A Farnesyltransferase Acts to Inhibit Ectopic Neurite Formation in C. elegans.

Genetic pathways that regulate nascent neurite formation play a critical role in neuronal morphogenesis. The core planar cell polarity components VANG-1/Van Gogh and PRKL-1/Prickle are involved in blocking inappropriate neurite formation in a subset of motor neurons in C. elegans. A genetic screen for mutants that display supernumerary neurites was performed to identify additional factors involved in this process. This screen identified mutations in fntb-1, the ß subunit of farnesyltransferase. We show that fntb-1 is expressed in neurons and acts cell-autonomously to regulate neurite formation. Prickle proteins are known to be post-translationally modified by farnesylation at their C-terminal CAAX motifs. We show that PRKL-1 can be recruited to the plasma membrane in both a CAAX-dependent and CAAX-independent manner but that PRKL-1 can only inhibit neurite formation in a CAAX-dependent manner.

Functional Brachyury binding sites establish a temporal read-out of gene expression in the Ciona notochord.

The appearance of the notochord represented a milestone in Deuterostome evolution. The notochord is necessary for the development of the chordate body plan and for the formation of the vertebral column and numerous organs. It is known that the transcription factor Brachyury is required for notochord formation in all chordates, and that it controls transcription of a large number of target genes. However, studies of the structure of the cis-regulatory modules (CRMs) through which this control is exerted are complicated in vertebrates by the genomic complexity and the pan-mesodermal expression territory of Brachyury. We used the ascidian Ciona, in which the single-copy Brachyury is notochord-specific and CRMs are easily identifiable, to carry out a systematic characterization of Brachyury-downstream notochord CRMs. We found that Ciona Brachyury (Ci-Bra) controls most of its targets directly, through non-palindromic binding sites that function either synergistically or individually to activate early- and middle-onset genes, respectively, while late-onset target CRMs are controlled indirectly, via transcriptional intermediaries. These results illustrate how a transcriptional regulator can efficiently shape a shallow gene regulatory network into a multi-tiered transcriptional output, and provide insights into the mechanisms that establish temporal read-outs of gene expression in a fast-developing chordate embryo.

VANG-1 and PRKL-1 cooperate to negatively regulate neurite formation in Caenorhabditis elegans.

Neuritogenesis is a critical early step in the development and maturation of neurons and neuronal circuits. While extracellular directional cues are known to specify the site and orientation of nascent neurite formation in vivo, little is known about the genetic pathways that block inappropriate neurite emergence in order to maintain proper neuronal polarity. Here we report that the Caenorhabditis elegans orthologues of Van Gogh (vang-1), Prickle (prkl-1), and Dishevelled (dsh-1), core components of planar cell polarity (PCP) signaling, are required in a subset of peripheral motor neurons to restrict neurite emergence to a specific organ axis. In loss-of-function mutants, neurons display supernumerary neurites that extend inappropriately along the orthogonal anteroposterior (A/P) body axis. We show that autonomous and non-autonomous gene activities are required early and persistently to inhibit the formation or consolidation of growth cone protrusions directed away from organ precursor cells. Furthermore, prkl-1 overexpression is sufficient to suppress neurite formation and reorient neuronal polarity in a vang-1- and dsh-1-dependent manner. Our findings suggest a novel role for a PCP-like pathway in maintaining polarized neuronal morphology by inhibiting neuronal responses to extrinsic or intrinsic cues that would otherwise promote extraneous neurite formation.

Kaede-centrin1 labeling of mother and daughter centrosomes in mammalian neocortical neural progenitors.

The importance of the centrosome in regulating basic cellular processes and cell fate decisions has become increasingly evident from recent studies tracing the etiology of developmental disorders to mutations in genes encoding centrosomal proteins. This unit details a protocol for a fluorescence-based pulse labeling of centrioles of neural progenitor cells in the developing neocortex of mice. In utero electroporation of Kaede-Centrin1 followed by in utero or ex vivo photoconversion allows a direct monitoring of the inheritance of centrosomes containing centrioles of different ages in dividing neocortical neural progenitors (i.e., radial glial cells). This is achieved by combining the irreversible photoconversion capacity of the Kaede protein from green to red fluorescence with the faithful duplication of the centrosome during each cell cycle. After two mitotic divisions following photoconversion, mother centrosomes containing the original labeled centriole appear in both red and green fluorescence, and can be distinguished from daughter centrosomes which appear in green fluorescence only. This facilitates the study of the inheritance and behavior of the mother and daughter centrosomes in asymmetric cell divisions in the developing mammalian neocortex.

Asymmetric centrosome inheritance maintains neural progenitors in the neocortex.

Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. Here we show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex.

Neurons of the ascidian larval nervous system in Ciona intestinalis: I. Central nervous system.

The tadpole larva of ascidians, basal living relatives of vertebrates, has a chordate body plan. The CNS has many homologies with that of vertebrates yet only about 100 neurons. These few, possibly fixed in number and composition, nevertheless govern a diverse repertoire of behaviors. To elucidate the circuits of the CNS first requires that we recognize each neuron type, for which we used electroporation to transfect precleavage embryos with a plasmid containing green fluorescent protein (GFP) driven by the promoter of the synaptotagmin gene. Hatched larvae were fixed and GFP 3-D reconstructions of confocal image stacks compiled into images of 31 whole or partial larvae, either with many GFP-labelled neurons or with few, each clearly visible. Neuron counts in the sensory vesicle (SV) and visceral ganglion (VG) indicated that between 75% (SV) and 69% (VG) of previously reported numbers of neurons were transfected. Based on their position, shape, and projections, the following neurons were identified in the SV: a prominent eminens neuron, possibly with direct input from papillar neurons, a large ventroposterior interneuron, photoreceptors of the ocellus, and putative antenna cells of the otolith. In the VG, we identified at least four subtypes of motor neuron, including an ovoid cell that may innervate distal tail muscle cells and contrapelo cells with ascending projections, unique among VG neurons. The caudal nerve cord contained the first reported neurons, the somata of planate neurons. These neurons are the first identified types, and will be used to construct a map of the nervous system for this model basal chordate.

Neurons of the ascidian larval nervous system in Ciona intestinalis: II. Peripheral nervous system.

The peripheral nervous system of the ascidian tadpole larva comprises a distributed population of isolated receptor neurons, most of unproved function, organized along the trunk or tail epithelium. Previous reports using immunocytochemical methods failed to resolve the detailed morphology of the neurons and their axon pathways. Precleavage embryos of Ciona intestinalis transfected with the promoter of the neuron-specific synaptotagmin gene fused to a green fluorescent protein (GFP) gene yielded clearly labelled GFP profiles. These we examined in confocal image stacks of 31 larvae. Anchor cells, at least eight in each adhesive apical papilla, contribute axons to the papillar nerves that terminate in the sensory vesicle of the central nervous system. Two nerve bundles projected from each papilla, suggesting that at least two subpopulations of papillar neurons exist. Each bundle fasciculated with axons of the rostral trunk epidermal neurons (RTEN) in a stereotyped pattern. The RTEN had a hitherto unreported elaborate arbor of sensory dendrites within the tunic, suggesting that each has an extended sensorial field. Two subpopulations of apical trunk epidermal neurons (ATEN), anterior and posterior, were distinguished. As with the RTEN, these neurons extended dendritic arbors into the tunic. Two additional types of tail neuron, the caudal epidermal neurons (dorsal and ventral) as well as a novel bipolar interneuron, were identified. These identified neuron types are the substrate for the ascidian larva's entire peripheral sensory input, important during larval swimming and settlement.