During embryonic development, axons from sensory neurons in the olfactory epithelium (OE) extend into the olfactory bulb (OB) where they synapse with projection neurons and form glomerular structures. To determine whether glycans play a role in these processes, we analyzed mice deficient for the glycosyltransferase ␤1,3-N-acetylglucosaminyltransferase 1 (␤3GnT1), a key enzyme in lactosamine glycan synthesis. Terminal lactosamine expression, as shown by immunoreactivity with the monoclonal antibody 1B2, is dramatically reduced in the neonatal null OE. Postnatal ␤3GnT1 Ϫ/Ϫ mice exhibit severely disorganized OB innervation and defective glomerular formation. Beginning in embryonic development, specific subsets of odorant receptor-expressing neurons are progressively lost from the OE of null mice, which exhibit a postnatal smell perception deficit. Axon guidance errors and increased neuronal cell death result in an absence of P2, I7, and M72 glomeruli, indicating a reduction in the repertoire of odorant receptor-specific glomeruli. By ϳ2 weeks of age, lactosamine is unexpectedly reexpressed in sensory neurons of null mice through a secondary pathway, which is accompanied by the regrowth of axons into the OB glomerular layer and the return of smell perception. Thus, both neonatal OE degeneration and the postnatal regeneration are lactosamine dependent. Lactosamine expression in ␤3GnT1 Ϫ/Ϫ mice is also reduced in pheromone-receptive vomeronasal neurons and dorsal root ganglion cells, suggesting that ␤3GnT1 may perform a conserved function in multiple sensory systems. These results reveal an essential role for lactosamine in sensory axon pathfinding and in the formation of OB synaptic connections.

Gonadotropin-releasing hormone (GnRH) neurons migrate from the vomeronasal organ (VNO) in the nasal compartment to the basal forebrain in mice, beginning on embryonic day 11 (E11). These neurons use vomeronasal axons as guides to migrate through the nasal mesenchyme. Most GnRH neurons then migrate along the caudal branch of the vomeronasal nerve to reach the hypothalamus. We show here that stromal cell-derived factor-1 [SDF-1, also known as chemokine C-X-C motif ligand 12 (CXCL12)] is expressed in the embryonic nasal mesenchyme from as early as E10 in an increasing rostral to caudal gradient that is most intense at the border of the nasal mesenchyme and the telencephalon. Chemokine C-X-C motif receptor 4 (CXCR4), the receptor for SDF-1, is expressed by neurons in the olfactory epithelium and VNO. Cells derived from these sensory epithelia, including migrating GnRH neurons and ensheathing glial precursors of the migrating mass (MM), also express CXCR4, suggesting that they may use SDF-1 as a chemokine. In support of this, most GnRH neurons of CXCR4 Ϫ/Ϫ mice fail to exit the VNO at E13, and comparatively few GnRH neurons reach the forebrain. There is also a significant decrease in the total number of GnRH neurons in CXCR4 Ϫ/Ϫ mice and an increase in cell death within the VNO relative to controls. The MM is smaller in CXCR4 Ϫ/Ϫ mice, suggesting that some MM cells also require SDF-1/CXCR4 function for migration and survival.

The distinction between the different classes of glycolipids is conditioned by the action of specific core transferases. The entry point for lacto-series glycolipids is catalyzed by the ␤1,3 N-acetylglucosaminyltransferase GlcNAc(␤1,3)Gal(␤1,4)Glc-ceramide (Lc3) synthase enzyme. The Lc3 synthase activity has been shown to be regulated during development, especially during brain morphogenesis. Here, we report the molecular cloning of a mouse gene encoding an Lc3 synthase enzyme. The mouse cDNA included an open reading frame of 1131 base pairs encoding a protein of 376 amino acids. The Lc3 synthase protein shared several structural motifs previously identified in the members of the ␤1,3 glycosyltransferase superfamily. The Lc3 synthase enzyme efficiently utilized the lactosyl ceramide glycolipid acceptor. The identity of the reaction products of Lc3 synthase-transfected CHOP2/1 cells was confirmed by thin-layer chromatography immunostaining using antibodies TE-8 and 1B2 that recognize Lc3 and Gal(␤1,4)GlcNAc(␤1,3)Gal(␤1,4)Glcceramide (nLc4) structures, respectively. In addition to the initiating activity for lacto-chain synthesis, the Lc3 synthase could extend the terminal N-acetyllactosamine unit of nLc4 and also had a broad specificity for gangliosides GA1, GM1, and GD1b to generate neolacto-ganglio hybrid structures. The mouse Lc3 synthase gene was mainly expressed during embryonic development. In situ hybridization analysis revealed that that the Lc3 synthase was expressed in most tissues at embryonic day 11 with elevated expression in the developing central nervous system. Postnatally, the expression was restricted to splenic B-cells, the placenta, and cerebellar Purkinje cells where it colocalized with HNK-1 reactivity. These data support a key role for the Lc3 synthase in regulating neolacto-series glycolipid synthesis during embryonic development.

The glycosylation enzyme alpha 1,3 galactosyltransferase, which synthesizes the carbohydrate Gal alpha 1-3Gal beta 1-4GlcNAc-R, is active in non-primate mammals, prosimians and New World monkeys, but not in Old World monkeys, apes and humans. In this study, we have cloned and sequenced the enzyme expressed in a New World monkey, determined the exact size of the stem region and assessed the minimal size of catalytically active alpha 1,3 galactosyltransferase (alpha 1,3GT). Various primer sets were used in the polymerase chain reaction to generate cDNAs which coded for forms of alpha 1,3GT with deletions at the N- or C-terminal domains. The cDNA was inserted into the expression vector pPROTA which contains the coding sequence for protein A, and subsequently transfected into COS cells. The soluble chimeric products (truncated enzyme and protein A) were harvested from the cell culture medium using IgG-Sepharose beads and assayed for enzymatic activity. As many as 67 amino acids could be truncated at the amino terminal region of the luminal portion of the enzyme without affecting its catalytic activity. Truncation of 68, 69 and 74 amino acids resulted in a 50, 75 and > 95% loss in the in vitro catalytic actively, respectively. Introduction of a frameshift mutation which is characteristic of apes and human alpha 1,3GT gene resulted in the complete loss of enzyme activity. Moreover, truncation of as few as three amino acids at the carboxyl end of alpha 1,3GT resulted in complete loss of the catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)