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Carrillo, R. A.

Publications and source records attributed to Carrillo, R. A..

8 recordsLinked to original sources

Structural insights into wiring specificity in the neuromuscular system through the Beat-Side complex

Nervous system assembly is guided by the actions of cell surface receptors. In Drosophila, members of the Beaten Path (Beat) and Sidestep (Side) protein families have been described as axon guidance receptor-cue pairs, in addition to roles in specifying synaptic connectivity in the optic lobe. To understand the molecular basis and specificity of Beat-Side interactions, we report here the first Beat-Side structure, Beat-Vc bound to Side-VI. The structure showed a binding topology similar to other neuronal immunoglobulin superfamily receptors, especially Nectins, SynCAMs, Dprs and DIPs, despite lack of established evolutionary relationships. Using a structure-based rational approach, we engineered and validated point mutations to break the binding between Beats and Sides. Using these mutant variants, we demonstrated in developing Drosophila larvae that the interaction between Beat-Ia and Side is required for establishing proper connectivity of motor neurons with muscles.

neuroscience↗

hkb is required for DIP-α expression and target recognition in the Drosophila neuromuscular circuit

Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins (CSPs). In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth and cell survival. However, the upstream regulation and downstream signaling mechanisms of Dprs and DIPs are not clear. In the Drosophila larval neuromuscular system, DIP- is expressed in the dorsal and ventral type-Is motor neurons (MNs). We conducted an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We found that the transcription factor, huckebein (hkb), genetically interacts with DIP- and is important for target recognition specifically in the dorsal Is MN, but not the ventral Is MN. Loss of hkb led to complete removal of DIP- expression. We then confirmed that this specificity is through the dorsal Is MN specific transcription factor, even-skipped (eve), which acts downstream of hkb. Genetic interaction between hkb and eve revealed that they act in the same pathway to regulate dorsal Is MN connectivity. Our study provides insight into the transcriptional regulation of DIP- and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons.

neuroscience↗

Glial Draper signaling triggers cross-neuron plasticity in bystander neurons after neuronal cell death

Neuronal cell death and subsequent brain dysfunction are hallmarks of aging and neurodegeneration, but how the nearby healthy neurons (bystanders) respond to the cell death of their neighbors is not fully understood. In the Drosophila larval neuromuscular system, bystander motor neurons can structurally and functionally compensate for the loss of their neighbors by increasing their axon terminal size and activity. We termed this compensation as cross-neuron plasticity, and in this study, we demonstrated that the Drosophila engulfment receptor, Draper, and the associated kinase, Shark, are required in glial cells. Surprisingly, overexpression of the Draper-I isoform boosts cross-neuron plasticity, implying that the strength of plasticity correlates with Draper signaling. Synaptic plasticity normally declines as animals age, but in our system, functional cross-neuron plasticity can be induced at different time points, whereas structural cross-neuron plasticity can only be induced at early stages. Our work uncovers a novel role for glial Draper signaling in cross-neuron plasticity that may enhance nervous system function during neurodegeneration and provides insights into how healthy bystander neurons respond to the loss of their neighboring neurons.

neuroscience↗

Neuronal wiring receptors Dprs and DIPs are GPI anchored and this modification contributes to their cell surface organization

The Drosophila Dpr and DIP proteins belong to the immunoglobulin superfamily of cell surface proteins (CSPs). Their hetero- and homophilic interactions have been implicated in a variety of neuronal functions, including synaptic connectivity, cell survival, and axon fasciculation. However, the signaling pathways underlying these diverse functions are unknown. To gain insight into Dpr-DIP signaling, we sought to examine how these CSPs are associated with the membrane. Specifically, we asked whether Dprs and DIPs are integral membrane proteins or membrane anchored through the addition of glycosylphosphatidylinositol (GPI) linkage. We demonstrate that Dprs and DIPs are GPI anchored to the membrane of insect cells and validate these findings for some family members in vivo using Drosophila larvae, where GPI anchor cleavage results in loss of surface labeling. Additionally, we show that GPI cleavage abrogates aggregation of insect cells expressing cognate Dpr-DIP partners. To test if the GPI anchor affects Dpr-DIP localization, we replaced it with a transmembrane domain and observed perturbation of sub-cellular localization on motor neurons and muscles. These data suggest that membrane anchoring of Dprs and DIPs through GPI linkage is required for localization and that Dpr-DIP intracellular signaling likely requires transmembrane co-receptors.

neuroscience↗

Systematic expression profiling of dprs and DIPs reveals cell surface codes in Drosophila larval peripheral neurons

In complex nervous systems, neurons must identify their correct partners to form synaptic connections. The prevailing model to ensure correct recognition posits that cell surface proteins (CSPs) in individual neurons act as identification tags. Thus, knowing what cells express which CSPs would provide insights into neural development, synaptic connectivity, and nervous system evolution. Here, we investigated expression of dprs and DIPs, two CSP subfamilies belonging to the immunoglobulin superfamily (IgSF), in Drosophila larval motor neurons (MNs), sensory neurons (SNs), peripheral glia and muscles using a collection of GAL4 driver lines. We found that dprs are more broadly expressed than DIPs in MNs and SNs, and each examined neuron expresses a unique combination of dprs and DIPs. Interestingly, many dprs and DIPs are not robustly expressed, but instead, are found in gradient and temporal expression patterns. Hierarchical clustering showed a similar expression pattern of dprs and DIPs in neurons from the same type and with shared synaptic partners, suggesting these CSPs may facilitate synaptic wiring. In addition, the unique expression patterns of dprs and DIPs revealed three uncharacterized MNs - MN23-Ib, MN6-Ib (A2) and MN7-Ib (A2). This study sets the stage for exploring the functions of dprs and DIPs in Drosophila MNs and SNs and provides genetic access to subsets of neurons.

neuroscience↗

Structural and functional synaptic plasticity induced by convergent synapse loss requires co-innervation in the Drosophila neuromuscular circuit

Throughout the nervous system, the convergence of two or more presynaptic inputs on a target cell is commonly observed. The question we ask here is to what extent converging inputs influence each others structural and functional synaptic plasticity. In complex circuits, isolating individual inputs is difficult because postsynaptic cells can receive thousands of inputs. An ideal model to address this question is the Drosophila larval neuromuscular junction where each postsynaptic muscle cell receives inputs from two glutamatergic types of motor neurons (MNs), known as 1b and 1s MNs. Notably, each muscle is unique and receives input from a different combination of 1b and 1s motor neurons. We surveyed synapses on multiple muscles for this reason. Here, we identified a cell-specific promoter to ablate 1s MNs after innervation. Additionally, we genetically blocked 1s innervation. Then we measured 1b MN structural and functional responses using electrophysiology and microscopy. For all muscles, 1s MN ablation resulted in 1b MN synaptic expansion and increased basal neurotransmitter release. This demonstrates that 1b MNs can compensate for the loss of convergent inputs. However, only a subset of 1b MNs showed compensatory evoked activity, suggesting spontaneous and evoked plasticity are independently regulated. Finally, we used DIP- mutants that block 1s MN synaptic contacts; this eliminated robust 1b synaptic plasticity, raising the possibility that muscle co-innervation may define an activity "set point" that is referenced when subsequent synaptic perturbations occur. This model can be tested in more complex circuits to determine if co-innervation is fundamental for input-specific plasticity. SIGNIFICANCE STATEMENTIn complex neural circuits, multiple converging inputs contribute to the output of each target cell. Thus, each input must be regulated, but whether adjacent inputs contribute to this regulation is unclear. To examine input-specific synaptic plasticity in a structurally and functionally tractable system, we turn to the Drosophila neuromuscular circuit. Each muscle is innervated by a unique pair of motor neurons. Removal of one neuron after innervation causes the adjacent neuron to increase synaptic outgrowth and functional output. However, this is not a general feature since each MN differentially compensates. Also, robust compensation requires co-innervation by both neurons. Understanding how neurons respond to perturbations in adjacent neurons will provide insight into nervous system plasticity in both healthy and diseased states.

neuroscience↗

Temporal transcription factors determine circuit membership by altering motor neuron-to-muscle synaptic partnerships

Previously, using the Drosophila motor system as a model, we found the classic temporal transcription factor, Hunchback acts in NB7-1 neuronal stem cells as a molecular switch to control which circuits are populated by NB7-1 neuronal progeny (Meng et al., 2019). Here, we manipulate cardinal transcription factors, Nkx6 and Hb9, which are candidate effectors of Hunchback and which alter axon pathfinding in embryos. Yet manipulation of these cardinal transcription factors does not permanently alter neuromuscular synaptic partnerships. This demonstrates that compensation can correct early defects. We perform additional temporal transcription factor manipulations, precociously expressing Pdm and Castor in NB7-1 and prolonging expression of Hunchback in NB3-1. In every case, we find permanent alterations in neuromuscular synaptic partnerships. These data support the idea that temporal transcription factors are uniquely potent determinants of circuit membership, which do not trigger compensatory programs because they act to establish the expected pattern of wiring for the motor system.

developmental biology↗

Transsynaptic interactions between IgSF proteins DIP-α and Dpr10 are required for motor neuron targeting specificity in Drosophila

The Drosophila larval neuromuscular system provides an ideal context in which to study synaptic partner choice, because it contains a small number of pre- and postsynaptic cells connected in an invariant pattern. The discovery of interactions between two subfamilies of IgSF cell surface proteins, the Dprs and the DIPs, provided new candidates for cellular labels controlling synaptic specificity. Here we show that DIP- is expressed by two identified motor neurons, while its binding partner Dpr10 is expressed by postsynaptic muscle targets. Removal of either DIP- or Dpr10 results in loss of specific axonal branches and NMJs formed by one motor neuron, MNISN-1s, while other branches of the MNISN-1s axon develop normally. The temporal and spatial expression pattern of dpr10 correlates with muscle innervation by MNISN-1s during embryonic development. We propose a model whereby DIP- and Dpr10 on opposing synaptic partners interact with each other to generate proper motor neuron connectivity.

neuroscience↗