Clonal Coat Color Variation Due to a Transforming Gene Expressed In
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Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6447-6451, August 1991 Developmental Biology Clonal coat color variation due to a transforming gene expressed in melanocytes of transgenic mice (simian virus 40 large tumor antigen/tyrosinase promoter/pigmentary patterns/melanocyte development/phenoclones) MONIKA BRADL, LIONEL LARUE, AND BEATRICE MINTZ* Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 Contributed by Beatrice Mintz, May 8, 1991 ABSTRACT Transgenic mice of an inbred black strain from genetically black eggs, as proved to be the case in the were previously produced with the Tyr-SV40E transgene, majority of mice in the present study. It could also occur by comprising simian virus 40 transforming sequences driven by differential induction of pigment in parts of the coat of mice the tyrosinase promoter, in order to obtain melanomas; the from genetically albino eggs, as was the case in an unrelated animals were found to be lighter than normal in coat color, to series injected with another transgene (B.M. and M.B., various degrees. As described here, hypopigmentation resulted unpublished data). from diminished differentiation ofmelanized pigment granules In natural genotypes of mice with a coat color pattern due in the melanocytes of the hair bulbs in vivo and occurred to gene expression in melanocytes, the patterns are ideal or autonomously in cultured melanocytes. Whereas some of the modified versions of the clonal developmental history of mice had single-color coats, most (7/13) had coats of two or melanoblasts, in which separate clones may be of different three colors; in addition, one single-color founder produced a colors or color intensities. This conclusion was drawn from two-color descendant. These eight mice had patterns seen in models in allophenic mice experimentally produced from natural genotypes; the most striking were transversely striped conjoined early embryo cells of dissimilar color genotypes to various extents, with regions of left-right asymmetry on (5-8). Melanoblasts originating in the neural crest (9), in either side of the dorsal midline. The patterns visualized the predetermined numbers, migrate dorsoventrally on each side same clonal developmental territories of coat melanocytes and form transverse mitotic clones (5). In the allophenic displayed in allophenic mice that are formed from conjoined animals, clonal color differences are due to genotypic differ- early embryo cells of different color genotypes. Some of the ences (10). This is not the case in nonexperimental mice of Tyr-SV40E transgenics were also cellular genotypic mosaics, natural genotypes. Among the latter, single-allele activation probably arising by late integration ofthe transgene. However, per cell, and per clone, explains the patterns in X chromo- one founder with a coat some-linked heterozygotes (11). Among other genotypes, the transgenic completely striped proved autosomal homozygotes are the most baffling. Their patterns to be true-breeding, with autosomal inheritance of the pattern. are apparently due to phenoclones-phenotypically different The inherited striped pattern thus exemplifies the formation of but genetically identical clones in which the same gene must phenotypically different but genetically identical developmen- be yielding more than one kind or amount of product in tal clones, or phenoclones, among cells of the same type. This mitotic lineages ofthe same cell type (6-8). The phenomenon line of transgenic mice provides exceptional material for ex- is presumably not limited to pigment cells. perimental analysis of the molecular basis for clonal variation We report here that the multicolored patterns in our in gene expression and of the fate of oncogenic phenoclones of Tyr-SV40E transgenic mice resemble those previously seen melanocytes occurring in the same individual. in allophenic mice and further confirm the developmental lineages of melanocytes. Some of the transgenic cases are Transgenic mice were recently described that are prone to clearly due to genetic mosaicism, possibly resulting from late melanoma in the eyes and skin (1) and to other tumors integration of the transgene. At least one autosomal trans- associated with melanosis (2). The melanomas arose through genic case is clearly an example of phenoclones and thus expression ofthe transgene in pigment cells, as expected, due provides unique material for analyses ofgene expression and to its tyrosinase gene promoter controlling expression of the of susceptibility to melanomas. simian virus 40 (SV40) early region transforming sequences, including the large tumor antigen (T antigen). Reduced coat pigmentation occurred, to varying degrees, reflecting the MATERIALS AND METHODS relative numbers of transgene copies in many but not all The group of transgenic mice previously described (1) pro- cases. As reported here, the light coat colors were found to vided the material for this study. The animals were produced arise from subnormal and sporadic melanization of pigment by injecting Tyr-SV40E recombinant DNA into fertilized granules in the melanocytes of the hair bulbs. This result is eggs of the C57BL/6 (black nonagouti) inbred strain. The consistent with incomplete differentiation of the cells and fusion construct contains the SV40 early region genes, in- may have arisen either by impairment of their maturation or cluding the coding sequences ofthe transforming large tumor by reinforcement of their proliferative potential (3), rather (T) and small tumor (t) antigens, under the control of 2.53 than by a direct effect of the transgene on melanin synthesis. kilobases (kb) of mouse tyrosinase gene promoter sequence. Given the fact that many single-gene mutations in mice lead Positive animals were identified, and the copy number of to multicolored coats (4), one would expect that any trans- the transgene determined, by Southern blot analysis of DNA gene capable of affecting color would also produce some from tail biopsies, tissue samples collected at autopsy, or skin multicolored individuals. This could occur by differential melanocytes grown in culture as described (12-14). Genomic reduction of pigment in various areas of the coat of animals DNA was digested with Pst I to determine whether single or multiple integration of the transgene had occurred and to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: SV40, simian virus 40; T antigen, large tumor antigen. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6447 6448 Developmental Biology: Bradl et al. Proc. Natl. Acad Sci. USA 88 (1991) distinguish between head-to-tail and tail-to-tail integration. The restriction fragments were resolved by gel electropho- resis, transferred to Nytran (Schleicher & Schuell), and hybridized with a SV40 T-antigen-specific RNA probe. Known amounts of the donor gene were used as a standard. Founder animals, which were hemizygous for the transgene, were in some cases mated to C57BL/6 controls. A RESULTS Of the 13 original Tg(Tyr-SV40E) mice in the series, 6 had single-color coats, ranging from a light black or dark brown- ish grey to pale grey; these were the founders of lines 5 and 9-13. The remaining 7, the founders or potential founders of lines 1-4 and 6-8, had coats of two colors (black or blackish and grey, two shades of grey, or grey and white) or three colors (black, grey, and or grey white, two shades of plus B white). In addition, a new two-color case arose spontane- ously in a descendant of the all-grey line 5; her coat had two shades of grey and she is included here as the potential founder of line 5a. These eight multicolored cases are grouped in Table 1 according to three general classes representing different, or partly different, developmental modes oforigin to which their patterns are here ascribed. The three groups are as follows: striped patterns with white, striped patterns without white, C and headspot patterns. The cases will be individually char- acterized in the order in which they are listed. FIG. 1. The coats of transgenic mice of lines 1, 2, and 3 display Striped Patterns with White (Lines 1-3). The original mice a few transverse stripes against a darker background. (A) The line 1 designated as line 1 (a male) and line 2 (a female) were not mouse was medium grey and had a "belt" of two light grey stripes, previously reported; they were severely retarded and were one on each side, meeting in the dorsal midline and merging ventrally with a large white belly spot; a barely visible transverse stripe at the sacrificed at 3-4 weeks of age. The line 3 animal was a sterile level of the left shoulder; a light grey stripe behind the right ear and male with undescended testes and was aspermia; his growth on the left front face; and white feet and tail tip. (B) The line 2 mouse somewhat retarded and he was killed at 15 weeks of age had a black background with a prominent grey stripe on the left side, because of advanced metastatic ocular melanoma (1). Mul- flanked by two small white spots, and merging ventrally with a large tiple copies of the transgene, from approximately 15 to 25, white belly spot; grey areas near the left ear and front ofthe face; and were present in these mice (Table 1). Integration had oc- white feet and tail tip. (C) The line 3 mouse was light grey and had curred in multiple sites, thus augmenting the chances for a sharp white stripe on each side, placed asymmetrically; a white insertional mutagenesis. Each founder had tandem copies of belly spot; and white feet and tail tip. the transgene oriented head-to-tail; tail-to-tail array may have occurred at some sites in lines 1 and 2 (data not shown). was pale grey with a sharp white stripe on each side, The mouse of line 1, shown in Fig.