Modulatory neurons project widely throughout the brain, dynamically altering network processing based on an animal's physiological state. The connectivity of individual modulatory neurons can be complex, as they often receive input from a variety of sources and are diverse in their physiology, structure, and gene expression profiles. To establish basic principles about the connectivity of individual modulatory neurons, we examined a pair of identified neurons, the "contralaterally projecting, serotonin-immunoreactive deutocerebral neurons" (CSDns), within the olfactory system of Specifically, we determined the neuronal classes providing synaptic input to the CSDns within the antennal lobe (AL), an olfactory network targeted by the CSDns, and the degree to which CSDn active zones are uniformly distributed across the AL. Using anatomical techniques, we found that the CSDns received glomerulus-specific input from olfactory receptor neurons (ORNs) and projection neurons (PNs), and networkwide input from local interneurons (LNs). Furthermore, we quantified the number of CSDn active zones in each glomerulus and found that CSDn output is not uniform, but rather heterogeneous, across glomeruli and stereotyped from animal to animal. Finally, we demonstrate that the CSDns synapse broadly onto LNs and PNs throughout the AL but do not synapse upon ORNs. Our results demonstrate that modulatory neurons do not necessarily provide purely top-down input but rather receive neuron class-specific input from the networks that they target, and that even a two cell modulatory network has highly heterogeneous, yet stereotyped, pattern of connectivity. Modulatory neurons often project broadly throughout the brain to alter processing based on physiological state. However, the connectivity of individual modulatory neurons to their target networks is not well understood, as modulatory neuron populations are heterogeneous in their physiology, morphology, and gene expression. In this study, we use a pair of identified serotonergic neurons within the olfactory system as a model to establish a framework for modulatory neuron connectivity. We demonstrate that individual modulatory neurons can integrate neuron class-specific input from their target network, which is often nonreciprocal. Additionally, modulatory neuron output can be stereotyped, yet nonuniform, across network regions. Our results provide new insight into the synaptic relationships that underlie network function of modulatory neurons.
Drosophila Enhancer of split M8, an effector of Notch signaling, is regulated by protein kinase CK2. The phosphatase PP2A is thought to play an opposing (inhibitory) role, but the identity of the regulatory subunit was unknown. The studies described here reveal a role for the PP2A regulatory subunit widerborst (wdb) in three developmental contexts; the bristle, wing and the R8 photoreceptors of the eye. wdb overexpression elicits bristle and wing defects akin to reduced Notch signaling, whereas hypomorphic mutations in this PP2A subunit elicit opposite effects. We have also evaluated wdb functions using mutations in Notch and E(spl) that affect the eye. We find that the eye and R8 defects of the well-known Nspl mutation are enhanced by a hypomorphic allele of wdb, whereas they are strongly rescued by wdb overexpression. Similarly, ectopic wdb rescues the eye and R8 defects of the E(spl)D mutation, which affects the m8 gene. In addition, wdb overexpression also rescues the bristle defects of ectopically expressed M8, or the eye and R8 defects of its CK2 phosphomimetic variant M8-S159D. The latter finding suggests that PP2A may target M8 at highly conserved residues in the vicinity of the CK2 site, whose phosphorylation controls repression of Atonal and the R8 fate. Together, the studies identify PP2A-Wdb as a participant in Notch signaling, and suggest that M8 activity is controlled by phosphorylation and dephosphorylation. The conservation of the phosphorylation sites between Drosophila E(spl) and the HES/HER proteins from mammals, reptiles, amphibians, birds and fish raises the prospect that this mode of regulation is widespread.
Drosophila atonal (ato) is required for the specification of founding R8 photoreceptors during retinal development. ato is regulated via dual eye-specific enhancers; ato-3’ is subject to initial induction whereas 5’-ato facilitates Notch-mediated autoregulation. Notch is further utilized to induce bHLH repressors of the E(spl) locus to restrict Ato from its initial broad expression to individual cells. Although Notch operates in two, distinct phases, it has remained unclear how the two phases maintain independence from one another. The difference in these two phases has attributed to the hypothesized delayed expression of E(spl). However, immunofluorescence data indicate that E(spl) are expressed during early Ato patterning, suggesting a more sophisticated underlying mechanism. To probe this mechanism, we provide evidence that although E(spl) exert no influence on ato-3’, E(spl) repress 5’-ato and deletion of the E(spl) locus elicits precocious 5’-ato activity. Thus, E(spl) imposes a delay to the timing in which Ato initiates autoregulation. We next sought to understand this finding in the context of E(spl)D, which encodes a dysregulated variant of E(spl)M8 that perturbs R8 patterning, though, as previously reported, only in conjunction with the mutant receptor Nspl. We established a genetic interaction between E(spl)D and roughened eye (roe), a known modulator of Notch signaling in retinogenesis. This link further suggests a dosage-dependence between E(spl) and the proneural activators Ato and Sens, as indicated via interaction assays in which E(spl)D renders aberrant R8 patterning in conjunction with reduced proneural dosage. In total, the biphasicity of Notch signaling relies, to some degree, on the post-translational regulation of individual E(spl) members and, importantly, that post-translational regulation is likely necessary to modulate the level of E(spl) activity throughout the progression of Ato expression.
On the roles of Drosophila Notch signaling and atonal autoregulation in the specification of R8 photoreceptors.
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