Our understanding of many fundamental questions of development has benefited from the study of Dictyostelium discoideum7,8, a forest-floor-dwelling social amoeba with the rare feature of its morphogenesis occurring almost entirely in the absence of cell division. Cellular differentiation and morphogenetic redistributions can therefore be conveniently followed. D. discoideum’s multicellular, developmental phase begins when up to 105 free-living cells (amoebae) aggregate together, a process initiated, when the supply of the food source, soil bacteria, is depleted. The amoebae move up a concentration gradient of extracellular cAMP and form a multicellular mound, within which an axis of polarity becomes macroscopically evident when a tip arises from the centre of the mound. The mound next transforms into a motile, slug-like body that can migrate through the soil, or leaf litter, in response to light and other cues; after culmination, it finally forms an aerial, spore-filled fruiting body9. For all but the last stage of development, the cells of the original tip act in similar ways to the classical organizers of chordate development: the tip cells remain the same, and regulate the organisation of tissues along one of the organism’s axes; additionally, they exert control upon the overall size of all later stages5,10. If a tip from one slug is transplanted onto another, the second slug will transform into two smaller, but properly organized slugs within four hours4. As to the identity of the persistent signal emanating from the tip that maintains tip dominance, adenosine has been suggested, on the basis of the smaller slugs formed when endogenous levels of adenosine are reduced11. But size regulation in D. discoideum is also influenced by processes that act prior to tip formation12, and it is possible that adenosine’s contribution is restricted to the earlier, aggregation stage. Here, we chose to investigate whether tip dominance involves serotonin, previously unreported in D. discoideum, but which, in addition to its well-known role as a neurotransmitter, is an important developmental regulator in organisms across many phyla13–15. We present multiple lines of evidence that indicate that serotonin does contribute to tip dominance in D. discoideum, and that the breaking of this dominance, and the enablement of the mound’s original tip, involves monoamine amine oxidase A, which degrades serotonin.
MAOA and serotonin in D. discoideum
To our knowledge, neither monoamine oxidases (MAOs) nor serotonin have been previously reported in D. discoideum, nor more widely, in protozoans and slime molds, respectively. According to its annotated genome, however, D. discoideum is predicted to encode four different isoforms of monoamine oxidase16 of which, maoA expression (DDB_G0288541) is particularly high during development17 (Extended Data Fig. 1a, b). Also, MAOA of D. discoideum has the conserved amine oxidase domain (Extended Data Fig. 2a) and its predicted tertiary structure is similar to that of humans MAOA (Extended Data Fig. 2b). The different levels of MAO activity in wildtype, [RNAi]maoA and maoA overexpressing clones confirm amine oxidase activity in D. discoideum (Extended Data Fig. 1c, d).
In spite of the absence of the gene, aromatic amino acid decarboxylase (AAAD), responsible for serotonin production18, D. discoideum cells produce serotonin, as ascertained by liquid chromatography-mass spectrometry (LC-MS) (Extended Data Fig. 3a, b). Tryptophan, a precursor for serotonin synthesis, when tagged with a methyl group, is expected to have a higher mass. MS confirmed that cells cultured with alpha-methyl tryptophan synthesize alpha-methyl serotonin (Fig. 1a), suggesting a novel serotonin biosynthesis pathway in D. discoideum. Serotonin levels in the wildtype mound were found to be 0.321 ng/108 cells (Extended Data Fig. 3c).
MAOA enables the tip organizer
To understand the function of MAOA, an [RNAi]maoA line was generated (Extended Data Fig. 4a, b). Development was arrested at the mound stage, and these mounds did not form tips (Fig. 2b and Supplementary Video 2). This phenotype could be mimicked by adding MAO specific inhibitors (isocarboxazid or nialamide) to wildtype cells (Fig. 2c, d and Supplementary Video 3, 4). Thus, inhibiting MAOA activity or reducing the level of MAOA blocks tip formation. Tip formation in [RNAi]maoA clones could be restored by adding human MAOA (hMAOA) enzyme directly onto them, and each mound then proceeded to form a slug (Fig. 2e). Conversely, when maoA was overexpressed in the wildtype (Extended Data Fig. 4c), each mound had multiple tips (Fig. 2f). Taken together, these results suggest that the increased level of maoA activity has a role in tip enablement.
Serotonin blocks tip formation
To examine whether the function of MAOA is related to its degradation of serotonin, [RNAi]maoA lines were treated with either methiothepin, a serotonin antagonist, or antibodies against serotonin. In each case, the wildtype phenotype was restored and [RNAi]maoA mounds were transformed into slugs (Fig. 3a). Further, when maoAOE lines (which normally form multiple tips) were treated with 1 mM serotonin, development was arrested at the mound stage (Extended Data Fig. 5d); a lower dose (500 µM) had no effect (Extended Data Fig. 5c). Conversely, the development of wildtype cells treated with 500 µM methiothepin was arrested at the mound stage (Extended Data Fig. 5b). These results suggest that serotonin’s normal role might be to block tip formation in the mound. Indeed, when the wildtype was treated with the serotonin antagonist, methiothepin, this resulted in multiple tips emerging from each mound, a phenotype similar to maoA overexpressing clones (Fig. 3b). When applied at 15 µM (minimal inhibitory dose) this gave rise to 1.34 ± 0.09 tips/ mound, and increasing the dose to 20 µM resulted in 2.17 ± 0.02 tips/mound (Fig. 3c). Thus, MAOA and serotonin appear to have opposite roles in both the enabling of the tip within a mound, and in suppressing the formation of any further tips, that is, tip dominance.
Serotonin and MAOA act on cAMP and cell-cell adhesion
To investigate the mechanisms by which MAOA/serotonin could regulate tip formation, we began by looking at whether extracellular serotonin affects cAMP levels. cAMP in D. discoideum plays a role in tip formation and cell fate determination11,19, and extracellular serotonin is known to trigger intracellular cAMP signalling in neurons20. LC-MS analysis of concentrated conditioned media (CM) (Fig. 1b), and the rescue of [RNAi]maoA by either extracellular hMAOA enzyme or by serotonin antibodies, are all consistent with the secretion of serotonin in D. discoideum. Further, in the maoA RNAi lines (which have high serotonin), the levels of adenyl cyclase A gene expression (which converts ATP to cAMP) were high, as were the total cAMP levels (Fig. 4a, b). Also, adding the aca specific inhibitor SQ22536, rescues the mound arrest phenotype of the maoA RNAi lines (Fig. 4d), which also supports the idea that increased serotonin inhibits tip formation by raising cAMP levels.
As to what might occur downstream, cAMP has previously been shown to regulate tip formation by modulating PKA activity, adenosine signalling and morphogenetic cell movements11,21,22. To ascertain which of these three pathways is significantly affected by the observed serotonin-induced cAMP increase, the following compounds were each applied to [RNAi]maoA developmentally-arrested mounds: an activator of PKA (8-Br-cAMP, cAMP) or its inhibitor (H89); or adenosine deaminase; or adenosine. Each treatment failed to restore tip formation (Extended Data Fig. 6, 7), suggesting that PKA and adenosine signalling act independently or upstream of serotonin.
In investigating whether it is morphogenetic cell movements that are affected by serotonin, our starting point was that their contribution to normal tip formation is known to depend upon chemotaxis and cell-cell adhesion23. Surprisingly, in [RNAi]maoA mounds, we found that chemotaxis is not affected (Extended Data Fig. 8c) but cell-cell adhesion is drastically reduced (Extended Data Fig. 8a). Thus, increased cAMP in [RNAi]maoA mounds possibly decreases cell-cell adhesion and thus impairs wave propagation, leading to inhibition of tips. In support of this, when [RNAi]maoA mounds were treated with the aca inhibitor, SQ22536 (which, as shown above, rescues developmental arrest) cell-cell adhesion was higher (Extended Data Fig. 8b). Further, we compared22 collective cell movements in the wildtype and [RNAi]maoA lines: in the former, the wave propagation pattern was spiral, and towards the tip, while in the knockdown, it was circular (Supplementary Video. 5, 6).
To now probe the impact of MAOA/serotonin on wave propagation, we treated [RNAi]maoA mounds with caffeine or IBMX (PDE4 inhibitor), both of which are known to induce tips24. Adding either one of the compounds to [RNAi]maoA mounds resulted in a cAMP wave propagation pattern that was closer to that of the wildtype, and restored tip formation to a more normal pattern (Fig. 4e, f and Supplementary Video. 7, 8). Interestingly, caffeine is known to reduce intracellular cAMP25 whereas PDE4 inhibitor increases extracellular cAMP26.
A model for tip enablement and dominance
Considering all the results together, we can propose the following model for the involvement of MAOA/serotonin in normal development of D. discoideum: (1) Prior to tip enablement, serotonin levels are uniformly high throughout a mound. High serotonin leads to decreased levels of extracellular cAMP (perhaps relative to intracellular cAMP), thus reducing cell-cell adhesion and blocking spiral wave propagation. Tip formation is thus inhibited. (2) This inhibition is subsequently released, but only at the site of future tip formation, by a localized increase in MAOA which degrades serotonin locally; and (3) In a mound, serotonin contributes to tip dominance, helping the mound to retain the size specified by processes that act during the earlier, aggregation stage27. One would expect, however, that another diffusible factor, with a tip-high concentration gradient, must also be involved. It is possible this second factor is adenosine, operating independently of MAOA/serotonin, and thus adding to its earlier role in size regulation, during the late aggregation stage11,28. Apart from size regulation, serotonin is likely to affect the cell type ratio along with free Ca2+ and cAMP29,30. Differences in serotonin levels between the tip and the rest of the mound possibly affect serotonylation driving differences in gene expression thereby establishing the prestalk-prespore identity.
The roles we have ascribed to serotonin and MAOA in D. discoideum are also indirectly supported by their developmental roles in other organisms. Serotonin has been specifically shown to regulate some features of animal morphogenesis, particularly gastrulation, via its effects on cell-cell adhesion31,32; and, more generally, differential cell adhesion is considered an important physical cue in the generation of self-organising structures33. MaoA is also expressed during gastrulation in many vertebrates and it is likely that it helps to regulate morphogenesis in these organisms34–36 (Extended Data Fig. 9a-c), perhaps also by its dampening of serotonin’s effects. MAO is also present in various ascomycota37 (spore forming fungi) and colony forming bacteria (Extended Data Fig. 10), where it could perhaps play a conserved role in regulating group or colony size.
The synthesis of serotonin in D. discoideum was surprising given the absence of the AAAD gene required for serotonin synthesis in humans. Entamoeba histolytica, a pathogenic amoeba, synthesizes and secretes serotonin38 without the canonical AAAD gene and thus, both D. discoideum and E. histolytica possibly synthesize serotonin through similar non-canonical pathways. While we have demonstrated the synthesis of serotonin, a BLAST analysis indicated the absence of the established serotonin receptors in D. discoideum. However, adding serotonin receptor specific antagonists confers the expected phenotype suggesting the presence of novel, but structurally overlapping, receptor(s). Such receptors are likely to be conserved across protozoa that infect humans, and identifying these novel serotonin receptors may point to useful drug targets.