Randall R. Reed

Identification of Ligands for Olfactory Receptors by Functional Expression of a Receptor Library

The recognition of odorants by olfactory receptors represents the first stage in odor discrimination. Here, we report the generation of an expression library containing a large and diverse repertoire of mouse olfactory receptor sequences in the transmembrane II-VII region. From this library, 80 chimeric receptors were tested against 26 odorants after transfection into HEK-293 cells. Three receptors were identified to respond to micromolecular concentrations of carvone, (-) citronellal, and limonene, respectively. We also found that the mouse I7 receptor, unlike the rat I7 receptor, prefers heptanal instead of octanal, as a result of a single valine-to-isoleucine substitution. This finding represents the beginning of a molecular understanding of odorant recognition. The identification, on a large scale, of cognate receptor-odorant interactions should provide insight into olfactory coding mechanisms.

Transcriptional control of preadipocyte determination by Zfp423

The worldwide epidemic of obesity has increased the urgency to develop a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARγ and several other transcription factors, but the molecular basis for preadipocyte determination is not understood. Using a new method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of Pparg in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. Short hairpin RNA (shRNA)-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte Pparg expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is markedly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates Pparg expression, in part, through amplification of the BMP signalling pathway, an effect dependent on the SMAD-binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.

Contribution of olfactory neural stem cells to tissue maintenance and regeneration

The olfactory neuroepithelium undergoes continual neurogenesis and, after extensive lesions, fully regenerates to maintain sensory function. The stem cell population underlying this regenerative capacity remains elusive. Here we show that mouse horizontal basal cells (HBCs) function as adult olfactory neuroepithelium neural stem cells and examine their distinct dynamics in olfactory neuroepithelium maintenance and regeneration. Fate-mapping analysis after olfactory neuroepithelium lesioning shows that HBCs are competent to regenerate both neuronal and non-neuronal olfactory neuroepithelium lineages. HBCs serve as a reservoir of long-lived progenitors that remain largely quiescent during normal neuronal turnover or even after acute, selective loss of mature neurons. Under these conditions, previously identified progenitors are largely responsible for tissue maintenance. Yet after extensive injuries that deplete resident neuronal precursors, HBCs transiently proliferate and their progeny fully reconstitute the neuroepithelium. Our data support a new model of adult neurogenesis in which distinct cell populations mediate normal neuronal turnover and neuronal replacement upon traumatic injury.

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in dictyostelium development

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.

Preferential expression of the Drosophila rutabaga gene in mushroom bodies, neural centers for learning in insects.

Seven lines were isolated with P element insertions in the cytogenetic vicinity of the learning and memory gene, rutabaga, from an enhancer detector screen designed to mark genes preferentially expressed in mushroom bodies. Six of these lines performed poorly in learning and memory tests, and several failed to complement an existing rutabaga allele. Molecular cloning revealed that the P elements were inserted in the putative promoter of the rutabaga gene. RNA in situ hybridization and immunohistochemistry demonstrated that the expression of the rutabaga gene, which encodes a Ca2+/calmodulin-responsive adenylyl cyclase, is markedly elevated in the mushroom bodies of normal flies and that the insertion elements compromised its expression in the new rutabaga mutants. The reisolation of a known learning and memory gene, but with a heretofore unknown expression pattern, strongly supports the postulate that mushroom bodies are principal sites mediating olfactory learning and memory.

Signaling Pathways in Odorant Detection

The application of molecular genetic techniques has led to the identification of olfactory-specific proteins that represent each component in a second messenger cascade. Our current understanding of signaling in the olfactory system suggests that receptor proteins of a large family, responsible in part for the specificity of the system, converge on a relatively small number of second messenger systems. The ability to express these elements in heterologous systems should allow for the reconstitution of the signaling cascade and provide insight into the specificity of ligand binding, pathway activation, and signal termination.

Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium

The mammalian main olfactory epithelium (MOE) recognizes and transduces olfactory cues through a G protein-coupled, cAMP-dependent signaling cascade. Additional chemosensory transduction mechanisms have been suggested but remain controversial. We show that a subset of MOE neurons expressing the orphan receptor guanylyl cyclase GC-D and the cyclic nucleotide-gated channel subunit CNGA3 employ an excitatory cGMP-dependent transduction mechanism for chemodetection. By combining gene targeting of Gucy2d, which encodes GC-D, with patch clamp recording and confocal Ca2+ imaging from single dendritic knobs in situ, we find that GC-D cells recognize the peptide hormones uroguanylin and guanylin as well as natural urine stimuli. These molecules stimulate an excitatory, cGMP-dependent signaling cascade that increases intracellular Ca2+ and action potential firing. Responses are eliminated in both Gucy2d- and Cnga3-null mice, demonstrating the essential role of GC-D and CNGA3 in the transduction of these molecules. The sensitive and selective detection of two important natriuretic peptides by the GC-D neurons suggests the possibility that these cells contribute to the maintenance of salt and water homeostasis or the detection of cues related to hunger, satiety, or thirst.

EBF2 determines and maintains brown adipocyte identity

The master transcription factor Pparγ regulates the general differentiation program of both brown and white adipocytes. However, it has been unclear whether Pparγ also controls fat lineage-specific characteristics. Here, we show that early B cell factor-2 (Ebf2) regulates Pparγ binding activity to determine brown versus white adipocyte identity. The Ebf DNA-binding motif was highly enriched within brown adipose-specific Pparγ binding sites that we identified by genome-wide ChIP-Seq. Of the Ebf isoforms, Ebf2 was selectively expressed in brown relative to white adipocytes and was bound at brown adipose-specific Pparγ target genes. When expressed in myoblasts or white preadipose cells, Ebf2 recruited Pparγ to its brown-selective binding sites and reprogrammed cells to a brown fat fate. Brown adipose cells and tissue from Ebf2-deficient mice displayed a loss of brown-specific characteristics and thermogenic capacity. Together, these results identify Ebf2 as a key transcriptional regulator of brown fat cell fate and function.

Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression

Phototransduction in retinal rods involves a G-protein-mediated signaling cascade that leads to cGMP hydrolysis and the closure of a cGMP-gated channel. This channel has recently been purified from bovine retina and molecularly cloned (Kaupp et al., 1989). We report here the cloning of cDNA and genomic DNA encoding the human rod cGMP-gated channel, based upon its homology to the bovine counterpart. The human mRNA structure differs from the bovine in containing an Alu repetitive element spliced into the 5′ untranslated region. The human cGMP-gated channel gene (CNCG) is located on chromosome 4 and contains at least 10 exons. One large exon encodes the carboxy-terminal two-thirds of the protein, whereas seven small exons encode the amino-terminal one-third of the protein. Alternative splicing removes one of the small exons in a subset of transcripts in the human retina, producing an internal in- frame deletion of 36 codons. When expressed in a human embryonic kidney cell line (293S), the full-length cDNA clone, but not the differentially spliced variant, produced functional ion channels broadly similar to the native channels in vertebrate rods.

Identification of DNA Recognition Sequences and Protein Interaction Domains of the Multiple-Zn-Finger Protein Roaz

ABSTRACT Roaz, a rat C2H2 zinc finger protein, plays a role in the regulation of olfactory neuronal differentiation through its interaction with the Olf-1/EBF transcription factor family. An additional role for the Roaz/Olf-1/EBF heterodimeric protein is suggested by its ability to regulate gene activation at a distinct promoter lacking Olf-1/EBF-binding sites. Using an in vitro binding-site selection assay (Selex), we demonstrate that Roaz protein binds to novel inverted perfect or imperfect repeats of GCACCC separated by 2 bp. We show that Roaz is capable of binding to a canonical consensus recognition sequence with high affinity (Kd = 3 nM). Analysis of the structural requirement for protein dimerization and DNA binding by Roaz reveals the role of specific zinc finger motifs in the Roaz protein for homodimerization and heterodimerization with the Olf-1/EBF transcription factor. The DNA-binding domain of Roaz is mapped to the N-terminal 277 amino acids, containing the first seven zinc finger motifs, which confers weak monomeric binding to a single half site and a stronger dimeric binding to the inverted repeat in a binding-site-dependent manner. Full-length protein can form dimers on both the inverted repeat and direct repeat but not on a single half site. These findings support the role of the TFIIIA-type Zn fingers in both protein-protein interaction and protein-DNA interaction and suggest distinct functions for specific motifs in proteins with a large number of zinc finger structures.