The D alpha 2 gene encodes a ligand-binding subunit of nicotinic acetylcholine receptors (nAChRs) from Drosophila melanogaster. We have studied the distribution of D alpha 2 transcripts and protein by in situ hybridization and immunohistochemistry, respectively, as well as the regulation of D alpha 2 gene expression in vivo using D alpha 2 promoter fragments fused to the Escherichia coli lacZ gene. Transcripts and protein from the D alpha 2 gene were detected exclusively in the central nervous system. Both in late embryos and adults D alpha 2-like immunoreactivity is widely but not uniformly distributed in the synaptic neuropil, suggesting that the D alpha 2 protein is a subunit of a synaptic nicotinic receptor. Its distribution resembles that of ALS and ARD proteins, two other nAChR subunits of the fly. Five different D alpha 2-lacZ fusion gene constructs were introduced into the Drosophila genome by P-element-mediated gene transfer to identity functional elements of the D alpha 2 promoter. All constructs produce a basic lacZ expression pattern that is compatible with the distribution of D alpha 2 transcripts and protein. A 880 bp upstream fragment harbors the cis elements for the expression of a weak but specific basic D alpha 2 pattern. The next 350 bp further upstream significantly enhance beta-galactosidase expression without influencing the pattern of expression. Between 1.7 and 7.3 kb upstream of the transcription start site one or more elements that are required for D alpha 2 expression in optic lobe tangential cells are located.

Specific recognition molecules ("homing receptors") on lymphocytes are thought to direct selective entry of cells into different organs. The lectin-related cell adhesion molecule LECAM-1 has previously been supposed to mediate lymphocyte entry into peripheral lymph nodes and, partially, mesenteric nodes but not into Peyer's patches. Here we present evidence that in vivo the molecule is also implicated in homing of mouse lymphocytes to Peyer's patches and may have a more general role as homing receptor for high endothelial venules-bearing lymphoid tissue, but not for most non-lymphoid tissue.

To analyze the role of a4-integrins in lymphoma metastasis, sublines of the T-cell lymphoma LB were generated by retrovirus-mediated gene transfer that differ exclusively in the expression of a4-integrins. Using LB-a4 and control LB-NTK cells, we demonstrate that expression of a4-integrins strongly suppresses metastasis formation of LB lymphoma cells in secondary lymphoid organs (15)(16)(17)20).In the present study, the role of a4-integrins in lymphoma metastasis was studied by using the murine T-cell lymphoma LB as a model. We demonstrate that expression of a4-integrins strongly reduces metastasis formation by LB cells after i.v. injection. On the basis of the route of injection, in vivo migration experiments, and analysis of intrinsic growth rates, we conclude that metastasis formation of disseminated lymphoma cells is inhibited at a stage subsequent to infiltration of target organs. MATERIALS AND METHODSCells and Antibodies. Antibodies used included rat antimurine integrin a4, PS/2 (American Type Culture Collection); rat anti-murine LFA-1 a chain, FD441.8 (American Type Culture Collection); rat anti-murine integrin ,B7, Fib3O and Fib 504 (21); rat anti-murine integrin aE, M290 (22); rat antimurine integrin a5, 5H10-27 (PharMingen); rat anti-murine integrin a6, EA-1 (23); rat anti-murine L-selectin, MEL-14 (American Type Culture Collection); rat anti-murine CD44, IM781 (24); and rat anti-murine T-cell receptor Vf37, TR310 as negative control (25). For flow cytometry analysis, cells were incubated with saturating amounts of mAbs, washed, and stained with fluorescein isothiocyanate-conjugated mouse F(ab)2 fragments reacting with rat Ig (Dianova, Hamburg, Germany) and analyzed on a Coulter EPICS XL cytometer.The T-cell lymphoma LB spontaneously developed in a BALB/c mouse (26). LB cells express cell surface CD8 and CD25 but are negative for CD3 and CD4 (26).Generation of LB-a4 and LB-NTK Cell Lines. Expression plasmid pNTK-a4 was constructed by cloning the murine a4 cDNA (27) into the retroviral vector pNTK (28). Recombinant retroviruses were prepared from the mouse ecotropic, helper virus-free producer line . LB cells were infectedAbbreviations: VCAM-1, vascular cell adhesion molecule 1; MAd-CAM-1, mucosal addressin cell adhesion molecule 1. 4821The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Nicotinic acetylcholine receptors (nAChRs) represent a heterogeneous group of excitatory neurotransmitter receptors in the insect brain. We have characterized the Da2 gene of Drosophila melanoguster, a new member of the nAChR gene family. The protein coding region is interrupted by six introns. The positions of three of these introns are shared with all other nAChR genes. The deduced Da2 protein shows the structural features of ligand-binding nAChR a-subunits. Cytogenetically, the Da2 gene maps at position 96A of the 3rd chromosome, close to the ALS gene which also encodes an a-like nAChR subunit. Da2 transcripts are predominantly expressed in late embryos and in fly heads.