wishful thinking: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - wishful thinking

Synonyms -

Cytological map position - 64A5

Function - receptor

Keywords - neuromuscular synapse

Symbol - wit

FlyBase ID: FBgn0024179

Genetic map position -

Classification - type II transforming growth factor beta receptor, protein kinase

Cellular location - surface transmembrane

NCBI link: Entrez Gene
wit orthologs: Biolitmine
Recent literature
Lee, S.H., Kim, Y.J. and Choi, S.Y. (2016). BMP signaling modulates the probability of neurotransmitter release and readily releasable pools in Drosophila neuromuscular junction synapses. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 27671198
The structure and function of synapses is modulated by the interaction of presynaptic and postsynaptic neurons via cell adhesion molecules or secreted signal molecules. Bone morphogenic protein (BMP) is a secreted molecule mediating retrograde signaling that is involved in the formation and maintenance of synaptic structure throughout many animal species. However, how BMP signaling modulates presynaptic neurotransmitter release is not yet clear. This study analyzed the function of BMP signaling factors in neurotransmitter release in Drosophila neuromuscular synapses using loss-of-function mutants in genes for BMP modulators, Wit, Mad, and Dad. Larvae with mutations in wit and mad commonly show a decreased synaptic bouton number in neuromuscular synapses. Larvae with dad mutations show an increased bouton number. The amplitudes of miniature EJC (mEJC) are normal for these mutants. Wit and mad mutants show decreased evoked EJC (eEJC) amplitude and increased paired pulse facilitation, implying impaired presynaptic neurotransmitter release. A reduction in readily releasable neurotransmitters pool sizes in wit and mad mutants was found. However, dad mutants show a normal probability of neurotransmitter release and readily releasable pool sizes and normal eEJC amplitude even with clear abnormalities in synaptic structure. These results suggest that BMP signaling is critical for each step of presynaptic neurotransmission. Also, that BMP signaling modulates both synaptic structure and function independently and specifically.

Lee, S. H., Kim, Y. J. and Choi, S. Y. (2016). BMP signaling modulates the probability of neurotransmitter release and readily releasable pools in Drosophila neuromuscular junction synapses. Biochem Biophys Res Commun 479: 440-446. PubMed ID: 27671198
The structure and function of synapses is modulated by the interaction of presynaptic and postsynaptic neurons via cell adhesion molecules or secreted signal molecules. Bone morphogenic protein (BMP) is a secreted molecule mediating retrograde signaling that is involved in the formation and maintenance of synaptic structure throughout many animal species. However, how BMP signaling modulates presynaptic neurotransmitter release is not yet clear. This study examined the function of BMP signaling factors in neurotransmitter release in Drosophila neuromuscular synapses using loss-of-function mutants in genes for BMP modulators, Wit, Mad, and Dad. Larvae with mutations in wit and mad commonly showed a decreased synaptic bouton number in neuromuscular synapses. Larvae with dad mutations showed an increased bouton number. The amplitudes of miniature EJC (mEJC) were normal for these mutants. wit and mad mutants showed decreased evoked EJC (eEJC) amplitude and increased paired pulse facilitation, implying impaired presynaptic neurotransmitter release. A reduction was found in readily releasable neurotransmitters pool sizes in wit and mad mutants. However, dad mutants showed a normal probability of neurotransmitter release and readily releasable pool sizes and normal eEJC amplitude even with clear abnormalities in synaptic structure. These results suggested that BMP signaling is critical for each step of presynaptic neurotransmission. The results also suggested that BMP signaling modulates both synaptic structure and function independently and specifically.
Marmion, R. A. and Yakoby, N. (2018). In locus analysis of patterning evolution in the BMPR2 Wishful thinking. Development 145(13). PubMed ID: 29884674
Proper tissue patterning is an essential step during organ formation. During this process, genes are expressed in distinct patterns, defining boundaries for future functional domains. The bone morphogenetic protein (BMP) signaling pathway sets the anterior domain during eggshell patterning. Previously, the Drosophila melanogaster BMPR2, Wishful thinking (WIT), was shown to be required for BMP signaling and patterning during eggshell formation. Expressed in a conserved anterior pattern, the width of wit patterning in the follicular epithelium is evolutionally divergent between Drosophila species. This study used genome editing to demonstrate how the gene pattern divergence is controlled in cis within the wit locus of D. virilis. Furthermore, unlike direct targets of BMP signaling, this study demonstrated how one transcription factor binding site shapes the pattern of WIT in D. melanogaster by negative regulation. However, changes in this site are not sufficient to explain the evolution of wit patterning, suggesting that a yet undiscovered positive regulatory element controls pattern divergence.
Peterson, A. J., Murphy, S. J., Mundt, M. G., Shimell, M., Leof, E. B. and O'Connor, M. B. (2022). A juxtamembrane basolateral targeting motif regulates signaling through a TGF-beta pathway receptor in Drosophila. PLoS Biol 20(5): e3001660. PubMed ID: 35594316
In polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. This study explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-β family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. The Dpp Type II receptor Punt was found to be enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. This study found that basolateral presentation of Punt is required for optimal signaling. Finally, evidence is presented that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.
Chen, S., Venkatesan, A., Lin, Y. Q., Xie, J., Neely, G., Banerjee, S. and Bhat, M. A. (2022). Drosophila Homolog of the Human Carpenter Syndrome Linked Gene, MEGF8, Is Required for Synapse Development and Function. J Neurosci 42(37): 7016-7030. PubMed ID: 35944997
Drosophila multiple epidermal growth factor-like domains 8 (dMegf8) is a homolog of human MEGF8. MEGF8 encodes a multidomain transmembrane protein which is highly conserved across species. In humans, MEGF8 mutations cause a rare genetic disorder called Carpenter syndrome, which is frequently associated with abnormal left-right patterning, cardiac defects, and learning disabilities. MEGF8 is also associated with psychiatric disorders. The presence of intellectual disabilities in Carpenter syndrome patients and association of MEGF8 with psychiatric disorders indicate that mutations in MEGF8 cause underlying defects in synaptic structure and functions. This study investigated the role of Drosophila dMegf8 in the larval neuromuscular junctions (NMJ). dMegf8 localizes to NMJ synapses and is required for proper synaptic growth. dMegf8 mutant larvae and adults show severe motor coordination deficits. At the NMJ, dMegf8 mutants show altered localization of presynaptic and postsynaptic proteins, defects in synaptic ultrastructure, and neurotransmission. Interestingly, dMegf8 mutants have reduced levels of the Type II BMP receptor Wishful thinking (Wit). dMegf8 displays genetic interactions with neurexin-1 (dnrx) and wit, and in association with Dnrx and Wit plays an essential role in synapse organization. These studies provide insights into human MEGF8 functions and potentially into mechanisms that may underlie intellectual disabilities observed in Carpenter syndrome as well as MEGF8-related synaptic structural and/or functional deficits in psychiatric disorders.
Zhou, X., Gan, G., Sun, Y., Ou, M., Geng, J., Wang, J., Yang, X., Huang, S., Jia, D., Xie, W. and He, H. (2022). GTPase-activating protein TBC1D5 coordinates with retromer to constrain synaptic growth by inhibiting Bone Morphogenetic Protein signaling. J Genet Genomics. PubMed ID: 36473687
Formation and plasticity of neural circuits rely on precise regulation of synaptic growth. At Drosophila neuromuscular junction (NMJ) Bone Morphogenetic Protein (BMP) signaling is critical for many aspects of synapse formation and function. The evolutionarily-conserved retromer complex and its associated GTPase-activating protein TBC1D5 are critical regulators of membrane trafficking and cellular signaling. However, their functions in regulating the formation of NMJ are less understood. This study reports that TBC1D5 is required for inhibition of synaptic growth, and loss of TBC1D5 leads to abnormal presynaptic terminal development, including excessive satellite boutons and branch formation. Ultrastructure analysis reveals that the size of synaptic vesicles and the density of subsynaptic reticulum are increased in TBC1D5 mutant boutons. Disruption of interactions of TBC1D5 with Rab7 and retromer phenocopies the loss of TBC1D5. Unexpectedly, this study found that TBC1D5 is functionally linked to Rab6, in addition to Rab7, to regulate synaptic growth. Mechanistically, this study showed that loss of TBC1D5 leads to upregulated BMP signaling by increasing the protein level of BMP type II receptor Wit at NMJ. Overall, these data establish that TBC1D5 in coordination with retromer constrains synaptic growth by regulating Rab7 activity, which negatively regulates BMP signaling through inhibiting Wit level.

Proper synaptic development is critical for establishing all aspects of neural function including learning, memory, and locomotion. wishful thinking (wit) gene is the Drosophila homolog of the vertebrate BMP type II receptor. Mutations in wit result in pharate lethality that can be rescued by expression of a wit transgene in motor neurons but not in muscles. Mutant larvae exhibit small synapses, severe defects in evoked junctional potentials, a lower frequency of spontaneous vesicle release, and an alteration in the ultrastructure of synaptic active zones. wit NMJs have decreased levels of the synaptic cell adhesion molecule Fasciclin II, and synaptic membrane detachment at active zones. Wit is expressed by a subset of neurons, including motoneurons. The neuromuscular junction (NMJ) phenotype is specifically rescued by transgenic expression of Wit only in motoneurons. Thus, Wit appears to function as a presynaptic receptor that regulates synaptic size at the Drosophila NMJ (Marqués, 2002 and Aberle, 2002).

All three BMP ligands, Decapentaplegic, Gbb and Screw, appear to share a common set of receptors that include the type II receptor Punt and the type I receptors Thick veins and Saxophone. Activation of these receptors leads to phosphorylation and nuclear translocation of Mad, the Drosophila homolog of Smad1, in a complex with Medea, the Drosophila Smad4 homolog. In contrast, the activin type I receptor Baboon signals in conjunction with Punt through dSmad2 in response to an as yet uncharacterized ligand (Marqués, 2002 and Aberle, 2002 and references therein).

Based on sequence information, the only principal TGF-ß type signal transduction component remaining to be described in Drosophila is predicted to code for a type II receptor. This receptor, Wishful thinking, is most closely related to the vertebrate BMPRII and Müllerian Inhibitory Substance (MIS) type II receptors (Baarends, 1994; Kawabata, 1995; Liu, 1995), both of which are characterized by presence of a large carboxy-terminal extension distal to the kinase domain that is not found in the ActRII and TGF-ß type II receptors. BMPRII is the only vertebrate type II receptor that specifically binds BMP type ligands and not other members of the TGF-ß family. Knockout of this receptor in mice results in embryonic lethality, suggesting that it is the type II receptor that functions with ALK-3 to convey the BMP-4 signal during gastrulation (Beppu, 2000). Studies in Xenopus indicate that BMPRII is required for mesodermal patterning and suppression of neural fate in the ectoderm (Frisch, 1998). In humans, mutations in BMPRII cause primary pulmonary hypertension (PPH) (Deng, 2000; Lane, 2000; Thomson, 2000).

In mice, BMPRII is expressed in a dynamic pattern in many tissues of all three germ layers during embryonic development. In the nervous system, prominent expression is seen in the hippocampus, the cerebellum, motoneurons, and sensory neurons of the dorsal root ganglia (Charytoniuk, 2000). Due to a widespread expression and requirement during early developmental stages, BMPRII-deficient mice die shortly after implantation, thus far precluding the study of the function of BMPRII in vivo during synapse development (Beppu, 2000). A regulatory function for vertebrate BMPs in dendritic growth has been suggested by in vitro results on cultured sympathetic neurons. Exposure of these neurons to recombinant BMP7 rapidly induces the upregulation of dendritic marker proteins and the outgrowth of new dendrites (Lein, 1995). BMP2, BMP6, and Drosophila Gbb (60A) have been shown to stimulate similar growth effects (Guo, 1998). The dendritic growth-promoting activity of BMP7 is also effective on cultured hippocampal neurons. Because BMP7 and other BMPs are expressed in the hippocampus, it has been suggested that they might play a developmental role in dendritic growth and synapse formation (Withers, 2000).

TGF-ß family members may also have a regulatory function in the development of the vertebrate NMJ. A ligand of the TGF-ß superfamily, Myostatin (GDF-8), is expressed exclusively in developing and adult somatic muscles and negatively regulates muscle growth. Mice mutant for the myostatin gene are significantly larger than their littermates due to a 2- to 3-fold increase in their skeletal muscle size and mass (McPherron, 1997). In addition, TGF-ß2 is thought to act as a target-derived neurotrophic factor for motoneurons because it is localized at the postsynaptic side of the NMJ, and its receptors are expressed in motoneurons (Jiang, 2000). These results show that ligands and receptors of the TGF- superfamily are present in both muscles and motoneurons and suggest that they might function in regulating the growth of the neuromuscular synapses. However, a functional requirement for BMP signaling in the growth of any synapse in vivo has not yet been demonstrated (Marqués, 2002 and Aberle, 2002 and references therein).

In Drosophila embryos and third instar larvae, wit is heavily expressed in a subset of neuronal cells. Consistent with a role for Wit as a BMP type II receptor that modulates neuronal function, wit null mutant embryos are found show a specific loss of phosphorylated-Mad (P-Mad) staining within motor neurons but not in other tissues. Using various Gal4 drivers, it has been demonstrated that Wit expression in motor neurons is sufficient to recover viability. Electrophysiological analysis of the neuromuscular junction (NMJ) synapses in third instar larvae demonstrates that wit mutant animals have severely reduced evoked junctional potentials and display a high rate of failures at low extracellular [Ca2+]. The frequency of spontaneous vesicle release is significantly reduced as are synapse size and bouton number. In contrast, quantal size appears relatively normal. Ultrastructural analysis of mutant boutons reveals alterations in the morphology of the active zones, suggesting that reduced quantal transmission results from a presynaptic active zone defect. These results highlight a role for BMP signaling in regulating Drosophila synapse assembly and/or maintenance. Furthermore, they raise the possibility that, like TGF-ß, BMP signaling may regulate additional aspects of synaptic plasticity such as those associated with long-term facilitation and memory (Marqués, 2002 and Aberle, 2002).

Defects in synaptic structure in wit mutants suggest that BMP signaling plays a key role in regulating presynaptic morphology and function at the Drosophila neuromuscular junction. Synapse formation and maturation are relatively late steps in neural development and are preceded by neural induction, neurogenesis, neural migration, and axonal pathfinding. Previously, BMPs and their cognate receptors have been shown to be widely expressed in the developing and mature vertebrate nervous system (Lorentzon, 1996; Soderstrom, 1996), and numerous lines of evidence have implicated them in controlling several different aspects of neural development and function, including neurulation, morphogenesis, lineage decisions, and cellular maturation. While wit is expressed in a subset of neurons beginning at stage 12, wit is probably not required during early neurogenesis since no defect in patterning or cell fate changes could be detected during the development of the embryonic CNS, and wit mutants could be rescued with either the elav-Gal4 or nervana-Gal4 drivers, the expression of these drivers being restricted to mature neurons after most differentiation has taken place (Marqués, 2002).

The results reveal a role for BMP type II receptors in regulating the assembly or maintenance of presynaptic active zones and synaptic transmission. In wit mutants, transmitter release is severely reduced. While this exocytotic defect could be explained by a potential endocytotic abnormality, wit mutant boutons do not show depletion of synaptic vesicles. Further, quantal size remains largely unaffected by the mutation, suggesting that postsynaptic receptors are normal. These results are consistent with the observation that presynaptic expression of Wit can completely rescue lethality and also significantly rescues both the frequency of spontaneous release and the EJC amplitude. Finally, ultrastructural studies indicate that defects in active zones may underlie the severe reduction in transmitter release (Marqués, 2002).

wit mutants exhibit a specific loss of P-Mad accumulation in a subset of embryonic neurons, including most motoneurons, implying that Wit mediates a BMP type signal. This suggests that Sax, Tkv, or both are likely to be the type I partners for Wit function. Consistent with this view, tkv, sax, and mad mutants show NMJ phenotypes similar to wit mutants and combinations of activated Sax and Tkv receptors, as well as chimeric combinations of Tkv and Wit, can rescue many aspects of the wit phenotype (McCabe, unpublished data; J. Rawson and S. Selleck, personal communication, cited in Marqués, 2002). At this point, the possibility that Wit might also participate with Babo in mediating a dSmad2 type signal or may participate in a type I receptor independent mechanism cannot be excluded (Marqués, 2002).

One other key issue that remains to be resolved is the identity and source of the ligand or ligands that bind to Wit. Potential ligands could be synthesized in the neurons themselves and provide an autocrine or paracrine type signal for presynaptic differentiation, or they could be secreted from the muscles and provide a retrograde signal. In Drosophila, motoneurons can differentiate and form active synapses in the complete absence of target muscles. This suggests that intrinsic cues guide most of the basic processes that regulate NMJ synaptogenesis. Drosophila dActivin ßB (Myoglianin) is expressed exclusively in the embryonic and larval CNS (Lo, 1999 and T.E.H. and M.B.O., unpublished data cited in Marqués, 2002) and therefore might provide an intrinsic cue that helps regulate synapse formation, maintenance, and function. However there is no evidence as yet that Wit participates in an activin-like pathway (Marqués, 2002).

Although the initial formation of synapses in embryos might be driven by intrinsic cues, during larval stages the muscle volume increases over 150-fold and the synapse must grow in a regulated manner to maintain proper synaptic strength. Since in wit mutants, synapse size is close to normal at the end of embryogenesis but is abnormal soon after, it is inferred that wit is required for synaptic growth. Whether it is required continuously during all larval stages or only at a defined time during late embryogenesis and the early 1st instar to set in motion a particular developmental program is difficult to determine. No P-Mad accumulation is found after the first instar stage. However it is possible that a lower level of BMP signaling is required for synapse growth throughout the larval stages (Marqués, 2002).

Since Wit function is required in the presynaptic cell, it is speculated that Wit could transduce a retrograde signal from the muscle to the nerve cell that provides a means of coordinating synapse growth with muscle growth. Evidence supporting a retrograde signal that modulates presynaptic transmitter release at the NMJ in Drosophila has come from experiments in which glutamate receptor levels were manipulated in postsynaptic cells. In vertebrates, recent evidence indicates that Wnt7a is a target-derived synaptogenic signal at the mossy fiber-granule cell synapse. While no direct evidence linking BMP signaling to synaptogenesis has been described previously, it is interesting to note that TGF-ß2 has been found to be associated with the subsynaptic muscle nuclei of mature rat neuromuscular junctions, and that TGF-ß receptors are transported in an anteriograde fashion and inserted in the nerve terminal (Jiang, 2000; McLennan, 1994). Tkv is specifically localized to the NMJ synapse, suggesting that a similar mechanism could operate in Drosophila (McCabe, unpublished data cited in Marqués, 2002). Candidate ligands for Wit would then be TGF-ßs expressed in the muscle (Marqués, 2002).

The targets of wit signaling are also unknown. The primary defect could be in electrical activity. It is well known that electrical activity influences synapse plasticity and produces changes in both bouton number and morphology. It is interesting to note in this regard that target-derived TGF-ß1 has been observed to stimulate the functional expression of Ca+2-activated K+ channels in developing chick ciliary ganglion. In this case, however, the effect appears to be mediated posttranslationally and therefore does not involve transcriptional responses by Mad type factors (Cameron, 1998, 1999, Marqués, 2002).

Although alterations in electrical activity per se could be responsible for some of the morphological and functional defects observed, it is more likely that wit mutants alter the expression of other targets. One gene whose expression level is known to influence synapse morphology is the adhesion molecule Fas II. Fas II is expressed both pre- and post-synaptically and is required in both the muscle and neuron for proper synapse development. Hypomorphic mutants of Fas II exhibit either an increase or a decrease in bouton number at the third larval instar stage depending on the level of the residual Fas II protein, and Fas II is downregulated in wit mutants (Aberle, 2002). However even if there is a direct effect of wit signaling on Fas II expression, Fas II is not likely to be the only Wit target since synaptic efficacy is not altered in fas II mutants, whereas in wit mutants there is a severe reduction in synaptic strength. Thus, if wit mutants do affect Fas II expression, then they must also alter the homeostatic compensation mechanism that normally acts to maintain constant synaptic strength despite differences in pre- and post-synaptic levels of Fas II protein in various combinations of fas II alleles (Marqués, 2002).

Other possible targets for wit signaling include components of the exocytotic machinery that controls vesicle targeting, docking, fusion, or release. Certain allelic combinations of mutations in the rop gene, for example, cause severe reductions in both EJPs and the frequency of spontaneous fusion events similar to what has been found for wit mutants. Rop codes for a member of the Sec1 family of proteins thought to regulate secretion by modulating syntaxin, SNAP-25, and synaptobrevin complex function. Unlike wit mutants however, mutations in rop do not affect overall synapse morphology (Marqués, 2002).

Perhaps the most interesting candidate targets for Wit signaling are those genes whose products reside in or contribute to active zone assembly. Very little is known about the composition or mechanism of assembly of this specialized structure. On the postsynaptic side, glutamate receptors are highly concentrated at active zone sites. However, it is unlikely that receptor clustering is the primary defect because the amplitude distribution of spontaneous vesicle releases is normal (Marqués, 2002).

The finding that a BMP pathway modulates synaptic structure and function at the Drosophila NMJ is particularly intriguing in light of other recent reports implicating TGF-ß type components in modulating neuronal plasticity (Chin, 1999; Zhang, 1997). Equally appealing in terms of a potential BMP connection is the observation that long-term sensitization training in Aplysia induces expression of a Tld/BMP-1-like product (Liu, 1997). Tld/BMP-1 type proteins encode metalloproteases that cleave the BMP inhibitors Sog and Chordin, respectively. Thus, the complex regulatory circuit that modulates the activity of BMP type ligands during early embryonic development might also govern the activities of these ligands during neuronal development and may play specific roles in regulating synaptic plasticity associated with long-term learning and memory (Marqués, 2002).

Retrograde BMP signaling controls Drosophila behavior through regulation of a peptide hormone battery

Retrograde BMP signaling in neurons plays conserved roles in synaptic efficacy and subtype-specific gene expression. However, a role for retrograde BMP signaling in the behavioral output of neuronal networks has not been established. Insect development proceeds through a series of stages punctuated by ecdysis, a complex patterned behavior coordinated by a dedicated neuronal network. In Drosophila, larval ecdysis sheds the old cuticle between larval stages, and pupal ecdysis everts the head and appendages to their adult external position during metamorphosis. This study found that mutants of the type II BMP receptor wit exhibited a defect in the timing of larval ecdysis and in the completion of pupal ecdysis. These phenotypes largely recapitulate those previously observed upon ablation of CCAP neurons, an integral subset of the ecdysis neuronal network. This study establish that retrograde BMP signaling in only the efferent subset of CCAP neurons (CCAP-ENs) is required to cell-autonomously upregulate expression of the peptide hormones CCAP, Mip and Bursicon β. In wit mutants, restoration of wit exclusively in CCAP neurons significantly rescued peptide hormone expression and ecdysis phenotypes. Moreover, combinatorial restoration of peptide hormone expression in CCAP neurons in wit mutants also significantly rescued wit ecdysis phenotypes. Collectively, these data demonstrate a novel role for retrograde BMP signaling in maintaining the behavioral output of a neuronal network and uncover the underlying cellular and gene regulatory substrates (Veverytsa, 2011).

Retrograde BMP signaling is required to maintain the behavioral output of neuronal networks. Collectively, these data show that retrograde BMP signaling upregulates the expression of a combination of peptide hormones, exclusively in the CCAP-EN subset of CCAP neurons and to a level required for those neurons to contribute to the normal execution of ecdysis behaviors. These findings in relation to the function of CCAP-ENs in ecdysis, as well as the utility of retrograde signaling as a conserved mechanism for differentiating neuronal identity and regulating behavior (Veverytsa, 2011).

A feed-forward peptide hormone cascade coordinates ecdysis. Larval and pupal pre-ecdysis is initiated by Ecdysis triggering hormone (ETH) from peripheral Inka cells stimulating Eclosion hormone (EH) secretion from brain Vm neurons. ETH and EH then act together on CCAP neurons to stimulate CCAP and Mip release. Work on the isolated Manduca central nervous system demonstrates that CCAP and MIP synergistically terminate pre-ecdysis and initiate ecdysis proper motor rhythm. This is supported by Drosophila studies; CCAP neuron ablation prolongs pre-ecdysis and ecdysis proper in larvae, and results in a deficit in the execution of the ecdysis program in pupae that reduces head and appendage eversion and extension. This role for CCAP neurons has largely been attributed to abdominal CCAP-INs acting locally on motoneurons. However, these observations indicate an essential role for BMP-dependent peptide hormone expression in CCAP-ENs. A detailed analysis of ETH-driven neuronal activity during Drosophila pupal ecdysis supports these conclusions. This study shows that T3 and A8/A9 CCAP neurons are active at the start of ecdysis proper, coincident with head eversion, and that A1-A4 CCAP neurons are active secondarily and throughout the remainder of ecdysis proper, coincident with appendage and head extension. It is suggested that the A1-A4 CCAP neurons active during pupal ecdysis proper and required for leg extension are CCAP-ENs. How would CCAP-ENs that secrete hormones into the hemolymph regulate ecdysis? It has been argued that hemolymph-borne CCAP, Mip and bursicon regulate heart rate, hemolymph pressure and cuticle expansion. However, these peptide hormones might also regulate the activity of central circuits, either indirectly or directly, as established for ETH. Genetic analysis of CCAP, Mip and bursicon peptide hormones and their receptors would provide valuable answers to these questions (Veverytsa, 2011).

CCAP-ENs require peripherally derived Gbb for BMP signaling and enhanced peptide hormone expression. CCAP-EN axons terminate on muscle 12. Muscle expresses Gbb and this study found that muscle-derived (but not neuronal-derived) Gbb significantly rescued BMP signaling and peptide hormone expression in CCAP-ENs. pMad immunoreactivity and GFP-Tkv (expressed from Ccap-GAL4) were also observed within type III boutons, indicative of local BMP signaling. Thus, together with reports that muscle-derived Gbb is sufficient for retrograde BMP signaling in motoneurons, the weight of evidence supports the somatic muscle as a primary target for Gbb access for CCAP-ENs. However, the possibility cannot be ruled out that other sources for Gbb exist, perhaps secreting the ligand into the circulating hemolymph. In this regard, it has been reported that, in gbb mutants, restoration of Gbb in another peripheral tissue, the fat body, failed to rescue BMP signaling in neurons, suggesting that distant signaling via the hemolymph is not sufficient. Further detailed analysis will be required to identify necessary and/or redundant roles for other tissues in neuronal BMP signaling (Veverytsa, 2011).

Although muscle is the likeliest target with respect to gbb, the muscle is unlikely to be the primary target for CCAP-EN peptide hormones. Ultrastructural analysis shows that type III boutons lie superficially on the muscle surface and that dense core vesicles exocytose towards the hemolymph and muscle. Furthermore, bursicon immunoreactivity is detectable in the hemolymph. CCAP-EN peptide hormones are known to target the wing, cuticle and cardiac and visceral muscle, but not the somatic muscle. This situation is unusual, as target-derived factors are typically viewed as influencing neuronal gene expression profiles pertinent to the target itself. Footpad-derived cytokines induce cholinergic differentiation of sympathetic neurons required for footpad sweat secretion. Axial differences in BMP4 ligand expression in the murine face direct subset-specific gene expression in innervating trigeminal neurons that shapes the formation of somatosensory maps. Activin and nerve growth factor in the developing skin induce expression of the hyperalgesic neuropeptide calcitonin gene-related peptide (CGRP) in nociceptive afferents (Veverytsa, 2011).

Without evidence for such a mutualistic relationship, what purpose could retrograde BMP-dependent gene expression play in CCAP-ENs? The tremendous cellular diversity of the nervous system is achieved through the progressive refinement of transcriptional cascades within increasingly diversified neuronal progenitor populations . Subsequently, retrograde signaling further differentiates the expression profile in postmitotic neurons. In such cases, unique access to extrinsic ligands allows for a certain mechanistic economy, enabling a somewhat common regulatory landscape to be adapted towards distinct gene expression profiles. In this context, it is postulated that retrograde BMP signaling functions to diversify the expression levels of peptide hormones in CCAP neurons. Drosophila interneurons and efferents can be sharply distinguished on the basis of BMP activity. Moreover, this study shows that BMP activation in CCAP-INs is capable of enhancing their peptide hormone expression, implicating a similar gene regulatory landscape in CCAP-ENs and CCAP-INs. Thus, the BMP dependence of CCAP, Mip and Bursß offers a simple solution to the problem of how to selectively enhance peptide hormone expression in CCAP-ENs (Veverytsa, 2011).

BMP signaling offers an additional advantage to neuronal diversification. Studies of axial patterning in Drosophila have unveiled a wealth of mechanisms that diversify and gauge transcriptional responses to BMP signaling. These mechanisms revolve around the outcome of pMad/Medea activity at a gene's cis-regulatory sequence, as influenced by their affinity for specific cis-regulatory sequences and local interactions with other transcription factors, co-activators and co-repressors. As a result, pMad/Medea activity can be extensively shaped to generate gene- and cell-specific responses and determine whether genes are on or off or up- or downregulated. This flexibility is likely to underpin the differential sensitivity of CCAP, Mip and Bursß to a common retrograde BMP signal within a single cell, as well as the utility of BMP signaling as a common retrograde regulator of subset-specific gene expression in distinct neuronal populations (Veverytsa, 2011).

Finally, the differential regulation of Bursα and Bursβ is intriguing because they are believed to only function as a heterodimer. Although the possibility of functional homodimers cannot be discounted, it is postulated that the selective BMP dependence of Bursβ might be an efficient mechanism for modulating the activity of the active bursicon hormone. This would be analogous, and perhaps orthologous, to the regulation of follicle-stimulating hormone in mammals. Its cyclical upregulation during the oestrous cycle is dictated by the regulation of only one of its subunits, FSHβ, by the TGFβ family ligand activin (Veverytsa, 2011).

Numerous studies have described the impact of retrograde signaling on neuronal network formation and function. During spinal sensory motor circuit development, retrograde neurotrophin signaling induces specific transcription factor expression in motoneurons and Ia afferents that is required for appropriate motor sensory central connectivity, which, when inoperative, results in ataxic limb movement. Similarly, murine trigeminal neurons utilize spatially patterned BMP4 expression in the developing face to target their centrally projecting axons in a somatotopically appropriate manner. Retrograde signaling also modulates physiologically responsive neuronal gene expression. In vertebrates, skin injury induces cutaneous activin and nerve growth factor expression, which retrogradely upregulates sensory neuron expression of CGRP, which mediates hyperalgesia. In sensory motor circuits of Aplysia, retrograde signals are required to upregulate presynaptic sensorin, a neuropeptide required for long-term facilitation of the sensorimotor synapse (Veverytsa, 2011).

The current evidence suggests that the function of BMP signaling is not mediated within a specific developmental window, but is required on an ongoing basis. The Ccap-GAL4 transgene is not active until late larval stage L1, after CCAP neuron network assembly and peptide hormone initiation. Yet, wit phenotypes were significantly rescued using Ccap-GAL4. Together with observation of persistent pMad immunoreactivity in CCAP-ENs, it is concluded that BMP signaling acts permissively to maintain the capacity of CCAP-ENs to contribute to ecdysis, rather than acting phasically at ecdysis to instructively activate ecdysis behaviors or enable CCAP-ENs to contribute. Such a maintenance role is supported by previous work showing that maintained expression of the neuropeptide FMRFa requires persistent retrograde BMP signaling. It was also found that type III synapses on muscle 12 have significantly fewer boutons and shorter branches in wit mutants, implicating a role for BMP signaling in CCAP-EN synaptic morphology, as first described for type I neuromuscular junctions in wit mutants. It will be of interest to investigate whether dense core vesicle exocytosis is also perturbed in wit mutants, akin to the reduced synaptic vesicle exocytosis at type I boutons in wit mutants (Veverytsa, 2011).

The Drosophila BMPRII, wishful thinking, is required for eggshell patterning

The Drosophila eggshell is an elaborate structure that is derived from a monolayer of follicular epithelium surrounding the developing oocyte within the female ovary. The bone morphogenetic protein (BMP) signaling pathway is essential for controlling the patterning and morphogenesis of the eggshell. During oogenesis, the roles of patterning and morphogenesis by the BMP type I receptor thickveins (tkv) have been studied extensively. However, signaling through this pathway requires both type I and II receptors, and the latter has yet to be established in oogenesis. This study focused on wishful thinking (wit), the Drosophila homolog to the mammalian BMP type II receptor, BMPRII. It was found that wit is expressed dynamically in the FCs of D. melanogaster in an evolutionary conserved pattern. The expression patterns are highly correlated with the dynamics of the BMP signaling, consistent with the finding that wit is a target of BMP signaling. Furthermore, it was established that WIT is necessary for BMP signaling, and loss of WIT is associated with cell autonomous loss of BMP responses. Of importance, it was demonstrated that perturbations in WIT led to changes in eggshell morphologies in domains that are patterned by BMP signaling. Previous studies have shown a role for WIT in BMP signaling during neurogenesis; however, these results reveal a role for WIT in epithelial cells development (Marmion 2013).

Several laboratories have established that BMP signaling is required for eggshell morphogenesis. The early phase of BMP signaling maintains the anterior domain of the operculum and prevents it from being fated as dorsal appendage materia. The late phase of signaling is associated with the eggshell's morphogenetic process. Modifications in eggshell formation are associated with changes in BMP signaling components including the levels of ligands, BMP inhibitors, and receptors. Consistent with these results, changes in the levels of WIT led to morphological deformities throughout the eggshell (Marmion 2013).

The loss of BMP signaling in FCs null for wit is consistent with the loss of P-MAD in wit null neurons. In neurons, BMP signals through the BMP ligand Glass bottom boat (Gbb). However, in the FCs, BMP signaling is induced by an anteriorly emanating DPP that signals through the type I BMP receptor thickveins (Tkv). It was shown that BMP-2/4, the mammalian homolog of DPP, can signal through WIT and TKV. Therefore, it is proposed that DPP can signal through the WIT/TKV complex in the FCs. Alternatively, gbb is expressed uniformly in all FCs, thus, GBB homodimers and heterodimers with DPP may contribute to signaling in the FCs. However, the contribution of ligands other than DPP to FC patterning is still unknown (Marmion 2013).

The disruption of the cell adhesion molecule Cad74A gave rise to eggshell defects that are similar to the phenotypes obtained by the disruption of wit. These phenotypes are also similar to the eggshells obtained by perturbations in tkv. In the retina of D. melanogaster pupae, BMP signaling regulates the expression of cadherins. In the FCs, other cadherins are expressed in patterns that are considered targets of BMP signaling. Thus, perturbations in BMP signaling may contribute to eggshell defects by altering the expression of adhesion molecules. Interestingly, in D. melanogaster, it was suggested that detachments of pre and postsynaptic membranes are caused by the loss or aberrant function of cell adhesion molecules in cells null for wit (Marmion 2013).

Upon the disruption of wit, the two dorsolateral patches of Broad (BR) appear closer to each other. While lateral shifts in BR patterning along the dorsoventral axis are associated with EGFR signaling, these shifts also reflect changes in the levels of BMP signaling. For example, when Dad was used to inhibit BMP signaling in all FCs, the two BR patches appeared closer to each other and in a more anterior domain in comparison to the WT pattern. Consistent with these results, an increase in the levels of BMP signaling led to a greater distance between the BR patches (Marmion 2013).

>Notably, at early stage 10B, the pattern of BR in egg chambers perturbed for wit did not differ from the pattern found in the wild type. Thus, the changes in BR patterning found at stage 11 may reflect function of WIT near the beginning stages of dorsal appendage morphogenesis at the transition from stage 10B into 11. At these stages, the cells adjacent to the BR cells, called floor cells, extend under the BR cells. Thus, the disruption of WIT may alter the morphogenetic processes in the FCs, and consequently the two patches of BR appear closer to each other and more anteriorly. These results are in agreement with the suggested role of TKV during eggshell morphogenesis (Marmion 2013).

There is conflicting evidence regarding the regulation of BR by BMP signaling. It was shown to be necessary for BR expression or to act as a repressor. In this report, the disruption of WIT generated a reduction in the levels of late BR expression on the dorsolateral patches, which supports a positive role for WIT in BR regulation. However, in the FCs, clones are generated at early stages of egg development; thus, information about the temporal necessity of WIT in BR patterning cannot be deduced without an adequate marker. Recently, the intricate expression of BR was analyzed with two reporter constructs that reflect the early and late patterns of BR. It was demonstrated how a network of EGFR regulated transcription factors differentially regulate the two reporters. These reporter constructs of BR can now be used to analyze the effects of BMP signaling components on the expression of BR (Marmion 2013).

Previous studies have been demonstrated that in large clones null for punt, the second Drosophila type II BMP receptor, Brk-LacZ was ectopically expressed in the anterior follicle cells of young egg chambers (stage 7/8). It was reasoned that the deletion of punt alleviates the repression of the reporter by BMP signaling. Interestingly, while punt was null throughout the follicle cells, the Brk-LacZ was expressed in an anterior to posterior intensity gradient. Furthermore, the most anterior follicle cells did not express the LacZ, suggesting that the anterior follicle cells have sufficient signal to repress the reporter. It is proposed that WIT may provide this signal. Thus, in the follicle cells, Punt plays a role in BMP signaling, but the spatiotemporal division of labor between the two receptors still needs to be further investigated (Marmion 2013).

To date, the function of WIT has been associated with synaptic growth and synaptic transmission in motoneuron. This report has established a role for WIT during epithelial cells’ development. Since wit is expressed in other tissues, it is proposed that WIT may have a role in the development of other organs, and it is suggested that WIT's function may be obscured by the action of Punt in these tissues (Marmion 2013).

Coordinated regulation of axonal microtubule organization and transport by Drosophila Neurexin and BMP pathway

Neurexins are well known trans-synaptic cell adhesion molecules that are required for proper synaptic development and function across species. Beyond synapse organization and function, little is known about other roles Neurexins might have in the nervous system. This study reports novel phenotypic consequences of mutations in Drosophila neurexin (dnrx), which alters axonal microtubule organization and transport. dnrx mutants display phenotypic similarities with the BMP receptor wishful thinking (wit) and one of the downstream effectors, futsch, which is a known regulator of microtubule organization and stability. dnrx has genetic interactions with wit and futsch. Loss of dnrx also results in reduced levels of other downstream effectors of BMP signaling, phosphorylated-Mad and Trio. Interestingly, postsynaptic overexpression of the BMP ligand, Glass bottom boat, in dnrx mutants partially rescues the axonal transport defects but not the synapse undergrowth at the neuromuscular junctions. These data suggest that dnrx and BMP signaling are involved in many diverse functions and that regulation of axonal MT organization and transport might be distinct from regulation of synaptic growth in dnrx mutants. Together, this work uncovers a novel function of Drosophila Neurexin and may provide insights into functions of Neurexins in vertebrates (Banerjee, 2018).

Neurexins are some of the most extensively studied synaptic cell adhesion molecules for their role in synapse formation and function. In Drosophila, dnrx mutants have reduced synaptic growth. This reduced synaptic growth is similar to what was observe in the BMP signaling mutants: wit, gbb, mad and trio. Recent studies have shown that dnrx regulates components of the BMP signaling pathway and directs synaptic growth and cytoarchitecture in conjunction with dnlg and wit. The data presented in this study shows that dnrx engages in a complex molecular machinery with the BMP receptor Wit, the ligand Gbb and other downstream effectors to allow smooth axonal transport and proper MT organization (Banerjee, 2018).

Both dnrx and wit mutants display phenotypic similarities with axonal MT organization and transport of various cargo along the segmental nerves. It is interesting to note that axonal MT and transport phenotypes resulting from gain of dnrx function are similar to loss of function mutants. This suggests that a fine balance in levels of dnrx is important, and either too little or too much of it is detrimental for MT organization and the transport machinery. Defects in axonal transport resulting from cell type specific reduction in motor neurons using dnrx-RNAi confirmed that the axon transport defect is autonomous to motor neurons. The defects resulting from the dnrx knockdown, however, were milder than dnrx loss-of-function possibly due to penetrance or expressivity factors. The Gal4-only controls (elav-Gal4 and OK6-Gal4) did not display any axonal transport or MT organization defects further confirming the specificity of the dnrx phenotypes. It is also worth noting that gain of wit did not disrupt MT organization or axonal transport (Banerjee, 2018).

The genetic interaction between dnrx and wit suggest that these proteins regulate axonal MT organization and transport. Recent studies have also showed that dnrx and wit coordinate synaptic organization and growth, which suggests that these proteins are needed for multiple processes during nervous system development. The ultrastructural phenotypes in dnrx and wit mutants reveal a structural disorganization in the arrangement of MT filaments along the axons. These datasets showed that there is indeed disorganization in MT morphology in the mutants and these findings are not artifacts from the altered localization of Futsch (Banerjee, 2018).

Both dnrx and wit are presynaptic proteins and their loss shows a multitude of defects at the axonal, cytoskeletal and synaptic levels. It was therefore important to examine the range of behavioral deficits in these mutant larvae. dnrx and wit mutants displayed defects both in peristalsis and locomotor behaviors. These behaviors are attributable, most likely at least in part, to the defects in axon transport, MT organization and possible dysfunction of the NMJ synapses. Behavior in animals could be a composite of defects at many levels of integration at the CNS, PNS, NMJ and circuits responsible for producing rhythmic patterns of movement and general locomotion. Defects in both peristalsis and wandering behavior in cysteine string protein (csp) mutant larvae, for example, are thought to be due to motor defects at the NMJ and at the levels of CNS output generation. The locomotor behaviors observed in dnrx and wit mutants also indicate that these proteins might play a boarder role in organization of motor circuits (Banerjee, 2018).

Recent studies have implicated BMP signaling in the regulation of synaptic strength, axonal MT organization and transport. While the current studies point to the reliance of MT organization and axonal transport on BMP signaling, there are other studies that show disruption of axonal transport perturbing BMP signaling contributes to synaptic abnormalities in neurodegenerative diseases. It is important to note that mutations that fall under the categories of both positive regulators of BMP, like dnrx8 and negative regulators of BMP such as, Spicthyin and Spartin both lead to MT disorganization. It is, therefore, interesting that while Spichthyin and Spartin function as negative regulators of the BMP signaling by regulating BMP receptor traffic and modulate regulation of MT cytoskeleton, loss of these proteins does not show impaired axonal transport. dnrx loss results in disorganized MTs, but unlike Spichthyin and Spartin, loss of dnrx shows transport defects. These findings suggest that MT cytoskeletal organization could be compromised by both positive and negative regulation of BMP signaling (Banerjee, 2018).

The regulation of microtubule stability is essential for axonal transport. The microtubule-associated protein, Futsch, recently implicated in the BMP pathway, has a role in the stabilization of MTs. futsch mutants display axonal transport defects consistent with altered MT organization similar to dnrx and wit mutants. A previous report, however, showed that futsch mutants do not show defects in axonal transport of Brp. This study examined futsch mutants for accumulation of Brp puncta along axons. Mild, yet significant, increase in accumulation of Brp was found along the axons in futsch mutants. These studies are not completely in agreement with the previous report. The discrepancies between the studies could be possibly due to: (1) differences in quantification parameters (Brp area/Hrp area as opposed to number of Brp puncta along nerve length); (2) difference in segmental nerves analyzed (the current analysis was restricted to segmental nerves 5-8); and (3) the region of nerve fiber analyzed (proximal or distal). It is also worth noting that light microscopy studies showed Syt and DCSP2 aggregation along mutant axons did not always co-localize with Brp puncta. These observations are consistent with recently published report that showed Syt co-traffic less efficiently with active zone scaffold proteins Brp and Basoon. Collectively, these findings suggest the possibility of the presence of distinct transport mechanisms for different synaptic cargoes. Future studies will be aimed at addressing the dynamics of axonal membrane traffic in mutants of dnrx and BMP pathway, and whether some synaptic proteins are co-transported and others are not, or whether transport mechanisms are able to discriminate synaptic cargo as it is transported along the axons (Banerjee, 2018).

futsch and dnrx interact genetically as double heterozygotes display axonal transport defects consistent with dose-dependent genetic interactions. This data further corroborates the idea that dnrx is not only necessary for trans-synaptic adhesion and apposition of the pre- and post-synaptic compartments, but also for maintaining the stability of the BMP receptor wit for optimal BMP signaling. Future studies will be aimed at addressing the link between dnrx and Futsch. dnrx is a transmembrane protein with a PDZ domain binding motif and Futsch is a MT-binding protein. Both of these proteins are highly expressed along the axons and could potentially exist as a complex. It is also possible that dnrx might interact with MT motor proteins such as Kinesin or Dynein. The Kinesin superfamily protein, KIF 17, was previously shown to interact with PDZ domain of mLin-10 (Mint1/X11) to transport NMDA receptor 2B. mLin-10 is part of a larger protein complex that includes mLin-2 (Cask) and mLin-7 (Veli). It is likely that the intracellular PDZ domain of dnrx is capable of recruiting protein complexes such as these to form a link between axonal membranes to the axonal MT cytoskeleton (Banerjee, 2018).

While it was not a surprise that levels of the downstream effectors of the BMP cascade, pMad and Trio, would be reduced in dnrx mutant BL/VNC given that previous studies showed reduced levels of Wit, Tkv and pMad at the dnrx mutant NMJ synapses. It is interesting that postsynaptic overexpression of the BMP ligand, Gbb, partially rescues the axonal transport phenotypes in dnrx mutant background but not the NMJ bouton undergrowth phenotype. This indicates that distinct regulatory mechanisms might be involved in proper axonal MT organization and transport and growth during NMJ development, which are still not well understood. While this finding may suggest that Wit/Gbb signaling does not require dnrx to regulate axon transport, it is important to note that the rescue of the axonal transport phenotype was partial and did not reach wild type levels, suggesting that dnrx is required to allow optimal levels of axonal transport to occur. This finding also indicates: (1) that possibly it is the retrograde transport that gets somewhat corrected while there might still be defects in anterograde transport of cargoes in dnrx mutants; (2) that BMP signaling solely is not responsible for trafficking of cargoes and overall MT health in dnrx mutants; and (3) that there might be additional proteins involved in forming a cascade to link dnrx from axonal membrane to MT cytoskeleton. Given the findings reported in this study on Drosophila Nrx, it would be of immense interest to investigate whether mammalian Neurexins will have similar functions separate from their role in synapse formation and their functional modulation, and also axonal microtubular organization. Future studies will further address other aspects of Neurexin functions in the nervous system (Banerjee, 2018).


cDNA clone length - 4034

Bases in 5' UTR - 208

Exons - 6

Bases in 3' UTR - 1113


Amino Acids - 903

Structural Domains

Conceptual translation of the wishful thinking ORF reveals a protein with a signal peptide, a single membrane-spanning domain, and a Ser-Thr kinase domain, containing an ATP binding site and a highly conserved coiled-coil/leucine zipper motif, followed by a carboxy-terminal tail of 378 aa with no distinctive features other than an abundance of Ser and Thr residues. The overall domain structure, including the presence of the carboxy-terminal tail, is a characteristic feature of the BMPRII and MIS type II receptors (Baarends, 1994; Kawabata, 1995; Liu, 1995). Phylogenetic analysis of the kinase domain clearly places Wit as a close homolog of the vertebrate BMPR-II receptor. However, despite the similarity within the kinase domain, it is noted that the C-terminal tail of Wit shows very little identity to the tails of either BMPRII or the MISR other than a short stretch of 7 aa shared with BMPRII within the C-terminal region of the tail. At the amino acid level, the overall identity of Wit to human BMPRII is 30% with the strongest homology in the kinase domain (Marqués, 2002 and Aberle, 2002).

In vertebrates, two alternatively spliced isoforms of the BMP type II receptor have been reported (Kawabata, 1995). The shorter version ends 28 aa after the kinase domain and does not contain the carboxy-terminal extension. By developmental Northern analysis a single 4.5 kb transcript was detected at all stages, suggesting that only one isoform of wit is expressed in Drosophila (Marqués, 2002).

wishful thinking: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 23 June 2023

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