Tyrosinase gene expression is not detected in mouse brain outside the retinal pigment epithelium cells

Introduction

Tyrosinase, the rate-limiting enzyme for melanin biosynthesis, is encoded in the mouse by a gene located in chromosome seven which is expressed in two cellular types: melanocytes, derived from migrating neural crest cells, that colonize iris, cochlea, skin and choroid and retinal pigment epithelium (RPE) cells, derived from the optic cup. During mouse embryogenesis, tyrosinase gene can first be detected from +10.5 days postcoitum (d.p.c.) onwards in the RPE cells and from +16.5 d.p.c. in skin melanocytes (Beermann et al., 1992; Steel et al., 1992). These results were established by in situ hybridization on mouse embryos. No direct systematic assessment was reported on tyrosinase in mouse developing brain in these earlier studies.

In contrast to the above, a series of studies using transgenic tyrosinase reporter mice has been published documenting lacZ expression in developing and adult mouse brain, under the control of mouse tyrosinase gene promoter and 5′ upstream regulatory sequences (Tief et al., 1996a,b, 1998; Camacho-Hubner & Beermann, 2001). These data had been taken as an indirect indication for endogenous tyrosinase expression in the mouse developing brain.

However, to date, endogenous tyrosinase expression had not been specifically analysed in developing or adult mouse brain by direct means, namely in situ hybridization or real-time reverse transcription–polymerase chain reaction (RT–PCR) techniques. It is important that the original observations using indirect methods are confirmed, as tyrosinase plays a key role in axon guidance in the visual system (Jeffery et al., 1994; Jeffery, 1997) and may have a wider role regulating such mechanisms in the brain.

Materials and methods

Results and discussion

We have used in situ hybridization and real-time RT–PCR techniques to examine the expression of the mouse tyrosinase gene. Outbred albino NMRI mice were used for all the analyses. The tyrosinase protein made by the albino allele (Tyr^c; Jackson & Bennett, 1990) is not functional and, hence, pigment is not accumulated, avoiding interference with in situ hybridization signals. However, transcription is not affected and tyrosinase mRNA can be detected at wild-type levels (Beermann et al., 1992; Schedl et al., 1993).

First, using a tyrosinase-specific antisense RNA probe, we systematically hybridized whole-mount mouse embryos from early to mid-embryogenesis (+8.5 to +14.5 d.p.c.). We could only detect tyrosinase-positive signals from day +10.5 d.p.c. in the RPE cells (Fig. 1), as reported previously (Beermann et al., 1992). None of these embryos showed any sign of tyrosinase expression in developing brain outside the RPE cells. In particular, no sign of tyrosinase expression was observed in telencephalic vesicles from +10.5 d.p.c. (Fig. 1a), +11.5 d.p.c. (Fig. 1b), +12 d.p.c. (Fig. 1c) or +13.5 d.p.c. (Fig. 1f) where strong lacZ staining had been reported (Tief et al., 1996a; Camacho-Hubner & Beermann, 2001). Hybridization with tyrosinase sense control probes did not show any specific signal (Fig. 1d).

Second, we carried out in situ hybridization of tyrosinase RNA probes on histological sections. The expected tyrosinase-specific labelling of RPE cells (Fig. 2a and c) was found in mouse developing eyes at +13.5 and +14.5 d.p.c. whereas no tyrosinase-specific expression was detected in the telencephalon of +12.5 d.p.c. (Fig. 2e), +13.5 d.p.c. (Fig. 2f) and +14.5 d.p.c. (Fig. 2g) embryos. No specific signals were observed with the control probe (Fig. 2b, d, h–j). Tyrosinase-specific expression was also not detected in the telencephalon of +16.5 d.p.c. mouse embryos (not shown).

Third, we evaluated tyrosinase gene expression using real-time RT–PCR. The presence of tyrosinase transcripts in ectopic body locations had been addressed previously by standard PCR methods (Battyani et al., 1993; Xu et al., 1997; Tief et al., 1996b). However, those approaches were, at best, semiquantitative. In contrast, we decided to run real-time RT–PCR experimental assays using three experimental approaches. First, we quantified the relative tyrosinase gene expression in relation to an endogenous gene (β-actin), using the comparative C_T method (Applied Biosystems). Second, we calculated the tyrosinase gene expression applying a reference made up of different amounts of tyrosinase mRNA molecules and third, we considered the number of tyrosinase mRNA molecules in relation to the number of β-actin mRNA molecules. Specific tyrosinase gene expression was measured in RNAs obtained from skin and eyes of +18.5 d.p.c. fetuses, postnatal day 3 pups and adults, whereas very low or undetectable values, similar to those obtained in blank nontemplate control, were measured in ectopic (brain and liver) samples (Fig. 3).

The strong ectopic brain-specific expression of tyrosinase-lacZ transgenes, as previously reported, did not appear in all transgenic mouse lines analysed carrying different tyrosinase promoter fragments (reviewed in Camacho-Hubner & Beermann, 2001). In contrast, the addition of different regulatory sequences derived from the locus control region of the mouse tyrosinase gene (Montoliu et al., 1996) resulted in the loss of lacZ staining in the brain in all lines analysed (Camacho-Hubner & Beermann, 2001). These results prompted the authors to suggest the presence of a repressor sequence within the locus control region that could be responsible for the loss of brain-specific expression of tyrosinase-lacZ transgenes.

Alternatively, it might be argued that the addition of endogenous regulatory sequences, such as the locus control region, restores rather than alters the endogenous-specific tyrosinase gene expression pattern, overcoming the common position effects observed with standard transgenesis (Gimenez et al., 2001; Giraldo & Montoliu, 2001, 2002). Indeed, the use of lacZ reporter transgenes in mice has been often associated with ectopic expression (MacKenzie et al., 1997; Zhao & Overbeek, 1999), variegation (Montoliu et al., 2000; Ramirez et al., 2001), interference or silencing (Cohen-Tannoudji et al., 2000) in adults and during development, due to the prokaryotic nature of lacZ sequences.

The putative existence of tyrosinase-like activity in some areas of the brain has been controversial, considering catecholamine synthesis in tyrosine hydroxylase-deficient mice (Rios et al., 1999) or explaining the presence of neuromelanin (Xu et al., 1997; Ikemoto et al., 1998; Tief et al., 1998; Matsunaga et al., 1999). However, it has not been possible to document tyrosinase-specific activity in brain protein extracts (Tief et al., 1996b).

In summary, we could not find detectable levels of tyrosinase gene expression in developing, perinatal or adult mouse brain, other than RPE cells or skin melanocytes, after evaluation by in situ hybridization and real-time RT–PCR techniques. The lack of endogenous regulatory sequences plus the intrinsic nature of lacZ sequences might have triggered the brain-specific expression of previously reported tyrosinase-lacZ constructs. This unexpected pattern disappeared on the addition of the endogenous tyrosinase locus control region sequences, thereby counteracting the position effects of unprotected transgenes and restoring what appears to be the normal endogenous expression pattern of the mouse tyrosinase gene, as we have documented here.

Acknowledgements

This work was supported by funds from the Spanish Ministry of Science and Technology Bio97-0628, Bio2000-1653, FEDER 2FD1997-2059 and Laboratorios Dr Esteve S.A. to L.M. The authors are grateful to F. Solano, J.C. García-Borrón, M. Torres and G. Jeffery for useful comments and N. Mercader, M. Cantero and P. Cozar for technical assistance.

Abbreviations