NUDC is critical for rod photoreceptor function, maintenance, and survival.

NUDC (nuclear distribution protein C) is a mitotic protein involved in nuclear migration and cytokinesis across species. Considered a cytoplasmic dynein (henceforth dynein) cofactor, NUDC was shown to associate with the dynein motor complex during neuronal migration. NUDC is also expressed in postmitotic vertebrate rod photoreceptors where its function is unknown.

NUDC is critical for rod photoreceptor function, maintenance, and survival.

Unlabelled: NUDC ( nu clear d istribution protein C) is a mitotic protein involved in nuclear migration and cytokinesis across species. Considered a cytoplasmic dynein (henceforth dynein) cofactor, NUDC was shown to associate with the dynein motor complex during neuronal migration. NUDC is also expressed in postmitotic vertebrate rod photoreceptors where its function is unknown.

Arf-like Protein 2 (ARL2) Controls Microtubule Neogenesis during Early Postnatal Photoreceptor Development.

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In a cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αβ-heterodimers, the building blocks of microtubules. To evaluate this function in the retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during the embryonic development () or after ciliogenesis specifically in rods ().

Deletion of CEP164 in mouse photoreceptors post-ciliogenesis interrupts ciliary intraflagellar transport (IFT).

Centrosomal protein of 164 kDa (CEP164) is located at distal appendages of primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of photoreceptor CEP164 before and after BB docking, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction (Prom1-ETCre). BBs dock to the cell cortex during postnatal day 6 (P6) to extend a connecting cilium (CC) and an axoneme.

Deletion of the phosphatase INPP5E in the murine retina impairs photoreceptor axoneme formation and prevents disc morphogenesis.

INPP5E, also known as pharbin, is a ubiquitously expressed phosphatidylinositol polyphosphate 5-phosphatase that is typically located in the primary cilia and modulates the phosphoinositide composition of membranes. Mutations to or loss of INPP5E is associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans-a syndromic ciliopathy affecting multiple tissues including the brain, liver, kidney, and retina.

Effect of conditional deletion of cytoplasmic dynein heavy chain DYNC1H1 on postnatal photoreceptors.

Cytoplasmic dynein (dynein 1), a major retrograde motor of eukaryotic cells, is a 1.4 MDa protein complex consisting of a pair of heavy chains (DYNC1H1) and a set of heterodimeric noncatalytic accessory components termed intermediate, light intermediate and light chains. DYNC1H1 (4644 amino acids) is the dynein backbone encoded by a gene consisting of 77 exons.

Diffuse or hitch a ride: how photoreceptor lipidated proteins get from here to there.

Photoreceptors are polarized neurons, with specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment (OS) where vision begins, an inner segment (IS) where protein synthesis occurs and a synaptic terminal for signal transmission to second-order neurons. The OS is a large, modified primary cilium attached to the IS by a slender connecting cilium (CC), the equivalent of the transition zone (TZ).

Deletion of both centrin 2 (CETN2) and CETN3 destabilizes the distal connecting cilium of mouse photoreceptors.

Centrins (CETN1-4) are ubiquitous and conserved EF-hand-family Ca-binding proteins associated with the centrosome, basal body, and transition zone. Deletion of CETN1 or CETN2 in mice causes male infertility or dysosmia, respectively, without affecting photoreceptor function. However, it remains unclear to what extent centrins are redundant with each other in photoreceptors.

Insights into photoreceptor ciliogenesis revealed by animal models.

Photoreceptors are polarized neurons, with very specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment, the site of photon capture that initiates vision, an inner segment that houses the biosynthetic machinery and a synaptic terminal for signal transmission to downstream neurons. Outer segments and inner segments are connected by a connecting cilium (CC), the equivalent of a transition zone (TZ) of primary cilia.

The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium.

RAB28, a member of the RAS oncogene family, is a ubiquitous, farnesylated, small GTPase of unknown function present in photoreceptors and the retinal pigmented epithelium (RPE). Nonsense mutations of the human gene cause recessive cone-rod dystrophy 18 (CRD18), characterized by macular hyperpigmentation, progressive loss of visual acuity, RPE atrophy, and severely attenuated cone and rod electroretinography (ERG) responses. In an attempt to elucidate the disease-causing mechanism, we generated mice by deleting exon 3 and truncating RAB28 after exon 2.

Small GTPases Rab8a and Rab11a Are Dispensable for Rhodopsin Transport in Mouse Photoreceptors.

Rab11a and Rab8a are ubiquitous small GTPases shown as required for rhodopsin transport in Xenopus laevis and zebrafish photoreceptors by dominant negative (dn) disruption of function. Here, we generated retina-specific Rab11a (retRab11a) and Rab8a (retRab8a) single and double knockout mice to explore the consequences in mouse photoreceptors. Rhodopsin and other outer segment (OS) membrane proteins targeted correctly to OS and electroretinogram (ERG) responses in all three mutant mouse lines were indistinguishable from wild-type (WT).

Centrin 2 is required for mouse olfactory ciliary trafficking and development of ependymal cilia planar polarity.

Centrins are ancient calmodulin-related Ca(2+)-binding proteins associated with basal bodies. In lower eukaryotes, Centrin2 (CETN2) is required for basal body replication and positioning, although its function in mammals is undefined. We generated a germline CETN2 knock-out (KO) mouse presenting with syndromic ciliopathy including dysosmia and hydrocephalus.

Germline deletion of Cetn1 causes infertility in male mice.

Centrins are calmodulin-like Ca(2+)-binding proteins that can be found in all ciliated eukaryotic cells from yeast to mammals. Expressed in male germ cells and photoreceptors, centrin 1 (CETN1) resides in the photoreceptor transition zone and connecting cilium. To identify its function in mammals, we deleted Cetn1 by homologous recombination.

Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase.

Dendritic patterning and spine morphogenesis are crucial for the assembly of neuronal circuitry to ensure normal brain development and synaptic connectivity as well as for understanding underlying mechanisms of neuropsychiatric diseases and cognitive impairments. The Rho GTPase family is essential for neuronal morphogenesis and synaptic plasticity by modulating and reorganizing the cytoskeleton. Here, we report that protocadherin (Pcdh) clusters and cell adhesion kinases (CAKs) play important roles in dendritic development and spine elaboration.

The protocadherin gene Celsr3 is required for interneuron migration in the mouse forebrain.

Interneurons are extremely diverse in the mammalian brain and provide an essential balance for functional neural circuitry. The vast majority of murine cortical interneurons are generated in the subpallium and migrate tangentially over a long distance to acquire their final positions. By using a mouse line with a deletion of the Celsr3 (Flamingo, or Fmi1) gene and a knock-in of the green fluorescent protein reporter, we find that Celsr3, a member of the nonclustered protocadherin (Pcdh) family, is predominantly expressed in the cortical interneurons in adults and in the interneuron germinal zones in embryos.

A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond.

We describe here a streamlined procedure for targeting vector construction, which often is a limiting factor for gene targeting (knockout) technology. This procedure combines various highly efficient recombination-based cloning methods in bacteria, consisting of three steps. First step is the use of Red-pathway-mediated recombination (recombineering) to capture a genomic fragment into a Gateway-compatible vector.

Toward simpler and faster genome-wide mutagenesis in mice.

Here we describe a practical Cre-loxP and piggyBac transposon-based mutagenesis strategy to systematically mutate coding sequences and/or the vast noncoding regions of the mouse genome for large-scale functional genomic analysis. To illustrate this approach, we first created loxP-containing loss-of-function alleles in the protocadherin alpha, beta and gamma gene clusters (Pcdha, Pcdhb and Pcdhg). Using these alleles, we show that, under proper guidance, Cre-loxP site-specific recombination can mediate efficient trans-allelic recombination in vivo, facilitating the generation of large germline deletions and duplications including deletions of Pcdha, and Pcdha to Pcdhb, simply by breeding (that is, at frequencies of 5.

Lesion-induced gelsolin upregulation in the hippocampus following entorhinal deafferentation.

Gelsolin is an actin-binding protein that regulates actin filament-severing and capping activity in the various processes of cell motilities. Here, we report the expression of gelsolin mRNA and protein in the hippocampus following transections of the entorhinal afferents. Northern blot analysis showed that transcript of gelsolin was upregulated in a transient manner in the deafferented hippocampus by 1.

Expression of thymosin beta4 mRNA by activated microglia in the denervated hippocampus.

Thymosin beta4 is a major actin-sequestering molecule. Here, we report a prominent upregulation of thymosin beta4 in the hippocampus following entorhinal deafferentation. Northern blotting displayed a transient increase of thymosin beta4 mRNA in the deafferented hippocampus by 1.

Entorhinal deafferentation induces upregulation of SPARC in the mouse hippocampus.

SPARC is a matricellular protein that modulates cell-cell and cell-matrix interactions by virtue of its antiproliferative and counteradhesive properties. Here, we report the denervation-induced upregulation of SPARC mRNA and protein in the mouse hippocampus following transections of the entorhinal afferents. Northern blot analysis showed that SPARC mRNA was upregulated in a transient manner in the deafferented mouse hippocampus.

Induction of ephrin-B1 and EphB receptors during denervation-induced plasticity in the adult mouse hippocampus.

Abstract It has been widely demonstrated that Eph receptors and their ephrin ligands play multiple pivotal roles in the development of the nervous system. However, less is known about their roles in the adult brain. Here we reported the expression of ephrin-B1 and its cognate EphB receptors in the adult mouse hippocampus at 3, 7, 15, 30 and 60 days after transections of the entorhinal afferents.

Regulated transcripts in the hippocampus following transections of the entorhinal afferents.

Based on the data from a cDNA microarray experiment which was carried out to screen the differential expressed genes in the rat hippocampus 10 days after removal of the entorhinal afferents, we confirmed the increase of expression of eight transcripts encoding protein osteonectin, thymosin-beta4, gelsolin, MHC I, MHC II, beta2-microglobulin, and interferon-gamma receptor using Northern blot. In situ hybridization revealed that the up-regulation of all these 8 transcripts localized specifically in the denervated target areas, the hippocampal stratum lacunosum-moleculare, and the dentate outer molecular layer. The results suggest that these molecules may have roles in the plasticity events in the hippocampus after entorhinal deafferentation.

Entorhinal deafferentation induces the expression of profilin mRNA in the reactive microglial cells in the hippocampus.

Profilin has been identified as an actin monomer sequestering protein and is thought to be a key regulator of actin polymerization in many fundamental cellular processes. We report the expression of profilin mRNA in the murine hippocampus following transections of the entorhinal afferents. Northern blot analysis showed that transcript of profilin was upregulated in a transient manner in the deafferented rat hippocampus by 1.

Semi-quantitative expression analysis of ephrin mRNAs in the deafferented hippocampus.

To search for gene expression changes probably responsible for deafferentation-induced reorganization in the brain, we have analyzed the expression of mRNAs for ephrin-A1, -A2, -A3, -A5 and -B1 in the rat hippocampus following transection of the entorhinal afferents by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). We found that their expression in the deafferented hippocampus increased significantly by 7 days, reached the maximum at 14 days and almost recovered to control levels by 60 days post-lesion. It is notable that the up-regulation of ephrin mRNAs occurs during the reorganization in the deafferented hippocampus, suggesting that ephrins may be involved in the plasticity events of the adult brain after lesion.