Transcription factors in melanocyte development ... - Semantic Scholar

Mechanisms of Development 101 (2001) 47±59

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Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf Thomas J. Hornyak a,b,*, Daniel J. Hayes a,b, Ling-Yu Chiu c, Edward B. Ziff c a

The Ronald O. Perelman Department of Dermatology, New York University Medical Center, New York, NY 10016, USA b Department of Dermatology, Henry Ford Health Sciences Center, One Ford Place 5D, Detroit, MI 48202, USA c Department of Biochemistry and Howard Hughes Medical Institute, New York University Medical Center, New York, NY 10016, USA Received 20 April 2000; received in revised form 17 November 2000; accepted 20 November 2000

Abstract A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing b-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf mi, with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of b-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf mi/1 embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quanti®cation and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf mi/Mitf mi embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Transcription factors; Melanocyte development: Pax-3; Mitf

1. Introduction Mutations in the transcription factor genes Pax3 and Mitf are responsible for the murine coat color mutants of the Splotch (Sp) and microphthalmia classes, respectively. The classic Splotch mutant, Sp (Russell, 1947; Auerbach, 1954) is a semidominant mutation which, when heterozygous, consists of white spotting of the tail, and/or abdomen and feet (Auerbach, 1954) and, when homozygous, features neural tube defects (Auerbach, 1954; Silvers, 1979) as well as abnormalities of development of neural crest-derived structures such as melanocytes, spinal ganglia, Schwann cells, and cardiac structures (Auerbach, 1954; Franz, 1989, 1990). The molecular mutation resulting in these

* Corresponding author. Tel.: 11-313-874-6376; fax: 11-313-876-2380. E-mail address: [email protected] (T.J. Hornyak).

defects is a complex mutation within intron 3 of the Pax3 gene resulting in the production of four distinct, aberrantly spliced mRNA transcripts. Three of these transcripts result in premature termination with the absence of an intact paired domain, octapeptide motif, and paired homeodomain. The fourth lacks exon 4, resulting in the loss of the C-terminal part of the paired domain as well as loss of the octapeptide motif (Epstein et al., 1993). Embryos homozygous for the Sp allele and the Sp 2H allele have similar phenotypes including death in utero by embryonic day (E) 16 of development. The mutation responsible for the Sp 2H phenotype is a 32 bp deletion within the paired homeodomain region of Pax3 (Epstein et al., 1991). Another splotch allele, Splotch-delayed (Sp d), features a less severe phenotype, with homozygous mutant embryos surviving until birth (Dickie, 1964). Splotch-delayed results from a point mutation in the paired domain of the Pax3 gene (Vogan et al.,

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T.J. Hornyak et al. / Mechanisms of Development 101 (2001) 47±59

1993). The shared severity of the homozygous phenotypes of the Sp and Sp 2H alleles, though resulting from distinct molecular mutations, has led to the suggestion that each represents a loss-of-function mutation in the Pax3 gene (Epstein et al., 1993). Supporting the contention that mutations in regions of the gene encoding distinct DNA-binding regions can abrogate Pax3 function is the observation that the presence of both paired and paired homeodomain binding sites, as well as appropriate spacing between the sites, has been shown to be important for high af®nity binding of recombinant Pax-3 to DNA oligonucleotides (Phelan and Loeken, 1998). The set of mouse mutants comprising the murine microphthalmia class of coat color mutants results from mutations in a basic-helix-loop-helix-leucine zipper transcription factor gene, Mitf (Hodgkinson et al., 1993), discovered on the basis of a transgenic insertional mutant. The Mitf mi mutant allele, the classic radiation-induced mouse microphthalmia mutation (Hertwig, 1942), results in the deletion of one of four arginine residues in the basic region of the gene (Hodgkinson et al., 1993). The Mitf mi mutation is semidominant, in that mice heterozygous for the mutation exhibit white spotting on the belly, head, and/or tail (Silvers, 1979), but no other apparent abnormalities. However, mice homozygous for the Mitf mi mutation feature total absence of coat pigmentation, severe microphthalmia, osteopetrosis, failure of incisor eruption, and deafness. Mutant Mitf mi protein can heterodimerize with TFE3, another member of the same transcription factor class, but cannot bind DNA as a homodimer or as a heterodimer with TFE3. Moreover, mutant Mitf mi protein inhibits the binding of TFE3 homodimer to DNA, exhibiting a dominant-negative effect in vitro (Hemesath et al., 1994). However, the undetectability of mutant Mitf mi protein in vivo in neural crest-derived cells (Nakayama et al., 1998), combined with the predominantly cytoplasmic localization of mutant Mitf mi protein observed following transfection (Takebayashi et al., 1996), suggests that in vivo this mutant may represent a loss-of-function, rather than a dominant-negative, phenotype. Several lines of evidence suggest relationships between Pax-3 and Mitf activities during melanocyte development. The similarities between the phenotypes of the Splotch heterozygotes and the Mitf semidominant heterozygotes, with white spotting in characteristic locations, suggest that these distinct transcription factors may regulate common mechanisms promoting melanocyte development. In humans, mutations in MITF and PAX3 cause different types of the congenital disorder of pigmentation Waardenburg syndrome, with PAX3 mutations causing types more severe than types caused by MITF mutations (Read and Newton, 1997). The regions of Pax3 and Mitf expression in the truncal neural tube overlap, with Mitf expression con®ned to a small group of cells in the dorsal midline (Nakayama et al., 1998), and Pax3 more widely expressed dorsal to the sulcus intermedius from earlier in development (Goulding et al., 1991). Additionally, human PAX3 has

been shown in cotransfection experiments to upregulate expression of a region of the human MITF promoter when that region contains consensus sequences for binding the PAX3 paired domain and paired homeodomain (Watanabe et al., 1998). To assess the roles of the Pax-3 and Mitf transcription factors in melanocyte development, we have utilized a transgenic mouse expressing b-galactosidase (lacZ) from the promoter of the dopachrome tautomerase gene (Dct). Dct, also known as tyrosinase-related protein-2 or TRP-2, is an early melanoblast marker (Steel et al., 1992), and transgenic mice expressing lacZ from the Dct promoter have been used previously to examine the effect of a c-kit receptor tyrosine kinase mutation upon melanocyte development (Mackenzie et al., 1997), as well as the effect of the Patch (Ph) mutation upon melanocyte development in Ph/1 heterozygotes (Zhao and Overbeek, 1998). We have generated a similar transgenic mouse line and intercrossed it with the Sp and Mitf mi murine coat color mutants to examine the effects of Pax-3 and Mitf on melanocyte development in vivo. The results suggest, in agreement with previous reports (Opdecamp et al., 1997), that Mitf plays a critical role in early melanocyte development, particularly by facilitating the survival of melanoblasts up to the migration staging area of neural crest cell development during early migration. We also demonstrate that heterozygosity for the Mitf mi mutation results in reduced melanoblast numbers during early melanocyte development, a result that may in part explain the semidominant phenotype of certain Mitf alleles. Paradoxically, analysis of melanoblast numbers at successive developmental stages during dorsolateral pathway migration shows that heterozygosity for the Mitf mi mutation increases the rate by which melanoblasts increase in number in the heterozygous embryo, suggesting that Mitf may exhibit different activities during early melanoblast development than during melanoblast migration and differentiation. In contrast, we show that Pax-3 exerts its major effect upon the generation of melanoblasts or melanoblast precursors in early melanocyte development, with little or no effect upon melanoblast migration, since melanoblasts are observed in characteristic locations in the embryo during development, albeit greatly reduced in number. Together, these results support a model for melanocyte development whereby factors other than Pax-3 are required to specify either melanoblasts or restricted progenitors of melanoblasts within the neural tube, with Pax-3 activity necessary to expand this initial speci®ed population of cells. Mitf activity in parallel appears to promote the survival of these precursor cells in a gene-dosage dependent manner both within the neural tube and immediately following delamination from it. Proper gene dosage of Mitf then appears to be necessary to regulate, directly or indirectly, the rate of melanoblast accrual in the dorsolateral pathway, a function that may ensure proper timing between continued cell proliferation and migration to facilitate the distribution of melanoblasts to the entire murine integument.


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Fig. 1. Expression pattern of the PDct/lacZ transgene. (A,B) Whole-mount photomicrographs of 28-somite, TS 16 (E9.5±9.75) embryo showing transgene expression in optic cup (oc, A), a small island of expression in dorsal telencephalon (t, A), and in individual cells overlying (small arrowhead, B) and lateral to (large arrowhead, B) the neural tube at and caudal to the hindlimb bud (hl, B). Cells lateral to the neural tube correspond to the neural crest migration staging area. (C,D) Whole-mount photomicrographs of E10.5 transgenic embryos showing continued expression in the optic cup; increased expression in the dorsal telencephalon (t, C); expression in individual cells in the cephalic, trunk, and tail regions of the embryo; expression in cells of the roof of the hindbrain (short arrow, C); and ectopic, segmental expression in cells associated with the dorsal root ganglion (arrowheads, C and D) most prominent at and caudal to the level of the hindlimb bud (hl, D). Expression in a discrete region of the ventral mesencephalon (long arrow, C) is observed. (E) Expression in the dorsal telencephalon (t), optic cup, in individual cells throughout the cephalic region and around the otic vesicle (ov) in E12.5 transgenic embryo. (F) Cross-section of trunk of E11.5 embryo in a region lateral to the neural tube (nt) shows individual cells present in the dorsolateral pathway of neural crest cell migration. Dorsal root ganglion (drg) is present immediately lateral to neural tube. (G) Melanoblasts surrounding otic vesicle (ov) near cephalic region in E11.5 transgenic embryo. (H) Transgene expression at E11.5 is con®ned to the dorsal aspect of the telencephalic neuroepithelium as shown by this coronal cross-section. (I,J) Transgene expression in the eye at E10.5 (I) and E11.5 (J) is con®ned to the retinal pigmented epithelium (RPE) layer and optic stalk (os, J), and restricted from the neural retina (nr) and lens vesicle (lv). (K) High-magni®cation view of melanoblasts overlying the neural tube at E11 with ectopic expression in dorsal root ganglia. These melanoblasts have a more spindled and dendritic shape than those of the E9.5±9.75 embryo in (B) which are rounder. (L) Groups of melanocytes expressing the transgene (arrowheads) among cells of the hair follicle (hf) in the skin of post-natal day 1 (P1) transgenic mice. Scale bar, 0.1 mm (G,I,L), 0.2 mm (F,J,K), 0.4 mm (B,H), 0.7 mm (D), 1 mm (A,E), 1.7 mm (C).

2. Results 2.1. Expression pattern of the PDct/lacZ transgene and marker analysis The transgene expression pattern was similar to a previously reported lacZ transgenic line generated with Dct upstream regulatory sequence (MacKenzie et al., 1997). Expression was observed in the developing eye, the

telencephalic buds, and in individual cells overlying and lateral to the neural tube in the caudal region of the embryos as early as E9.5±9.75 (28 somites, Theiler stage (TS) 15) (Fig. 1A,B). At E10.5, expression was observed in the eye, the telencephalon, the roof of the hindbrain (Fig. 1C), and in individual cells throughout the embryo at this point (Fig. 1C,D) and at E12.5 (Fig. 1E). Truncal sections of E11.5 embryos reveal that these cells are present in the dorsolateral pathway of neural crest cell migration (Fig. 1F), consis-

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tent with the location of migratory melanoblasts. We also noted individual cells surrounding the otic vesicle at this stage (Fig. 1G), again consistent with the location of migratory melanoblasts in previous studies (Steel et al., 1992). Expression of the transgene in the telencephalon at E11.5 was con®ned to the dorsal aspect (Fig. 1H). Consistently noted was expression in the ventral mesencephalon (Fig. 1C); this expression has not yet been further characterized. Expression in the eye was restricted to the presumptive retinal pigment epithelium (RPE) layer at E10.5 (Fig. 1I) and extended to the optic stalk at E11.5 (Fig. 1J). Migratory melanoblasts were observed overlying the neural tube at E11 (Fig. 1K), as was expression associated with dorsal root ganglia (Fig. 1C,D,K). As expected, expression was con®ned to cells amid the follicular epithelium in the skin of post-natal day 1 (P1) mice (Fig. 1L). In a previous study with a similar transgene (Zhao and Overbeek, 1998), expression of endogenous Dct was compared with expression of Dct promoter-driven lacZ. Expression of the lacZ transgene was noted in all areas of endogenous Dct expression as well as in areas such as the dorsal root ganglia (DRG) that we have noted. To provide further evidence that transgene-expressing cells present in the characteristic location of neural crest-derived melanoblasts indeed represent these cells, we performed double-label immuno¯uorescence using the rat monoclonal antibody ACK2 (Ogawa et al., 1991) to label the alternative melanoblast marker c-kit (Opdecamp et al., 1997) and a rabbit polyclonal antibody to label b-galactosidase. Examination of cryosections from E15 PDct/lacZ transgenic embryos revealed coexpression of c-kit and b-galactosidase in the epidermal layer, the expected location of melanoblasts at this stage of develop-

Fig. 2. Co-expression of c-kit and b-galactosidase in epidermal melanoblasts of E15 PDct/lacZ transgenic embryos. (A,D) Expression of b-galactosidase (green) in embryonic skin of E15 PDct/lacZ embryos. (B,E) Expression of c-kit (red) in same sections. (C,F) Merged images demonstrate co-expression of markers in same cells (yellow). Scale bar, 10 mm.

ment (Fig. 2); similar coexpression was observed in E13.5 embryos (data not shown). Although not all ACK2-labeled cells exhibited b-galactosidase labeling, a fact that may be due to the presence of mast cells in the skin that express ckit but not b-galactosidase (Kim et al., 1999), differential af®nities of primary antibodies for their respective antigens, and possible differences between the subcellular localization of antigens in different cells, nevertheless 23/23 cells identi®ed initially as positive for b-galactosidase expression also labeled positive for c-kit in one section of epidermis examined, indicating that in this transgenic line transgene expression most likely does not occur in other cell types that are in the characteristic location of melanoblasts. Taken together, these results and the results of others show that

Fig. 3. Melanoblast location and number in E10 transgenic progeny of Mitf mi/1 heterozygous cross. Transgenic 1/1 (A,D), Mitf mi/1 (B,E), and Mitf mi/Mitf mi (C,F) embryos from the same embryonic litter are shown at E10 with comparisons between cephalic views (A±C) and views of the dorsal trunk (dorsal neural tube) (D±F) at the level of the hindlimb. (A) Cephalic view of wild-type embryo with expression in melanoblasts, dorsal telencephalon, optic cup, and roof of hindbrain. (B) Reduced number of melanoblasts in cephalic region of heterozygous embryo. (C) No melanoblasts in cephalic region of homozygous mutant embryo. Expression in dorsal telencephalon, optic cup, and roof of hindbrain is unaffected. Note expression in tail region overlying neural tube most caudally (arrowhead) and in migration staging area more rostrally (arrow). (D) View of dorsal embryo at level of the hindlimb (hl) with transgene-expressing cells overlying neural tube (nt) and in migration staging area (msa). (E) Reduced numbers of transgene-expressing cells (committed melanoblasts or restricted progenitors) overlying neural tube and in migration staging area of heterozygous embryo at level of hindlimb. (F) Reduced numbers of transgene-expressing cells overlying neural tube and in migration staging area of homozygous embryo at level of hindlimb. Scale bar, 0.5 mm (A±C) and 0.3 mm (D±F).


the PDct/lacZ transgene, as noted with similar transgenes (MacKenzie et al., 1997; Zhao and Overbeek, 1998), can function as a marker of migratory melanoblasts and postmigratory melanocytes, and that small differences between the regulatory units chosen for transgene construction do not signi®cantly affect the transgene expression pattern. 2.2. Mitf mi/Mitf mi embryos do not have melanoblasts entering the dorsolateral pathway of neural crest cell migration Embryos resulting from transgenic heterozygous crosses could be analyzed by staining for lacZ expression to determine the presence of and number of melanoblasts in embryos at different stages of development. At E10 (TS 16), 1/1 embryos featured a signi®cant number of migratory melanoblasts in the cranial region of embryos (Fig. 3A) as well as overlying the neural tube at the level of the hindlimb (Fig. 3D). In contrast, an Mitf mi/ Mitf mi embryo at this stage contained no detectable migratory melanoblasts in the cranial region (Fig. 3C), with only a few scattered cells detectable overlying the neural tube at the level of the hindlimb (Fig. 3F). Transgene expression in cells of the dorsal telencephalon, the optic cup, and the roof of the hindbrain is unaffected in the Mitf mi/Mitf mi embryo at E10 (Fig. 3C). At E11.5 (TS 19) and E12.5 (TS 21), the ®ndings are comparable. At E11.5 (TS 19), numerous melanoblasts are present in the truncal region and cranial region of a 1/1 embryo, whereas no melanoblasts are seen in these regions of an Mitf mi/Mitf mi transgenic littermate at this stage (data not shown). At E12.5 (TS 21), evidence of further melanoblast proliferation and migration is apparent by the greater number of cells in the truncal and cranial regions and, in the cranial region, by the presence of melanoblasts in more frontal locations of 1/1 embryos (Fig. 4A,D). No melanoblasts are apparent in these regions of an Mitf mi/Mitf mi embryo despite persistent transgene expression in the telencephalon, DRG, and RPE (Fig. 4C,F). Although at E10 (Fig. 3F), a few cells could be observed immediately overlying the neural tube of Mitf mi/ Mitf mi embryos, at no developmental stage were melanoblasts of Mitf mi/Mitf mi embryos observed lateral to the migration staging area or to the groups of transgene-expressing cells associated with the DRGs, suggesting that they were either not capable of entering or had not survived to be able to enter the dorsolateral pathway of neural crest cell migration. These results show that Mitf appears to be required to promote the development of melanoblasts in vivo at developmental stages prior to entry of melanoblasts to the dorsolateral pathway of neural crest cell migration. Although we did not examine Mitf mi/Mitf mi embryos directly with other melanoblast markers to substantiate further the absence of melanoblasts, there is substantial evidence from the study of Dct expression in human (Yasumoto et al., 1997) and murine (Hemesath et al., 1998; Hornyak, unpublished observations) melanocytes and melanoma cells to suggest

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that Mitf is not required for Dct expression in these cell types. The development of melanoblasts is not merely delayed in Mitf mi/Mitf mi embryos, since embryos as advanced as E15 in additional experiments did not contain any detectable melanoblasts. Although fewer numbers of cells are observed overlying the neural tube in the E10 Mitf mi/Mitf mi embryo relative to the 1/1 embryos, suggesting that fewer precursors are produced within the neural tube, the inability to visualize the precursors within the neural tube imposes limitations upon the ability of the PDct/lacZ transgene to provide information about numbers of melanocyte precursors prior to emergence from the neural tube. These results also show that Mitf is not absolutely required for Dct expression, since Dct expression can be observed both in the RPE and in the telencephalon in the absence of functional Mitf activity that causes a failure of melanoblast development.

Fig. 4. Melanoblast location and number in E12.5 transgenic progeny of Mitf mi/ 1 heterozygous cross. Transgenic 1/1 (A,D), Mitf mi/ 1 (B,E), and Mitf mi/Mitf mi (C,F) embryos from the same embryonic litter are shown at E12.5 with comparisons between whole-mount (A±C) and cephalic (D±F) views. (A) Whole-mount photomicrograph of wild-type transgenic embryo. Transgene expression in melanoblasts is visualized in the cephalic region, near the otic vesicle (ov), and throughout the trunk. (B) Reduced numbers of melanoblasts in heterozygous embryo. (C) No melanoblasts in homozygous mutant embryo. Expression in telencephalon and optic cup is unaffected. (D) Higher magni®cation view of cephalic region of wild-type embryo in (A), with expression in melanoblasts, telencephalon, and optic cup. (E) Reduced numbers of melanoblasts in cephalic region of heterozygous embryo. A greater number of melanoblasts is present compared to the heterozygous E11.5 transgenic embryo in Fig. 3E. (F) No melanoblasts in homozygous mutant embryo. Scale bar, 1.5 mm (A± C) and 0.8 mm (D±F).

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2.3. Heterozygosity for the Mitf mi allele results in a reduction of melanoblasts but a higher rate of increase in melanoblast number during dorsolateral pathway migration Sequence length polymorphisms resulting from the ampli®cation of both pedigree tail DNA and embryo yolk sac DNA from mouse chromosome 6 permitted distinguishing between embryos homozygous and heterozygous for the Mitf mi mutation. Comparison of melanoblast number in Mitf mi/1 embryos at different stages (Figs. 3B and 4B,E) with 1/1 littermates (Figs. 3A and 4A,D) revealed decreased melanoblast numbers at each developmental stage in Mitf mi/1 embryos compared with 1/1 littermates. This difference in melanoblast number is evident from the earliest stages of melanoblast delamination from the neural tube, since comparison of Fig. 3D,E suggests that fewer transgene-expressing cells delaminate from the dorsal neural tube at E10 in Mitf mi/1 embryos at the level of the hindlimb. The increase in melanoblast number in migratory cells seen in Mitf mi/1 embryos occurring between E10 (Fig. 3B) and E12.5 (Fig. 4E), and con®rmed by counting melanoblasts in these embryos and in additional embryo sets (Fig. 5), shows that these cells are capable of proliferation, suggesting that the effect of Mitf mi heterozygosity upon melanoblast number occurs prior to, or at, the earliest stages of migration. The presence of a limited number of melanoblasts in a well-de®ned region, the cephalic region, of E10±E12.5 embryos rendered it possible to count them to con®rm the visual impression that melanoblast number was greater in 1/1 embryos compared to Mitf mi/1 embryos. Melanoblast counts of embryos with 1/1 and Mitf mi/1 genotypes at developmental ages E10, E10.5, E11.5, and E12.5 were performed as described in Experimental Procedures and

mean and standard deviation determined for each category. The results of these tabulations are shown in Fig. 5, and con®rm the visual impression that the melanoblast number is lower at each of the above developmental ages in the Mitf mi/1 embryos compared to the 1/1 embryos. The higher numbers of cells noted at advancing gestational ages in both the 1/1 and Mitf mi/1 backgrounds in the cephalic region indicated that melanoblasts proliferate in each genetic background during their migratory phase. However, other factors such as programmed cell death, cell migration to and from the dorsolateral pathway, gain of transgene expression by recruitment of precursor cells, or loss of transgene expression could also affect the number of labeled cells present in an embryo at a given developmental stage, hence modifying how the rate of increase of melanoblasts might otherwise re¯ect absolutely a rate of melanoblast proliferation during initial migration. Nonetheless, determining the rate at which melanoblast numbers increase in each genetic background may provide insight into how different levels of Mitf affect these activities that contribute to the overall number of melanoblasts present in the dorsolateral pathway at a given stage of development. To estimate a rate at which melanoblast numbers increase in each genetic background, the cell count data was ®t by non-linear regression to the equation nt n0 eln2=td ´t

which describes proliferative cell growth, where nt is the number of cells present at time t; n0 is the number of cells at zero time, here de®ned as E10; and td is the doubling time of the cells, here taken as the doubling time of transgeneexpressing cells wherein competing rates of programmed cell death, gain and loss of transgene expression, and migratory events modify the ideal rate of cell proliferation. Regression analysis to the general form of the Eq. (1) y Aebt

Fig. 5. Quanti®cation and modeling of melanoblast numbers in 1/1 and Mitf mi/1 embryos. Melanoblasts in the cephalic region of E10 through E12.5 embryos were counted and means and standard deviations calculated for each developmental stage. Data points were analyzed by non-linear regression to Eq. (2), and Eq. (1) subsequently used to determine the cell doubling time, or td, for each genotype. Symbols and sample size for embryos of each genotype: 1/1 (O), n 3 (E10), 4 (E10.5), 4 (E11.5), and 2 (E12.5); Mitf mi/1 (X), n 2 (E10), 5 (E10.5), 7 (E11.5), and 1 (E12.5). Error bars represent standard deviation.

1

2

yielded coef®cients for t of 0.56 ^ 0.11 and 0.81 ^ 0.13 for 1/1 and Mitf mi/1 embryos, respectively, where the error represents the standard error of measurement. This results in calculated doubling times of 1.2 days (range, 1.03±1.54 days) and 0.85 days (range, 0.73±1.01 days), respectively, or a 41% greater doubling time for cephalic migratory melanoblasts in 1/1 embryos compared to Mitf mi/1 embryos. These data support the notion that Mitf has a gene dosagedependent effect upon melanoblast survival during the earliest stages of melanocyte development, prior to entry to the dorsolateral pathway, when committed melanoblasts and/or restricted precursors are present within and are delaminating from the neural tube. During dorsolateral pathway migration, the higher rate of increase of melanoblast number observed in heterozygous embryos may re¯ect a distinct activity of Mitf that relates differentiation and/or proliferation during the migratory phase of melanocyte development, an indirect compensatory phenomenon in melanoblasts


lacking the full complement of Mitf, or other factors such as programmed cell death as described above. Despite the lower rate of proliferation in 1/1 embryos, melanoblast numbers are not normalized between embryos of the two genotypes, since signi®cant differences in absolute melanoblast number persist at E12.5, as seen in Fig. 5, as well as at E15, as seen below. 2.4. Ventral migration of melanoblasts and belly spotting Mitf mi/1 heterozygote adults exhibit a characteristic ventral belly patch, and may also feature white spotting on the head and tail. Examination of the ventral trunk of 1/1 and Mitf mi/1 embryos at E15, just prior to involution of the umbilical hernia, shows that the aberrant distribution of melanoblasts in the integument responsible for the white spotting phenotype is apparent already at this stage, since the region of abdominal integument adjacent to the umbilical hernia is densely and uniformly populated with melanoblasts in 1/1 embryos (Fig. 6A), whereas melanoblasts are more sparse and less uniformly distributed in Mitf mi/1 embryos (Fig. 6B). Although these results do not demonstrate a cellular mechanism for the development of the ventral white spot in heterozygous mice, they suggest an embryonic origin of ventral white spotting that is correlated with a more irregular, sparse distribution of melanoblasts in this area as early as E15, a distribution that may be linked to the differential rate of melanoblast increase noted between 1/1 embryos and Mitf mi/1 embryos.

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all, similar expression patterns in the optic cup and dorsal telencephalon, showing that expression of the transgene is not absolutely dependent upon Pax-3 function. Additional comparison revealed that migratory melanoblasts were indeed present in the Sp/Sp embryo at this stage, in contrast to the Mitf mi/Mitf mi embryos considered earlier, which do not feature migratory melanoblasts at this stage. Melanoblasts in Sp/Sp embryos were predominantly limited to the upper trunk near the forelimb (Fig. 7F), the region of the otic vesicle (Fig. 7H), and the caudal trunk, although they are greatly reduced in number in these regions compared to control transgenic littermates (Fig. 7E,G). Similar ®ndings are present in an E12.5 litter (TS 21), with a viable Sp/Sp embryo containing migratory melanoblasts in the trunk, near the otic vesicle, and in the cranial region (Fig. 7J). However, the numbers of melanoblasts in these areas in a littermate exhibiting a wild-type expression pattern (Fig. 7I) are much greater. Close examination of the region of the neural tube defect in a Sp/Sp embryo at this

2.5. Sp/Sp mutant embryos show markedly reduced melanoblast number, but normal migration in regions of embryos unaffected by neural tube defects Embryonic litters were obtained from heterozygous crosses of Sp/1 mice after intercrossing the PDct/lacZ transgene into the male member of the cross. Litters were obtained prior to the point of Sp/Sp embryonic demise and transgene expression visualized by lacZ staining. Homozygous Sp/Sp embryos could be identi®ed by the presence of neural tube defects (Tajbakhsh et al., 1997). Examination of the small region of the neural tube affected by the neural tube defect in Sp/Sp embryos at E11 (TS 18) and E12.5 (TS21) revealed individual transgene-expressing cells lined at the lateral margins of the neural tube defect (Fig. 7A,B). Comparisons of melanoblast location and number were made between transgenic embryos with and without neural tube defects in embryonic litters at different developmental stages. In these experiments, we did not distinguish between embryos of the 1/1 and Sp/1 genotypes, but simply compared embryos lacking visible neural tube defects, all of which exhibited the normal transgene expression pattern and may be either 1/1 or Sp/1, with Sp/Sp embryos that bear neural tube defects. In an E12 (TS 20) litter containing both transgenic control littermates (Fig. 7C) and Sp/Sp (Fig. 7D) embryos, comparison of these embryos showed, ®rst of

Fig. 6. Melanoblast location and number on ventral integument of E15 1/1 and Mitf mi/1 transgenic embryos. Transgenic 1/1 (A) and Mitf mi/1 (B) embryos are shown at E15. Black lines have been added to demarcate the ventral integument from the umbilical hernia. (A) Ventral integument of 1/ 1 embryos (below black border) adjacent to umbilical hernia (above black border) shows dense, uniformly distributed melanoblasts. (B) Ventral integument of Mitf mi/1 embryos (below black border) in similar location shows sparse, irregularly distributed melanoblasts. Views are representative of two 1/1 embryos and seven Mitf mi/1 embryos in this transgenic litter. Scale bar, 0.2 mm.

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reduces melanocyte number compared to embryos with at least one normal Pax-3 allele, suggesting that the major effect of Pax-3 activity upon the early melanoblast population is to foster proliferation of either restricted progenitor cells or committed melanoblasts, most likely within the dorsal neural tube where Pax-3 expression has been described during development (Goulding et al., 1991). The developmental defects in other cells and structures derived from the neural crest in Sp/Sp mutant embryos, such as dorsal root ganglia (Auerbach, 1954) and Schwann cells (Franz, 1990), most strongly suggests an effect by Pax3 upon restricted precursor cells that might give rise to cells of the glial lineage as well as melanocytes (Climent, 1990; Lahav et al., 1998). The substantial decrease in segmental ectopic transgene expression around the dorsal root ganglion in Sp/Sp embryos (Fig. 7D,J) is consistent with this idea. In contrast to Mitf mi/Mitf mi embryos, some melanoblasts were observed in characteristic locations of the melanoblast migratory pathway in Sp/Sp embryos, demonstrating the continued survival of melanoblasts able to commence migration in a Pax-3 homozygous mutant background.

3. Discussion 3.1. Mitf and melanocyte survival

Fig. 7. Melanoblast location and number in transgenic progeny of Sp/1 heterozygous cross. (A) E11 Sp/Sp embryo, neural tube defect (NTD) in caudal neural tube at the level of hindlimb. Melanoblasts are at the lateral margins of the neural tube defect (arrowheads). (B) E12.5 Sp/Sp embryo, view of caudal neural tube in region of NTD. Melanoblasts are aligned at lateral edge of NTD (arrowheads), but are observed entering the dorsolateral migratory pathway rostral to the defect (arrow). (C,D) Control (C) and Sp/Sp (D) transgenic littermates at E12 (TS20). Sp/Sp embryo with NTD (ntd, D) shows reduction in number of migratory melanoblasts compared to control. (E±H) Control (E,G) and Sp/Sp (F,H) transgenic littermates at E11.5. Sp/Sp embryo has fewer melanoblasts (arrowheads) in the areas of the rostral trunk near the forelimb (F) and the otic vesicle (H) compared to the corresponding areas (E,G, respectively) of a control littermate. (I,J) Control (I) and Sp/Sp (J) transgenic littermates at E12.5 (TS21). Similar pattern as in (C) and (D) with fewer melanoblasts in regions of Sp/Sp embryo such as near the otic vesicle (arrowhead) and locations on the trunk (arrowhead). Scale bar, 0.5 mm (A); 0.4 mm (B); 1 mm (C,D,I,J); 0.3 mm (E±H).

stage shows continued presence of melanoblasts at the lateral margin of the defect but normal migration in a region immediately rostral to the defect (Fig. 7B), thus emphasizing the distinction between the majority of regions of the embryo where the neural tube is intact and melanoblast migration occurs normally, and the focal regions containing neural tube defects where melanoblast migration is affected. These results show that loss of Pax-3 activity greatly

The importance of Mitf in melanocyte development was ®rst established by the correlation of the cloned gene with the microphthalmia phenotype via a transgenic insertional mutant lacking coat pigmentation (Hodgkinson et al., 1993). The importance was further demonstrated in experiments with Mitf homozygous mutant embryos that showed an absence of Mitf-expressing cells in Mitf vga-9 mice, the transgenic insertional mutant null allele, as well as a paucity of Mitf-expressing cells in the embryos of Mitf mi-ew mice. The Mitf-expressing cells observed in vivo in Mitf mi-ew homozygous mutant embryos did not express Dct, although a very small number of cells in a single neural crest cell culture from an Mitf mi-ew/Mitf mi-ew embryo were found to express Dct (Opdecamp et al., 1997). In another study, similar observations were made in cross-sections of embryos of Mitf mi mice, where Mitf expression is observed in a limited number of neural crest-derived cells with no expression of Mitf protein or Dct detected (Nakayama et al., 1998). In the aforementioned study, expression of Mitf and Dct was also studied in retinal pigmented epithelial (RPE) cells, a nonneural crest-derived pigment cell, in both wild-type mice and Mitf mutant embryos. Expression of both Mitf and Dct was maintained in RPE cells in wild-type and homozygous mutant embryos throughout development, thus demonstrating a difference between Mitf effects in neural crest-derived and non-neural crest-derived pigment cells. Our results with Mitf mi embryos transgenic for the PDct/ lacZ transgene support and extend these ®ndings. We observe strong expression of the PDct/lacZ transgene in


cells of the optic cup in wild-type, heterozygous and homozygous mutant Mitf mi embryos through E12.5 as shown here, and through E15 in additional mutant embryo sets (data not shown). However, in neural crest-derived cells in transgenic, Mitf mi/Mitf mi embryos, expression of the transgene is observed only in a small number of cells overlying the neural tube and immediately lateral to the neural tube, at least to the migration staging area. Further work will be required to classify these cells, using endogenous Dct and Mitf as markers to determine whether they are committed melanoblasts or restricted progenitor cells that show evidence of transgene expression either because of increased sensitivity of the lacZ reporter relative to an in situ hybridization probe, because of aberrant transgene expression due to incomplete promoter-enhancer elements, or because of stability of the lacZ transcript or protein. However, it seems apparent from our studies that expression of Mitf is required for the presence of committed melanoblasts along the dorsolateral pathway of neural crest cell migration, and that function-blocking mutations in Mitf compromise melanoblast survival up to this point. It is dif®cult to exclude, on the basis of the existing data, a separate activity for Mitf that promotes melanoblast proliferation, as opposed to survival, in a gene dosage-dependent manner either within the neural tube or immediately following delamination from the neural tube prior to migration. However, this interpretation would require postulating a separate mechanism for melanoblast or restricted progenitor cell increase that becomes inversely dependent upon Mitf gene dosage once cell migration commences. The effects of heterozygous mutations in Mitf upon melanocyte development have not previously been reported, though the semidominant nature of alleles such as Mitf mi, with characteristic white spotting in the adult heterozygote, implies that there exists some effect upon melanocyte development in the heterozygous state. Our characterization of heterozygous Mitf mi embryos from E10 to E12.5, comprising most of the period of melanoblast proliferation and migration in the dorsolateral pathway during embryogenesis prior to epidermal entry, shows that heterozygotes consistently feature decreased numbers of melanoblasts throughout this stage of development, demonstrating the need for proper Mitf gene dosage prior to this stage to generate an appropriate number of melanoblasts. Surprisingly, the result of nonlinear regression analysis of melanoblast numbers in different genetic backgrounds showed that migratory melanoblasts in Mitf mi/1 embryos, despite being less numerous, increase in number more rapidly than melanoblasts in 1/1 embryos, implying that Mitf may possess a different activity in migratory melanoblasts than in premigratory melanoblasts or restricted progenitor cells. It is not unreasonable to suggest that Mitf is a transcription factor that may have different roles at distinct stages in melanoblast development and melanocyte differentiation. For example, our studies and others (Opdecamp et al., 1997; Nakayama et al., 1998) support an important role for Mitf in melanoblast survival prior to migra-

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tion, but Mitf has also been found to regulate the promoters of the Tyr and Tyrp1 genes (Bentley et al., 1994; Yasumoto et al., 1994; Yavuzer et al., 1995), genes important for the differentiated phenotype of the melanocyte that are not expressed until well after dorsolateral pathway migration begins (Steel et al., 1992). Mitf as a transcription factor may be responsible for inducing a set of genes required for melanoblast survival during cell fate determination and delamination from the neural tube, yet switch roles to induce expression of a set of differentiation genes, perhaps by interacting with other cofactors, during dorsolateral pathway migration. Melanoblasts during dorsolateral pathway migration may represent `transit amplifying cells' (Hall and Watt, 1989; Potten and Loef¯er, 1990), cells that have the ability to expand initially the number of differentiating cells but exhibit diminishing self-renewal capabilities as differentiation proceeds. Mitf may have a dosage-dependent, differentiation-promoting activity during dorsolateral pathway migration that, directly or indirectly, results in a limitation of the proliferation rate of melanoblasts in wild-type embryos relative to heterozygous embryos. Interestingly, the apparent doubling time in vivo in the wild-type background of 1.2 days, or 29 h, is quite similar to the doubling time of 32 h reported for Dct- expressing melanoblasts in heterogeneous neural crest cell primary cultures in the presence of endothelin 3, which supports melanoblast proliferation under these conditions (Opdecamp et al., 1998). 3.2. Pax-3 and melanoblast development The small number of melanoblasts observed in characteristic locations in Sp/Sp embryos has dual implications. First of all, it implies that proper migration of melanoblasts can occur in a Pax3 homozygous mutant background. This is in contrast to the Mitf homozygous mutants, in which melanoblasts were not observed to enter the dorsolateral pathway of neural crest cell migration. The conclusion is that Pax3 is not absolutely required for melanoblast survival and entry to the dorsolateral pathway. However, the small number of melanoblasts also implies that Pax3 has a profound effect upon the generation of melanoblasts or restricted progenitor cells early in development. This effect is probably not limited to melanoblasts, since the analysis of Sp/Sp embryos has shown that the development of other neural crest derivatives is severely affected in Sp/Sp mutants. In this same study, pigment cell development was not observed, but it is possible that the techniques used in this study, involving the transplantation of murine neural tube into either chick embryonic coelom or mouse eye anterior chamber followed by sustained culture periods (Auerbach, 1954), were not sensitive enough to detect the small number of melanoblasts we observe in our study. In a more recent study utilizing transgenic mice expressing lacZ from the Wnt1 enhancer, thereby marking neural crest cells migrating in the ventromedial pathway, the number of visible neural crest-derived cells in transgenic Sp/Sp embryos was also greatly reduced,

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although migration of these cells from Sp/Sp embryos was unaffected when transplanted into a similar location of a chick embryo (Serbedzija and McMahon, 1997). These results in conjunction with ours suggest that the primary effect of Pax3 upon neural crest cells is upon a neural crest progenitor cell early in neural crest development, probably within the dorsal neural tube, with the generation of these precursor cells greatly dependent upon Pax3 activity. In regions of Sp/Sp embryos affected by neural tube defects, entry of melanoblasts into the dorsolateral pathway was compromised. Cells were lined up at the lateral margins of such defects. Since the migration of neural crest-derived cells is likely to be dependent upon the interaction of integrin-receptor-expressing neural crest cells with speci®c extracellular matrix proteins (Henderson and Copp, 1997), this observation may re¯ect a disruption of the normal distribution of extracellular matrix components in the region of the dorsal neural tube in Sp/Sp embryos, a disruption that results in an inability of melanoblasts to enter the dorsolateral pathway.

namely survival of the melanoblast prior to entry to the dorsolateral pathway. Alternatively, PAX3 may be required for MITF activation in the human but not in the mouse.

3.3. Pax-3 and Mitf: relationship during melanocyte development Although the transactivation of the human MITF promoter by human PAX3 protein that has been demonstrated (Watanabe et al., 1998) supports a role for PAX3 in the induction of MITF expression early in development, the ®ndings of the present study suggest a more complicated relationship than a straightforward requirement for initial activation of Mitf expression by Pax3 during melanocyte development. If Pax3 is required for Mitf expression and subsequent melanocyte development, it is dif®cult to explain why some melanocyte development should proceed in the Pax3 mutant background when none does beyond the migration staging area in Mitf mutant backgrounds. It is more consistent with the data in our study that Pax3 and Mitf act in parallel to promote different aspects of melanocyte development, with Mitf promoting survival of committed melanoblasts and affecting the rate of increase of differentiating melanoblasts and Pax3 promoting expansion of a pool of restricted progenitor cells, as proposed in Fig. 8. The regulation of the Mitf gene that has been demonstrated on the molecular level may be important for other aspects of Mitf activity apart from its role in initial melanoblast survival. Mitf is a transcription factor that may have multiple roles in the development and differentiation of the melanocyte, as demonstrated by its regulation of the tyrosinase and TRP-1 promoters, promoters for genes expressed by differentiated melanocytes (Bentley et al., 1994; Yasumoto et al., 1994, 1997; Yavuzer et al., 1995; Bertolotto et al., 1996; Hemesath et al., 1998), and by its role in survival of the adult cell, as judged from the phenotype of the Mitf mi-vit mouse, with loss of coat pigmentation beginning later in adult life (Lerner et al., 1986). Pax3 may regulate Mitf in these stages which are distinct from the stage where we have shown Mitf to be initially important,

Fig. 8. Model summarizing melanocyte development with results from wild-type and mutant embryos examined. Schematic diagrams represent cross-sections through vertebrate embryos during the developmental time period studied. (A) Melanocyte development in wild-type embryos. Unknown factors (?) specify a small group of cells in the dorsal neural tube (nt) as committed melanoblasts or restricted progenitor cells, an event that is independent of Pax-3 activity. Mitf is important initially for promoting initial survival and/or increase in this cell population during delamination from the neural tube, movement to the migration staging area (msa), and entry to the dorsolateral migratory pathway. During migration, the level of Mitf affects the rate of increase in melanoblast number via a separate, direct or indirect activity, here depicted as Mitf 0 . (B) Melanocyte development in Mitf mi/1 embryos. A fewer number of melanoblasts are present early in migration, but these increase in number approximately 8-fold from E10 to E12.5, as determined from Fig. 5, in contrast to the 4-fold increase observed in wild-type embryos depicted in (A). (C) Melanocyte development in Mitf mi/Mitf mi embryos. A limited number of melanoblasts or restricted progenitors survive to the migration staging area, but do not enter the dorsolateral migratory pathway. (D) Melanocyte development in Sp/Sp embryos. A limited number of melanoblasts are generated with absence of Pax-3 activity, but these, in contrast to the cells in Mitf mi/Mitf mi embryos, are capable of migration to the characteristic locations of melanoblasts. Other abbreviation: l, lumen of neural tube.


4. Experimental procedures 4.1. Transgene construction and generation of transgenic mice The linearized transgene was the 8.3 kb fragment from the plasmid pPDct/lacZ. Plasmid pPDct/lacZ was generated from the expression plasmid pPDct. Plasmid pPDct was constructed by ligating pcDNA3DCMV, the 4.5 kb fragment lacking the cytomegalovirus (CMV) promoter obtained from a BglII ± EcoRV digest of the eukaryotic expression vector pcDNA3 (Invitrogen), with the 3.4 kb BamHI ± Eco47III Dct fragment from the clone l B plasmid (Budd and Jackson, 1995). This clone l B fragment contains nucleotides 23240 to 1148 relative to the transcriptional start site in the EMBL/GenBank version of the sequence (X85126 M. Musculus Tyrp2 gene) published previously (Budd and Jackson, 1995). However, a comparison of the electronic version of the sequence and the published version reveals that the second and third nucleotides of the hexanucleotide recognition sequence for Eco47III (GCGCTC) are missing in the published version. Nonetheless, Eco47III still cleaves at this location. Plasmid pPDct/luc was generated by subcloning the 2.7 kb XhoI ± SalI fragment, containing the luciferase cDNA linked to an SV40 polyadenylation sequence, from the plasmid pGL2-Basic (Promega) into the XhoI site 3 0 to the EcoRV/Eco47III linkage in pPDct. Plasmid pPDct/lacZ was generated by subcloning the SalI fragment from the plasmid pNL-bgal (gift of Dr Bob Schneider, Department of Biochemistry, NYU Medical Center), that contains the lacZ fragment from the plasmid pCH110 (Pharmacia), into the XhoI site of pPDct. Transgenic mice were generated of fertilized outbred CD1 zygotes by the Skirball Institute Transgenic Animal Facility, NYU Medical Center. Of 42 founder mice screened, ®ve exhibited insertion of the transgene with Southern blot. Three of the ®ve founders were found to express the transgene at signi®cant level, with two exhibiting an identical expression pattern similar to that reported previously (Mackenzie et al., 1997). 4.2. Generation of transgenic coat color mutants and embryos Two Mitf mi/ 1 B6C3Fe-a/a female mice were obtained from The Jackson Laboratory and mated with a single transgenic founder. Mitf mi/ 1 heterozygotes produced from these matings were used to generate a colony of mice for use in the experiments. Heterozygotes were identi®ed by coat color spots and/or tail bands and transgene presence determined by PCR. Transgenic, heterozygote males were mated with transgenic or non-transgenic heterozygote females to obtain timed pregnancies. Four C57BL6 Sp/ 1 male mice were obtained from The Jackson Laboratory and rederived into speci®c-pathogen free C57BL6 mice to obtain Sp/ 1 females. A single transgenic male from the

57

same transgenic line was mated to a Sp/ 1 female to obtain transgenic heterozygotes. One male transgenic heterozygote was subsequently mated to another Sp/ 1 female to generate the two male transgenic heterozygotes used for this study. These males were mated with C57BL6 Sp/ 1 females to obtain timed pregnancies. Embryos and yolk sacs were harvested at the indicated gestational ages, ®xed for 2 h in 0.4% paraformaldehyde in PBS, stained overnight with X-gal solution, and post®xed in 4% paraformaldehyde for 4 h prior to whole-mount photomicroscopy or cryosectioning. Embryos were incubated overnight in 50% glycerol/PBS for whole-mount photomicroscopy. Embryos were cryoprotected in 30% sucrose/PBS prior to freezing in OCT and cryosectioning. 4.3. Identi®cation and characterization of embryos Mitf mi/ 1 and Mitf mi/Mitf mi embryos were identi®ed by genotyping both embryos and ancestors with the D6MIT102 PCR primer pair, combined with pedigree analysis. Sp/Sp homozygotes were identi®ed visually by the presence of neural tube defects. For characterization of the transgenic line, embryonic age was determined either by designating the morning that a vaginal plug was discovered as embryonic day 0.5 (E0.5), or by the Theiler system as described (Kaufman and Baird, 1999). For staging embryos resulting from matings of heterozygous coat color mutants, the Theiler system was utilized (Kaufman and Baird, 1999). 4.4. Quanti®cation and analysis of migratory melanoblasts Melanoblasts were counted visually in the cephalic regions of 1/1 and Mitf mi/ 1 embryos. The cephalic region was de®ned by an imaginary line connecting the lateral corner of the roof of the hindbrain and the ®rst branchial cleft, and individual, lacZ-staining cells not associated with patches of telencephalic, diencephalic, or RPE expression were visualized under a dissecting microscope (Leica MZFLIII), counted on both sides of the embryos, and the counts summed. For embryos with larger numbers of melanoblasts, the embryo was positioned in the center of the viewing ®eld and counts were performed in individual quadrants which were then summed. Regression to the exponential equation y Aebx was performed with SigmaPlot 5.0 (SPSS Inc.), and resulted in coef®cients of A 232 ^ 54 and 38 ^ 12 and b 0.56 ^ 0.11 and 0.81 ^ 0.13 for 1/1 and Mitf mi/ 1 embryos, respectively, where the indicated errors are the standard errors of measurement. 4.5. Immuno¯uorescence studies For immuno¯uorescence experiments, E15 embryos from a cross between a PDct/lacZ hemizygous transgenic male and a CD1 female were embedded immediately after dissection in OCT medium and ¯ash frozen on dry ice. Cryosections (10 mm) were cut and ®xed in 100% methanol at 2208C for 15 min followed by 2% paraformaldehyde in

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PBS for 2 min. Sections were blocked with 10% goat serum/ 3% BSA/0.1% Triton X-100 in PBS for 1 h, then sequentially incubated with ACK2 (Gibco) at 1:50 overnight at 48C, Alexa Fluor goat anti-rat 546 (Molecular Probes) at 1:500 for 1 h at room temperature, rabbit anti-b-galactosidase (Chemicon) at 1:400 for 1 h at room temperature, and Alexa Fluor goat anti-rabbit 488 (Molecular Probes) at 1:500 for 1 h at room temperature. Controls lacking each primary antibody were also performed to ensure speci®city of each signal. Images were collected using a BioRad MRC 1024 Laser Scanning Confocal Microscope. Color lookup tables were added to the non-merged images using Adobe Photoshop 5.0. Acknowledgements We would like to acknowledge Jaclyn Gawel for technical assistance and Ian Jackson for supplying the Dct promoter clone used to generate the transgenic construct. We would also like to thank Anna Auerbach and members of the Skirball Institute Transgenic Facility for generating the transgenic mice for this study, Cindy Loomis for advice about embryonic dissections and analysis, Bill Pavan for suggestions about genotyping mice, Grant Blouse for suggestions about non-linear regression analysis, and Jeff Loeb for advice on immuno¯uorescence studies. This work was supported by National Institutes of Health Grants AR01992 and AR45001 to T.J.H. E.B.Z. is an Investigator of the Howard Hughes Medical Institute. References Auerbach, R., 1954. Analysis of the developmental effects of a lethal mutation in the house mouse. J. Exp. Zool. 127, 305±329. Bentley, N.J., Eisen, T., Goding, C.R., 1994. Melanocyte-speci®c expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator. Mol. Cell. Biol. 14, 7996± 8006. Bertolotto, C., Bille, K., Ortonne, J.-P., Ballotti, R., 1996. Regulation of tyrosinase gene expression by cAMP in B16 melanoma cells involves two CATGTG motifs surrounding the TATA box: implication of the microphthalmia gene product. J. Cell Biol. 134, 747±755. Budd, P.S., Jackson, I.J., 1995. Structure of the mouse tyrosinase-related protein-2/dopachrome tautomerase (Tyrp2/Dct) gene and sequence of two novel slaty alleles. Genomics 29, 35±43. Ciment, G., 1990. The melanocyte Schwann cell progenitor: a bipotent intermediate in the neural crest lineage. Commun. Dev. Neurobiol. 1, 207±223. Dickie, M.M., 1964. New splotch alleles in the mouse. J. Hered. 55, 97± 101. Epstein, D.J., Vekemans, M., Gros, P., 1991. splotch (Sp 2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3. Cell 67, 767±774. Epstein, D.J., Vogan, K.J., Trasler, D.G., Gros, P., 1993. A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch (Sp) mouse mutant. Proc. Natl. Acad. Sci. USA 90, 532±536. Franz, T., 1989. Persistent truncus arteriosus in the splotch mutant mouse. Anat. Embryol. 180, 457±464.

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