Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis.

The human RUNX3/AML2 gene belongs to the 'runt domain' family of transcription factors that act as gene expression regulators in major developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression pattern of Runx1. E10.5 and E14.5-E16.5 embryos were analyzed using both immunohistochemistry and beta-galactosidase activity of targeted Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them plays a role in embryogenesis.

Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin.

In skin, multipotent stem cells generate the keratinocytes of the epidermis, sebaceous gland, and hair follicles. In this paper, we show that Tcf3 and Lef1 control these differentiation lineages. In contrast to Lef1, which requires Wnt signaling and stabilized beta-catenin to express the hair-specific keratin genes and control hair differentiation, Tcf3 can act independently of its beta-catenin interacting domain to suppress features of epidermal terminal differentiation, in which Tcf3 is normally shut off, and promote features of the follicle outer root sheath (ORS) and multipotent stem cells (bulge), the compartments which naturally express Tcf3. These aspects of Tcf3's action are dependent on its DNA binding and Groucho repressor-binding domains. In the absence of its beta-catenin interacting domain, Lef1's behavior (Delta NLef1) seems to be markedly distinct from that of Delta NTcf3. Delta NLef1 does not suppress epidermal differentiation and promote ORS/bulge differentiation, but rather suppresses hair differentiation and gives rise to sebocyte differentiation. Taken together, these findings provide powerful evidence that the status of Tcf3/Lef complexes has a key role in controlling cell fate lineages in multipotent skin stem cells.

A common human skin tumour is caused by activating mutations in beta-catenin.

WNT signalling orchestrates a number of developmental programs. In response to this stimulus, cytoplasmic beta-catenin (encoded by CTNNB1) is stabilized, enabling downstream transcriptional activation by members of the LEF/TCF family. One of the target genes for beta-catenin/TCF encodes c-MYC, explaining why constitutive activation of the WNT pathway can lead to cancer, particularly in the colon. Most colon cancers arise from mutations in the gene encoding adenomatous polyposis coli (APC), a protein required for ubiquitin-mediated degradation of beta-catenin, but a small percentage of colon and some other cancers harbour beta-catenin-stabilizing mutations. Recently, we discovered that transgenic mice expressing an activated beta-catenin are predisposed to developing skin tumours resembling pilomatricomas. Given that the skin of these adult mice also exhibits signs of de novo hair-follicle morphogenesis, we wondered whether human pilomatricomas might originate from hair matrix cells and whether they might possess beta-catenin-stabilizing mutations. Here, we explore the cell origin and aetiology of this common human skin tumour. We found nuclear LEF-1 in the dividing tumour cells, providing biochemical evidence that pilomatricomas are derived from hair matrix cells. At least 75% of these tumours possess mutations affecting the amino-terminal segment, normally involved in phosphorylation-dependent, ubiquitin-mediated degradation of the protein. This percentage of CTNNB1 mutations is greater than in all other human tumours examined thus far, and directly implicates beta-catenin/LEF misregulation as the major cause of hair matrix cell tumorigenesis in humans.

De Novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin.

An effector of intercellular adhesion, beta-catenin also functions in Wnt signaling, associating with Lef-1/Tcf DNA-binding proteins to form a transcription factor. We report that this pathway operates in keratinocytes and that mice expressing a stabilized beta-catenin controlled by an epidermal promoter undergo a process resembling de novo hair morphogenesis. The new follicles formed sebaceous glands and dermal papilla, normally established only in embryogenesis. As in embryologically initiated hair germs, transgenic follicles induce Lef-1, but follicles are disoriented and defective in sonic hedgehog polarization. Additionally, proliferation continues unchecked, resulting in two types of tumors also found in humans. Our findings suggest that transient beta-catenin stabilization may be a key player in the long-sought epidermal signal leading to hair development and implicate aberrant beta-catenin activation in hair tumors.

Overexpression, solubilization and purification of rat and human olfactory receptors.

The superfamily of olfactory receptor genes, whose products are thought to be activated by odorant ligands, is critical for odor recognition. Two olfactory receptors, olp4 from rat and OR17-4 from human, were overexpressed in Sf9 insect cells. The presence of the proteins in cell membranes was monitored by immunoblotting with peptide-specific polyclonal antibodies directed against the C-terminal sequences of these receptors and with a mAb against an N-terminal octapeptide epitope tag. A DNA sequence that codes for a His6 tag, which binds tightly to a Ni2+-chelate-affinity column, was incorporated into the N-termini of both genes. The expressed olfactory receptors were found mainly in the cell-membrane fraction. The proteins were difficult to solubilize by many detergents and only lysophosphatidylcholine was found to be both suitable for efficient solubilization of the overexpressed olfactory receptors and compatible with the purification system used. After solubilization, the olfactory receptors were purified to near homogeneity by affinity chromatography on nickel nitrilotriacetic acid resin and by cation-exchange chromatography. Electrophoresis of the purified proteins and visualization with Coomassie Blue staining or by immunoblotting with specific antibodies, revealed bands of 32, 69 and 94 kDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins. The oligomeric forms were resistant to reduction and alkylation, and are therefore thought to be held together by non-covalent hydrophobic interactions that are resistant to SDS. This finding is similar to previous observations for other guanine-nucleotide-binding-regulatory-protein-coupled receptors. Reconstitution in phospholipid vesicles showed that the purified olfactory receptors insert specifically into the lipid bilayer. This provides a means to study functional reconstitution with putative transduction components such as olfactory guanine-nucleotide-binding-regulatory protein.

Olfactory receptor proteins. Expression, characterization and partial purification.

A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation was a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a specific polyclonal antibody against a C-terminal peptide of olp4. Translation in vitro, followed by immunoprecipitation, showed a major olp4-specific band of 27-29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag-olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag-olp4 product on the cell surface of the infected host Sf9 cells, with the N-terminus and C-terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.

Olfactory receptors: transduction, diversity, human psychophysics and genome analysis.

The emerging understanding of the molecular basis of olfactory mechanisms allows one to answer some long-standing questions regarding the complex recognition machinery involved. The ability of the olfactory system to detect chemicals at sub-nanomolar concentrations is explained by a plethora of amplification devices, including the coupling of receptors to second messenger generation through GTP-binding proteins. Specificity and selectivity may be understood in terms of a diverse repertoire of olfactory receptors of the seven-transmembrane-domain receptor superfamily, which are probably disposed on olfactory sensory neurons according to a clonal exclusion rule. Signal termination may be related to sets of biotransformation enzymes that process odorant molecules, as well as to receptor desensitization. Many of the underlying molecular components show specific expression in olfactory epithelium, with a well-orchestrated developmental sequence of emergence, possibly related to sensory neuronal function and connectivity requirements. A general model for molecular recognition in biological receptor repertoires allows a prediction of the number of olfactory receptors necessary to achieve efficient detection and sheds light on the analogy between the immune and olfactory systems. The molecular cloning and mapping of a human genomic olfactory receptor cluster on chromosome 17 provides insight into olfactory receptor diversity, polymorphism and evolution. Combined with future genotype-phenotype correlation, with particular reference to specific anosmia, as well as with computer-based molecular modelling, these studies may provide insight into the odorant specificity of olfactory receptors.