Developmental Neurobiology

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.

In adult superficial dorsal horn, 90% of Reelin (Reln+) and 70% of Disabled-1 (Dab1+) neurons co-express the transcription factor LIM-homeobox 1-beta (Lmx1b+) and therefore are glutamatergic neurons. Here we asked if embryonic Reln+Lmx1b+ and Dab1+Lmx1b+ dorsal horn neurons are derived from Lmx1b-expressing early-born dI5 or late-born dILB dorsal neurons. On Embryonic day (E)11.5, Reln+ and Dab1+ neurons appear to be part of the migration of early-born dI5 Lmx1b-expressing neurons. Between E12.5-E15.5, the lateral Reln+Lmx1b+ and Dab1+Lmx1b+ neurons migrate circumferentially along the rim of what will become the superficial dorsal horn, whereas medial Reln+Lmx1b+ and Dab1+Lmx1b+ neurons move into the dorsal midline and then migrate into lamina V. The small, late-born dILB Reln+Lmx1b+ and Dab1-Lmx1b+ neurons fill the superficial dorsal horn. In Reln mutants, large Dab1+Lmx1b+ neurons were mispositioned in lamina I and at the border between the superficial and deep dorsal horn. To confirm the identity of the circumferential and midline Reln+Lmx1b+ and Dab1+Lmx1b+ neurons, we asked if they expressed the transcription factor Zfhx3, a marker of dI5 projection neurons. We detected examples of Reln+Lmx1b+Zfhx3+ and Dab1+Lmx1b+Zfhx3+ projection neurons that migrated along the outer rim of the superficial dorsal horn and others that migrated from the midline into lamina V. Taken together, our study demonstrates that the larger Reln+Lmx1b+Zfhx3+ and Dab+Lmx1b+Zfhx3+ neurons represent two subsets of dI5 projections neurons, whereas smaller Reln+Lmx1b+ and Dab1+Lmx1b+ neurons concentrated in lamina II are likely dILB interneurons.

AO_SCPLOWBSTRACTC_SCPLOWThe laminins are a family of extracellular matrix proteins that regulate numerous cellular processes, including adhesion, neurite outgrowth, and axon guidance. However, it remains unclear whether laminin regulates axon guidance through local translation. Here, we show that laminin is necessary for local translation in axonal growth cones. Local translation is significantly increased in growth cones of embryonic day 17 mouse cortical neurons, either cultured on or acutely stimulated with soluble laminin 111, in the presence of BDNF. When cultured on laminin isoforms 211 or 221 in the presence of BDNF, there was a remarkable decrease in local translation in growth cones. Using a puromycin-proximity ligation assay to examine newly synthesized {beta}-actin specifically, we find a significant increase in growth cones of neurons cultured on laminin 111 in the presence of BDNF. However, soluble laminin 111 alone results in a significant reduction in nascent {beta}-actin protein synthesis. These results indicate that laminin isoforms can act in multiple ways, including synergistically with guidance cues and independently, to modulate local mRNA translation, thereby differentially influencing axon growth and guidance during development. SO_SCPLOWUMMARYC_SCPLOW SO_SCPLOWTATEMENTC_SCPLOWLocal translation in axons is critical for axon guidance. Laminin, a key component of the extracellular matrix, is necessary to induce local translation and thus mediate axon growth and guidance.

Voltage-gated sodium channels (VGSCs) are conventionally described as heterotrimers composed of one alpha and two beta subunits. However, the patterns of co-expression of alpha- and beta-subunits in neurons remain unclear. In the present study, we report that alpha- (Nav1.1, Nav1.2, and Nav1.6) and beta- (beta-1 and beta-2) subunits are densely expressed in axon initial segments (AISs) of neurons in the neocortex, hippocampus and cerebellum at postnatal days 14-15 (P14-15) and 8-9 weeks (8-9W). These distributions are largely unique and partially overlapping among brain regions. Notably, in the neocortex and hippocampus, AISs of presumptive parvalbumin-positive inhibitory neurons are positive for Nav1.1 and beta-1, whereas those of excitatory ones are positive for Nav1.2 and beta-2. Similarly, AISs of cerebellar basket cells, which are inhibitory neurons, are positive for Nav1.1 and beta-1, whereas those of granule cells, which are excitatory neurons, are positive for Nav1.2 and beta-2. Nav1.6 is expressed in many of these neurons. Some subunits exhibited distinct distribution patterns at the two postnatal stages analyzed, possibly because of their developmental changes of subcellular localizations. Taken together, these results indicate that combinations of VGSC subunits are largely unique among different neuronal subpopulations. These findings provide a useful reference for understanding the distribution and interactions of VGSC subunits in the brain.

Neurophysiologists have discovered many mechanisms underlying the production of animal behaviors in specific species; these involve a collection of neuromuscular systems, neuronal membrane and neural network properties, as well as the hormones and neuromodulators known to modify them. However, the mechanistic basis of behavioral evolution is less well-studied, and causal links between differences in gene expression, cellular mechanisms and species-typical behaviors are rare. Vertebrate vocal behaviors are an excellent system for studying the evolution of behaviors because they are ancient, diverse and readily quantifiable. Xenopus frogs are particularly well-suited to the study of vocal evolution due to the temporal diversity of male advertisement calls between closely related species and the well-described vocal pattern generating circuitry. Here we focus on two species, X. laevis and X. petersii, that diverged 8.5 million years ago and produce advertisement calls with distinct timing. To begin bridging the gap between behavioral and mechanistic diversity in Xenopus vocal behaviors, we performed RNA sequencing of the parabrachial nucleus, a vocal premotor hindbrain area known to encode species-typical temporal patterns in X. laevis and X. petersii. We identified hundreds of differentially expressed genes between the two species, including many genes related to hormone signaling, neuromodulation, neuronal and synaptic functions, ion channels and neurotransmitter receptors. We explore several testable hypotheses emerging from these results that may explain mechanisms by which candidate genes and gene families may contribute to vocal pattern differences between X. laevis and X. petersii.

Adult hydrozoan cnidarians undergo extensive tissue turnover, generating neural cell types including nematocytes (stinging cells) and gland cells from interstitial stem cells (i-cells) expressing stemness proteins such as Piwi and Nanos. The contribution of i-cells during embryogenesis, however, has been unclear. Here we address the origin of neural cells during development of the Clytia hemisphaerica planula larva. Marker gene in situ hybridisation revealed that Piwi/Nanos1-expressing cells within the early gastrula presumptive endoderm generate a substantial pool of nematoblasts, a few of which migrate and differentiate in the planula ectoderm. Some neurogenic and neuronal markers, however, showed a markedly distinct expression profile, developing within a basal layer of the aboral/lateral ectoderm during gastrulation. Embryo bisection and lineage tracing experiments confirmed that sensory neurons and secretory cell types derive from gastrula ectoderm, while nematocytes and at least some ganglionic neurons derive from i-cells. Knockdown and inhibitor treatments revealed steps in neuron and nematocyte development regulated by Wnt-{beta}-catenin. We conclude that two distinct neurogenesis pathways operate during Clytia embryogenesis, one involving aboral ectoderm delamination, and one generating mainly nematocytes from i-cell-like precursors. Summary statementDuring embryogenesis in the hydrozoan Clytia neural cell types derive both from Piwi/Nanos expressing "i-cells" and from ectodermal delamination during gastrulation.

Animals produce different vocalization types, which differ in their acoustic features and are produced in different behavioral contexts. How vocalization-related brain circuits are organized to enable the production of different vocalization types remains poorly understood. The nucleus retroambiguus is a hindbrain premotor region that regulates the production of both ultrasonic vocalizations (USVs) and distress calls (squeaks) in adult mice, but whether distinct or overlapping populations of RAm neurons are recruited during the production of these two vocalization types is unknown. In the current study, we used Fos immunohistochemistry to compare the counts and spatial distributions of Fos-positive RAm neurons in males and females that produced USVs and females that produced courtship squeaks. We also combined in vivo activity-dependent (TRAP2) labeling with Fos immunohistochemistry to directly compare Fos expression associated with the production of USVs and courtship squeaks in the same females. Our findings suggest that RAm contains three vocalization-related populations of neurons: squeak-related neurons, USV-related neurons, and shared neurons that are recruited during both vocalization types. These findings refine current models of the premotor control of vocalization and set the stage for future work to explore anatomical and functional heterogeneity within RAm.

The Lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) superfamily (LU super family) of proteins are involved in diverse biological processes. In Drosophila melanogaster, members of the LU superfamily have undergone lineage-specific gene duplication and acquired specialized functions in distinct tissues. A glycosylphosphatidylinositol (GPI)-anchored LU family protein Belly roll (Bero) has recently been shown to regulate larval escape behavior; however, its cellular expression profile and potential roles remain incompletely understood. In this study, we generated a bero-GAL4T2A transgenic line to delineate endogenous bero expression. This analysis revealed that bero is expressed in the peptidergic neurons in the central nervous system (CNS) that had not been documented in previous studies, as well as in the peripheral nervous system (PNS) and non-neuronal tissues, such as the anal pad and epidermis. Reanalysis of publicly available single-cell RNA sequencing (scRNA-seq) datasets demonstrated that bero is expressed in several peptidergic neurons. These findings suggest that Bero is specifically expressed in diverse peptidergic neurons and may play important roles in coordinating hormonal and neural regulation in D. melanogaster.

The functional organization of the teleost telencephalic pallium remains poorly understood, particularly regarding the presence of modality-specific sensory domains and their topographic arrangement. Here, we used in vivo wide-field voltage-sensitive dye imaging to map sensory-evoked neural activity across the dorsal surface of the telencephalic pallium of adult goldfish. Somatosensory, auditory, gustatory, and visual stimulation revealed distinct, modality-specific domains primarily located within the dorsomedial (Dm) and dorsolateral (Dl) pallium. Within Dm, somatosensory and auditory stimuli activated partially overlapping territories in the caudal subregion (Dm4), exhibiting clear somatotopic and tonotopic organization along the mediolateral axis. Gustatory stimulation selectively engaged Dm3, where different tastants activated spatially distinct but partially overlapping domains. A more rostral subregion (Dm2) responded only to high-intensity somatosensory stimulation, suggesting involvement in processing negatively valenced inputs. Visual stimulation activated a circumscribed area within the dorsolateral pallium (Dld2),that closely matched cytoarchitectural boundaries. Pharmacological blockade of ionotropic glutamate receptors markedly reduced sensory-evoked responses, indicating that these maps depend on glutamatergic synaptic transmission. Together, these findings show that the goldfish pallium contains distinct, spatially organized sensory representations and a refined internal functional architecture. This organization suggests that pallial topographic sensory maps may not be exclusive to mammals and birds. Based on these results, we propose that dorsomedial and dorsolateral pallial regions may be functionally comparable to components of the mammalian mesocortical network, more than to the pallial amygdala or the neocortex. This framework provides a new perspective on pallial organization in teleosts and contributes to understanding the evolutionary origins of the vertebrate pallium. HIGHLIGHTSO_LIVoltage-sensitive dye imaging was used to map sensory responses in the goldfish pallium. C_LIO_LIDistinct sensory areas for somatosensory, auditory, gustatory, and visual modalities were identified. C_LIO_LISome sensory regions in Dm show topographically organized maps. C_LIO_LIFunctional segregation suggests a complex, non-diffuse pallial organization. C_LIO_LIFindings support a novel hypothesis linking Dm and Dld to mammalian mesocortical regions. C_LI

BackgroundThe trigeminal ganglion (TG) is a structure of the peripheral nervous system, composed of neuronal and non-neuronal cell types, that integrates sensory input from the face and jaw. The developing TG is derived from two embryonic cell populations: neural crest and cranial placode. Both populations play critical roles in TG development and must interact to coordinate changes in gene expression that regulate specification, differentiation, and organization. However, the molecular characteristics of the heterogeneous cell populations within the developing TG remain poorly defined. ResultsWe performed single-cell RNA-sequencing (scRNA-seq) on TG from developing chick embryos at HH17. Our high-resolution dataset (14 clusters, [~]87000 cells) provides insight into cellular diversity within the developing TG. As expected, we identified placode-derived neurons as well as neural crest cells prior to neuronal differentiation. In addition to classic markers, we identified novel transcripts with unknown roles in TG development, including several long non-coding RNAs (lncRNAs). ConclusionsWe generated a single-cell atlas of the developing chick trigeminal ganglion during early axonogenesis and defined the transcriptomic states of its diverse cell populations. Our results provide a useful resource for better understanding the cell populations contributing to TG development and gene expression that drives cell identity and differentiation.

BackgroundChoroid plexus (ChP) produces cerebrospinal fluid (CSF), and regulates brain development and adult subventricular zone (SVZ) neurogenesis, but its role in hippocampal subgranular zone (SGZ) neurogenesis in adulthood and early postnatal stages is not well understood. Current tools to directly manipulate neonatal ChP/CSF volume are very limited, representing an urgent need in the field. MethodsWe first discovered the specific "leaky" expression of DTR gene in the ChP of adult ROSA26-iDTR mice which can be used to specifically ablate ChP in adult brain that generated robust and long-lasting ablation of ChP and reduction of CSF volume. In this study, we the effectiveness of ROSA26-iDTR allele in ablating neonatal ChP. We also developed a novel AAV2/5-CMV-DTR vector with validated ChP tropism in both neonatal and adult mice, which induces substantial CSF loss in both neonates and adult mice. With both the ROSA26-iDTR genetic and AAV2/5-DTR viral-mediated ChP ablation in young adults and at defined postnatal ages, we quantified ventricular CSF volume by MRI and characterized postnatal neurogenesis. Doublecortin-positive (DCX+) neuroblasts, Ki67+ proliferating cells, and TUNEL+ apoptotic cells were quantified in SVZ and SGZ using confocal microscopy and machine learning-assisted cell counting. ResultsWe show that ROSA26-iDTR-mediated ChP ablation is inefficient before postnatal day 10, suggesting that this line may be of limited utility for CSF reduction in the early neonatal period before P10. P3-5 Dtx treatment of a previously used dosage of 20ng/g dosage did not lead to a reduction in CSF volume. Higher dosage of 40ng/gX3 Dtx dosage at p3-5 generated only moderate partial reduction of CSF in third ventricle and total CSF volume, with indication of toxicity associated with high Dtx dosage in general. In contrast, p10-12 injection of 20ng/gX3 Dtx led to robust CSF reduction. To target early neonatal days, AAV2/5 CMV-DTR virus shows high tropism for ChP epithelial cells and leads to near-complete ablation of CSF in neonatal brains. ChP/CSF loss in neonates or young adult mice leads to a substantial reduction of DCX+ cells at the SVZ but a moderate but significant reduction of SGZ DCX+ neuroblasts, without changes in Ki67+ or TUNEL+ cells. ConclusionsThis study reports a novel role of the ChP/CSF in maintaining the neuroblast pool in the neurogenic niches in both early postnatal and adult stages. Moreover, we expand the available tools to target the ChP and CSF production in the neonate, with potential uses in treating conditions such as neonatal hydrocephalus.

Receptor internalization regulates the duration and qualitative output of intracellular signaling. Although generic mechanisms of receptor internalization are well characterized, how these are deployed and regulated in different cell types remains much less understood. Here we show that the p75 neurotrophin receptor (p75NTR), a key regulator of neuron survival and function, internalizes in hippocampal (HCNs)and cerebellar granule (CGNs)neurons with very different kinetics, regulated by distinct mechanisms. Compared to HCNs, p75NTR internalizes at a much slower rate and shows stronger interaction with caveolin in CGNs. In both cell types, p75NTR internalization was enhanced by nerve growth factor (NGF)but reduced by inhibitors of the RhoA and PKC pathways. In line with this, internalization of a p75NTR mutant specifically impaired in RhoA signaling was significantly reduced and insensitive to NGF in both neuron types. Accordingly, this mutant showed a much stronger interaction with caveolin than wild type p75NTR. On the other hand, internalization of a mutant specifically impaired in coupling to the NF-kB signaling pathway was greatly accelerated in CGNs but unaffected in HCNs. These results reveal the crucial role of intracellular signaling in p75NTR internalization and demonstrate that the receptor is differentially wired to the endocytosis machinery in different neuron types, leading to distinct internalization behaviors.

In mammals, a dysregulated immune response is detrimental to spinal cord repair. In zebrafish, which are capable of spinal cord regeneration, the immune response promotes regeneration. Neutrophils are the first immune cells to arrive at a spinal cord injury site, but their role in successful regeneration is not fully understood. Here we show that ablating neutrophils, including a subpopulation that expresses the cytokine il4, increases expression of il1b (coding for Il-1{beta}) in macrophages/microglia and impairs anatomical and functional recovery after a spinal cord injury in larval zebrafish. Regeneration is fully rescued by over-expression of il4 alone or experimentally reducing Il-1{beta} levels. Disruption of il4 mimics the detrimental effect of neutrophil ablation for axonal regeneration and is also rescued by reducing Il-1{beta} levels. Hence, after spinal cord injury, a pro-regenerative neutrophil subpopulation promotes spinal cord regeneration in larval zebrafish by controlling expression of il1b in macrophages/microglia. For this neutrophil action, il4 expression is necessary and sufficient. HIGHLIGHTS- Neutrophil ablation impairs spinal cord repair in zebrafish - The neutrophil response can be replaced by reducing Il-1{beta} levels - A pro-regenerative subpopulation of neutrophils expresses il4 - il4 overexpression fully rescues effects of neutrophil ablation

Nervous systems display extensive diversity in structure and organization, yet a broadly conserved set of signaling pathway components and transcription factors is consistently associated with early neurogenesis in many animal lineages. Determining how these conserved markers map onto the spatiotemporal organization of neurogenic territories across phylogenetically informative but underrepresented lineages, particularly within Spiralia, is critical for refining inferences about the evolutionary origins and diversification of nervous systems. Chaetognaths, a spiralian lineage frequently recovered close to Gnathifera, have a compact and centralized nervous system but lack detailed molecular descriptions of early neural development. Here, we generate an expression-based developmental map of early neurogenesis in the chaetognath Spadella cephaloptera by combining nuclear-staining-based anatomical staging with spatiotemporal analyses of conserved developmental genes associated with early neurogenesis and axial patterning from gastrulation through early post-embryonic stages. Sce-soxB1-like1 and Sce-neuroD expressions mark a lateral neuroectodermal territory during gastrulation. Notably, Sce-neuroD is activated early in a broad ectodermal domain and is expressed within mitotically active neuroectodermal cells, consistent with early deployment in proliferative neurogenic territories. Sce-soxB1 and Sce-soxB2 show delayed and more spatially restricted expression relative to Sce-soxB1-like1, suggesting a paralog-specific partitioning of SoxB deployment during chaetognath neurogenesis. Sce-bmp2/4 and Sce-chd exhibit reciprocal dorsoventral expression during gastrulation that coincides with early neurogenic territory formation, before transitioning to more localized expression later in development. Sce-nk6 and Sce-hb9 reveal early ventral regionalization of the developing ventral nerve center (VNC), with Sce-hb9 occupying a subset of a broader Sce-nk6 domain, in line with conserved ventral subtype-associated regionalization. Sce-th (tyrosine hydroxylase) is detected in a small bilateral subset of hatchling VNC cells, while Sce-dbh (dopamine beta-hydroxylase) is first detected only in early juveniles in the anterior VNC and head domains, suggesting stage-dependent and region-specific deployment of catecholamine-pathway components. Together, these expression-based datasets provide a comparative reference for early neurogenesis in chaetognaths and a framework for assessing conserved and lineage-specific features of early neurogenic patterning across Spiralia.

Calcium functions as an important second messenger in a wide variety of intracellular processes. In photoreceptor cells, calcium is involved in activation, deactivation, and adaptation in response to light stimuli. Calcium-binding protein 53E (Cbp53E, also known as calbindin-32 or cbn), a protein with 6 EF-Hand domains thought to act as a calcium buffer, was previously identified to have elevated expression levels in the eye of drosophila. While a recent study showed that transgenic flies lacking Cbp53E have aberrant axonal arborization at the neuromuscular junction, nothing is known about the role of Cbp53E in the visual system. We performed electroretinogram (ERG) recordings on Cbp53E mutant flies to test whether eye function was affected. Here, we report that Cbp53E null mutants exhibit a prolonged repolarization (or slow termination) phenotype which can be rescued by expressing Cbp53E in photoreceptor cells. The human homologs Calbindin 2, Calbindin 1, and S100G also rescue the Drosophila ERG phenotype. This supports a role for Cbp53E in regulating intracellular calcium levels of photoreceptor cells and contributing to normal sensory neuron response dynamics in vivo in Drosophila and suggests a similar function in human photoreceptor cells as well.

Amacrine cells (ACs) are retinal interneurons that regulate synaptic transmission from bipolar cells to retinal ganglion cells (RGCs) and play essential roles in object motion detection, contrast sensitivity, and light adaptation. A subtype of GABAergic ACs identified using a tyrosine hydroxylase (TH) promoter-driven green fluorescent protein (GFP) mouse line has been termed TH2 amacrine cells (TH2-ACs). Although TH2-ACs contribute to the feature selectivity of object-motion signals in the adult retina, their functional properties during early postnatal development remain unclear. Using genetic mouse models, electrophysiology, immunohistochemistry, and calcium imaging, we show that TH2-ACs exhibit spontaneous rhythmic depolarizations during development. In the first postnatal week, these depolarizations were abolished by acetylcholine receptor antagonists, indicating that TH2-ACs are excited by starburst amacrine cells (SACs) via spontaneous cholinergic retinal waves. During the second postnatal week, rhythmic depolarizations persisted but were blocked by glutamate receptor antagonists, demonstrating that TH2-ACs are subsequently driven by bipolar cells through glutamatergic waves. Calcium imaging further revealed that this activity propagates across the TH2-AC network in a wave-like manner, potentially resulting in spatially and temporally patterned GABA release. Pharmacological blockades of GABAA receptors significantly enhanced glutamatergic wave activity in SACs and RGCs, indicating that GABAergic signaling from TH2-ACs participates in exerting inhibitory control over retinal waves. Together, these findings identify TH2-ACs as active participants in the development of retinal wave circuits and suggest that this participation via GABA signaling could contribute to activity-dependent refinement of retinal circuits underlying object motion processing. Key pointsO_LITH2 amacrine cells are excited by starburst amacrine cells through cholinergic retinal wave activity during the first postnatal week. C_LIO_LIDuring the second postnatal week, TH2 amacrine cells are driven by bipolar cells via glutamatergic retinal wave activity. C_LIO_LIThe dense dendritic arborization of TH2 amacrine cells enables their participation in the propagation of both cholinergic and glutamatergic waves. C_LIO_LIWave-like GABA release from TH2 amacrine cells contributes to the modulation of retinal wave activity through activation of GABAA receptors. C_LI

Strabismus, or misalignment of the eyes, is a heritable disorder frequently associated with vision loss and decreased quality of life. Incomitant strabismus, where the degree of misalignment differs based on gaze angle, can arise from mutations in genes that regulate the development of extraocular motor neurons. To date, few such genes have been identified. The extraocular motor system is highly conserved across vertebrates, suggesting a comparative transcriptomic discovery approach would be fruitful. Using bulk and single-cell sequencing in a small accessible vertebrate, the larval zebrafish, we identified genes expressed in subpopulations of extraocular motor neurons in cranial nuclei nIII/nIV. We next assessed extraocular motor neuron number and vestibulo-ocular reflex performance after CRISPR/Cas9-mediated mutagenesis of three genes with suggestive expression patterns: sim1a, nav2a, one-cut1, and one known to disrupt nIII/nIV motor neuron specification: phox2a. Loss of sim1a impaired the vestibulo-ocular reflex without change to nIII/nIV motor neuron number. Our data suggest that constitutive disruptions to sim1a can impair nIII/nIV-dependent eye movements. More broadly, our work illuminates considerable transcriptomic diversity among extraocular motor neuron subpopulations, and establishes a pipeline to identify genes relevant to ocular motor disease etiology.

The signaling pathways that control embryonic development, migration, and differentiation of gonadotropin-releasing hormone (GnRH) neurons, as well as the postnatal fate, function, and survival of differentiated cells, are the subject of ongoing research. Here, we examined the role of phosphoinositides in this complex multistep process by generating GnRH neuron-specific phosphatidylinositol 4-kinase alpha knockout mice. These mice were healthy and indistinguishable from their control littermates in size. However, adult knockout females and males were infertile due to underdeveloped gonads and reproductive organs. Furthermore, hypothalamic GnRH immunoreactivity was absent, and expression of the hypothalamic Gnrh1 gene and pituitary gonadotroph-specific genes was reduced. In contrast, hypothalamic kisspeptin immunoreactivity was preserved, and Kiss1 expression was modified in a nuclei specific-manner, consistent with the loss of circulating sex steroid hormones. Embryonic neurogenesis and migration of GnRH neurons were not impaired, as evidenced by normal Gnrh1 expression in the hypothalamus of neonatal animals and the presence of immunoreactive GnRH neurons in infantile mice in comparable number and distribution to age-matched controls. However, their cellular degeneration was evident, accompanied by reduced Gnrh1 expression. GnRH neuron-specific tdTomato expression confirmed their postnatal degeneration and death, whereas ectopic tdTomato cells located in the lateral septum remained unaffected. Together, these findings indicate that phosphoinositides dependent on phosphatidylinositol 4-kinase alpha activity are not critical for embryonic steps in the development of the GnRH neuronal network, but are essential for the postnatal function and survival of these cells. Significance StatementDifferentiation of neuroendocrine GnRH cells involves neurogenesis in the olfactory placodes, migration to the hypothalamus, projection to the median eminence, and connections with upstream neurons, including kisspeptin neurons. Here we show that knockout of phosphatidylinositol 4-kinase alpha in GnRH neurons does not affect these strps of embryonic development. However, the activity of this enzyme is essential for postnatal survival of GnRH neurons; in the absence of this gene, the neurons die, causing infertility in both female and male mice.

Mutations in human LIS1 cause lissencephaly, a severe developmental brain malformation. Although most stud-ies focus on development, LIS1 is also expressed in adult mouse tissues. We previously induced LIS1 knockout (iKO) in adult mice using a Cre-Lox approach with an actin promoter driving CreERT2 expression. This proved to be rapidly lethal, with evidence pointing toward nervous system dysfunction. CreERT2 activity was observed in astrocytes, brainstem and spinal motor neurons, and axons and Schwann cells in the sciatic and phrenic nerves, suggesting dysfunctional cardiorespiratory and motor circuits. However, it is unclear how LIS1 knockout in these different cell types contributes to the lethal phenotype. We now report that LIS1 depletion from astro-cytes is not lethal to mice (male or female), although glial fibrillary protein (GFAP) expression is increased in all LIS1-depleted astrocytes. In contrast, LIS1 depletion from projection neurons causes motor deficits and rapid lethality in both males and females. This is accompanied by progressive, widespread axonal degeneration along the entire length of both motor and sensory axons. Interestingly, sensory neurons harvested from iKO mice ini-tially extend axons in culture but soon develop axonal swellings and fragmentation, indicating axonal degenera-tion. LIS1 is a prominent regulator of cytoplasmic dynein 1 (dynein, hereafter), a microtubule motor whose dis-ruption can cause both cortical malformations and later-onset neurodegenerative diseases, such as Charcot-Marie-Tooth disease. Our results raise the possibility that LIS1 depletion, through disruption of dynein function in mature axons, may lead to Wallerian-like axon degeneration without traumatic nerve injury.

Unraveling how adult neurons reshape their architecture is key to understanding post-developmental plasticity. Drosophila clock neurons, which remodel their terminals on a daily basis, offer a unique model to examine the mechanisms underlying structural plasticity. In this study, we examine the impact of the experimental design on the remodeling process. We established a simple fixation protocol that preserves tissue integrity and prevents its deformation while enabling the fixation of a larger number of individuals within the appropriate time window. We show that intrinsic (i.e., targeting fluorescent reporters to the membrane) or extrinsic (i.e., temperature) variables may influence this dynamic process. Examining ex vivo preparations, we found that the s-LNv terminals display numerous thin filopodia extending from their synaptic boutons. However, these fine membrane protrusions are lost upon fixation, as they could only be accurately visualized ex vivo. Finally, we present MorphoScope, a Python-based interface that eliminates observer bias in complexity measurements. Altogether, we present a powerful and robust model to investigate the principles of adult neuronal plasticity, with implications extending beyond circadian biology.