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PHM is required for normal developmental transitions and for biosynthesis of secretory peptides in Drosophila
N Jiang1, A S Kolhekar, P S Jacobs
1Department of Anatomy & Neurobiology, Washington University School of Medicine, Saint Louis 63130, USA.
Insights
Mutations in peptidylglycine alpha-hydroxylating monooxygenase (PHM) disrupt secretory peptide signaling, impacting developmental transitions in Drosophila. PHM is crucial for neuropeptide amidation, essential for insect development and survival.
Area of Science:
- Developmental Biology
- Neuroendocrinology
- Molecular Genetics
Background:
- Secretory peptides are vital signaling molecules in development.
- C-terminal alpha-amidation is a critical post-translational modification for many secretory peptides, particularly neuropeptides in insects.
- The peptidylglycine alpha-hydroxylating monooxygenase (PHM) is the rate-limiting enzyme for this amidation process.
Purpose of the Study:
- To investigate the role of PHM and peptide amidation in Drosophila development and signaling.
- To elucidate the specific functions of amidated neuropeptides during developmental transitions.
Main Methods:
- Generated and analyzed Drosophila mutants lacking PHM activity (null and hypomorphic alleles).
- Utilized a PHM transgene for rescue experiments to assess developmental rescue windows.
- Examined neuropeptide biosynthesis and levels in PHM mutants using biochemical and genetic approaches.
- Observed and characterized developmental phenotypes, including molting and metamorphosis.
Main Results:
- PHM mutants exhibit lethality, primarily during embryonic or larval stages, with specific defects in molting.
- Phenotypes of PHM mutants resemble those of mutations affecting the ecdysone regulatory circuit.
- Amidated neuropeptides are largely absent in strong PHM mutants, while precursors and non-amidated forms accumulate.
- Rescue experiments demonstrate that PHM activity is essential during specific developmental windows, particularly early post-embryonic stages.
Conclusions:
- Secretory peptide amidation, mediated by PHM, is critical for successful transitions between developmental stages in Drosophila.
- PHM is required throughout the lifespan for maintaining peptide amidating activity.
- These findings provide new genetic tools and insights into the in vivo study of neural and endocrine peptide biosynthesis and function.
Abstract:
To understand the roles of secretory peptides in developmental signaling, we have studied Drosophila mutant for the gene peptidylglycine alpha-hydroxylating monooxygenase (PHM). PHM is the rate-limiting enzyme for C-terminal alpha-amidation, a specific and necessary modification of secretory peptides. In insects, more than 90% of known or predicted neuropeptides are amidated. PHM mutants lack PHM protein and enzyme activity; most null animals die as late embryos with few morphological defects. Natural and synthetic PHM hypomorphs revealed phenotypes that resembled 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; approximately 50% hatch and initially display normal behavior, then die as young larvae, often while attempting to molt. PHM mutants were rescued with daily induction of a PHM transgene and complete rescue was seen with induction limited to the first 4 days after egg-laying. The rescued mutant adults produced progeny which survived to various stages up through metamorphosis (synthetic hypomorphs) and displayed prepupal and pupal phenotypes resembling those of ecdysone-response gene mutations. Examination of neuropeptide biosynthesis in PHM mutants revealed specific disruptions: Amidated peptides were largely absent in strong hypomorphs, but peptide precursors, a nonamidated neuropeptide, nonpeptide transmitters, and other peptide biosynthetic enzymes were readily detected. Mutant adults that were produced by a minimal rescue schedule had lowered PHM enzyme levels and reproducibly altered patterns of amidated neuropeptides in the CNS. These deficits were 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.