Homologous elements hs3a and hs3b in the 3' regulatory region of the murine immunoglobulin heavy chain (Igh) locus are both dispensable for class-switch recombination.

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

Ephrin-B2 and EphB1 mediate retinal axon divergence at the optic chiasm.

In animals with binocular vision, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. Here, we show that ephrin-Bs in the chiasm region direct the divergence of retinal axons through the selective repulsion of a subset of RGCs that express EphB1. Ephrin-B2 is expressed at the mouse chiasm midline as the ipsilateral projection is generated and is selectively inhibitory to axons from ventrotemporal (VT) retina, where ipsilaterally projecting RGCs reside. Moreover, blocking ephrin-B2 function in vitro rescues the inhibitory effect of chiasm cells and eliminates the ipsilateral projection in the semiintact mouse visual system. A receptor for ephrin-B2, EphB1, is found exclusively in regions of retina that give rise to the ipsilateral projection. EphB1 null mice exhibit a dramatically reduced ipsilateral projection, suggesting that this receptor contributes to the formation of the ipsilateral retinal projection, most likely through its repulsive interaction with ephrin-B2.

EphB2 and ephrin-B2 regulate the ionic homeostasis of vestibular endolymph.

The ability to transport cations and anions across epithelia is critical for the regulation of pH, ionic homeostasis, and volume of extracellular fluids. Although the transporters and channels that facilitate ion and water movement across cell membranes are well known, the molecular mechanisms and signal transduction events that regulate these activities remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are well known to transduce bidirectional signals that control axon guidance and other cell migration/adhesion events during development. However, these molecules are also expressed in non-motile epithelial cells, including EphB2 in K(+)-secreting vestibular dark cells and ephrin-B2 in the adjacent transitional cells of the inner ear. Consistent with these expression patterns, mice with cytoplasmic domain mutations that interfere with EphB2 forward signaling or ephrin-B2 reverse signaling exhibit a hyperactive circling (waltzing) locomotion associated with a decreased amount of endolymph fluid that normally fills the vestibular labyrinth. Endolymph is unusual as an extracellular fluid in that it is normally high in K(+) and low in Na(+). Direct measurement of this fluid in live animals revealed significant decreases in K(+) concentration and endolymphatic potential in both EphB2 and ephrin-B2 mutant mice. Our findings provide evidence that bidirectional signaling mediated by B-subclass Ephs and ephrins controls the production and ionic homeostasis of endolymph fluid and thereby provide the first evidence that these molecules can control the activities of mature epithelial cells.

SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity.

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function.