Transcription Factor Acj6 Controls Dendrite Targeting via Combinatorial Cell-Surface Codes
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ABSTRACT: Transcription factors specify the fate and connectivity of developing neurons. We investigate how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs) by regulating the expression of cell-surface proteins. Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and proteins previously not associated with wiring, such as Piezo, whose mechanosensitive ion channel activity is dispensable for its function in PN dendrite targeting. Comprehensive genetic analyses revealed that Acj6 employs unique sets of cell-surface proteins in different PN types for dendrite targeting. Combinatorial expression of Acj6 wiring executors rescued acj6 mutant phenotypes with higher efficacy and breadth than expression of individual executors. Thus, Acj6 controls wiring specificity of different neuron types by specifying distinct combinatorial expression of cell-surface executors.
Project description:Xie Q, Li J1, Li H, Udeshi ND, Svinkina T, Kohani S, Guajardo R, Mani DR, Xu C, Li T, Han S, Wei W, Shuster SA, Luginbuhl DJ, Ting AY, Carr SA, Luo L. 2022.
Transcription factors specify the fate and connectivity of developing neurons. To understand how distal neurites execute commands from these nucleus-residing factors to specify their connectivity, we investigated how a lineage-specific transcription factor, Acj6, controls the precise dendrite targeting of Drosophila olfactory projection neurons (PNs). Quantitative cell-surface proteomic profiling of wild-type and acj6 mutant PNs in intact developing brains and a proteome-informed genetic screen identified PN surface proteins that execute Acj6-regulated wiring decisions. These include canonical cell adhesion molecules and molecules previously not associated with wiring, such as the mechanosensitive ion channel Piezo. Ion channel activity is dispensable for Piezo's function in PN dendrite targeting. Combinatorial expression of Acj6 executors more strongly rescued specific acj6 mutant phenotypes than expression of individual executors. Together, our findings reveal that a key transcription factor controls wiring specificity of different neuron types by regulating expression of unique combinations of cell-surface executors.
Project description:How a neuronal cell type is defined and how this relates to its transcriptome are still open questions. The Drosophila olfactory projection neurons (PNs) are among the bestcharacterized neuronal types: Different PN classes target dendrites to distinct olfactory glomeruli and PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA-sequencing, we comprehensively characterized the transcriptomes of 40 PN classes and unequivocally identified transcriptomes for 6 classes. We found a new lineage-specific transcription factor that instructs PN dendrite targeting. Transcriptomes of closely-related PN classes exhibit the largest difference during circuit assembly, but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Genes encoding transcription factors and cell-surface molecules are the most differentially expressed, indicating their central roles in specifying neuronal identity. Finally, we show that PNs use highly redundant combinatorial molecular codes to distinguish subtypes, enabling robust specification of cell identity and circuit assembly.
Project description:Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues, and thus govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally-resolved approach to profile cell surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed a global downregulation of wiring molecules and an up-regulation of synaptic molecules in the transition from developing to mature PNs. To compare the RNA and protein dynamics of PN surface molecules in the developing-to-mature transition, we also profiled the PN transcriptomes from 36hAPF pupae and 5d adults. We performed bulk RNA-sequencing of PNs with 3 samples for each stage and 2k cells for each sample.
Project description:Li J, Han S, Li H, Udeshi ND, Svinkina T, Mani DR, Xu C, Guajardo R, Xie Q, Li T, Luginbuhl DJ, Wu B, McLaughlin CN, Xie A, Kaewsapsak P, Quake sR, Carr SA, Ting AY, Luo L. 2019.
Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues, and thus govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally-resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed a global down-regulation of wiring molecules and an up-regulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 new cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally-resolved in situ cell-surface proteomic profiling in discovering new regulators of brain wiring.
Project description:Patterns of synaptic connectivity are remarkably precise and complex. Single-cell RNA sequencing has revealed a vast transcriptional diversity of neurons. Nevertheless, a clear logic underlying the transcriptional control of neuronal connectivity has yet to emerge. Here, we focused on Drosophila T4/T5 neurons, a class of closely related neuronal subtypes with different wiring patterns. Eight subtypes of T4/T5 neurons are defined by combinations of two patterns of dendritic inputs and four patterns of axonal outputs. Single-cell profiling during development revealed distinct transcriptional programs defining each dendrite and axon wiring pattern. These programs were defined by the expression of a few transcription factors and different combinations of cell surface proteins. Gain and loss of function studies provide evidence for independent control of different wiring features. We propose that modular transcriptional programs for distinct wiring features are assembled in different combinations to generate diverse patterns of neuronal connectivity.
Project description:Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with advanced virus- and genetic tracings, we have molecularly identified the 3 main types of PNs (Ia, Ib and II) and unexpectedly found that they segregate into 8 subgroups. Our data further reveal a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles, that together contribute to the sensory monitoring of complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later along with increased motor activity and show versatility in the adult following exercise training, suggesting adaptive proprioceptive function.
Project description:The formation of the mammalian neocortex during development requires coordinated establishment of functional regions at proper anterior-posterior and medial-lateral positions – area patterning, as well as neocortical layers. Although key transcription factors (TFs) were known to specify and maintain cell fates, mechanisms underlying how TFs are precise expressed and repressed are largely elusive. Here we showed that NSD1, the methyltransferase for histone H3 lysine 36 dimethylation (H3K36me2), controls both areal and layer identities of the neocortex. Nsd1-ablated neocortex showed prominent areal shift of all four primary functional regions and aberrant wiring of major cortico-thalamic-cortical projections. Nsd1 conditional knockout mice displayed defects in spatial memory, motor learning and coordination, reminiscent of patients with the Sotos syndrome carrying NSD1 mutations. Although projection neurons (PN) of neocortical layers were mostly properly produced and positioned upon Nsd1 deletion, post-mitotic PNs could not establish their layer-specific identities. More strikingly, in adult Nsd1 conditional knockout neocortices, superficial-layer PNs progressively mis-expressed markers for deep-layer PNs. Moreover, neocortical PNs ablated with Nsd1 remained immature morphologically and electrophysiologically. Loss of NSD1 in post-mitotic PNs causes genome-wide loss of H3K36me2 and re-distribution of DNA methylation, which accounts for diminished expression of neocortical layer specifiers but ectopic expression of non-neural genes. Our findings revealed that H3K36me2 mediated by NSD1 is required for establishment and maintenance of region- and layer-specific neocortical identities.
Project description:The formation of the mammalian neocortex during development requires coordinated establishment of functional regions at proper anterior-posterior and medial-lateral positions – area patterning, as well as neocortical layers. Although key transcription factors (TFs) were known to specify and maintain cell fates, mechanisms underlying how TFs are precise expressed and repressed are largely elusive. Here we showed that NSD1, the methyltransferase for histone H3 lysine 36 dimethylation (H3K36me2), controls both areal and layer identities of the neocortex. Nsd1-ablated neocortex showed prominent areal shift of all four primary functional regions and aberrant wiring of major cortico-thalamic-cortical projections. Nsd1 conditional knockout mice displayed defects in spatial memory, motor learning and coordination, reminiscent of patients with the Sotos syndrome carrying NSD1 mutations. Although projection neurons (PN) of neocortical layers were mostly properly produced and positioned upon Nsd1 deletion, post-mitotic PNs could not establish their layer-specific identities. More strikingly, in adult Nsd1 conditional knockout neocortices, superficial-layer PNs progressively mis-expressed markers for deep-layer PNs. Moreover, neocortical PNs ablated with Nsd1 remained immature morphologically and electrophysiologically. Loss of NSD1 in post-mitotic PNs causes genome-wide loss of H3K36me2 and re-distribution of DNA methylation, which accounts for diminished expression of neocortical layer specifiers but ectopic expression of non-neural genes. Our findings revealed that H3K36me2 mediated by NSD1 is required for establishment and maintenance of region- and layer-specific neocortical identities.
Project description:The formation of the mammalian neocortex during development requires coordinated establishment of functional regions at proper anterior-posterior and medial-lateral positions – area patterning, as well as neocortical layers. Although key transcription factors (TFs) were known to specify and maintain cell fates, mechanisms underlying how TFs are precise expressed and repressed are largely elusive. Here we showed that NSD1, the methyltransferase for histone H3 lysine 36 dimethylation (H3K36me2), controls both areal and layer identities of the neocortex. Nsd1-ablated neocortex showed prominent areal shift of all four primary functional regions and aberrant wiring of major cortico-thalamic-cortical projections. Nsd1 conditional knockout mice displayed defects in spatial memory, motor learning and coordination, reminiscent of patients with the Sotos syndrome carrying NSD1 mutations. Although projection neurons (PN) of neocortical layers were mostly properly produced and positioned upon Nsd1 deletion, post-mitotic PNs could not establish their layer-specific identities. More strikingly, in adult Nsd1 conditional knockout neocortices, superficial-layer PNs progressively mis-expressed markers for deep-layer PNs. Moreover, neocortical PNs ablated with Nsd1 remained immature morphologically and electrophysiologically. Loss of NSD1 in post-mitotic PNs causes genome-wide loss of H3K36me2 and re-distribution of DNA methylation, which accounts for diminished expression of neocortical layer specifiers but ectopic expression of non-neural genes. Our findings revealed that H3K36me2 mediated by NSD1 is required for establishment and maintenance of region- and layer-specific neocortical identities.