera. Based on position, SDNFMRFamide immunoreactive material is expressed in a limited number of cells that contain the FMRFamide polypeptide precursor. This finding suggests that the Drosophila FMRFamide precursor undergoes differential post-translational processing (Nichols, 1995b).

The Drosophila FMRFamide gene encodes multiple FMRFamide-related peptides. These peptides are expressed by neurosecretory cells and may be released into the blood to act as neurohormones. The effects of eight of these peptides were examined on nerve-stimulated contraction (twitch tension) of Drosophila larval body-wall muscles. Seven of the peptides strongly enhance twitch tension, and one of the peptides is inactive. Their targets are distributed widely throughout the somatic musculature. The effects of one peptide, DPKQDFMRFamide, are unchanged after the onset of metamorphosis. The seven active peptides show similar dose-response curves. Each has a threshold concentration near 1 nM, and the EC50 for each peptide is approximately 40 nM. At concentrations of <0.1 microM, the responses to each of the seven excitatory peptides follows a time course that matches the fluctuations of the peptide concentration in the bath. At higher concentrations, twitch tension remains elevated for 5-10 min or more after wash-out of the peptide. When the peptides are presented as mixtures predicted by their stoichiometric ratios in the dFMRFamide propeptide, the effects are additive, and there are no detectable higher-order interactions among them. One peptide was tested and found to enhance synaptic transmission. At 0.1 microM, DPKQDFMRFamide increases the amplitude of the excitatory junctional current to 151% of baseline within 3 min. Together, these results indicate that the products of the Drosophila FMRFamide gene function as neurohormones to modulate the strength of contraction at the larval neuromuscular junction. In this role these seven peptides appear to be functionally redundant (Hewes, 1998).

Fragile X syndrome (FXS) is the most common form of inherited mental disability and known cause of autism. It is caused by loss of function for the RNA binding protein FMRP, which has been demonstrated to regulate several aspects of RNA metabolism including transport, stability and translation at synapses. Recently, FMRP has been implicated in neural stem cell proliferation and differentiation both in cultured neurospheres as well as in vivo mouse and fly models of FXS. Previous studies have shown that FMRP deficient Drosophila neuroblasts upregulate Cyclin E, prematurely exit quiescence, and overproliferate to generate on average 16% more neurons. This study further investigated FMRP's role during early development using the Drosophila larval brain as a model. Using tissue specific RNAi it was found that FMRP is required sequentially, first in neuroblasts and then in glia, to regulate exit from quiescence as measured by Cyclin E expression in the brain. Furthermore, the hypothesis was tested that FMRP controls brain development by regulating the insulin signaling pathway, which has been recently shown to regulate neuroblast exit from quiescence. The data indicate that phosphoAkt, a readout of insulin signaling, is upregulated in dFmr1 brains at the time when FMRP is required in glia for neuroblast reactivation. In addition, dFmr1 interacts genetically with dFoxO, a transcriptional regulator of insulin signaling. These results provide the first evidence that FMRP is required in vivo, in glia for neuroblast reactivation and suggest that it may do so by regulating the output of the insulin signaling pathway (Callan, 2012).

Although there is a clear role for FMRP in the glia, its precise function in these cells remains poorly understood. It was previously shown that FMRP is expressed in glia during embryonic development but appears downregulated postnatally in the mouse brain. Co-culture of glia and hippocampal neurons demonstrated that glial cells contribute to the neuroanatomical defects found in the Fragile X brain, albeit the factors responsible are yet to be identified. This work provides the first in vivo evidence for FMRP's requirement in glial cells, and future work will focus on using more restricted glial Gal4 drivers to dissect the contribution of different glial types to regulating neuroblast reactivation in the dFmr1 mutant brains (Callan, 2012).

Several mRNA targets have been predicted or confirmed for FMRP. Given FMRP's complex tissue specific and temporal requirements during development it is likely that more remain to be identified and confirmed in vivo. Some clues as to the possible pathways and targets regulated by FMRP in the developing brain come from recent reports that glia can provide cues (in the form of secreted dILPs) to neighboring neuroblasts awaiting a reactivation signal. These findings together with the current data showing that FMRP is required sequentially in neuroblasts, then in glia, for proper neuroblast reactivation suggest a model whereby FMRP may control the timing and/or levels of insulin signaling in the brain by acting in different tissues at different times during development. While more work is needed to fully validate this model and to identify the direct mRNA targets of FMRP during brain development, initial tests of the model were carried out by evaluating the levels of pAkt, a readout of insulin signaling in the brain. This work shows that indeed, at the time when FMRP is required in glia (12-18 h ALH), more cells belonging to neuroblast lineages express pAkt. Coupled with the genetic interaction discovered between dFmr1 and dFoxO, a downstream effector as well as inhibitor of insulin signaling, it is suggested that FMRP controls neural stem cell behavior by directly regulating components of insulin signaling. Notably, PI3K, an upstream activator of Akt has been previously shown to be a target of FMRP in the context of mGluR signaling at synapses. Thus for its autonomous function in neuroblasts, FMRP could regulate PI3K directly, while later, in glia, for its nonautonomous function, FMRP could control (directly or indirectly) the expression of the dILPs secreted by glial cells. Notably, in vertebrates, Insulin Growth Factor-1 (IGF-1) and PI3K/Akt can also promote cell-cycle progression in neural stem cells, thus raising the possibility that the current findings in the fly model may be highly relevant to the molecular mechanisms underlying FXS. While more work is needed to elucidate FMRP's role in the communication between the glial niche and neuroblasts, it is tempting to speculate that FMRP may regulate similar signaling cassettes and molecules (i.e., PI3K) in different developmental contexts. The Drosophila model offers unique opportunities to dissect tissue specific regulation such as glial versus neuroblast specific mRNA targets in future experiments (Callan, 2012).

Adult

Changes in the pattern of specific neuropeptide gene expression were studied during the metamorphosis of the Drosophila nervous system. Prior to metamorphosis, the Drosophila FMRFamide gene is expressed exclusively within the central nervous system in a stereotyped pattern that comprises roughly 60 neurons. The FMRFamide gene is continuously expressed throughout all stages examined: at each of 15 stages of adult development and through at least the first 10 days of adult life. There are no differences between the results observed with 2 different exon-specific hybridization probes, thus indicating little if any alternative splicing during postembryonic development. Despite many changes in the positions of individual hybridization signals due to the large-scale reorganization of the nervous system, the continuous pattern of gene expression through adult development permits many adult signals to be identified as larval signals. It is concluded that the adult pattern of FMRFamide gene expression is largely derived from persistent larval neurons. Adult-specific hybridization signals in the brain and ventral ganglion are also detected and these correspond to many of the approximately 40 adult-specific FMRFamide-immunoreactive neurons. One specific larval signal is lost during adult development and the intensities of other signals fluctuate in reproducible manners. These stereotyped differences in hybridization signal intensity resemble similar observations made in larval stages and support the hypothesis that the steady-state levels of FMRFamide transcripts are differentially regulated among the diverse neurons that express the gene (O'Brien, 1991).

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 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).

Mode of action of a Drosophila FMRFamide in inducing muscle contraction

Drosophila is a model system for examining mechanisms of action of neuropeptides. FMRFamide (DPKQDFMRFamide) has been shown to induce contractions in Drosophila body wall muscle fibers in a Ca2+ -dependent manner. The present study examined the possible involvement of a G-protein coupled receptor and second messengers in mediating this myotropic effect after removing the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf Receptor (FR) either ubiquitously or specifically in muscle tissue. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor, KN-93. Peptide-induced contractions in the mutants of the phosholipase C-beta (PLCbeta) gene (norpA larvae) and in IP3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-Isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP3, PLC, CaMKII, or arachidonic acid (Milakovic, 2014).


FMRFamide: Biological Overview | Evolutionary Homologs | Regulation | References

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