Misregulation of cell adhesion molecules in the Ciona neural tube closure mutant bugeye.

Neural tube closure (NTC) is a complex multi-step morphogenetic process that transforms the flat neural plate found on the surface of the post-gastrulation embryo into the hollow and subsurface central nervous system (CNS). Errors in this process underlie some of the most prevalent human birth defects, and occur in about 1 out of every 1000 births. Previously, we discovered a mutant in the basal chordate Ciona savignyi (named bugeye) that revealed a novel role for a T-Type Calcium Channel (Cav3) in this process. Moreover, the requirement for CAV3s in Xenopus NTC suggests a conserved function among the chordates. Loss of CAV3 leads to defects restricted to anterior NTC, with the brain apparently fully developed, but protruding from the head. Here we report first on a new Cav3 mutant in the related species C. robusta. RNAseq analysis of both C. robusta and C. savignyi bugeye mutants reveals misregulation of a number of transcripts including ones that are involved in cell-cell recognition and adhesion. Two in particular, Selectin and Fibronectin leucine-rich repeat transmembrane, which are aberrantly upregulated in the mutant, are expressed in the closing neural tube, and when disrupted by CRISPR gene editing lead to the open brain phenotype displayed in bugeye mutants. We speculate that these molecules play a transient role in tissue separation and adhesion during NTC and failure to downregulate them leads to an open neural tube.

Photoreceptor specialization and the visuomotor repertoire of the primitive chordate Ciona.

The swimming tadpole larva of Ciona has one of the simplest central nervous systems (CNSs) known, with only 177 neurons. Despite its simplicity, the Ciona CNS has a common structure with the CNS of its close chordate relatives, the vertebrates. The recent completion of a larval Ciona CNS connectome creates enormous potential for detailed understanding of chordate CNS function, yet our understanding of Ciona larval behavior is incomplete. We show here that Ciona larvae have a surprisingly rich and dynamic set of visual responses, including a looming-object escape behavior characterized by erratic circular swims, as well as negative phototaxis characterized by sustained directional swims. Making use of mutant lines, we show that these two behaviors are mediated by distinct groups of photoreceptors. The Ciona connectome predicts that these two behavioral responses should act through distinct, but overlapping, visuomotor pathways, and that the escape behavior is likely to be integrated into a broader startle behavior.

ACAM, a novel member of the neural IgCAM family, mediates anterior neural tube closure in a primitive chordate.

The neural IgCAM family of cell adhesion molecules, which includes NCAM and related molecules, has evolved via gene duplication and alternative splicing to allow for a wide range of isoforms with distinct functions and homophilic binding properties. A search for neural IgCAMs in ascidians (Ciona intestinalis, Ciona savignyi, and Phallusia mammillata) has identified a novel set of truncated family members that, unlike the known members, lack fibronectin III domains and consist of only repeated Ig domains. Within the tunicates this form appears to be unique to the ascidians, and it was designated ACAM, for Ascidian Cell Adhesion Molecule. In C. intestinalis ACAM is expressed in the developing neural plate and neural tube, with strongest expression in the anterior sensory vesicle precursor. Unlike the two other conventional neural IgCAMs in C. intestinalis, which are expressed maternally and throughout the morula and blastula stages, ACAM expression initiates at the gastrula stage. Moreover, C. intestinalis ACAM is a target of the homeodomain transcription factor OTX, which plays an essential role in the development of the anterior central nervous system. Morpholino (MO) knockdown shows that ACAM is required for neural tube closure. In MO-injected embryos neural tube closure was normal caudally, but the anterior neuropore remained open. A similar phenotype was seen with overexpression of a secreted version of ACAM. The presence of ACAM in ascidians highlights the diversity of this gene family in morphogenesis and neurodevelopment.

CellECT: cell evolution capturing tool.

BACKGROUND: Robust methods for the segmentation and analysis of cells in 3D time sequences (3D+t) are critical for quantitative cell biology. While many automated methods for segmentation perform very well, few generalize reliably to diverse datasets. Such automated methods could significantly benefit from at least minimal user guidance. Identification and correction of segmentation errors in time-series data is of prime importance for proper validation of the subsequent analysis. The primary contribution of this work is a novel method for interactive segmentation and analysis of microscopy data, which learns from and guides user interactions to improve overall segmentation. RESULTS: We introduce an interactive cell analysis application, called CellECT, for 3D+t microscopy datasets. The core segmentation tool is watershed-based and allows the user to add, remove or modify existing segments by means of manipulating guidance markers. A confidence metric learns from the user interaction and highlights regions of uncertainty in the segmentation for the user's attention. User corrected segmentations are then propagated to neighboring time points. The analysis tool computes local and global statistics for various cell measurements over the time sequence. Detailed results on two large datasets containing membrane and nuclei data are presented: a 3D+t confocal microscopy dataset of the ascidian Phallusia mammillata consisting of 18 time points, and a 3D+t single plane illumination microscopy (SPIM) dataset consisting of 192 time points. Additionally, CellECT was used to segment a large population of jigsaw-puzzle shaped epidermal cells from Arabidopsis thaliana leaves. The cell coordinates obtained using CellECT are compared to those of manually segmented cells. CONCLUSIONS: CellECT provides tools for convenient segmentation and analysis of 3D+t membrane datasets by incorporating human interaction into automated algorithms. Users can modify segmentation results through the help of guidance markers, and an adaptive confidence metric highlights problematic regions. Segmentations can be propagated to multiple time points, and once a segmentation is available for a time sequence cells can be analyzed to observe trends. The segmentation and analysis tools presented here generalize well to membrane or cell wall volumetric time series datasets.

A transiently expressed connexin is essential for anterior neural plate development in Ciona intestinalis.

A forward genetic screen in the ascidian Ciona intestinalis identified a mutant line (frimousse) with a profound disruption in neural plate development. In embryos with the frimousse mutation, the anteriormost neural plate cells, which are products of an FGF induction at the blastula and gastrula stages, initially express neural plate-specific genes but fail to maintain the induced state and ultimately default to epidermis. The genetic lesion in the frimousse mutant lies within a connexin gene (cx-11) that is transiently expressed in the developing neural plate in a temporal window corresponding to the period of a-lineage neural induction. Using a genetically encoded calcium indicator we observed multiple calcium transients throughout the developing neural plate in wild-type embryos, but not in mutant embryos. A series of treatments at the gastrula and neurula stages that block the calcium transients, including gap junction inhibition and calcium depletion, were also found to disrupt the development of the anterior neural plate in a similar way to the frimousse mutation. The requirement for cx-11 for anterior neural fate points to a crucial role for intercellular communication via gap junctions, probably through mediation of Ca(2+) transients, in Ciona intestinalis neural induction.

A one-dimensional model of PCP signaling: polarized cell behavior in the notochord of the ascidian Ciona.

Despite its importance in development and physiology the planar cell polarity (PCP) pathway remains one of the most enigmatic signaling mechanisms. The notochord of the ascidian Ciona provides a unique model for investigating the PCP pathway. Interestingly, the notochord appears to be the only embryonic structure in Ciona activating the PCP pathway. Moreover, the Ciona notochord as a single-file array of forty polarized cells is a uniquely tractable system for the study of polarization dynamics and the transmission of the PCP pathway. Here, we test models for propagation of a polarizing signal, interrogating temporal, spatial and signaling requirements. A simple cell-cell relay cascading through the entire length of the notochord is not supported; instead a more complex mechanism is revealed, with interactions influencing polarity between neighboring cells, but not distant ones. Mechanisms coordinating notochord-wide polarity remain elusive, but appear to entrain general (i.e., global) polarity even while local interactions remain important. However, this global polarizer does not appear to act as a localized, spatially-restricted determinant. Coordination of polarity along the long axis of the notochord requires the PCP pathway, a role we demonstrate is temporally distinct from this pathway's earlier role in convergent extension and intercalation. We also reveal polarity in the notochord to be dynamic: a cell's polarity state can be changed and then restored, underscoring the Ciona notochord's amenability for in vivo studies of PCP.

Deep homologies in chordate caudal central nervous systems.

Invertebrate chordates, such as the tunicate Ciona, can offer insight into the evolution of the chordate phylum. Anatomical features that are shared between invertebrate chordates and vertebrates may be taken as evidence of their presence in a common chordate ancestor. The central nervous systems of Ciona larvae and vertebrates share a similar anatomy despite the Ciona CNS having ~180 neurons. However, the depth of conservation between the Ciona CNS and those in vertebrates is not resolved. The Ciona caudal CNS, while appearing spinal cord-like, has hitherto been thought to lack motor neurons, bringing into question its homology with the vertebrate spinal cord. We show here that the Ciona larval caudal CNS does, in fact, have functional motor neurons along its length, pointing to the presence of a spinal cord-like structure at the base of the chordates. We extend our analysis of shared CNS anatomy further to explore the Ciona "motor ganglion", which has been proposed to be a homolog of the vertebrate hindbrain, spinal cord, or both. We find that a cluster of neurons in the dorsal motor ganglion shares anatomical location, developmental pathway, neural circuit architecture, and gene expression with the vertebrate cerebellum. However, functionally, the Ciona cluster appears to have more in common with vertebrate cerebellum-like structures, insofar as it receives and processes direct sensory input. These findings are consistent with earlier speculation that the cerebellum evolved from a cerebellum-like structure, and suggest that the latter structure was present in the dorsal hindbrain of a common chordate ancestor.

doublesex/mab3 related-1 (dmrt1) is essential for development of anterior neural plate derivatives in Ciona.

Ascidian larvae have a hollow, dorsal central nervous system that shares many morphological features with vertebrate nervous systems yet is composed of very few cells. We show here that a null mutation in the gene dmrt1 in the ascidian Ciona savignyi results in profound abnormalities in the development of the sensory vesicle (brain), as well as other anterior ectodermal derivatives, including the palps and oral siphon primordium (OSP). Although the phenotype of the mutant embryos is variable, the majority have a complete loss of the most anterior structures (palps and OSP) and extensive disruption of sensory structures, such as the light-sensitive ocellus, in the sensory vesicle. dmrt1 is expressed early in the blastula embryo in a small group of presumptive ectodermal cells as they become restricted to anterior neural, OSP and palp fates. Despite the early and restricted expression of dmrt1, we were unable, using several independent criteria, to observe a defect in the mutant embryos until the early tailbud stage. We speculate that the variability and late onset in the phenotype may be due to partially overlapping activities of other gene products.

A single oscillating proto-hypothalamic neuron gates taxis behavior in the primitive chordate Ciona.

Ciona larvae display a number of behaviors, including negative phototaxis. In negative phototaxis, the larvae first perform short spontaneous rhythmic casting swims. As larvae are cast in a light field, their photoreceptors are directionally shaded by an associated pigment cell, providing a phototactic cue. This then evokes an extended negative taxis swim. We report here that the larval forebrain of Ciona has a previously uncharacterized single slow-oscillating inhibitory neuron (neuron cor-assBVIN78) that projects to the midbrain, where it targets key interneurons of the phototaxis circuit known as the photoreceptor relay neurons. The anatomical location, gene expression, and oscillation of cor-assBVIN78 suggest homology to oscillating neurons of the vertebrate hypothalamus. Ablation of cor-assBVIN78 results in larvae showing extended phototaxis-like swims, even in the absence of phototactic cues. These results indicate that cor-assBVIN78 has a gating activity on phototaxis by projecting temporally oscillating inhibition to the photoreceptor relay neurons. However, in intact larvae, the frequency of cor-assBVIN78 oscillation does not match that of the rhythmic spontaneous swims, indicating that the troughs in oscillations do not themselves initiate swims but rather that cor-assBVIN78 may modulate the phototaxis circuit by filtering out low-level inputs while restricting them temporally to the troughs in inhibition.

A single oscillating proto-hypothalamic neuron gates taxis behavior in the primitive chordate Ciona.

Ciona larvae display a number of behaviors, including negative phototaxis. In negative phototaxis, the larvae first perform short spontaneous rhythmic casting swims. As larvae cast in a light field, their photoreceptors are directionally shaded by an associated pigment cell, providing a phototactic cue. This then evokes an extended negative taxis swim. We report here that the larval forebrain of Ciona has a previously uncharacterized single slow-oscillating inhibitory neuron (neuron cor-assBVIN78 ) that projects to the midbrain, where it targets key interneurons of the phototaxis circuit known as the photoreceptor relay neurons . The anatomical location, gene expression and oscillation of cor-assBVIN78 suggest homology to oscillating neurons of the vertebrate hypothalamus. Ablation of cor-assBVIN78 results in larvae showing extended phototaxis-like swims, but which occur in the absence of phototactic cues. These results indicate that cor-assBVIN78 has a gating activity on phototaxis by projecting temporally-oscillating inhibition to the photoreceptor relay neurons. However, in intact larvae the frequency of cor-assBVIN78 oscillation does not match that of the rhythmic spontaneous swims, indicating that the troughs in oscillations do not themselves initiate swims, but rather that cor-assBVIN78 may modulate the phototaxis circuit by filtering out low level inputs while restricting them temporally to the troughs in inhibition.

Key steps in the morphogenesis of a cranial placode in an invertebrate chordate, the tunicate Ciona savignyi.

Tunicates and vertebrates share a common ancestor that possessed cranial neurogenic placodes, thickenings in embryonic head epidermis giving rise to sensory structures. Though orthology assignments between vertebrate and tunicate placodes are not entirely resolved, vertebrate otic placodes and tunicate atrial siphon primordia are thought to be homologous based on morphology and position, gene expression, and a common signaling requirement during induction. Here, we probe key points in the morphogenesis of the tunicate atrial siphon. We show that the siphon primordium arises within a non-dividing field of lateral-dorsal epidermis. The initial steps of atrial primordium invagination are similar to otic placode invagination, but a placode-derived vesicle is never observed as for the otic vesicle of vertebrates. Rather, confocal imaging reveals an atrial opening through juvenile stages and beyond. We inject a photoactivatable lineage tracer to show that the early atrial siphon of the metamorphic juvenile, including its aperture and lining, derives from cells of the atrial placode itself. Finally, we perturb the routing of the gut to the left atrium by laser ablation and pharmacology to show that this adaptation to a sessile lifestyle depends on left-right patterning mechanisms present in the free-swimming chordate ancestor.

The ascidian mouth opening is derived from the anterior neuropore: reassessing the mouth/neural tube relationship in chordate evolution.

The relative positions of the brain and mouth are of central importance for models of chordate evolution. The dorsal hollow neural tube and the mouth have often been thought of as developmentally distinct structures that may have followed independent evolutionary paths. In most chordates however, including vertebrates and ascidians, the mouth primordia have been shown to fate to the anterior neural boundary. In ascidians such as Ciona there is a particularly intimate relationship between brain and mouth development, with a thin canal connecting the neural tube lumen to the mouth primordium at larval stages. This so-called neurohypophyseal canal was previously thought to be a secondary connection that formed relatively late, after the independent formation of the mouth primordium and the neural tube. Here we show that the Ciona neurohypophyseal canal is present from the end of neurulation and represents the anteriormost neural tube, and that the future mouth opening is actually derived from the anterior neuropore. The mouth thus forms at the anterior midline transition between neural tube and surface ectoderm. In the vertebrate Xenopus, we find that although the mouth primordium is not topologically continuous with the neural tube lumen, it nonetheless forms at this same transition point. This close association between the mouth primordium and the anterior neural tube in both ascidians and amphibians suggests that the evolution of these two structures may be more closely linked than previously appreciated.

The C. elegans Opa1 homologue EAT-3 is essential for resistance to free radicals.

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants.

Antagonistic Inhibitory Circuits Integrate Visual and Gravitactic Behaviors.

Larvae of the tunicate Ciona intestinalis possess a central nervous system of 177 neurons. This simplicity has facilitated the generation of a complete synaptic connectome. As chordates and the closest relatives of vertebrates, tunicates promise insight into the organization and evolution of vertebrate nervous systems. Ciona larvae have several sensory systems, including the ocellus and otolith, which are sensitive to light and gravity, respectively. Here, we describe circuitry by which these two are integrated into a complex behavior: the rapid reorientation of the body followed by upward swimming in response to dimming. Significantly, the gravity response causes an orienting behavior consisting of curved swims in downward-facing larvae but only when triggered by dimming. In contrast, the majority of larvae facing upward do not respond to dimming with orientation swims-but instead swim directly upward. Under constant light conditions, the gravity circuit appears to be inoperable, and both upward and downward swims were observed. Using connectomic and neurotransmitter data, we propose a circuit model that can account for these behaviors. The otolith consists of a statocyst cell and projecting excitatory sensory neurons (antenna cells). Postsynaptic to the antenna cells are a group of inhibitory primary interneurons, the antenna relay neurons (antRNs), which then project asymmetrically to the right and left motor units, thereby mediating curved orientation swims. Also projecting to the antRNs are inhibitory photoreceptor relay interneurons. These interneurons appear to antagonize the otolith circuit until they themselves are inhibited by photoreceptors in response to dimming, thus providing a triggering circuit.

Parallel visual circuitry in a basal chordate.

A common CNS architecture is observed in all chordates, from vertebrates to basal chordates like the ascidian Ciona. Ciona stands apart among chordates in having a complete larval connectome. Starting with visuomotor circuits predicted by the Ciona connectome, we used expression maps of neurotransmitter use with behavioral assays to identify two parallel visuomotor circuits that are responsive to different components of visual stimuli. The first circuit is characterized by glutamatergic photoreceptors and responds to the direction of light. These photoreceptors project to cholinergic motor neurons, via two tiers of cholinergic interneurons. The second circuit responds to changes in ambient light and mediates an escape response. This circuit uses GABAergic photoreceptors which project to GABAergic interneurons, and then to cholinergic interneurons. Our observations on the behavior of larvae either treated with a GABA receptor antagonist or carrying a mutation that eliminates photoreceptors indicate the second circuit is disinhibitory.

Reciprocal and dynamic polarization of planar cell polarity core components and myosin.

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

Genetic redundancy in endoderm specification within the genus Caenorhabditis.

Specification of the endoderm precursor, the E cell, in Caenorhabditis elegans requires a genomic region called the Endoderm Determining Region (EDR). We showed previously that end-1, a gene within the EDR encoding a GATA-type transcription factor, restores endoderm specification to embryos deleted for the EDR and obtained evidence for genetic redundancy in this process. Here, we report molecular identification of end-3, a nearby paralog of end-1 in the EDR, and show that end-1 and end-3 together define the endoderm-specifying properties of the EDR. Both genes are expressed in the early E lineage and each is individually sufficient to specify endodermal fate in the E cell and in non-endodermal precursors when ectopically expressed. The loss of function of both end genes, but not either one alone, eliminates endoderm in nearly all embryos and results in conversion of E into a C-like mesectodermal precursor, similar to deletions of the EDR. While two putative end-1 null mutants display no overt phenotype, a missense mutation that alters a residue in the zinc finger domain of END-3 results in misspecification of E in approximately 9% of mutant embryos. We report that the EDR in C. briggsae, which is estimated to have diverged from C. elegans approximately 50--120 myr ago, contains three end-like genes, resulting from both the ancient duplication that produced end-1 and end-3 in C. elegans, and a more recent duplication of end-3 in the lineage specific to C. briggsae. Transgenes containing the C. briggsae end homologs show E lineage-specific expression and function in C. elegans, demonstrating their functional conservation. Moreover, RNAi experiments indicate that the C. briggsae end genes also function redundantly to specify endoderm. We propose that duplicated end genes have been maintained over long periods of evolution, owing in part to their synergistic function.