BIOLOGICAL OVERVIEW

Hormonal structure and function have been evolutionarily conserved to a remarkable degree: this is true for insects as a class as well as a wide variety of other metazoans. At least five insect peptide hormones are known to have been structurally and/or functionally conserved: prothoracicotropic hormone and bombyxin (induce release of ecdysteroid by the prothoracic glands) allatotropin, allatostatin (regulate production of juvenile hormone by the corpora allata), and diuretic hormone. None of these have been cloned in Drosophila, but use of antibodies against the hormones of other insect species reveals hormone presence in distinct sets of cells in the central nervous system of Drosophila larvae, pupae, and adults. Brain neurons synthesizing bombyxin, PTTH (see Bombyx and Manduca prothoracicotropic hormone), and DH are in strikingly similar positions when compared with their lepidopteran counterparts, indicating that at least some Drosophila neuroendocrine cells are homologous to those of lepidopterans. Allatotropin and allatostatin-immunopositive neurons of Drosophila differ from those of lepidopterans, but many of them are identical to neurons that express the FMRFamide gene. Antibodies to bombyxin, PTTH, allatostatin, and DH also stain axons and axon terminals in the neurohemal part of the ring gland, and all tested antibodies except that against bombyxin show positive reaction in the neurohemal area of the ventral ganglion (Zitnan, 1993).

The Drosophila FMRFamide gene was identified by its homology to a gene first sequenced in the marine snail Aplysia. FMRFamide was first purified as a cardioregulatory tetrapeptide from the central nervous system of the clam. In molluscs, it modulates both cardiac output and the actions of neurons, as well as regulating evoked muscle tension. In cattle, two peptides immunoreactive to a related lobster hormone have been identified; the cattle proteins have anti-analgesic properties. FMRFamide genes of Drosophila and Aplysia share sequence homologies with mammalian genes encoding the opioid peptide and corticotrophin-releasing factor. To confuse matters, multiple RFamide-containing peptides are present in Drosophila, encoded by three genes: drosulfakinin (Nichols, 1988 and 1992b), dromyosuppressin, and FMRFamide (Taghert, 1992 and references).

There is a remarkably reproducable distribution of FMRFamide neuropeptide in identifiable neuroendrocrine cells and interneurons in Drosophila and other insects. The expression of the FMRFamide gene is a cell specific phenotype confined to about 30 regions within the larval CNS. Among this set of neurons, there is a reproducible and systematic variation in the intensity of hybridization signals and in the time during development when transcripts are first detectable (Schneider, 1993a).

Representative of the distribution of products of the FMRFamide gene is the distribution of DPKQDFMRFamide, one of the 13 peptides coded for by the FMRFamide gene. The earliest observed DPKQDFMRFamide is found in neural tissue from stage 16 embryos. Faint staining is observed in one cell in the subesophageal ganglion from which an immunoreactive fiber projects into the ventral ganglion and to one cell in each of the three thoracic ganglia. In larval neural tissue, staining is observed in two bilaterally paired cells in the subesophageal ganglia (SE2) and SV, and in bilaterally paired cells in each of the thoracic ganglia (T1, T2 and T3). Three bilaterally paired cells in the superior protocerebrum (SP1, SP2 and SP3) stain for DPKQDFMRFamide. In pupae, SP1 remains stained, and a new bilaterally paired cell in the lateral protocerebrum (LP1) stains. The T1-3 staining remains and an additional bilaterally paired cell in the second thoracic ganglion (T2dm) stains as well as a bilaterally paired cell in the abdominal ganglion (A8). In the adult, staining is apparent is SP1, SP2, SP3, LP1, cells of the optic lobe (OL2), cells of the subeosophageal ganglion (SV and SE2), cells in the three thoracic ganglia (T1-3 and T2dm) and in the abdominal ganglion. These cells are a subset of cells that stain with an FMRFamide antiserum that stains for the polypeptide precursor. These data suggest that the FMRFamide polypeptide precursor undergoes differential processing to produce DPKQDFMRFamide immunoreactive material in a limited number of cells expressing the FMRFamide precursor (Nichols, 1995a).

What is the cellular target of FMRFamide? Is it a serpentine seven pass transmembrane receptor, the classic targets of peptide hormones, or is there another target? Although FMRFamides constitute a major class of invertebrate peptide neurotransmitters, the molecular structure of their receptors has not yet been identified. In neurons of the snail Helix aspersa, as well as in the bursting and motor neurons of Aplysia, FMRFamide induces a fast excitatory depolarizing response due to direct activation of an amiloride-sensitive Na+ channel. A complementary DNA has been isolated from Helix nervous tissue; when expressed in Xenopus oocytes, it encodes an FMRFamide-activated Na+ channel (FaNaCh) that can be blocked by amiloride. The protein shares a very low sequence identity with epithelial Na+ channel subunits and C. elegans degenerins, but it displays the same overall structural organization. This is the first characterization of a peptide-gated ionotropic receptor (Lingueglia, 1995).

Another study implicates G protein coupled receptors as mediators of FMRFamide signals. PDVDHVFLRFamide acts in the locust oviduct. Inhibitory peptides and stimulatory peptides share a single receptor by having the same binding sequence, VFLRFamide, but are able to produce opposite muscle responses due to the differences in activation sites. The receptors for the inhibitory and stimulatory FMRFamide-related peptides (FaRPs) are coupled with G proteins and both inhibitory and excitatory effects of FaRPs on locust oviduct occur through the activation of G proteins. It is very likely that the receptor is coupled with two different G proteins. The activation of one is responsible for the inhibitory effect and the activation of the other is responsible for the stimulatory effect (Wang, 1995b).

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 β-hydroxylase expression in mouse sympathetic neurons, Pet-1, Gata3 and Lmx1b for serotonergic marker expression in mouse serotonergic neurons, and Nurr1 for dopaminergic marker expression in murine dopaminergic neurons (Eade, 2012).

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

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

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

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

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

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

GENE STRUCTURE

Bases in 5' UTR - 106

Exons - 2

Bases in 3' UTR - 275

PROTEIN STRUCTURE

Amino Acids - 347

Structural Domains

The Drosophila FMRFamide neuropeptide gene contains two exons separated by an intron of approximately 2.5 kilobase pairs. The promoter region contains a TATA box 30 nucleotides upstream from a consensus transcription start site. The open reading frame (encoding the neuropeptide precursor) begins with the first nucleotide of exon II. The precursor protein contains 5 copies of DPKQDFMRFamide, as well as 8 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides (Chin, 1990, Nambu, 1988, Schneider, 1988 and 1990).

date revised: 5 August 2023

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