We report that EphB receptors direct unique axonal behaviors required for mapping the dorsal-ventral (D-V) retinal axis along the lateral-medial (L-M) axis of the superior colliculus (SC). EphBs are expressed in a D-V gradient, ephrin-B1 in a L-M gradient in SC, and ephrin-B3 at its midline. EphBs and ephrin-Bs are expressed in countergradients in retina and SC. Developmental analyses reveal that retinal axons lack D-V ordering along the L-M axis, but directionally extend branches along it to establish ordered arbors. Directed branch extension is disrupted in EphB2; EphB3-deficient mice resulting in lateral ectopic arbors. Mice with kinase-inactive EphB2 have similar D-V mapping defects indicating that forward signaling dominates over reverse signaling. Our data suggest that branches of EphB expressing axons are attracted medially by ephrin-B1, and provide molecular mechanisms for D-V mapping in visual centers.

To discover new genes involved in axon navigation, we conducted a forward genetic screen for recessive alleles affecting motor neuron pathfinding in GFP reporter mice mutagenized with ENU. In Magellan mutant embryos, motor axons were error prone and wandered inefficiently at choice points within embryos, but paradoxically responded to guidance cues with normal sensitivity in vitro. We mapped the Magellan mutation to the Phr1 gene encoding a large multidomain E3 ubiquitin ligase. Phr1 is associated with the microtubule cytoskeleton within neurons and selectively localizes to axons but is excluded from growth cones. Motor and sensory neurons from Magellan mutants display abnormal morphologies due to a breakdown in the polarized distribution of components that segregate between axons and growth cones. The Magellan phenotype can be reversed by stabilizing microtubules with taxol or inhibiting p38MAPK activity. Thus, efficacious pathfinding requires Phr1 activity for coordinating the cytoskeletal organization that distinguishes axons from growth cones.

Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+ -dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We recently showed that Scp1 is an evolutionarily conserved regulator of neuronal gene silencing. Here, we present the X-ray crystal structures of a dominant-negative form of human Scp1 (D96N mutant) bound to mono- and diphosphorylated peptides encompassing the CTD heptad repeat (Y1S2P3T4S5P6S7). Moreover, kinetic and thermodynamic analyses of Scp1-phospho-CTD peptide complexes support the structures determined. This combined structure-function analysis discloses the residues in Scp1 involved in CTD binding and its preferential dephosphorylation of P.Ser5 of the CTD heptad repeat. Moreover, these results provide a template for the design of specific inhibitors of Scp1 for the study of neuronal stem cell development.

The agent that causes prion diseases is thought to be identical with PrPSc, a conformer of the normal prion protein PrPC. PrPC‐deficient mice do not exhibit major pathologies, perhaps because they express a protein termed Dpl, which shares significant biochemical and structural homology with PrPC. To investigate the physiological function of Dpl, we generated mice harbouring a homozygous disruption of the Prnd gene that encodes Dpl. Dpl deficiency did not interfere with embryonic and postnatal development, but resulted in male sterility. Dpl protein was expressed at late stages of spermiogenesis, and spermatids of Dpl mutants were reduced in numbers, immobile, malformed and unable to fertilize oocytes in vitro. Mechanical dissection of the zona pellucida partially restored in vitro fertilization. We conclude that Dpl regulates male fertility by controlling several aspects of male gametogenesis and sperm–egg interaction.

Conversion of cellular prion protein (PrP(C)) into a pathological conformer (PrP(Sc)) is thought to be promoted by PrP(Sc) in a poorly understood process. Here, we report that in wild-type mice, the expression of PrP(C) rendered soluble and dimeric by fusion to immunoglobulin Fcgamma (PrP-Fc(2)) delays PrP(Sc) accumulation, agent replication, and onset of disease following inoculation with infective prions. In infected PrP-expressing brains, PrP-Fc(2) relocates to lipid rafts and associates with PrP(Sc) without acquiring protease resistance, indicating that PrP-Fc(2) resists conversion. Accordingly, mice expressing PrP-Fc(2) but lacking endogenous PrP(C) are resistant to scrapie, do not accumulate PrP-Fc(2)(Sc), and do not transmit disease to others. These results indicate that various PrP isoforms engage in a complex in vivo, whose distortion by PrP-Fc(2) affects prion propagation and scrapie pathogenesis. The unique properties of PrP-Fc(2) suggest that soluble PrP derivatives may represent a new class of prion replication antagonists.

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The agent that causes prion diseases is thought to be identical to PrP Sc , a conformer of the normal prion protein PrP C . Recently a novel protein, termed Doppel (Dpl), was identified that shares significant biochemical and structural homology with PrP C . To investigate the function of Dpl in neurogenesis and in prion pathology, we generated embryonic stem (ES) cells harbouring a homozygous disruption of the Prnd gene that encodes Dpl. After in vitro differentiation and grafting into adult brains of PrP C -deficient Prnp 0/0 mice, Dpl-deficient ES cell-derived grafts contained all neural lineages analyzed, including neurons and astrocytes. When Prnd-deficient neural tissue was inoculated with scrapie prions, typical features of prion pathology including spongiosis, gliosis and PrP Sc accumulation, were observed. Therefore, Dpl is unlikely to exert a cell-autonomous function during neural differentiation and, in contrast to its homologue PrP C , is dispensable for prion disease progression and for generation of PrP Sc .