Disorganized olfactory bulb lamination in mice deficient for transcription factor AP-2ɛ
Introduction
The olfactory bulb (OB) is a laminated structure, with layers representing specific limited neuron types, neuronal processes and connections required for processing and relaying odor information to the piriform (olfactory) cortex (Shepherd, 2004, Shepherd, 2007). Starting from the outermost layer, the murine OB contains the olfactory nerve layer (ONL), glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (MCL), internal plexiform layer (IPL), and granule cell layer (GCL). The projection neurons – mitral cells and tufted cells – receive, process, and relay odor information. Mitral cell bodies are normally confined to the MCL, whereas tufted cells bodies are located more superficially in the OB, either in the inner GL or dispersed within the EPL. The development of the ordered structure of the OB requires both innervation by OSN axons and appropriate neural interactions between different OB layers. In this regard, surgical manipulation and explant studies have demonstrated that the olfactory epithelium stimulates outgrowth of mitral and tufted cell projection neurons, possibly via a secreted ligand(s) of the TGFβ superfamily (Graziadei and Monti-Graziadei, 1992, Lopez-Mascaraque et al., 2005, Slotnick et al., 2004, Tran et al., 2008). Moreover, two groups of genes have been identified whose deletion in mouse affects the development of the OB layers. Disruption of the first group of genes, including Fez, Pax6, Emx1/2 and Dlx5, prevents the OSN axons from reaching the OB (Harrison et al., 2008, Hirata et al., 2006, Jimenez et al., 2000, Laub et al., 2006, Long et al., 2003, Nomura and Osumi, 2004, Yoshida et al., 1997). Mutations in the second group of genes, including Dlx1/2, Tbr1, Reelin, Lis1, and Arx, impact neurogenesis, neural migration, and terminal differentiation of interneurons in the OB (Albrecht et al., 1996, Andrade et al., 2007, Bulfone et al., 1995, Bulfone et al., 1998, Hallonet et al., 1998, Ko et al., 2001, Perez-Garcia et al., 2004, Soria et al., 2004, Tomasiewicz et al., 1993, Toresson and Campbell, 2001, Whitley et al., 2005, Wyss et al., 1980, Yoshihara et al., 2005, Yun et al., 2003).
However, many questions remain with regard to the formation and function of the OB laminar structure. In particular, while recent work has provided information on the migration of principal neuron precursors and formation of the MCL (Blanchart et al., 2006), little is known about the factors contributing to these developmental processes. Disruption of the MCL could potentially alter the development of adjacent layers, the EPL, which includes secondary dendrites from mitral/tufted cells, and the IPL. The IPL contains tufted cell axons, and recent reports suggest that these connect mirror symmetric glomeruli in each OB that receive input from neurons expressing the same olfactory receptor molecule (Liu and Shipley, 1994, Lodovichi et al., 2003, Marks et al., 2006). Since the postsynaptic targets of the tufted cells are inhibitory granule cells it has been hypothesized that the axons within the IPL form intrabulbar inhibitory circuits that are used to coordinate topographically organized neural networks in the OB (Liu and Shipley, 1994). However, as yet there are no suitable animal models to test this hypothesis directly.
The mammalian AP-2 transcription factor family consists of five members: AP-2α, AP-2β, AP-2γ, AP-2δ and AP-2ɛ (Eckert et al., 2005, Feng and Williams, 2003, Moser et al., 1995, Tummala et al., 2003, Wang et al., 2004, Zhao et al., 2001a). Three of the mouse AP-2 genes, Tcfap2a, Tcfap2b, and Tcfap2c – encoding AP-2α, AP-2β and AP-2γ, respectively – are essential for embryogenesis and post-natal viability (Auman et al., 2002, Brewer et al., 2004, Nottoli et al., 1998, Satoda et al., 2000, Schorle et al., 1996, Zhao et al., 2001b). Much less is known concerning the function of Tcfap2d and Tcfap2e, the mouse genes encoding AP-2δ and AP-2ɛ. These two genes are more restricted in their expression than the other three family members, with prominent expression detected mainly in the developing midbrain for AP-2δ and the forebrain for AP-2ɛ. Expression of Tcfap2e is first observed in the OB primordium at E11.5 and then becomes refined to the projection neurons prior to birth, before declining to undetectable levels by P14 (Feng and Williams, 2003, Tummala et al., 2003, Wang et al., 2004). The dynamic expression pattern of AP-2ɛ suggested a role for this transcription factor in early OB development (Feng and Williams, 2003). Here, we have investigated this possibility by generating mice containing a Tcfap2e-null allele (AK allele). Analysis of homozygous mutant mice demonstrates an essential role for AP-2ɛ in the establishment of OB lamination as well as for the appropriate orientation and connectivity of individual projection cells.
Section snippets
Generation and analysis of the Tcfap2e Cre recombinase knock-in AK allele
AP-2ɛ has a restricted expression pattern in the OB and represents the earliest known marker for the developing OB (Feng and Williams, 2003, Tummala et al., 2003, Wang et al., 2004). To understand the function of AP-2ɛ in OB development, we used ES cell gene targeting to create a null allele in the endogenous mouse Tcfap2e locus. This AK allele disrupts exon 4 of the gene, which is essential for the DNA binding activity of AP-2ɛ (Fig. 1A). Subsequent PCR and Southern blot analyses demonstrated
Discussion
Previous studies have indicated that Tcfap2e expression is one of the earliest events marking the formation of the specialized OB during mouse embryogenesis (Feng and Williams, 2003, Wang et al., 2004). Therefore, in this study, we have generated and characterized Tcfap2e-null mice to investigate the role of AP-2ɛ in the development and function of the olfactory system. Analysis of these mice reveals a specific requirement for AP-2ɛ in development of the OB, particularly in the normal
Generation of the Tcfap2e AK allele
The Tcfap2e AK allele was generated by targeting IRES-Cre and pGK-Neo sequences into exon 4 of Tcfap2e (Fig. 1A). Briefly, 5′ and 3′ homologous DNA fragments (4.8 and 4.5 kb respectively) flanking Tcfap2e exon 4 were PCR-derived from 129 ES cell genomic DNA using primer pairs AK1/2 and AK3/4 (Table 1), respectively. PCR products were sequenced, and cloned into a targeting vector SP73 (Promega). IRES-Cre and a pGK-Neo cassette flanked with Frt sites were both inserted between the 5′ and 3′
Acknowledgments
We thank Dr. Vida Melvin for her efforts in reading, revising and improving the writing of this manuscript. We are grateful to Bärbel Böttger, Robert Cornell, Brian Parr and Shane Rolen for discussion and assistance. The 5B8 (NCAM) and 2H3 (neurofilament) monoclonal antibodies developed by T.M. Jessell and J. Dodd were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa
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