InteractiveFly: GeneBrief

Peptidylglycine-alpha-hydroxylating monooxygenase: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Genetic analysis in Drosophila has been used to study the process of C-terminal peptide alpha-amidation. This process is a late event in the biosynthesis of secretory peptides and is likely, in many instances, to be the rate-limiting step. In insects, more than 90% of known or predicted neuropeptides are amidated. Peptidylglycine alpha-hydroxylating monooxygenase (Phm) carries out the penultimate step in alpha-amidation, hydroxylating intermediates from prohormone precursor cleavage products that terminate in glycine residues (Jiang, 2000).

Phm mutants lack Phm protein and enzyme activity; most null animals die as late embryos with few morphological defects. Natural and synthetic Phm hypomorphs reveal phenotypes that resemble those of animals with mutations in genes of the ecdysone-inducible regulatory circuit. Animals bearing a strong hypomorphic allele contain no detectable Phm enzymatic activity or protein; ~50% hatch and initially display normal behavior, then die as young larvae, often while attempting to molt. Phm mutants are rescued with daily induction of a Phm transgene and complete rescue is seen with induction limited to the first 4 days after egg-laying. The rescued mutant adults produce progeny that survive to various stages up through metamorphosis (synthetic hypomorphs) and display prepupal and pupal phenotypes resembling those of ecdysone-response gene mutations. Examination of neuropeptide biosynthesis in Phm mutants reveals specific disruptions: amidated peptides are largely absent in strong hypomorphs, but peptide precursors, a nonamidated neuropeptide, nonpeptide transmitters, and other peptide biosynthetic enzymes are readily detected. Mutant adults that are produced by a minimal rescue schedule have lowered Phm enzyme levels and reproducibly altered patterns of amidated neuropeptides in the CNS. These deficits are partially reversed within 24 h by a single Phm induction in the adult stage. These genetic results support the hypothesis that secretory peptide signaling is critical for transitions between developmental stages, without strongly affecting morphogenetic events within a stage. Further, they show that Phm is required for peptide alpha-amidating activity throughout the life of Drosophila. Finally, they define novel methods to study neural and endocrine peptide biosynthesis and functions in vivo (Jiang, 2000).

C-terminal alpha-amidation results from the sequential actions of two enzymes: Phm and Pal (Peptidyl-alpha-hydroxyglycine-alpha-amidating lyase). While Phm creates hydroxylated intermediates from prohormone precursor cleavage products, Pal cleaves the intermediates to produce the final amidated peptides and glyoxylate. In vertebrates, the two enzymes occupy adjacent domains of a bifunctional protein called PAM (Eipper, 1992); in Drosophila, the Phm and Pal enzyme activities are both present, yet they are physically and genetically distinct (Kolhekar, 1997). The Drosophila genome sequencing project predicts one unlinked Phm gene (CG3832 at 60B1-2) and two unlinked Pal genes (CG12130 at 46C6-7 and CG5472 at 59F4-6). A homozygous lethal transposon insertion (P[07623]) lies within the coding region of Phm and reduces Phm enzyme levels (as measured in heterozygous adults) without also reducing Pal levels (Kolhekar, 1997 and Jiang, 2000).

The nervous systems of Phm^P[07623] homozygous, hemizygous, and heterozygous first-instar larvae were examined to determine whether Phm mutants display alterations in neuropeptide processing. An immunological approach was used to distinguish between amidated and nonamidated products derived from the pro-dFMRF precursor. Antiserum PT2 was generated against the tetrapeptide FMRFamide and reveals a pattern of staining that is greater than that displayed by products of the dFMRFamide gene. That pattern likely includes products of related neuropeptide genes (other peptides that share a common '-RFamide' C terminus). The second antiserum was directed against the final 19 amino acids of the pro-dFMRF prohormone, which is not amidated. The pro-dFMRF antiserum produces a pattern highly similar to that displayed by FMRFamide-related mRNA and by large FMRFamide-related-lacZ reporter transgenes. The PT2 antiserum stained a robust pattern of ~26 neurons in the larval brain and ventral ganglion of first-instar Phm^P[07623] heterozygotes, but revealed virtually nothing in the CNS of Phm^P[07623] homozygotes or hemizygotes. On rare occasions, small spots of immunoreactivity were observed in the CNSs of homozygotes in locations normally occupied by prominent FMRFamide-positive neurons. These 'spots' may be explained by any of several possibilities: (1) an alternate (low level or highly inefficient) source of Phm-like enzyme activity, (2) the action of maternally derived Phm mRNA or Phm enzyme activity, or (3) a lack of specificity by the anti-FMRFamide antiserum such that it weakly detects nonamidated peptide forms. In contrast, no differences were seen in the pattern or intensity of the ~14 neurons immunostained with the pro-dFMRF antiserum between heterozygotes and mutant Phm^P[07623] animals. These results are consistent with the hypothesis that Phm mutant animals have defects in a posttranslational step(s) in neuropeptide biosynthesis (Jiang, 2000).

To extend the phenotypic analysis to other amidated neuropeptides, antibodies were used to Aplysia peptide myomodulin (MM); there are ~34 MM-immunoreactive neurons in the larval CNS; antibody to Leucophaea leukokinin I (LKI: induces Leucophaea maderae hindgut contraction) stains a different set of ~16 neurons. In neither case have the putative, homologous peptides of Drosophila been isolated. Likewise, antibodies to nonamidated versions of myomodulin or leukokinin I are not available. It was found that these two antisera produced strong staining reactions in Phm^P[07623] heterozygous animals and weak or negligible staining reactions in Phm^P[07623] homozygous or hemizygous animals (Jiang, 2000).

Weak signals (in positions of neurons that are normally strongly stained) were seen in fewer than half the specimens and in only certain positions; zero staining was seen in most specimens. The anti-LKI staining pattern is not as intense as the MM pattern in wild-type animals; zero staining was seen in homozygous and in hemizygous Phm^P[07623] mutant animals. Together these immunochemical results support the hypothesis that Phm is required for the normal production of amidated neuropeptides in Drosophila larvae. Additional observations indicate that mutation of the Phm locus does not overtly disrupt aminergic transmitter systems, or those few peptidergic transmitter systems that normally lack amidation, or other neuropeptide processing enzymes (Jiang, 2000).

In Drosophila, mutations that specifically affect genes encoding secretory peptides are rare. This scarcity is mainly due to the difficulty in predicting accurate phenotypes for gene products that encode multiple signals whose physiological actions display both intra- and intergenic redundancy. Genetic methods to ablate specific secretory cells represent an alternative approach in vivo to studying secretory peptide functions. An alternative genetic approach is the study of secretory peptide biosynthesis. Kolhekar (1997a) and Siekhaus (1999) analyzed two separate genes encoding different neuropeptide-processing enzymes, Phm and PC2 (amontillado, the Drosophila homolog of the neuropeptide precursor processing protease), respectively. The genetics of neuropeptide processing offers a broad-based approach to examining secretory peptide functions. Such phenotypes provide insight into functions that secretory molecules may perform as a class (Jiang, 2000).

The P(28) chromosome contains 1309 bp deleted to one side of the inserted P[07623] transposon. The deletion removes critical Phm sequences, but also truncates an overlapping gene, CG17263. This other gene encodes a LIM-only protein, and the P(28) chromosome removes two of its three LIM domains. Because of this, the P(28) chromosome should also be considered an allele of CG17263. It is suspected that some part of the P(28) mutant phenotype may derive from absence of CG17263 function, but the contribution of CG17263 to the phenotype cannot be defined at present. The analysis of P(28) mutant phenotypes has been included in this analysis of Phm based on a strict reliance upon rescue with wild-type Phm sequences (Jiang, 2000).

The reversal of mutant phenotypes following heat shock-Phm induction permitted the inference that the absence of Phm is responsible for particular deficits. Specifically, full rescue of embryonic, larval, pupal, and adult lethality is possible. In addition, hs-Phm is capable of reversing the aberrant processing of secretory peptides (amidation of FMRF peptides). Therefore despite an ambiguity derived from the closely overlapped nature of the Phm and CG17263 genes, it is felt that specific interpretations concerning Phm functions are conservative and appropriate (Jiang, 2000).

Phm mutant animals die early in development, either as late embryos or as young larvae. The earliest lethal phase is seen with the Phm^P(28) allele, which is a null by several measures. Most mutant animals (as homozygotes and hemizygotes) reach late stages of embryogenesis with relatively normal morphological appearance. The CNS is slightly smaller than normal, but its organization and complexity appear normal. Animals homozygous for the Phm^P[07623] allele survive to later larval stages, as compared to Phm^P(28) mutants. While they contained no detectable Phm protein, they display potentially trace amounts of Phm enzyme activity. It is proposed that Phm^P[07623] animals, despite the insertion of a large transposon in the Phm locus, contain low levels of zygotic Phm enzyme due to compensatory transcriptional and/or posttranscriptional mechanisms. In contrast, Phm^P(28) animals, lacking about half of the Phm coding sequence, contain no zygotic enzyme activity (Jiang, 2000).

The relatively normal growth of mutant animals indicates that zygotic Phm expression is largely dispensable to complete the generation and morphogenesis of embryonic tissues. The contribution of maternally derived Phm to early morphogenesis is not yet known. While some larval tissues in the most severe Phm mutant sometimes appear smaller (e.g., the brain), a quantitative analysis is required to determine if and when these differences are significant. These results suggest a general view that, in insects, large-scale alterations of secretory peptide biosynthesis do not produce large-scale morphogenetic defects (Jiang, 2000).

Many Phm^P[07623] homozygotes die as late stage embryos or in the midst of larval molts. In general, these results indicate that Phm mutant animals have difficulty at or near times of developmental transitions: embryonic hatching and/or larval molting. A similar conclusion was reached by Siekhaus (1999) in an analysis of the PC2/amontillado gene. PC2/amon encodes a potential prohormone convertase with many similarities to the mammalian enzyme PC2, which is known to be important for processing of neural and endocrine peptides. A disruption of molting processes in Phm mutant animals is consistent with a large body of evidence relating secretory peptides to the orchestration of molting events (reviewed by Henrich, 1999). In particular, ecdysis (which is a late event in the molting process) is coordinated and modified by cascades of homones. These hormone cascades include several amidated secretory peptides made in the CNS or peripheral endocrine centers. The GAL4 3' UAS system will be useful to create tissue-specific Phm mosaics and so the question becomes which tissues must produce amidated peptides to permit normal embryonic hatching and larval molting (Jiang, 2000)?

First generation rescued mosaic animals are normal in many respects, but also show stereotyped behavioral abnormalities. Rescued G1 adults are both fecund and fertile. Their G2 progeny could live at restrictive temperatures and a small percentage of these reach the adult stage. Thus a minimal rescue schedule provides the opportunity to study animals homozygous for the Phm^P(28) mutation past their normal lethal phase. A study of G2 animals has produced additional observations on the requirements for Phm activity during later (metamorphic) developmental stages. G2 animals show two prominent developmental defects that are ascribed to insufficient Phm activity -- a prevalent deformity of puparia and a developmental block that occurs during or just after head eversion. This suggests that events at or around this critical stage require Phm (and signaling by amidated peptides) for normal progression to form the puparium and to complete adult development (Jiang, 2000).

The poor disc and head eversion may be due to retained attachment of larval mouthparts: without an ability to move posteriorly within the puparium, the animal faces increased confinement and antagonism to the emergence of pupal tissue. If this explanation is correct, the defects involving Phm activity center more on postpupation events than on pupation itself. Secondary hs-Phm transgene inductions increased the percentage of animals successfully completing these developmental transitions from roughly 20% to nearly 100%. Likewise the form of animals receiving additional inductions more closely resembles that of wild-type animals. These observations support the hypothesis that lowered Phm levels are responsible for these 'late' mutant phenotypes (Jiang, 2000).

The phenotypes produced in hypomorphic Phm mutant animals closely resemble those produced by hypomorphic mutations in several of the ecdysone-response genes. In addition, they resemble those of genes implicated in ecdysone/steroid hormone production, including the dre4 and dare genes. Strong similarities between Phm mutant phenotypes and those of ecdysone production/signaling genes are also evident at larval developmental stages. In particular, the 'double mouthhook' phenotype seen in Phm^P[07623] animals (produced by a failure to complete larval molts) is also displayed by certain mutants of the EcR gene, the dare gene, and the developmental mutant cryptocephal (crc). Later in development, EcR, crc, dare, and Phm hypomorphic phenotypes include a failure to pupariate. Also, some Phm hypomorphs, like those of DHR3, survive to pupal stages but die around the time of head eversion with defects in puparial form, body shortening, and gas bubble movements. These defects suggest problems with proper activation of the muscles needed to produce shortening, puparium formation, and disc eversion (Jiang, 2000).

Together these observations strengthen the argument that amidated secretory peptides are required for signaling events that ensure progression through several critical developmental transitions. The inclusion of Phm phenotypes in this common list suggests that amidated secretory peptides are involved in many of the hormonal signaling events that are initially triggered by the steroid hormone ecdysone. In addition, amidated secretory peptides are likely involved in the signaling events that regulate ecdysone production and titers. It will be of interest therefore to test genetic interactions between the peptide and the steroid signaling pathways. Also, to place Phm defects within the framework of known regulatory pathways, it will be useful to measure the expression of RNAs for various steroid hormone response genes in Phm mutant animals. The paradigms established here should be useful for future screens that seek to identify genes needed to produce and to mediate peptide signaling. In general, such information will be useful in assigning functional roles to peptidergic systems in their interactions with steroid hormones and will further define the regulation of insect metamorphosis in molecular detail (Jiang, 2000).

These results provide in vivo evidence that Phm is required for peptide alpha-amidating activity throughout the life of Drosophila. Loss-of-function alleles show that this is true in larvae. Phm^P[07623] animals contain (at best) trace levels of Phm enzyme activity and of Phm protein. Further, when assayed using the expression of FMRFamide neuropeptides, Phm mutant animals display little if any staining for amidated neuropeptides, although staining for nonamidated peptides, for nonpeptide transmitters, and for neuropeptide processing enzymes appears normal. From the analysis of Phm mutant animals that were maintained beyond their normal lethal phase to reach pupal and adult ages, the same conclusion is drawn for late developmental stages as well. Limiting the induction of transgenic Phm to just the first larval days fully rescues Phm mutant lethality. However, such rescued adults are still abnormal, i.e., they contain ~20% of normal Phm levels and display abnormal cellular profiles of amidated FMRF peptides. The abnormal cellular pattern is highly reproducible because identified neurons (e.g., OL2 and MP2) lack staining, while other identified neurons (e.g., SP1) stain normally in all animals examined. It is speculated that these patterns reflect similar abnormalities in other amidated neuropeptides, expressed by other sets of neurons. It is concluded that a compensating activity does not appear later in development (at least not in the case of adult CNS neurons) and that the Phm gene represents the principle source of Phm enzyme activity at all developmental stages (Jiang, 2000).

Based on an immunological survey of three amidated peptide systems, it is inferred that Phm^P[07623] mutant animals lack most amidated peptides and therefore lack most functional neuropeptides. In that regard, their locomotor and feeding behaviors appear remarkably normal for the first hours after larval hatching. Many die, associated with a failure to thrive, and their decline probably reflects a loss of function in several systems. There may be a loss of neural drive that is normally modulated by neuropeptides. Also, the death of mutant animals may reflect the lack of organized digestive functions, since Phm and amidated peptides are abundant in midgut epithelia and are likely required for normal gut physiology. The mutant phenotype may reflect an imbalance in the maintenance or use of energy stores by factors such as Adipokinetic hormone or an absence of sufficient hemolymph regulation by cardioacceleratory peptide. The Phm mutant animals currently available do not allow for the destinction between these or other plausible explanations. However, the viability of the strong Phm hypomorphs, their effective lack of amidated peptide stores, and the availability of methods to create Phm mosaics will help to define specific roles for particular peptidergic systems (Jiang, 2000).

DEVELOPMENTAL BIOLOGY

Staining of larval CNS with the affinity-purified antisera indicates widespread expression of Phm-like protein throughout all levels of the CNS, as well as in other tissues, including endocrine glands and gut. Antibody specificity was deduced by comparison with tissues that were stained with preimmune serum. This expression is limited to a small number of CNS neurons (approximately a few hundred) that displayed very high levels of Phm-like immunoreactivity. Among stained neurons, immunoreactivity is seen both in cell bodies and within neuropil regions. The latter represents stained neuronal processes and also may include glial staining. Phm antibody staining is also prevalent in secretory cells of the Ring Gland, salivary gland, and in diverse cells at all levels of the midgut . In the CNS, several strongly stained cells were identifiable as neuroendocrine neurons because they project immunoreactive axons to defined neurohemal organs like the Ring Gland or the dorsal neurohemal organs of the ventral ganglion. Many Phm-positive neurons have positions similar to those of peptidergic cells. Such identity has been verified in the case of several FMRFamide-expressing neurons using a Drosophila stock containing a FMRFamide-beta-galactosidase construct called pWF3. The lacZ-positive SE2 interneurons of the subesophageal neuromeres and the neuroendocrine Tv and Tva neurons of thoracic neuromeres are among the strong Phm-staining neurons. Thus, in the case of certain identified neurons, high levels of Phm expression correlates with a peptidergic neuronal phenotype (Kolhekar, 1997a).

EFFECTS OF MUTATION

The P[07623] insertion lies within the coding region of the Phm gene and the lethality associated with the stock is reverted when the element undergoes precise excision (Kolhekar, 1997a). A screen was performed for imprecise excisions of the P[07623] element to generate a small deletion of the Phm locus. Revertants of the white⁺ phenotype were selected, and these stocks were analyzed by PCR and by Southern blotting, and one deletion was selected called Df(2R)Phm^P(29) [the P(29) allele]. Phm^P(29) animals retain homozygous lethality and fail to complement large deficiencies of the region. From blot analysis, it was inferred that the deletion lost part of Exon I, and all of Exons II, III, and IV, and that it ended within intron D. Sequence analysis confirms that the deletion extends over 1309 bp from the P[07623] insertion to 561 bp 59 of Exon V. There are 301 bp of the P[07623] element remaining within the P(29) allele. The initial segment of Exon I also remains, lying to the retained side of the P element. The portions of the Phm coding region lost in the P(29) deletion include motifs that are essential for activity of the enzyme (e.g., one of the two dihistidine motifs known to bind copper; Eipper, 1995). The sequence analysis indicates that at least one other predicted gene (CG17263) is mutated in the P(29) chromosome (Jiang, 2000).

The two mutant alleles of Phm -- the P[07623] insertion and the P(28) deletion -- both display complete lethality, as indicated by the absence of homozygous and hemizygous adults. Occasional putative adult escapers (~0.1% at 25^oC) were identified as balanced heterozygotes by multiplex, single-fly PCR. Marked mutant stocks were studied following various genetic crosses to determine when Phm mutant animals died. The hatching rate of mutant animals was first determined to evaluate their ability to complete embryogenesis. Compared to Mendelian predictions, ~58% of Phm^P[07623] homozygotes and ~76% of balanced Phm^P[07623] heterozygotes hatched from the egg. With very few exceptions, Phm^P(28) homozygotes failed to hatch; those that did were nearly immobile and died within a few hours. Phm^P[07623]/Phm^P(28) trans-heterozygotes hatched at a rate comparable to that of Phm^P[07623] homozygotes (55% of the predicted value). When analyzed in trans to a large deficiency of the region [Df(2R)or-Br11], Phm^P[07623] appeared less severe than did Phm^P(28). These results indicate that the Phm^P(28) allele produces nearly complete embryonic lethality (failure to hatch) and acts as a null in this assay. Phm^P[07623] acts like a strong hypomorph, with approximately 50% of animals failing to hatch (Jiang, 2000).

While the Phm^P(28) mutant animals fail to hatch, most complete the large part of embryogenesis prior to their death. Homozygotes and hemizygotes were scored by cuticular markers and/or by single embryo PCR: More than 70% of Phm^P(28) mutant embryos complete stage 16 and more than 40% reached stage 17. These embryos display little evidence of tissue disorganization or morphological abnormality. For example, BP102 staining of Phm^P(28) homozygotes reveals proper axonal organization within the CNS by stage 15. A minority of embryos (15/79) die at early stages without evidence of cellularization. Single-embryo PCR does not yield signals with these embryos, so they remain unidentified as to genotype. If these represent Phm mutant animals, the embryonic phenotype would include an early lethal phase. Those Phm mutant embryos that hatch as larvae display normal morphology when they first emerge, as indicated by anti-Synaptotagmin staining of neuropil tissues in Phm^P[07623] homozygotes (Jiang, 2000).

The ability of hs-Phm induction to rescue the lethality associated with Phm^P(28) homozygotes was tested. The effect of hs-Phm-A5 induction on Phm levels in WT animals was measured. Eggs were collected from balanced P[hs-Phm-A5];Phm^P(28)/SM6 adults and then given daily 30-min inductions through the embryonic and postembryonic stages, until the day the first adults emerged. Thirty-five percent (89 of 253) of adult flies emerging from cultures that had received this induction schedule were homozygous Phm^P(28) mutant animals. This indicates that the absence of Phm activity is responsible for the lethality associated with homozygous Phm^P(28) mutant animals (Jiang, 2000).

Phm immunoreactivity is present heterogeneously in most larval tissues (Kolhekar, 1997a). In heterozygous and wild-type animals, Phm-like immunostaining in the CNS consists of strong staining in about 200 scattered neuronal cell bodies and in widespread neuropil regions. Other cell bodies and neuropil regions display lower levels of staining. In both homozygous and hemizygous Phm^P[07623] and Phm^P(28) mutants, CNS staining is severely reduced although not absent. Neuropil staining is weak and diffuse. Most cell body staining is eliminated, with the exception of a few lightly stained cells: a single protocerebral brain cell and ~5 cells in lateral positions of anterior abdominal segments. The weak neuropil staining may represent unrelated, cross-reacting species similar to non-Phm proteins that are recognized by anti-Phm in tissue immunoblots. Moderate signals in specific abdominal neuronal cell bodies may represent maternally derived Phm that could be enriched and retained in peptide-containing secretory granules of certain peptidergic neurons in mutant animals. In the gut, wild-type and heterozygous Phm mutant animals display strong staining of scattered, putative endocrine cells in the anterior and middle regions of the midgut. In homozygous and hemizygous mutant animals, all gut staining was eliminated. Likewise, the normally strong Phm immunostaining of the salivary gland was eliminated in all Phm homozygous and hemizygous mutants (Jiang, 2000).

The survival of some Phm^P[07623] homozygotes into early larval stages afforded the opportunity to evaluate levels of Phm enzyme and protein in these mutants. Homozygous first-instar larvae contain an amount of Phm activity that is at least 10 times less than heterozygotes and that is equal to or just above control values. By Western blot analyses, anti-Phm recognizes a doublet of immunoreactive proteins of ~40 kDa. Phm^P[07623] homozygous animals contain no detectable ~40-kDa immunoreactive species compared to a sizable amount present in heterozygous animals. By both measures therefore, the Phm^P[07623] allele contains very little or no Phm protein in young larvae. In parallel experiments, Phm levels were measured in adult heads of wild-type animals that are transgenic for a Phm cDNA under the control of an hsp70 heat-inducible promoter. A 30-min 37°C induction in the P[hs-Phm-A5] line produces an ~10-fold induction of Phm enzyme levels within 4 h; 24 h after induction, induced levels remain about 5-fold higher than baseline. By Western blot analysis, the same induction paradigm also produced a sizable increase in staining of an ~40-kDa protein doublet (Jiang, 2000).

The behavior of the heterozygous Phm^P[07623] mutant larvae is normal with respect to their general coordination, their locomotory steps and feeding movements, and their response to tactile stimulation and molting behavior. By casual inspection, homozygous and hemizygous Phm^P[07623] larvae resemble their heterozygous siblings immediately following their emergence. However, over the course of the next 24-48 h, homozygous and hemizygous mutant animals become lethargic and typically die at or near the end of the first larval instar. When reared at room temperature, the longest-lived mutants reach 7 days of age and have just molted to the third larval instar. 780 Phm^P[07623] mutant larvae were segregated within 12 h of hatching and their lethal phase was examined by scoring the structures of mouthparts and of the anterior spiracles displayed by corpses. Among corpses recovered, more than 75% display a double-mouthparts phenotype, with tooth structures characteristic of animals molting to the second larval instar. Nearly 20% of the corpses were animals that had died as nonmolting first-instar larvae. A small number of animals were recovered that had successfully completed the first and/or second larval molts and that died as second-instar animals, as third-instar animals, or while attempting to molt to the third instar. These data indicate that more than half of the Phm^P[07623] mutants that survive embryogenesis die while attempting to molt to the second larval instar (Jiang, 2000).

Abbreviated schedules of hs-Phm transgene induction were tested to begin defining developmental stages when Phm expression was required to reverse lethality in Phm^P(28) homozygotes. Limiting inductions (one per day) to just the first 4 days after egg-laying is sufficient to rescue most or all mutant animals. Conversely, delaying inductions for as few as the first 2 days after egg-laying severely lowers the number of rescued Phm^P(28) homozygotes, and no rescue is observed when inductions are delayed until 5 days after egg-laying. Together these results suggest that a minimal Phm induction schedule limited to early developmental stages is both necessary and sufficient to rescue adult lethality (Jiang, 2000).

The minimal Phm induction schedule produced Phm^P(28) homozygous adults (here called G1 mosaic animals) that were normal in many respects, but that also showed stereotyped behavioral abnormalities. Rescued G1 adults were both fecund and fertile. Their G2 progeny could live at restrictive temperatures and a small percentage of these reached the adult stage. Thus a minimal rescue schedule provided the opportunity to study animals homozygous for the Phm^P(28) mutation past their normal lethal phase. Immunostaining with anti-FMRFamide revealed a largely normal pattern in the CNS of G2 larvae. G2 animals were produced and reared at 25°C and these G2 Phm^P(28) mutants remained homozygous for the inducible hs-Phm-A5 transgene. Approximately 76% of G2 Phm^P(28) homozygous animals completed embryogenesis and hatched, and approximately 61% of G2 Phm^P(28) first-instar larval homozygotes became pupae. Only 22% of G2 Phm^P(28) homozygous prepupae completed metamorphosis. Puparium formation was often aberrant: puparia typically displayed a slight curvature, a failure to shorten normally, and a failure to evert anterior spiracles properly. The gas bubble failed to move anteriorly, leaving pupae in anterior positions with abnormal posterior gas spaces. A subset of the noneclosing puparia were dissected to evaluate their lethal phases. The majority of these (28/57) died after completing pupation and proper eversion of the head, wings, and legs, but they revealed no further adult differentiation (e.g., died between stages P4ii and P7). Almost as many animals (25/57) reached this same stage, but displayed aberrant disc eversion, retention of larval mouthparts, and imprecise elimination of apolysed tracheal cuticle. In the entire group of Phm^P(28) homozygotes examined, only 2 of the 255 prepupae that failed to eclose differentiated past stage P7. Of these two, one had normal adult features, but was cryptocephalic. The other displayed heterochronic differentiation: scattered bristles were tanned prior to other developmental events that normally occur earlier (Jiang, 2000).

Daily 37°C inductions of Phm^P(28) G2 animals did not increase the percentage of mutant animals that completed larval development: the observed larval mortality appears due to the experimental manipulation of the young larvae. Inductions did rescue nearly all mutant prepupae through their transitions to adult eclosion. In addition, the puparial shapes of G2 animals receiving hs-Phm inductions were largely normal. This indicates that the defects of metamorphosis observed in G2 Phm^P(28) homozygotes reflect the absence of normal Phm functions (Jiang, 2000).

P[hs-Phm-A5]; Phm^P(28) homozygous G1 mutant adults, produced by the minimal abbreviated schedules of hs-Phm induction, contain lowered levels of Phm enzymatic activity. When animals experienced daily inductions through the day of adult eclosion, heads from P[hs-Phm-A5]; Phm^P(28)/SM6 animals (Phm heterozygotes) and P[hs-Phm-A5]; Phm^P(28)/Phm^P(28) (Phm homozygotes) both contained large amounts of enzyme activity on adult day 1. Levels subsequently declined in each genotype over the next 20 days, but homozygotes exhibited ~50% less enzyme than heterozygotes. A value of ~2.5 pmol/mg/h is found in Phm^P[07623] heterozygotes; the approximately twofold higher values seen in these Phm^P(28) heterozygotes animals presumably reflect the contributions of basal and induced (perduring) levels of P[hs-Phm-A5] transgene expression. Similar though more dramatic results were observed in animals that experienced the minimal rescue schedule (4 early inductions). Throughout all adult stages assayed, P[hs-Phm-A5];Phm^P(28)/SM6 animals that had received the minimal induction schedule displayed levels comparable to older P[hs-Phm-A5];Phm^P(07623)/SM6 adults that had received the long induction schedule (i.e., ~5 nmol/mg/h). In contrast, sibling P[hs-Phm-A5];Phm^P(28)/Phm^P(28) animals had reduced levels throughout all adult days tested (Jiang, 2000).

The expression of amidated neuropeptides was examined in G1 P[hs-Phm-A5];Phm^P(28) heterozygous and homozygous adults that had experienced the minimal induction schedule. The homozygotes are referred to as 'rescued adults.' In particular, the spatial profile of FMRFamide-like immunoreactivity, which in the wild-type adult brain has been categorized within 13 principal cell types, was studied. In adult brains (adult days 1 through 10), 6 of 13 FMRFamide-immunoreactive cell types were absent or in-frequently stained in rescued G1 Phm^P(28) homozygotes. Most of the 'missing cells' correspond to neurons that are 'adult-specific' for the FMRFamide neuropeptide phenotype, e.g., the MP2 and OL2 neurons that appear only during or after metamorphosis. To ask whether this mutant phenotype represents incomplete cellular differentiation or a defect in neuropeptide biosynthesis, comparable animals were examined 24 h after they received a single additional hs-Phm induction as adults. It was reasoned that a neuropeptide biosynthesis defect should be reversible within a short period following such a late transgene induction. In separate experiments, single inductions were given on adult days 4 and 10. After 24 h, tissues were analyzed by comparison to age-matched P[hs-Phm-A5]; Phm^P(28)/SM6 given the same induction schedule and to rescued P[hs-Phm-A5];Phm^P(28)/Phm^P(28) not given an additional induction. A reversal of the phenotype for OL2, MP2, and CC neuronal cell types was observed. For example, OL2 neurons were detected in 20 of 20 hemispheres of P[hs-Phm-A5]; Phm^P(28)/Phm^P(28) animals that received the additional induction. In 12 hemispheres of the age- and genotype-matched controls (day 5 rescued adults that did not experience additional induction), no OL2 neurons were seen (Jiang, 2000).

Following induction, OL2 immunolabeling included both cell bodies and axonal projections, indicating that previously 'missing' neurons displayed considerable morphological differentiation in rescued Phm mutant adults. A comparison to staining in age-matched P[hs-Phm-A5]; Phm^P(28)/SM6 animals suggests the phenotype reversal is not complete: OL2 neurons in Phm heterozygotes are more intensely stained in 18 of 18 hemispheres (Jiang, 2000).

In Drosophila, the amidated neuropeptide Pigment dispersing factor (PDF) is expressed by the ventral subset of lateral pacemaker neurons and is required for circadian locomotor rhythms. Residual rhythmicity in pdf mutants likely reflects the activity of other neurotransmitters. It was asked whether other neuropeptides contribute to such auxiliary mechanisms. The gal4/UAS system was used to create mosaics for the neuropeptide amidating enzyme PHM; amidation is a highly specific and widespread modification of secretory peptides in Drosophila. Three different gal4 drivers restrict PHM expression to different numbers of peptidergic neurons. These mosaics display aberrant locomotor rhythms to degrees that parallel the apparent complexity of the spatial patterns. Certain PHM mosaics are less rhythmic than pdf mutants and as severe as per mutants. Additional gal4 elements were added to the weakly rhythmic PHM mosaics. Although adding pdf-gal4 provided only partial improvement, adding the widely expressed tim-gal4 largely restored rhythmicity. These results indicate that, in Drosophila, peptide amidation is required for neuropeptide regulation of behavior. They also support the hypothesis that multiple amidated neuropeptides, acting upstream, downstream, or in parallel to PDF, help organize daily locomotor rhythms (Taghert, 2001).

These results support the hypothesis that daily locomotor rhythms in flies require signaling by neuropeptides that are C-terminally amidated. The gal4/UAS-PHM system predictably controls PHM spatial expression. It was found that certain PHM mosaic flies (e.g., 36Y-gal4- and c929-gal4-rescued) are largely arrhythmic under conditions of constant darkness. The working hypothesis is that such a behavioral disruption is attributable to changes in the normal patterns of peptide amidation. The last point has not been demonstrated directly. It is a premise based on the previous demonstration that manipulation of PHM produces large-scale changes in peptide amidation in both larval and adult stages (Taghert, 2001).

A second result supports the conclusion that amidated peptides contribute to daily locomotor rhythms; increasing PHM expression by combining pdf(N)-gal4 with c929-gal4 improves rhythmic behavior over that displayed by c929-gal4 alone. Although the combination does not completely restore wild-type behavior, the improvement indicates that PHM activity in PDF neurons does contribute to display of rhythmic daily locomotion. It is presumed that this indicates a requirement for amidation of PDF because the pigment-dispersing activity of PDH on crustacean melanophores is highly dependent (~300-fold) on the C-terminal amide. Whether PDF must be modified as such to display circadian signaling activity is unknown (Taghert, 2001).

The strongest evidence for this conclusion comes from the performance of 36Y-gal4- and c929-gal4-rescued flies under constant conditions. Unlike pdf⁰¹ flies, these populations displayed little rhythmicity behavior during the first cycle of constant conditions. Also, the average signal-to-noise ratio of 36Y-gal4-rescued flies was much less than that of pdf⁰¹ and very comparable with that of per⁰¹. Are such deficits attributable to a loss of PHM function or a gain of deleterious function? The pdf neuropeptide gene can produce a gain-of-function phenotype: When pdf was misexpressed by certain gal4 drivers in WT flies, rhythmic locomotor behavior was degraded. In the case of PHM, however, two results suggest it is the absence of the enzyme that causes arrhythmicity: (1) increasing PHM expression (by adding tim-gal4 to 36Y-gal4 or c929-gal4) restored near-normal rhythmicity to both arrhythmic lines; (2) misexpression of PHM by driving it with tim-gal4 or with c929-gal4 in a WT did not degrade behavioral rhythmicity. In general, the greater the extent of PHM gene expression in a PHM mutant background, the more predictable is the degree of behavioral improvement. Interestingly, some combinations restored considerable PHM expression (e.g., Appl-gal4) but improved behavioral performance only moderately. Together, those experiments suggest that normal PHM expression is required in specific neurons and/or secretory cells for the behavior examined. The tim-gal4 line produced a broad expression pattern of great complexity. That amount of expression precludes clear definition of places or times by which 'additional' PHM restores the functions of circadian regulatory circuits (Taghert, 2001).

The behavior of stocks which each contained multiple transposons was compared. To improve the scope of the study, five control genotypes were tested that combined subsets of the multiple transposons used in the experimental genotypes. In general, these controls displayed a level of rhythmicity lower than that of WT but greater than those of PHM mosaics. gal4 lines were used primarily to create spatial differences in gene expression. Three gal4 lines for this study (36Y, c929, and 386Y) were used because their expression patterns prominently feature peptidergic neurons of the CNS and secretory cells of peripheral tissues. The three patterns are very similar: the fact that all successfully reverted PHM lethality is probably a reflection of such anatomical similarities. The gal4 patterns also included clear differences in cell number (386Y > c929 > 36Y) (Taghert, 2001).

These pattern differences are of interest, because they may reveal specific neurons (or non-neuronal cells) that produce secretory peptides required for circadian behaviors. However, it is concluded that the interpretation of where 'critical PHM expression' occurs in these experiments is problematic, because there are several ways by which such patterns may defy simple interpretation. For example, two gal4 patterns could appear similar and stable in the adult stage, yet be different because of a transient event during development. In fact, the c929-gal4 pattern is relatively stable in the adult stage but transiently includes the VA neuroendocrine neurons for only a brief period during adult development. In such a case, behavioral rescue may reflect temporal, not spatial, differences in gal4-dependent gene expression. A separate problem in the interpretation could arise when two patterns are spatially similar but differ in levels of expression by specific neurons. In that case, the extent of behavioral rescue may reflect quantitative, not spatial, differences in gal4-dependent gene expression (Taghert, 2001).

Given these complexities, it is currently not possible to specify in which neurons, beyond the LN-V, PHM activity is required for daily locomotor rhythms. Instead, considering candidate amidated neuropeptides directly is favored for subsequent analysis. From scans of the Drosophila genome, there are at least 23 neuropeptide-encoding genes. This is likely an underestimate because of the difficulty in predicting neuropeptide precursor sequences with accuracy. Of the identified genes, ~20 encode peptides are known or are predicted to display C-terminal amidation. Thus, it may be reasonable to systematically address the roles of each of the ~20 precursors using Drosophila genetics (Taghert, 2001).

Genetic studies of circadian behaviors traditionally strive to establish that a mutant phenotype does not simply degrade the ability to produce movement. It this study, the behavior of animals with large-scale alterations in transmitter profiles throughout the entire nervous system has been analyzed. In one genotype (36Y-gal4/UAS-PHM), rhythmicity under constant conditions was extremely poor (as low as that of per⁰¹ animals); activity levels were also lower than in other genotypes tested. Nevertheless, when the locomotor rhythm of 36Y-gal4-rescued animals was restored by addition of tim-gal4, activity levels were not also increased. It is proposed that the 36Y-gal4 transmitter mosaic contains disruptions of distinct neural centers, ones that control the general level of activity and ones that organize rest-activity cycles. A similar point is made considering the results seen with tim-gal4. Addition of tim-gal4 to 36Y-gal4-rescued flies produced the greatest restoration of rhythmicity. However, tim-gal4 was by itself unable to revert the lethality of PHM mutants. Therefore, places and times of PHM expression that promote normal vitality do not necessarily equal those promoting circadian behavioral rhythmicity (Taghert, 2001).

Flies lacking clock gene function (e.g., per⁰) display light-driven behavior under cycling conditions, then become arrhythmic during the first cycle of constant conditions. Arrhythmic PHM mosaics are different. For example, 36Y-gal4-rescued flies, whose rhythmicity under constant conditions is quantitatively as weak as that of per⁰¹ animals, entrained well during LD. Therefore, it is concluded that even the most severely arrhythmic PHM mosaic animals studied have levels of circadian clock function and output greater than that present in authentic clock mutants (Taghert, 2001).

The average activity histograms for behavior under constant conditions indicates that different gal4 drivers produce graded levels in circadian locomotor performance. Evidence has been found for at least three levels. The lowest level is represented by single gal4 flies (e.g., 36Y); they have the weakest measures of rhythmicity (by periodogram or MESA) and display little reproducible variation in the average activity histogram. An intermediate level is seen in certain gal4-combination flies (e.g., c929/D42); these display moderate rhythmicity and an average activity peak during early subjective day. The strongest level is seen in other gal4-combination flies (e.g., c929/tim); these are strongly rhythmic, and they display a large average activity peak during late subjective day and a rapid decrease in activity during early subjective night. Presumably, these graded levels of performance reflect incremental contributions by different amidated peptides to one or more circuit components. Relating specific peptide systems to separate levels of behavioral performance represents a challenge for future studies (Taghert, 2001).

REFERENCES

Search PubMed for articles about Drosophila Peptidylglycine-alpha-hydroxylating monooxygenase

Ciccotosto, G. D., Hand, T. A., Mains, R. E. and Eipper, B. A. (2000). Breeding stock-specific variation in peptidylglycine {alpha}-amidating monooxygenase messenger ribonucleic acid splicing in rat pituitary. Endocrinology 141: 476-486. 10650926

Eipper, B. A., et al. (1992). Alternative splicing and endoproteolytic processing generate tissue-specific forms of pituitary peptidylglycine alpha-amidating monooxygenase (PAM). J. Biol. Chem. 267(6): 4008-15. 1740449

Eipper, B. A., Quon, A. S., Mains, R. E., Boswell, J. S., and Blackburn, N. J. (1995). The catalytic core of peptidylglycine alpha-hydroxylating monooxygenase: Investigation by site-directed mutagenesis, Cu X-ray absorption spectroscopy, and electron paramagnetic resonance. Biochemistry 34: 2857-2865. 7893699

Henrich, V. C., Rybczynski, R. and Gilbert, L. I. (1999). Peptide hormones, steroid hormones, and puffs: Mechanisms and models in insect development. Vitam. Horm. 55: 73-125. 9949680

Jiang, N., et al. (2000). Phm is required for normal developmental transitions and for biosynthesis of secretory peptides in Drosophila. Dev. Biol. 226: 118-136. 10993678

Kolhekar, A. S., Roberts, M. S., Jiang, N., Johnson, R. C., Mains, R. E., Eipper, B. A., and Taghert, P. H. (1997a). Neuropeptide amidation in Drosophila: Separate genes encode the two enzymes catalyzing amidation. J. Neurosci. 17: 1363-1376. 9006979

Kolhekar, A. S., et al. (1997b). Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core. Biochemistry 36(36): 10901-9. 9283080

Kolhekar, A. S., Quon, A. S. W., Berard, C. A., Mains, R. E. and Eipper, B. A. (1998). Post-translational N-glycosylation of a truncated form of a peptide processing enzyme. J. Biol. Chem. 273: 23012-23018. 9722525

Prigge, S. T., et al. (1997). Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase. Science 278(5341): 1300-5. 9360928

Prigge, S. T., et al. (1999). Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase. Nat. Struct. Biol. 6(10): 976-83. 10504734

Siekhaus, D. E. and Fuller, R. S. (1999). A role for amontillado, the Drosophila homolog of the neuropeptide precursor processing protease PC2, in triggering hatching behavior. J Neurosci. 19(16): 6942-54. 10436051

Spijker, S., et al. (1999). A molluscan peptide {alpha}-amidating enzyme precursor that generates five distinct enzymes. FASEB J. 13: 735-748. 10094934

Taghert, P. H., et al. (2001). Multiple amidated neuropeptides are required for normal circadian locomotor rhythms in Drosophila. J. Neurosci. 21(17): 6673-6686. 11517257

date revised: 5 December 2023

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