Developmental Neurobiology

Striatal neurons play a central role in vertebrate action selection; however, their location in larval zebrafish is not well defined. We assayed for conserved striatal markers in the zebrafish subpallium using fluorescent in situ hybridization (FISH) and immunohistochemistry. Whole mount FISH revealed an inhibitory neuronal cluster rostral to the anterior commissure that expresses tac1, a gene encoding substance P. This molecular profile is shared by mammalian striatal direct pathway neurons. A second partially overlapping population of inhibitory neurons was identified that expresses penka, a gene encoding enkephalin. This molecular profile is shared by striatal indirect pathway neurons. Immunostaining for substance P and enkephalin confirmed the presence of these peptides in the subpallium. The tac1 and penka populations were both found to increase linearly across larval stages. Together, these findings support the existence of a striatal homologue in larval zebrafish that grows to match the development and increasing behavioural complexity of the organism.

Construction and maturation of the postsynaptic apparatus are crucial for synapse and dendrite development. The fundamental mechanisms underlying these processes are most often studied in glutamatergic central synapses in vertebrates. Whether the same principles apply to excitatory cholinergic synapses in the insect central nervous system (CNS) is not known. To address this question, we investigated Drosophila ventral lateral neurons (LNvs) and identified nAchR1 (D1) and nAchR6 (D6) as the main functional nicotinic acetylcholine receptor (nAchR) subunits in these cells. With morphological and calcium imaging studies, we demonstrated their distinct roles in supporting dendrite morphogenesis and synaptic transmission. Furthermore, our analyses revealed a transcriptional upregulation of D1 and downregulation of D6 during larval development, indicating a close association between the temporal regulation of nAchR subunits and synapse maturation. Together, our findings show transcriptional regulation of nAchR composition is a core element of developmental and activity-dependent regulation of central cholinergic synapses.

Axon growth is an essential cellular process during neural development, and its dysregulation contributes to numerous neurodevelopmental disorders. During axon growth, extracellular signals direct neurons to extend projections that connect with their synaptic targets. Paxillin is a key member of adhesion sites that control motility by linking the intracellular actin cytoskeleton to the extracellular matrix. Paxillin also binds to the cytoskeletal protein, tubulin. However, little is known about the role of adhesion proteins in neurons. Here, we use conditional paxillin knockout mice to investigate how loss of paxillin in pyramidal cortical neurons affects developing neuron morphology. Surprisingly, loss of paxillin in pyramidal cortical neurons caused no change in axon length or soma area between control (PxnF/F) and conditional paxillin knockout (PxnF/F; Emx1-Cre) mice at basal conditions. Following brain-derived neurotrophic factor stimulation, the loss of paxillin resulted in no change in soma area or axonal {beta}-tubulin levels, but did result in a significant increase in axon length, as compared to control. Finally, the corpus callosum size was not significantly different between PxnF/F and PxnF/F; Emx1-Cre animals. In summary, these data suggest that paxillin is not required for axonal growth during neural development.

Glial cells perform many functions in the nervous system, including maintaining the blood-brain/nerve barriers and structurally supporting axons. While their functions are well-characterized, the complex molecular mechanisms important for their development are less known. Here, we investigated whether microRNA-mediated post-transcriptional regulation is involved during glial development, ensheathment and blood-nerve-barrier formation in Drosophila. In this study, we systematically knocked down 120 different microRNAs by competitive inhibition using microRNA-sponges and analyzed peripheral glial morphology. Knockdown of miRNA-125 in the blood-nerve barrier-forming glia (subperineurial glia) resulted in the most penetrant morphological defects. Since microRNA-125 is co-transcribed with miRNAs-let7 and -100 in a genetic cluster, our further verification for subperineurial glia function included miRNA-125 plus all other members of this cluster. However, the loss of each individual gene and the entire cluster did not lead to any morphological defects in the subperineurial glia. To test the efficiency of the microRNA sponge approach in subperineurial glia, we expressed a sponge targeting a microRNA established to be vital for blood-brain barrier formation (microRNA-285) and found no defects in brain lobes and peripheral nerves. Given that a scrambled-sponge control also generated morphological defects, this suggests that using miRNA sponge lines may not be an effective approach to study miRNA function in Drosophila peripheral glia.

Astrocytes are the major glial population of the brain and have been associated with a vast number of functions. To probe this diversity and to reach a similar level of understanding about astrocyte physiology that we have about neurons, we need genetic tools to target specific astrocytic subpopulations. In Drosophila, we are restricted to using driver lines that drive expression in astrocytes throughout the brain. To target specific astrocytes, we have optimized the genetic tool TRACT (and refer to it as astro-TRACT), allowing effector expression specifically in local astrocytes of a given neuronal circuit. We analyzed specificity, sensitivity and reproducibility of the tool across various MB split-Gal4 drivers. We found that the number of pre-synapses correlates positively with the success of the tool. Applying the tool to characterize morphology of individual astrocytes revealed that local astrocytes around MB medial compartments project into the ellipsoid body. Astro-TRACT will be a valuable resource to investigate both mechanistic astrocyte-neuron signaling and functional and structural astrocytic diversity across the adult Drosophila brain.

AbstractNeural stem cell (NSC) differentiation is controlled by cell-intrinsic and external signals from the stem cell niche including niche surface glia (SG). However, the mechanisms by which transcription factors drive NSC differentiation within the niche remain largely unknown. Here, we show that the Drosophila melanogaster transcription factor, Chromatin-linked adaptor for MSL proteins (CLAMP) is required for regulation of stemness and proliferation of NSCs, especially of the optic lobe (OL). CLAMP promotes transcription of genes involved in stemness, proliferation, and glial development and represses transcription of genes involved in neurogenesis and niche survival. Consistent with transcriptional changes, CLAMP promotes NSC proliferation and niche SG production, while lack of CLAMP severely and specifically impacts OL development. To identify potential mechanisms by which CLAMP may regulate brain development, we examined CLAMP motifs and available CLAMP ChIP-seq data to determine which genes may be direct versus indirect targets. CLAMP motifs are present at many target genes including the glial-determining gene, glial cells missing, while Tailless, the master regulator of OL-development is directly bound by CLAMP. In accordance to these results, in larval OL NSCs lacking CLAMP, Tailless levels are decreased dramatically, suggesting that CLAMP controls OL neurogenesis via Tailless. Overall, our results suggest that CLAMP regulates a transcriptional program which drives NSC proliferation and differentiation via cell-intrinsic and niche-dependent mechanisms that involve transcriptional regulation of Tailless and niche glia.

Glia are irreplaceable components for the nervous system development and function. However, the cellular mechanisms each glial layer utilizes to communicate with each other and the extracellular environment is not well characterized. Here, we investigated the role of a heparan-sulfate proteoglycan, Syndecan (Sdc), in regulating glial cell function and development in the Drosophila nervous system. Sdc is expressed throughout multiple glial layers and loss of Sdc in all glia resulted in disruption of both central and peripheral glia. Within the CNS loss of Sdc in all glia lead to reduced brain lobes and disruption of neuroblast proliferation. In the PNS, loss of Sdc in different glial layers resulted in impaired ensheathment in wrapping glia and abnormal septate junction morphology in subperineurial glia. We focused on the outer layer of perineurial glia and found ensheathment defects and a reduction in glial numbers with Sdc loss. These phenotypes mirror those previously observed with the loss of integrins and a mutation in the integrin {beta}-subunit enhanced the phenotypes observed with loss of Sdc within the perineurial. Thus, our results indicate Sdc has multiple roles in Drosophila nervous system development including as an integral component in regulating glial cell morphology, maintaining neuroblast populations within the optic lobe and in mediating glial-ECM interactions.

Neuronal loss caused by neurodegenerative diseases and traumatic brain injuries (TBI) often results in long-term disabilities, highlighting the urgent need for further research and effective regenerative strategies. In mammals, neurogenic capacity is inherently limited and declines further with age. In contrast, the young adult killifish demonstrates a remarkable ability to regenerate neurons in the telencephalon following TBI. However, it remains unknown whether and when these newly generated neurons functionally integrate into existing circuits, as traditional histological analysis of fixed tissue offers only static insights into this dynamic process. To this end, we optimized a retroviral vector strategy to label dividing stem cells and their progeny, including newborn neurons. By introducing a combination of novel approaches i.e., retroviral vector labeling, electrophysiology and a conditioned place avoidance test, we investigated the generation, morphology, and synaptic integration of newborn neurons following TBI in the dorsomedial (Dm) zone of the telencephalon, a region homologous to the mammalian amygdala in other teleost fish. Our results show that injury-induced adult-born neurons functionally integrate into existing circuits, and that killifish can achieve functional behavioral recovery after TBI. While previous histological assessments using a stab-wound injury suggested a 30-day recovery period, our functional data reveal that full behavioral recovery requires approximately 50 days. At this point, fish successfully relearn to avoid a conditioned place, and the new neurons exhibit mature morpho-electric characteristics, including abundant dendritic spines. Electrophysiological analysis revealed that newborn neurons in an injured environment take longer to mature when compared to neurons in naive killifish. Together, our findings demonstrate that structural regeneration aligns with functional recovery, and establish retroviral vectors as a powerful tool for birth dating injury-induced neurogenesis in teleosts. Killifish thus represent a promising model for studying interventions aimed at enhancing neuronal maturation and integration after brain injury. Key pointsO_LIA retroviral vector strategy allows specific and sparse labeling of adult-born neurons in the killifish brain. C_LIO_LIThe Dm zone in the telencephalon of the killifish is responsible for avoidance learning and memory and thus homologous to the mammalian amygdala. C_LIO_LIIn young adult killifish, upon Dm injury, adult-born neurons mature morphologically and functionally in 50 days, which is slower than in constitutive neurogenesis. C_LIO_LIThe timing and extent of behavioral recovery from such injury aligns with morpho-electric observations. C_LI

A role for axon guidance genes in the adult nervous system has not been fully elucidated. We performed an RNAi screen against guidance genes in the adult Drosophila melanogaster nervous system and identified fourteen genes required for adult survival and normal motility. Additionally, we show that adult expression of Semaphorins and Plexins in motoneurons is necessary for neuronal survival, indicating that guidance genes have critical functions in the mature nervous system.

Late in neural development, the expression of growth/differentiation factor (GDF) 15 increases in the germinal epithelium of the murine ganglionic eminence (GE), especially in progenitors with characteristics of neural stem cells (NSCs). However, the function of GDF15 in this region is still unknown. We here show that apical progenitors in the E18 GE also express the GDF15 receptor and that ablation of GDF15 promotes proliferation and cell cycle progression of apically and subapically dividing progenitors. A similar phenotype was also observed in the adult ventricular subventricular zone (V-SVZ). At both ages, increased proliferation leads to the transient generation of more neuronal progenitors, which is compensated by cell death, and to a permanent increase in the number of ependymal cells and apical NSCs. We also found that GDF15 receptor-expressing cells display immunoreactivity for the epidermal growth factor receptor (EGFR), which is also involved in progenitor proliferation, and that manipulation of GDF15 affects the expression of EGFR in mutant progenitors. Moreover, our data indicate that EGFR signalling in WT and mutant progenitors relies on distinct transduction modes. However, only exposure to exogenous GDF15, but not to EGF, normalized proliferation and the number of apical progenitors, indicating that alteration in EGFR signalling is not the main mechanism by which GDF15 affects proliferation in the embryonic GE. Taken together, GDF15 directly regulates proliferation of apical progenitors in the developing GE, thereby affecting the number of total ependymal cells and NSCs in this region.

Neuropeptides have been reported to regulate progenitor proliferation and neurogenesis in the central nervous system. However, these studies have typically been conducted using pharmacological agents in ex vivo preparations, and in vivo evidence for their developmental function is generally lacking. Recent scRNA-Seq studies have identified multiple neuropeptides and their receptors as being selectively expressed in neurogenic progenitors of the embryonic mouse and human retina. This includes Sstr2, whose ligand somatostatin is transiently expressed by immature retinal ganglion cells. By analyzing retinal explants treated with selective ligands that target these receptors, we found that Sstr2-dependent somatostatin signaling induces a dose-dependent inhibition of photoreceptor generation while increasing the relative fraction of primary progenitor cells. These effects were confirmed by scRNA-Seq analysis of retinal explants and abolished in Sstr2-deficient retinas. Although no changes in the relative fraction of primary progenitors or photoreceptor precursors were observed in Sstr2-deficient retinas in vivo, scRNA-Seq analysis demonstrated accelerated differentiation of neurogenic progenitors. We conclude that Sstr2 signaling may act to negatively regulate retinal neurogenesis in combination with other retinal ganglion cell-derived secreted factors such as Shh, although in vivo Sstr2 is dispensable for normal retinal development.

The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. Here, we demonstrate that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. We utilized the TARGET system to spatiotemporally knockdown Fas3 in selected populations of adult neurons. Our findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. We also demonstrated that Fas3 knockdown activates caspase-3 mediated cell death in olfactory local interneurons, which can be rescued by overexpressing p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a critical role for axon guidance proteins in the maintenance of neuronal circuits in adults. SUMMARY STATEMENTLittle is known about the maintenance of adult neural circuits. We show that the continuous expression of Fasciclin 3, a cell adhesion protein involved in axon guidance, is required for neuronal survival in the adult olfactory circuit.

The spinal dorsal horn comprises heterogeneous neuronal populations, that interconnect with one another to form neural circuits modulating various types of sensory information. Decades of evidence has revealed that transcription factors expressed in each neuronal progenitor subclass play pivotal roles in the cell fate specification of spinal dorsal horn neurons. However, the development of subtypes of these neurons is not fully understood in more detail as yet and warrants the investigation of additional transcription factors. In the present study, we examined the involvement of the POU domain-containing transcription factor Brn3a in the development of spinal dorsal horn neurons. Analyses of Brn3a expression in the developing spinal dorsal horn neurons in mice demonstrated that Brn3a was downregulated in majority of the Brn3a-lineage neurons during embryonic stages (Brn3a-transient neurons), whereas a limited population continued to express Brn3a after E18.5 (Brn3a-persistent neurons). Brn3a knockout disrupted the localization pattern of Brn3a-persistent neurons, indicating a critical role of this transcription factor in the development of these neurons. In contrast, Brn3a overexpression in Brn3a-transient neurons directed their localization in a manner similar to that in Brn3a-persistent neurons. Moreover, Brn3a-overexpressing neurons exhibited increased axonal extension to the ventral and ventrolateral funiculi, where the axonal tracts of Brn3a-persistent neurons reside. These results suggest that Brn3a controls the soma localization and axonal extension patterns of Brn3a-persistent spinal dorsal horn neurons.

The Wnt pathway plays critical roles in neurogenesis. The expression of Axin2 is induced by Wnt/{beta}-catenin signaling, making this gene a sensitive indicator of canonical Wnt activity. We employed pulse-chase genetic lineage tracing with the Axin2-CreERT2 allele to follow the fate of Axin2-positive cells in the adult hippocampal formation. We found Axin2 expressed in astrocytes, neurons and endothelial cells, as well as in the choroid plexus epithelia. Simultaneously with tamoxifen induction of Axin2 fate mapping, the dividing cells were marked with 5-ethynyl-2-deoxyuridine (EdU). Tamoxifen induction resulted in significant increase of dentate gyrus granule cells three months later; however, none of these neurons contained EdU signal. Conversely, six months after the tamoxifen/EdU pulse-chase labeling, EdU-positive granule neurons were identified in each animal. Our data imply that Axin2 is expressed at several different stages of adult granule neuron differentiation and suggest that the process of integration of the adult-born neurons from certain cell lineages may take longer than previously thought.

Clustered protocadherins (cPcdhs) are candidates for the neural circuit formation; however, the localization of cPcdhs in pre- and post-synaptic compartments has not been well characterized. Here we examined the localization of cPcdh{gamma} proteins in the mouse hippocampal CA1 region using light and electron microscopy. From postnatal day 7 to 21, cPcdh{gamma} immunosignals were detected in approximately 40-60% of spines of pyramidal cells. SDS-digested freeze-fracture replica labelling revealed that cPcdh{gamma} immunolabeling was found in 50% of PSD 95-positive postsynaptic profiles but only in less than 10% of vGluT1-positive pre-synaptic terminals. Interestingly, cPcdh{gamma}-positive pre-synaptic terminal was exclusively accompanied by cPcdh{gamma}-positive postsynaptic counterpart. In addition, electrophysiological investigations revealed that the miniature excitatory postsynaptic current frequency in cPcdh{gamma} cKO mice was significantly higher than that in wild-type mice. These results suggest that cPcdh{gamma} proteins are unequally distributed in the pre- and post-synaptic membrane during neural circuit development and regulate the number of excitatory synapses.

Neurod2 and Neurod6 (Neurod2/6) are key transcription factors that promote neuronal differentiation, however, their role in the developing neocortex is not fully understood. Both genes have similar expression patterns during development as well as binding motifs. In order to investigate their role in cortical neurogenesis, we generated a mouse model deficient for both Neurod2/6. Here we demonstrate that differentiation of Tbr2-positive (Tbr2+) basal progenitors (BPs), but not Pax6+ apical progenitors, was severely defected, resulting in ectopically expanded BPs in perinatal Neurod2/6 deficient brains. The sequential fate specification of cortical neurons was also impaired in the absence of Neurod2/6. Ectopic Tbr2+ BPs expressed multiple proliferation markers and were able to self-renew. Olig2+ glial precursors were consequently over-produced in Neurod2/6 deficient brains. Restoration of Neurod2/6 in the double deficient brains downregulated Tbr2 expression, and exhibited substantial rescue effects on defected laminar subtype specification and excessive gliogenesis. Our work indicates that Neurod2/6 regulate BP differentiation and sequential production of cortical cell subtypes via inhibiting Tbr2-dependent genetic program.

The proper formation and function of neural circuits is crucial for cognition, sensation, and behavior. Neural circuits are highly-specific, and this specificity is dependent on neurons developing key features of their individual identities: morphology, anatomical location, molecular expression and biophysiological properties. Previous research has demonstrated that a neurons identity is, in part, generated by the temporal transcription window the neuron is born in, and the homeodomain transcription factors expressed in the mature neuron. However, whether temporal transcription factors and homeodomain transcription factors regulate neural circuit formation, maintenance and function remains unknown. Here, we utilize a well-characterized neural circuit in the Drosophila larvae, the Pair1 neuron. We determined that in the Pair1 neuron, the temporal transcription factor Hunchback activates the homeodomain transcription factor Bicoid (Bcd). Both Hunchback and Bcd are expressed in Pair1 throughout larval development. Interestingly, Hunchback and Bcd were not required in Pair1 for neurotransmitter identity or axonal morphology, but were required for synapse density. We found that these transcription factors were functioning post-mitotically in Pair1 to regulate synapse density. Additionally, knocking down Hunchback and Bcd in Pair1 neurons disrupted the behavioral output of the circuit. We utilized the genetic tool TransTango to determine that Hunchback function in Pair1 is to repress forming synapses with erroneous neurons. To our knowledge, these data are the first to show Hunchback activating Bcd expression, as well as the first to demonstrate a role for Hunchback and Bcd post-mitotically.

Dendritic spines are the principal site of excitatory synapse formation in the human brain. Impaired formation of spines during development has been observed in several autism spectrum disorders (ASDs), including Fragile X syndrome. Fragile X is caused by transcriptional silencing of the Fmr1 gene encoding the RNA-binding protein FMRP (Fragile X mental retardation protein). While spine development has been well characterized in the mammalian CNS, spines are not unique to mammals. Pyramidal neurons (PyrNs) of the zebrafish optic tectum form an apical dendrite containing a dense array of dendritic spines. We employed a genetic labeling system to monitor PyrN dendritic spine development in larval zebrafish. Our findings identify a developmental window when PyrN dendrite growth is concurrent with spine formation. Throughout this period, motile, transient filopodia gradually transform into stable spines containing postsynaptic specializations. fmr1 mutant zebrafish larvae exhibit pronounced defects in both PyrN dendrite growth and the formation of morphologically mature spines. Live imaging of PyrN dendrites suggests these defects are caused by an inability to stabilize nascent contacts. These findings indicate spine stabilization is essential for PyrN dendritic arborization and establish zebrafish larvae as a model system to study spine development in vivo.

Morphology is a defining feature of neuronal identity. Like neurons, glia display diverse morphologies, both across and within glial classes, but are also known to be morphologically plastic. Here, we explored the relationship between glial morphology and transcriptional signature using the Drosophila central nervous system, where glia are categorized into five main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes), which show within-class morphological diversity. We analysed and validated single cell RNA sequencing data of Drosophila glia in two well-characterized tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the ventral nerve cord. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While we did detect extensive within-class transcriptomic diversity for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, we generated a single-cell transcriptomic atlas of glia in Drosophila, and our extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. This atlas will serve as a resource for the community to probe glial diversity and function.

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.