Sorting nexin-14, a gene expressed in motoneurons trapped by an in vitro preselection method.

A gene-trap strategy was set up in embryonic stem (ES) cells with the aim of trapping genes expressed in restricted neuronal lineages. The vector used trap genes irrespective of their activity in undifferentiated totipotent ES cells. Clones were subjected individually to differentiation in a system in which ES cells differentiated into neurons. Two ES clones in which the trapped gene was expressed in ES-derived neurons were studied in detail. The corresponding cDNAs were cloned, sequenced, and analysed by in situ hybridisation on wild-type embryo sections. Both genes are expressed in the nervous system. One gene, YR-23, encodes a large intracellular protein of unknown function. The second clone, YR-14, represents a sorting nexin (SNX14) gene whose expression in vivo coincides with that of LIM-homeodomain Islet-1 in several tissues. Sorting nexins are proteins associated with the endoplasmic reticulum (ER) and may play a role in receptor trafficking. Gene trapping followed by screening based on in vitro preselection of differentiated ES recombinant clones, therefore, has the potential to identify integration events in subsets of genes before generation of mouse mutants.

Juxtaposition of CNR protocadherins and reelin expression in the developing spinal cord.

The CNR (cadherin-related neuronal receptors) family of protocadherins is of great interest because of their potential roles as molecular tags in the formation of specific synaptic connections, and as receptors for reelin, during neuronal migration, and cell body positioning. In order to know more about potential functions of CNRs we have mapped their expression during mouse nervous system development and compared their expression with that of reelin and its intracellular effector Dab1 in several tissues. In spinal cord, CNRs and Dab1 are expressed in motoneurons, while reelin is located in adjacent cells. In the hindbrain, there is a differential expression of CNRs and Dab1 in various motor nuclei. In the retina and olfactory system, we observe CNR and reelin expression but not that of Dab1. These results provide new insights into the potential functions of CNRs and their possible integration in the reelin pathway during development.

Cardiotrophin-1 requires LIFRbeta to promote survival of mouse motoneurons purified by a novel technique.

The cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) signal through a receptor complex formed between two transmembrane proteins, gp130 and LIFRbeta. In addition, CNTF also uses a ligand-binding component which is anchored to the cell membrane. In the case of cardiotrophin-1 (CT-1), LIFRbeta is also required in cardiomyocytes, but this has not been proven in neurons, and published data suggest that motoneurons may use a different receptor complex. We used Lifrbeta knockout mice to assess the requirement for this receptor component in the signal transduction of CT-1 in motoneurons. To study purified motoneurons from such mutants, we have developed a method allowing for isolation of highly purified mouse motoneurons. This protocol is based on the immunoaffinity purification of motoneurons using antibodies against the extracellular domain of the neurotrophin receptor, p75, followed by cell sorting using magnetic microbeads. We show that CNTF, LIF, and CT-1 are unable to promote the survival of motoneurons derived from homozygous Lifrbeta-/- mutant embryos. Thus, LIFRbeta is absolutely required to transduce the CT-1 survival signal in motoneurons.

Stabilization of the large T protein in temperature-independent (type A) FR 3T3 rat cells transformed with the simian virus 40 tsA30 mutant.

The stabilities of in vivo [35S]methionine-labeled large T and small t proteins, synthesized in temperature-sensitive (type N) and temperature-insensitive (type A) FR 3T3 rat cells transformed by an early temperature-sensitive mutant of simian virus 40 (SV40), tsA30, were analyzed at the permissive and restrictive temperatures. The two polypeptides, detected in greatly reduced amounts in cells of the N type at the restrictive temperature, were also unstable at the permissive temperature. However, both were made in similar amounts and were apparently stable in cells of the A type, irrespective of the temperature. The structures of the viral RNAs present at the permissive temperature were analyzed for transformants representative of each type, and containing a single integration of viral DNA. The two cell lines synthesized transcripts identical to the large T and small t mRNAs identified in SV40-infected monkey cells. Similar amounts of viral RNA were found in A and N transformants in active growth at the permissive and restrictive temperatures, which argued against a control at a transcriptional level. Assay of a defined function of the protein, namely, the binding of nucleotide detected by affinity labeling with periodate-oxidized [alpha-32P]ATP, clearly showed that the large T proteins from both types of transformants exhibited, at least for that particular biochemical function, the same in vitro temperature sensitivity. In transformants of the A type only could a reduced binding activity be detected in extracts from cells grown at the restrictive temperature. Thus, the temperature-independent behavior of the A transformants may result from an in vivo partial stabilization of the newly synthesized large T protein, probably through interaction with a cellular component(s).