Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

The Role of Protocadherin γ in Adult Sensory Neurons and Skin Reinnervation.

The clustered protocadherins (cPcdhs) play a critical role in the patterning of several CNS axon and dendritic arbors, through regulation of homophilic self and neighboring interactions. While not explored, primary peripheral sensory afferents that innervate the epidermis may require similar constraints to convey spatial signals with appropriate fidelity. Here, we show that members of the γ-Pcdh (Pcdhγ) family are expressed in both adult sensory neuron axons and in neighboring keratinocytes that have close interactions during skin reinnervation. Adult mice of both sexes were studied. Pcdhγ knock-down either through small interfering RNA (siRNA) transduction or AAV-Cre recombinase transfection of adult mouse primary sensory neurons from floxed Pcdhγ mice was associated with a remarkable rise in neurite outgrowth and branching. Rises in outgrowth were abrogated by Rac1 inhibition. Moreover, AAV-Cre knock-down in Pcdhγ floxed neurons generated a rise in neurite self-intersections, and a robust rise in neighbor intersections or tiling, suggesting a role in sensory axon repulsion. Interestingly, preconditioned (3-d axotomy) neurons with enhanced growth had temporary declines in Pcdhγ and lessened outgrowth from Pcdhγ siRNA. In vivo, mice with local hindpaw skin Pcdhγ knock-down by siRNA had accelerated reinnervation by new epidermal axons with greater terminal branching and reduced intra-axonal spacing. Pcdhγ knock-down also had reciprocal impacts on keratinocyte density and nuclear size. Taken together, this work provides evidence for a role of Pcdhγ in attenuating outgrowth of sensory axons and their interactions, with implications in how new reinnervating axons following injury fare amid skin keratinocytes that also express Pcdhγ.SIGNIFICANCE STATEMENT The molecular mechanisms and potential constraints that govern skin reinnervation and patterning by sensory axons are largely unexplored. Here, we show that γ-protocadherins (Pcdhγ) may help to dictate interaction not only among axons but also between axons and keratinocytes as the former re-enter the skin during reinnervation. Pcdhγ neuronal knock-down enhances outgrowth in peripheral sensory neurons, involving the growth cone protein Rac1 whereas skin Pcdhγ knock-down generates rises in terminal epidermal axon growth and branching during re-innervation. Manipulation of sensory axon regrowth within the epidermis offers an opportunity to influence regenerative outcomes following nerve injury.

Sticking to your side: A niche for the development of ipsilateral retinal projections.

Visual impairments in albinism result from decreased ipsilateral retinal projections. In this issue of Neuron, Slavi, Balasubramanian, and colleagues1 demonstrate how low CyclinD2 in the ciliary marginal zone perturbs generation of ipsilaterally projecting RGCs and that restoring CyclinD2 improves vision in albino mice.

PV-IRES-Cre mouse line targets excitatory granule neurons in the cerebellum.

Parvalbumin-expressing inhibitory neurons (PV-INs) are critical for the balance and fine-tuning of complex neuronal circuits. Studies of PV-IN biology require tools for their specific labeling, targeting and manipulation. Among these, the Cre/LoxP system is the most popular in mice, with the two commonly used PV-Cre lines cited over 5600 times. Here we report in the mouse cerebellar cortex that PV-Cre activity is not restricted to inhibitory neurons. Imaging of Cre-activated reporters demonstrated recombination in excitatory granule cells. We present evidence that PV-Cre recombination is: (1) spatially regulated and lobule specific; (2) detected in granule cells in the external and internal granule cell layers arising from strong, but transient Pvalb expression in progenitors between E13-E15; and (3) delayed in a subset of inhibitory interneurons, asynchronous with PV protein expression. Together, our findings establish the spatio-temporal patterns PV-Cre activation in the mouse cerebellum, raising considerations for conditional targeting of Pvalb-expressing inhibitory populations.

Morphological pseudotime ordering and fate mapping reveal diversification of cerebellar inhibitory interneurons.

Understanding how diverse neurons are assembled into circuits requires a framework for describing cell types and their developmental trajectories. Here we combine genetic fate-mapping, pseudotemporal profiling of morphogenesis, and dual morphology and RNA labeling to resolve the diversification of mouse cerebellar inhibitory interneurons. Molecular layer interneurons (MLIs) derive from a common progenitor population but comprise diverse dendritic-, somatic-, and axon initial segment-targeting interneurons. Using quantitative morphology from 79 mature MLIs, we identify two discrete morphological types and presence of extensive within-class heterogeneity. Pseudotime trajectory inference using 732 developmental morphologies indicate the emergence of distinct MLI types during migration, before reaching their final positions. By comparing MLI identities from morphological and transcriptomic signatures, we demonstrate the dissociation between these modalities and that subtype divergence can be resolved from axonal morphogenesis prior to marker gene expression. Our study illustrates the utility of applying single-cell methods to quantify morphology for defining neuronal diversification.

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations.

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Molecular mechanisms that mediate dendrite morphogenesis.

Neurons develop dendritic morphologies that bear cell type-specific features in dendritic field size and geometry, branch placement and density, and the types and distributions of synaptic contacts. Dendritic patterns influence the types and numbers of inputs a neuron receives, and the ways in which neural information is processed and transmitted in the circuitry. Even subtle alterations in dendritic structures can have profound consequences on neuronal function and are implicated in neurodevelopmental disorders. In this chapter, I review how growing dendrites acquire their exquisite patterns by drawing examples from diverse neuronal cell types in vertebrate and invertebrate model systems. Dendrite morphogenesis is shaped by intrinsic and extrinsic factors such as transcriptional regulators, guidance and adhesion molecules, neighboring cells and synaptic partners. I discuss molecular mechanisms that regulate dendrite morphogenesis with a focus on five aspects of dendrite patterning: (1) Dendritic cytoskeleton and cellular machineries that build the arbor; (2) Gene regulatory mechanisms; (3) Afferent cues that regulate dendritic arbor growth; (4) Space-filling strategies that optimize dendritic coverage; and (5) Molecular cues that specify dendrite wiring. Cell type-specific implementation of these patterning mechanisms produces the diversity of dendrite morphologies that wire the nervous system.

The γ-Protocadherins Regulate the Survival of GABAergic Interneurons during Developmental Cell Death.

Inhibitory interneurons integrate into developing circuits in specific ratios and distributions. In the neocortex, inhibitory network formation occurs concurrently with the apoptotic elimination of a third of GABAergic interneurons. The cell surface molecules that select interneurons to survive or die are unknown. Here, we report that members of the clustered Protocadherins (cPCDHs) control GABAergic interneuron survival during developmentally-regulated cell death. Conditional deletion of the gene cluster encoding the γ-Protocadherins (Pcdhgs) from developing GABAergic neurons in mice of either sex causes a severe loss of inhibitory populations in multiple brain regions and results in neurologic deficits such as seizures. By focusing on the neocortex and the cerebellar cortex, we demonstrate that reductions of inhibitory interneurons result from elevated apoptosis during the critical postnatal period of programmed cell death (PCD). By contrast, cortical interneuron (cIN) populations are not affected by removal of Pcdhgs from pyramidal neurons or glial cells. Interneuron loss correlates with reduced AKT signaling in Pcdhg mutant interneurons, and is rescued by genetic blockade of the pro-apoptotic factor BAX. Together, these findings identify the PCDHGs as pro-survival transmembrane proteins that select inhibitory interneurons for survival and modulate the extent of PCD. We propose that the PCDHGs contribute to the formation of balanced inhibitory networks by controlling the size of GABAergic interneuron populations in the developing brain.SIGNIFICANCE STATEMENT A pivotal step for establishing appropriate excitatory-inhibitory ratios is adjustment of neuronal populations by cell death. In the mouse neocortex, a third of GABAergic interneurons are eliminated by BAX-dependent apoptosis during the first postnatal week. Interneuron cell death is modulated by neural activity and pro-survival pathways but the cell-surface molecules that select interneurons for survival or death are unknown. We demonstrate that members of the cadherin superfamily, the clustered γ-Protocadherins (PCDHGs), regulate the survival of inhibitory interneurons and the balance of cell death. Deletion of the Pcdhgs in mice causes inhibitory interneuron loss in the cortex and cerebellum, and leads to motor deficits and seizures. Our findings provide a molecular basis for controlling inhibitory interneuron population size during circuit formation.

Human iPSC-derived Down syndrome astrocytes display genome-wide perturbations in gene expression, an altered adhesion profile, and increased cellular dynamics.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

Combinatorial Effects of Alpha- and Gamma-Protocadherins on Neuronal Survival and Dendritic Self-Avoidance.

The clustered protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly arrayed gene clusters, Pcdh-α, Pcdh-ß, and Pcdh-γ (Pcdha, Pcdhb, and Pcdhg, respectively). Pcdh isoforms from different clusters are combinatorially expressed in neurons. They form multimers that interact homophilically and mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, axonal tiling, and dendritic self-avoidance. Most studies have analyzed clusters individually. Here, we assessed functional interactions between Pcdha and Pcdhg clusters. To circumvent neonatal lethality associated with deletion of Pcdhgs, we used Crispr-Cas9 genome editing in mice to combine a constitutive Pcdha mutant allele with a conditional Pcdhg allele. We analyzed roles of Pcdhas and Pcdhgs in the retina and cerebellum from mice (both sexes) lacking one or both clusters. In retina, Pcdhgs are essential for survival of inner retinal neurons and dendritic self-avoidance of starburst amacrine cells, whereas Pcdhas are dispensable for both processes. Deletion of both Pcdha and Pcdhg clusters led to far more dramatic defects in survival and self-avoidance than Pcdhg deletion alone. Comparisons of an allelic series of mutants support the conclusion that Pcdhas and Pcdhgs function together in a dose-dependent and cell-type-specific manner to provide a critical threshold of Pcdh activity. In the cerebellum, Pcdhas and Pcdhgs also cooperate to mediate self-avoidance of Purkinje cell dendrites, with modest but significant defects in either single mutant and dramatic defects in the double mutant. Together, our results demonstrate complex patterns of redundancy between Pcdh clusters and the importance of Pcdh cluster diversity in postnatal CNS development.SIGNIFICANCE STATEMENT The formation of neural circuits requires diversification and combinatorial actions of cell surface proteins. Prominent among them are the clustered protocadherins (Pcdhs), a family of ∼60 neuronal recognition molecules. Pcdhs are encoded by three closely linked gene clusters called Pcdh-α, Pcdh-ß, and Pcdh-γ. The Pcdhs mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, and spatial patterning of axons and dendrites. Most studies to date have been limited to single clusters. Here, we used genome editing to assess interactions between Pcdh-α and Pcdh-γ gene clusters. We examined two regions of the CNS, the retina and cerebellum and show that the 14 α-Pcdhs and 22 γ-Pcdhs act synergistically to mediate neuronal survival and dendrite patterning.

Neuronal territory formation by the atypical cadherins and clustered protocadherins.

Spatial patterns of neuronal connectivity are critical for neural circuit function and information processing. For many neuron types, the development of stereotyped dendritic and axonal territories involves reiterative contacts between neurites and successive re-calibration of branch outgrowth and directionality. Here I review emerging roles for members of the atypical cadherins (Fmi/Celsrs) and the clustered Protocadherins (Pcdhs) in neurite patterning. These cell-surface molecules have shared functions: they engage in homophilic recognition and mediate dynamic and contact-dependent interactions to establish reproducible and space-filling arborization patterns. As shown in genetic and molecular studies, the atypical cadherins and clustered Pcdhs serve in multiple contexts and signal diverse actions such as neurite repulsion or selective adhesion. In some cell types, they regulate the non-overlapping arrangement of branches achieved through homotypic interactions, such as in self-avoidance or tiling. In others, they promote dendritic complexity through cell-cell interactions. With critical roles in both the fine-scale arrangement of axonal and dendritic branching and the large-scale organization of axon tracts and neuronal networks, the atypical cadherins and clustered Pcdhs are key regulators of neural circuit assembly and function.

Development of dendritic form and function.

The nervous system is populated by numerous types of neurons, each bearing a dendritic arbor with a characteristic morphology. These type-specific features influence many aspects of a neuron's function, including the number and identity of presynaptic inputs and how inputs are integrated to determine firing properties. Here, we review the mechanisms that regulate the construction of cell type-specific dendrite patterns during development. We focus on four aspects of dendrite patterning that are particularly important in determining the function of the mature neuron: (a) dendrite shape, including branching pattern and geometry of the arbor; (b) dendritic arbor size;

Dendrite self-avoidance requires cell-autonomous slit/robo signaling in cerebellar purkinje cells.

Dendrites from the same neuron usually develop nonoverlapping patterns by self-avoidance, a process requiring contact-dependent recognition and repulsion. Recent studies have implicated homophilic interactions of cell surface molecules, including Dscams and Pcdhgs, in self-recognition, but repulsive molecular mechanisms remain obscure. Here, we report a role for the secreted molecule Slit2 and its receptor Robo2 in self-avoidance of cerebellar Purkinje cells (PCs). Both molecules are highly expressed by PCs, and their deletion leads to excessive dendrite self-crossing without affecting arbor size and shape. This cell-autonomous function is supported by the boundary-establishing activity of Slit in culture and the phenotype rescue by membrane-associated Slit2 activities. Furthermore, genetic studies show that they act independently from Pcdhg-mediated recognition. Finally, PC-specific deletion of Robo2 is associated with motor behavior alterations. Thus, our study uncovers a local repulsive mechanism required for self-avoidance and demonstrates the molecular complexity at the cell surface in dendritic patterning.

Functional significance of isoform diversification in the protocadherin gamma gene cluster.

The mammalian Protocadherin (Pcdh) alpha, beta, and gamma gene clusters encode a large family of cadherin-like transmembrane proteins that are differentially expressed in individual neurons. The 22 isoforms of the Pcdhg gene cluster are diversified into A-, B-, and C-types, and the C-type isoforms differ from all other clustered Pcdhs in sequence and expression. Here, we show that mice lacking the three C-type isoforms are phenotypically indistinguishable from the Pcdhg null mutants, displaying virtually identical cellular and synaptic alterations resulting from neuronal apoptosis. By contrast, mice lacking three A-type isoforms exhibit no detectable phenotypes. Remarkably, however, genetically blocking apoptosis rescues the neonatal lethality of the C-type isoform knockouts, but not that of the Pcdhg null mutants. We conclude that the role of the Pcdhg gene cluster in neuronal survival is primarily, if not specifically, mediated by its C-type isoforms, whereas a separate role essential for postnatal development, likely in neuronal wiring, requires isoform diversity.

Protocadherins mediate dendritic self-avoidance in the mammalian nervous system.

Dendritic arborizations of many neurons are patterned by a process called self-avoidance, in which branches arising from a single neuron repel each other. By minimizing gaps and overlaps within the arborization, self-avoidance facilitates complete coverage of a neuron's territory by its neurites. Remarkably, some neurons that display self-avoidance interact freely with other neurons of the same subtype, implying that they discriminate self from non-self. Here we demonstrate roles for the clustered protocadherins (Pcdhs) in dendritic self-avoidance and self/non-self discrimination. The Pcdh locus encodes 58 related cadherin-like transmembrane proteins, at least some of which exhibit isoform-specific homophilic adhesion in heterologous cells and are expressed stochastically and combinatorially in single neurons. Deletion of all 22 Pcdh genes in the mouse γ-subcluster (Pcdhg genes) disrupts self-avoidance of dendrites in retinal starburst amacrine cells (SACs) and cerebellar Purkinje cells. Further genetic analysis of SACs showed that Pcdhg proteins act cell-autonomously during development, and that replacement of the 22 Pcdhg proteins with a single isoform restores self-avoidance. Moreover, expression of the same single isoform in all SACs decreases interactions among dendrites of neighbouring SACs (heteroneuronal interactions). These results suggest that homophilic Pcdhg interactions between sibling neurites (isoneuronal interactions) generate a repulsive signal that leads to self-avoidance. In this model, heteroneuronal interactions are normally permitted because dendrites seldom encounter a matched set of Pcdhg proteins unless they emanate from the same soma. In many respects, our results mirror those reported for Dscam1 (Down syndrome cell adhesion molecule) in Drosophila: this complex gene encodes thousands of recognition molecules that exhibit stochastic expression and isoform-specific interactions, and mediate both self-avoidance and self/non-self discrimination. Thus, although insect Dscam and vertebrate Pcdh proteins share no sequence homology, they seem to underlie similar strategies for endowing neurons with distinct molecular identities and patterning their arborizations.

Wnt signals organize synaptic prepattern and axon guidance through the zebrafish unplugged/MuSK receptor.

Early during neuromuscular development, acetylcholine receptors (AChRs) accumulate at the center of muscle fibers, precisely where motor growth cones navigate and synapses eventually form. Here, we show that Wnt11r binds to the zebrafish unplugged/MuSK ectodomain to organize this central muscle zone. In the absence of such a zone, prepatterned AChRs fail to aggregate and, as visualized by live-cell imaging, growth cones stray from their central path. Using inducible unplugged/MuSK transgenes, we show that organization of the central muscle zone is dispensable for the formation of neural synapses, but essential for AChR prepattern and motor growth cone guidance. Finally, we show that blocking noncanonical dishevelled signaling in muscle fibers disrupts AChR prepatterning and growth cone guidance. We propose that Wnt ligands activate unplugged/MuSK signaling in muscle fibers to restrict growth cone guidance and AChR prepatterns to the muscle center through a mechanism reminiscent of the planar cell polarity pathway.

gamma-Protocadherins regulate neuronal survival but are dispensable for circuit formation in retina.

Twenty-two tandemly arranged protocadherin-gamma (Pcdh-gamma) genes encode transmembrane proteins with distinct cadherin-related extracellular domains and a common intracellular domain. Genetic studies have implicated Pcdh-gamma genes in the regulation of neuronal survival and synapse formation. Because mice lacking the Pcdh-gamma cluster die perinatally, we generated conditional mutants to analyze roles of Pcdh-gamma genes in the development and function of neural circuits. Retina-specific deletion of Pcdh-gammas led to accentuation of naturally occurring death of interneurons and retinal ganglion cells (RGCs) during the first two postnatal weeks. Nonetheless, many neuronal subtypes formed lamina-specific arbors. Blocking apoptosis by deletion of the pro-apoptotic gene Bax showed that even neurons destined to die formed qualitatively and quantitatively appropriate connections. Moreover, electrophysiological analysis indicated that processing of visual information was largely normal in the absence of Pcdh-gamma genes. These results suggest that Pcdh-gamma genes are dispensable for elaboration of specific connections in retina, but play a primary role in sculpting neuronal populations to appropriate sizes or proportions during the period of naturally occurring cell death.

Differential requirement for MuSK and dystroglycan in generating patterns of neuromuscular innervation.

Vertebrates display diverse patterns of neuromuscular innervation, but little is known about how such diversity is generated. In mammals, neuromuscular junctions form predominantly at equatorial locations, giving rise to a focal innervation pattern along a central endplate band. In addition, vertebrate striated muscles exhibit two nonfocal neuromuscular patterns, myoseptal and distributed innervation. Although agrin-MuSK-rapsyn signaling is essential for the focal innervation pattern, it is unknown whether the same genetic program also controls synaptogenesis at nonfocal innervation sites. Here we show that one of three transcripts generated by the zebrafish unplugged locus, unplugged FL, encodes the zebrafish MuSK ortholog. We demonstrate that UnpFL/MuSK is critical for the assembly of focal synapses in zebrafish and that it cooperates with dystroglycan in the formation of nonfocal myoseptal and distributed synapses. Our results provide the first genetic evidence that neuromuscular synapse formation can occur in the absence of MuSK and that the combinatorial function of UnpFL/MuSK and dystroglycan generates diverse patterns of vertebrate neuromuscular innervation.

FoxD3 regulation of Nodal in the Spemann organizer is essential for Xenopus dorsal mesoderm development.

Induction and patterning of the mesodermal germ layer is a key early step of vertebrate embryogenesis. We report that FoxD3 function in the Xenopus gastrula is essential for dorsal mesodermal development and for Nodal expression in the Spemann organizer. In embryos and explants, FoxD3 induced mesodermal genes, convergent extension movements and differentiation of axial tissues. Engrailed-FoxD3, but not VP16-FoxD3, was identical to native FoxD3 in mesoderm-inducing activity, indicating that FoxD3 functions as a transcriptional repressor to induce mesoderm. Antagonism of FoxD3 with VP16-FoxD3 or morpholino-knockdown of FoxD3 protein resulted in a complete block to axis formation, a loss of mesodermal gene expression, and an absence of axial mesoderm, indicating that transcriptional repression by FoxD3 is required for mesodermal development. FoxD3 induced mesoderm in a non-cell-autonomous manner, indicating a role for secreted inducing factors in the response to FoxD3. Consistent with this mechanism, FoxD3 was necessary and sufficient for the expression of multiple Nodal-related genes, and inhibitors of Nodal signaling blocked mesoderm induction by FoxD3. Therefore, FoxD3 is required for Nodal expression in the Spemann organizer and this function is essential for dorsal mesoderm formation.

Zebrafish unplugged reveals a role for muscle-specific kinase homologs in axonal pathway choice.

En route to their target, pioneering motor growth cones repeatedly encounter choice points at which they make pathway decisions. In the zebrafish mutant unplugged, two of the three segmental motor axons make incorrect decisions at a somitic choice point. Using positional cloning, we show here that unplugged encodes a homolog of muscle-specific kinase (MuSK) and that, unlike mammalian MuSK, unplugged has only a limited role in neuromuscular synaptogenesis. We demonstrate that unplugged is transiently expressed in cells adjacent to the choice point and that unplugged signaling before the arrival of growth cones induces changes in the extracellular environment. In addition, we find that the unplugged locus generates three different transcripts. The splice variant 1 (SV1) isoform lacks the extracellular modules essential for agrin responsiveness, and signaling through this isoform mediates axonal pathfinding, independent of the MuSK downstream component rapsyn. Our results demonstrate a new role for MuSK homologs in axonal pathway selection.