The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dct tm1(Cre)Bee ] lack Dct mRNA and dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dct slt /Dct slt ) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.

The terminal differentiation of melanocytes is associated with the transcriptional activation of genes responsible for pigment production such as tyrosinase. Pigment cell-specific transcription factors, such as Mitf, as well as specific proximal and distal regulatory elements (DRE) are implicated in the tight control of tyrosinase expression during development and adulthood. Proper tyrosinase expression in melanocytes depends upon the presence of a DRE that is located at -15 kb and provides enhancer activity via a central element termed core-enhancer. In this report, we show that the transcription factors Sox10, Mitf and USF-1 are able to activate the core-enhancer in luciferase reporter assays. Comparative sequence analysis identified evolutionarily motifs resembling Sox10 binding sites that were required for full enhancer activity in melanoma cells and in tyrosinase::lacZ transgenic mice. Sox10 was able to bind the DRE in vitro and mutation of the conserved motifs abolished the enhancer transactivation mediated by Sox10. In addition, two highly conserved CAGCTG E-box motifs were identified that were also required for enhancer activity and for transactivation by Mitf. The results suggest that Sox10 directly, and Mitf, most likely indirectly, activate the tyrosinase enhancer, underlining the contribution of Sox10 to tyrosinase gene regulation in melanocytes.

Pigment cells of mammals are characterized by two different developmental origins: cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain, whereas melanocytes arise from the neural crest. The pigmentation gene tyrosinase is expressed in all pigment cells but differentially regulated in melanocytes and RPE. The tyrosinase promoter does not confer strong expression in pigment cells in vivo, while inclusion of a distal regulatory element at position -15 kb is necessary and sufficient to provide strong expression in melanocytes. Nevertheless, the regulatory elements responsible for correct spatial and temporal tyrosinase expression in the RPE remained unidentified so far. In this report, we show that a 186 kb BAC containing the tyrosinase gene provides transgene expression in both RPE and melanocytes indicating the presence of regulatory sequences required for expression in the RPE. A deletion analysis of the BAC was performed demonstrating that a RPE-regulatory element resides between -17 and -75 kb. Using multi-species comparative genomic analysis we identified three conserved sequences within this region. When tested in transgenic mice one of these sequences located at -47 kb targeted expression to the RPE. In addition, deletion of this regulatory element within a tyrosinase::lacZ BAC provided evidence that this sequence is not only sufficient but also required for correct spatial and temporal expression in the RPE. The identification of this novel element demonstrates that tyrosinase gene expression is controlled by separate distal regulatory sequences in melanocytes and RPE.

Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal pigment epithelium (RPE) originate from the optic cup of the developing forebrain. Previous studies have suggested that pigmentation genes are controlled by different regulatory networks in melanocytes and RPE. The promoter of the tyrosinase-related family gene Tyrp1 has been shown to drive detectable transgene expression only to the RPE, even though the gene is also expressed in melanocytes as evident from Tyrp1-mutant mice. This indicates that the regulatory elements responsible for Tyrp1 gene expression in the RPE are not sufficient for expression in melanocytes. We thus searched for a putative melanocyte-specific regulatory sequence and demonstrate that a bacterial artificial chromosome (BAC) containing the Tyrp1 gene and surrounding sequences is able to target transgenic expression to melanocytes and to rescue the Tyrp1b (brown) phenotype. This BAC contains several highly conserved non-coding sequences that might represent novel regulatory elements. We further focused on a sequence located at -15 kb, which we identified as a melanocyte-specific enhancer as shown by cell culture and transgenic mice experiments. In addition, we show that the transcription factor Sox10 can activate this conserved enhancer. The presence of a distal Tyrp1 regulatory element, which specifies melanocyte-specific expression, supports the idea that separate regulatory sequences can mediate differential gene expression in melanocytes and RPE.

The secreted signaling molecule fibroblast growth factor 8 (Fgf8) is an essential component of certain embryonic signaling centers including the mid-hindbrain (isthmic) organizer, the first branchial arch (BA1), and the apical ectodermal ridge (AER). In these signaling centers Fgf8 transcripts are expressed in a dynamic and transient fashion, but the mechanism by which this highly specific expression pattern is established remains largely unknown. We used DNA sequence comparisons coupled to transgenic approaches to obtain insight into the structure and function of regulatory elements in the Fgf8 locus. First, a bacterial artificial chromosome (BAC) containing the mouse Fgf8 gene partially rescues the embryonic lethality of Fgf8-deficient mice and controls Fgf8-specific gene expression of a coinjected lacZ reporter transgene. Second, sequence comparison of vertebrate Fgf8 loci revealed evolutionarily highly conserved noncoding sequences that were unexpectedly located mainly 3' of the Fgf8 coding region. Third, in transgenic mice some of these elements were sufficient to target expression to the AER, tail bud, and brain, including the isthmic organizer, indicating that they may represent Fgf8 cis-acting elements. Collectively, these data identify novel regulatory elements of the Fgf8 gene sufficient to drive expression to regions of known Fgf8 activity.