Mutations in microphthalmia, the mouse homolog of the human deafness gene MITF, affect neuroepithelial and neural crest-derived melanocytes differently

Mechanisms of Development 70 (1998) 155–166

Mutations in microphthalmia, the mouse homolog of the human deafness gene MITF, affect neuroepithelial and neural crest-derived melanocytes differently Atsuo Nakayama1, Minh-Thanh T. Nguyen, Catherine C. Chen, Karin Opdecamp2, Colin A. Hodgkinson3, Heinz Arnheiter* Laboratory of Developmental Neurogenetics, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA Received 24 September 1997; accepted 11 November 1997

Abstract The mouse microphthalmia (Mitf) gene encodes a basic-helix-loop-helix-zipper transcription factor whose mutations are associated with abnormalities in neuroepithelial and neural crest-derived melanocytes. In wild type embryos, Mitf expression in neuropithelium and neural crest precedes that of the melanoblast marker Dct, is then co-expressed with Dct, and gradually fades away except in cells in hair follicles. In embryos with severe Mitf mutations, neural crest-derived Mitf-expressing cells are rare, lack Dct expression, and soon become undetectable. In contrast, the neuroepithelial-derived Mitf-expressing cells of the retinal pigment layer are retained, express Dct, but not the melanogenic enzyme genes tyrosinase and Tyrp1, and remain unpigmented. The results show that melanocyte development critically depends on functional Mitf and that Mitf mutations affect the neural crest and the neuroepithelium in different ways.  1998 Elsevier Science Ireland Ltd. Keywords: Retinal pigment epithelium; Stria vascularis; Basic-helix-loop-helix-zipper protein; DOPAchrome tautomerase; Kit

1. Introduction Mutations that are associated with pigment cell abnormalities occur in many species, including mouse and man. In the mouse, more than 80 different loci are known to influence the development or function of pigment cells (Green, 1989). Approximately one-quarter of these loci have been analyzed at the molecular level and a plethora of transcription factors, signaling systems, motor proteins and pigment enzymes have been identified that are required for normal melanogenesis (Spritz and Hearing, 1994; Mouse Genome * Corresponding author. LDN, NINDS, NIH, Building 36/Room 5D04, 36 Convent Drive MSC 4160, Bethesda, MD 20892-4160, USA. Tel.: +1 301 4961645; fax: +1 301 4960899; e-mail: [email protected] 1 Present address: Department of Pathology, Nagoya University School of Medicine, Nagoya 466, Japan. 2 Present address: CNRS EP 560/Institut Pasteur de Lille, Diffe´renciation Cellulaire et Mole´culaire 1, rue Calmette, B.P. 245, Lille Cedex, France. 3 Present address: Howard Hughes Medical Institute, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, MI 481090650, USA.

0925-4773/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0925-4773 (97 )0 0188-3

Database, 1997). Mutant alleles at these loci may not only affect pigment cells but also other cells, either because of shared expression of the mutant genes or because abnormal pigment cells affect the physiology of other cells. Consequently, disturbances of pigmentation are often part of syndromes that may include abnormalities in hematopoiesis, gametogenesis or sensory organs such as eyes or ears (Spritz and Hearing, 1994; Online Mendelian Inheritance in Man, 1997). Thus, the analysis of these mutations is important far beyond the goal of understanding pigment cell development. The melanocytes that make up the retinal pigment epithelium (RPE), and those that are found in part of the iris, Harderian gland, choroid, inner ear and skin have distinct developmental origins. The RPE cells are derived from the proximal parts of the budding diencephalic neural epithelium while the other pigment cells are derived from the neural crest. As a consequence, mutations in some genes, such as those encoding the tyrosine kinase receptor Kit or its ligand Mgf, may affect neural crest-derived melanocytes but spare neuroepithelial melanocytes (Geissler et al., 1988;

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Chabot et al., 1988; Copeland et al., 1990; Zsebo et al., 1990). Evidently, Kit signaling is only crucial for the development of neural crest-derived melanocytes. However, mutations in other genes, such as that encoding tyrosinase, which is involved in melanin synthesis, may affect melanocytes of either origin (Spritz and Hearing, 1994). Mutations in still others, such as that encoding the transcription factor Mitf, also affect both neuroepithelial and neural crestderived melanocytes but, as will be shown here, by pathogenetic mechanisms that differ between these two cell types. The Mitf gene, originally cloned from a transgenic insertional mutation at the microphthalmia locus (Hodgkinson et al., 1993; Hughes et al., 1993), encodes a basic-helix-loophelix-zipper protein that forms homodimers and heterodimers with related proteins, interacts with other transcription regulators and specifically binds E-box motifs present in the promoter elements of pigment cell-specific genes (Hodgkinson et al., 1993; Hughes et al., 1993; Hemesath et al., 1994; Yavuzer et al., 1995; Sato et al., 1997). In vitro co-transfection assays with Mitf expression and appropriate reporter plasmids suggest that Mitf serves as a positive transcription factor for tyrosinase (Tyr) and tyrosinase-related protein-1 (Tyrp1) (Bentley et al., 1994; Yasumoto et al., 1994, 1997). The human DOPAchrome tautomerase (Dct or Tyrp2 or tyrosinase-related protein-2) promoter also contains E-box motifs but, based on co-transfection experiments, Mitf does not transactivate the Dct promoter efficiently (Yokoyama et al., 1994; Yasumoto et al., 1997). Mutations in Mitf have occurred in several species. In humans, they are found in families with Waardenburg syndrome IIa whose key features are congenital deafness and pigment disturbances in skin and eye (Tassabehji et al., 1994). Mitf is also the gene mutated in mib (microphthalmie-blanc) rats (Moutier et al., 1989; Opdecamp et al., unpublished data) and Wh (anophthalmic white) hamsters (Knapp and Polivanov, 1958; Asher, 1968; Hodgkinson et al., unpublished data). In the mouse, there are over 20 different Mitf mutations, many of them leading to amino acid substitutions in critical molecular domains (Steingrı´msson et al., 1994; Mouse Genome Database, 1997). Common to all of these mutations is a deficiency in skin or coat melanocytes that in the mouse may range in severity from minor reductions in coat tyrosinase activity but with normal eyes (as with the allele Mitfmi-sp) to total lack of coat and eye pigmentation, small, colobomatous eyes, deafness and additional disturbances such as osteopetrosis (as in Mitfmi) (Moore, 1995). In fact, in the mouse alone, the series of independent alleles at Mitf is the largest collection of mutations in any member of this class of transcription factor. Here, we have analyzed the developmental profile of Mitf expression in the neural crest and neuroepithelium of wild type mouse embryos and embryos homozygous for several Mitf mutations. The analyses revealed crucial differences in the way the two types of melanocyte are affected by Mitf.

2. Results and discussion 2.1. Mitf expression in wild type neural crest 2.1.1. Rostro-caudal sequence of Mitf expression Recent experiments in cultured cells derived from trunk neural crest indicated that the Mitf transcription factor is required early on in development, before the generation of melanoblasts (Opdecamp et al., 1997). We now examined whether the in vivo Mitf expression pattern was consistent with such an early role in the trunk and other areas of the crest. Wild type embryos and embryos homozygous for several different Mitf mutations were harvested at different stages of development and serial cryostat sections were prepared. The sections were then processed for in situ hybridization or immunolabeling, using probes and antibodies specific for Mitf and a number of additional markers of the melanocyte lineage. Fig. 1 documents the early appearance of wild type Mitf + cells as identified by a digoxigenin-labeled Mitf riboprobe in four anatomical areas. The first Mitf + cells were seen at the 25–26 somite stage in the narrow space between the surface ectoderm and the neuroepithelium of the brain (marked head crest) and in the region immediately caudal to the otic vesicle (marked vagal crest) (Fig. 1A–C, arrow in B,C). In this region, they were soon seen in clusters close to the neural tube (arrow, Fig. 1H for the 29–30 somite stage). Sagittal sections of the head region showed cells in the mesenchyme at the forebrain/midbrain and midbrain/hindbrain boundaries (arrows, Fig. 1L for the 31–32 somite stage). In the trunk region, Mitf + cells were not seen until the 27–28 somite stage. Soon thereafter, some rare Mitf + cells appeared close to the dorsal midline of the neural tube (arrows, Fig. 1I for the 29–30 somite stage). Some of these cells directly overlay the roof plate of the neural tube as shown in the high power view of Fig. 2A. In the sacral area, the first Mitf + cells were observed at the 33–34 somite stage and were more numerous at the 40–45 somite stage. Interestingly, in this region, Mitf + cells were found in locations characteristic of the dorsolateral neural crest migration pathway (arrow in Fig. 10) as well as the ventro-medial pathway (arrowhead in Fig. 10) where they came to lie close to the dorsal root ganglia (dotted line in Fig. 2B). At these later stages, formerly clustered cells in the more rostral areas were now dispersed into individual cells and located more laterally from the dorsal midline (Fig. 1M,N, arrows). The above analysis revealed that Mitf is expressed early in development, first in the head and last in the tail, and at locations where previous work had identified neural crestderived cells. With the embryos’ increasing age, the number of Mitf + cells initially increased but then decreased such that at birth only hair bulb melanocytes were still positive for Mitf (see below). Mitf + cells likely are precursors to melanocytes since mice with severe Mitf mutations lack authentic melano-

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Fig. 1. Mitf expression in the neural crest as revealed by non-radioactive in situ hybridization. A2G (wild type, albino) embryos at the indicated somite stages were sectioned at the indicated anatomical levels (except for Fig. 1L, see below). (A,B) Boxed area in (A) magnified in (B), showing Mitf signal underneath the surface ectoderm in the head region (arrow). (C,D,E) Note Mitf signal only in section caudal to the otic vesicle (arrow in C). (F,G) Boxed area in (F) magnified in (G), showing Mitf + cells underneath the surface ectoderm in the head region. (H,I,K) Note clusters of Mitf + cells caudal to the otic vesicle (arrow in H) and individual Mitf + cells in the dorsal midline of the trunk neural tube (arrows in I). (L) 31–32 somite stage parasagittal section. Mitf + cells (arrows) in the mesenchyme are localized at the borders between telencephalic and the midbrain vesicle and between the midbrain vesicle and the fourth ventricle. Presumptive RPE (arrowhead) also expresses Mitf. (M,N,O) Note single cells in the dorso-lateral pathway caudal to the otic vesicle (arrows in M), trunk area (arrows in N), and sacral area (arrow in O). The arrow head in (O) points to an Mitf + cell in the ventro-medial pathway. Magnification bar: 400 mm for (A,L); 100 mm for (D,G,H,K,M,N,O); 65 mm for (B,C,E,I); 580 mm for (F).

blasts (Opdecamp et al., 1997; see below) and the melanocytes derived from them. Consequently, the cells in the dorsal midline seem to make their initial steps along the melanocyte differentiation pathway at a premigratory stage. That their number is small would be consistent with the interpretation of the phenotypes of allophenic chimeras presented earlier (Mintz, 1967). However, whether these few early cells are sufficient to give rise to all melanocytes remains to be determined. In the sacral region, although Mitf + cells were found in intimate association with dorsal root ganglia (Fig. 2B), they were not found within the ganglia. Future lineage-tracing experiments will reveal whether these cells, located in places not characteristic of melanocytes, may assume the fate of other neural crest derivatives, or die out. In any event, the results indicate that with respect to the migration pathways of early Mitf + cells, the head, trunk, and sacral regions differ, and suggest that depending on the region, Mitf + cells may have different developmental potentials. 2.1.2. Co-expression of Mitf and the melanoblast markers Dct and Kit To examine whether Mitf + cells were indeed part of the melanocyte lineage, we tested for co-expression of Mitf and

Dct or Kit, the latter two known markers of melanoblasts (Steel et al., 1992; Reid et al., 1995). A series of doublelabeling assays with a digoxigenin-labeled riboprobe for Mitf and a 35S-labeled riboprobe for Dct showed that in all anatomical areas examined, Mitf expression preceded that of Dct by 4–6 h, the time period it takes for 2–3 pairs of somites to develop (data not shown). Similar results were obtained when the Mitf riboprobe was radioactively labeled and the Dct riboprobe digoxigenin-labeled, thus excluding artifacts potentially caused by differences in sensitivities of detection of the two genes (data not shown). As shown in Fig. 3A for E11.5, sections through the area of the otic vesicle showed overlap of Mitf and Dct expression in the majority of Mitf + cells. In the tangential section through the trunk (Fig. 3B), there was overlap of the two markers in the majority of the more lateral cells but not in those still lying closer to the dorsal midline. Similarly, in the sacral region (Fig. 3C), cells close to the dorsal midline were Mitf single-positive whereas cells further away from the midline were Mitf/Dct double-positive. These results suggest that cells first express Mitf and later, having migrated further laterally, Dct. Mitf/Kit double-labeling experiments were performed by double indirect immunofluorescence as previously

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Motohashi et al., 1994). Lack of these melanocytes may lead to absence of endocochlear potential, secondary degeneration of hair cells and hearing impairment (Steel and Barkway, 1989; Cable et al., 1994). Fig. 5A shows wild type Mitf + cells at the 29–30 somite stage located between the otic vesicle and the neuroepithelium of the hindbrain. During the following days, these cells increased in number and became intimately associated with the otic vesicle (Fig. 5B, see also Fig. 3A). Three days later, they were concentrated in a small area that represented the presumptive stria vascularis (Fig. 5C); in a serial section, this area was also positive for Dct (Fig. 5D), consistent with earlier results (Steel et al., 1992). Later, the Mitf signal in the stria vascularis was less intense and at birth was undetectable while the Dct signal remained intact (data not shown). These observations suggest that in the area of the otic vesicle, Mitf + cells migrate on the ventro-medial pathway toward the otic vesicle where they give rise to pigmented cells in the inner ear, including the intermediate cells of the stria vascularis. In addition, they are also found in the dorso-lateral migration pathway where they give rise to skin melanocytes.

Fig. 2. (A) High power view of Fig. 1I. Single Mitf + cell in the dorsal midline of the trunk neural tube. The positive cell overlays the neural tube just underneath the surface ectoderm. (B) Sacral crest at E12.5. Mitf + cells are found on the dorso-lateral neural crest migration pathway (left-hand side of the figure) and on the ventro-medial pathway close to a dorsal root ganglion (d). Magnification bar: 8 mm for (A); 50 mm for (B).

described (Opdecamp et al., 1997). As shown in Fig. 4A,B, in a region caudal to the otic vesicle as well as in the trunk of an E10.5 embryo, there were many Mitf + cells (nuclear red fluorescence) that were also Kit+ (cytoplasmic green fluorescence). Unlike Dct expression which was delayed relative to that of Mitf, Kit expression was seen as soon as Mitf became detectable, i.e. from E10.5, consistent with previous Kit expression studies (Keshet et al., 1991; Matsui et al., 1990). As shown in Fig. 4C, the sacral area contained double-positive cells in locations corresponding to the dorsolateral migration pathway (arrows) but also close to the neural tube (arrowhead) where Mitf + cells have been observed by in situ hybridization (Fig. 2B). Thus, it appears that in the neural crest, the majority of Mitf + cells develop into melanoblasts – expressing at least two additional melanoblast markers. However there were also some cells that were single-labeled for any of the markers (see for instance Kit single-labeled cells in Fig. 4A). Whether these cells simply reflect dynamic changes in the sequence of gene expression or whether they belong to separate lineages awaits further analyses. 2.1.3. Expression in the otic vesicle It has been noted earlier that Mitf mutant mice may be deaf due to lack of neural crest-derived melanocytes in the stria vascularis of the cochlea (Tachibana et al., 1992;

2.1.4. Expression in the skin As shown in Fig. 1, Mitf + cells on the lateral pathway were seen beneath the surface ectoderm until the 40–45 somite stage. At E12.5, such cells became incorporated in the epithelial layer as shown for the forelimb bud area in Fig. 6A. Four days later, Mitf + cells were found in the dermis and epidermis (Fig. 6B). We cannot exclude, however, that some of these cells represented mast cells, which are known to express Mitf (Hodgkinson et al., 1993) and populate the embryonic skin (Isozaki et al., 1994). In newborn skin, Mitf expression was restricted to hair follicles (Fig. 6C) where it persisted postnatally (Fig. 6D). In fact, immunofluorescent assays showed that a subset of the pigmented cells in hair follicles stayed positive for Mitf (Fig. 6E), in contrast to pigmented cells in other locations. This is consistent with the observation that mice homozygous for the Mitfvit allele show an age-dependent progressive loss of coat pigmentation (Lerner et al., 1986; Boissy et al., 1991) but apparently no postnatal changes in the RPE (Boissy et al., 1987; Nir et al., 1995) where Mitf is not expressed postnatally (see below). 2.2. Mitf expression in mutant neural crest Mitf expression in the neural crest was examined in embryos homozygous for three different Mitf alleles: Mitfvga-9, a transgenic insertional allele that accumulates little if any Mitf mRNA (Hodgkinson et al., 1993), Mitfmi, an allele that encodes a protein with a deletion of a critical arginine in its DNA-binding domain (Steingrı´msson et al., 1994), and Mitfmi-ew, which encodes a protein in which 25 residues, including most of the basic DNA-binding domain, are replaced by a single valine (Steingrı´msson et al., 1994). Previous examination of cultured trunk neural crest cells of

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Fig. 3. (A,B,C) Mitf and Dct double-label in situ hybridization of wild type embryos at E11.5 at the indicated anatomical levels, except for (A) which is a parasagittal section through the otic vesicle. Mitf signal (digoxigenin-labeled probe) is in dark blue, Dct signal (35S-labeled probe) is in red (pseudocolor rendition of dark field image superimposed, slightly offset, on differential interference contrast image). Note the predominantly double-labeled cells in (A,B). (C) shows the sacral region where Mitf expression just begins at E11.5. Note that cells closer to the midline are Mitf single-positive whereas cells farther lateral are double-positive. (D,E,F) Mitf and Dct non-radioactive in situ hybridization of pairs of adjacent sections of E11.5 embryos homozygous for Mitfmiew . Note the reduced number of Mitf + cells and the absence of Dct signal. Magnification bar: 190 mm for (A,B); 50 mm for (C); 60 mm for (D–F).

embryos homozygous for Mitfmi-ew has revealed that mutant Mitf + cells are present, albeit only transiently. Furthermore, such mutant cells did not express Dct. The analysis of Mitf expression in several anatomic regions of embryos homozygous for Mitfmi-ew (Figs. 3D–F, 4D–F) indicated that mutant Mitf + cells were rare and seen no longer after E12.5. It is worth mentioning that immunofluorescence did not reveal detectable Mitfmi-ew protein in the neural crest (note the absence of red fluorescent signal in (Fig. 4D–F), despite the fact that Mitf mRNA could be detected (compare with Fig. 3D–F). It is possible that mutant protein, unable to move efficiently to the nucleus (Takebayashi et al., 1996; see below for RPE), was in these cells less stable than the wild type protein. Similar observations were made

with embryos homozygous for Mitfmi, and in embryos homozygous for the null allele Mitfvga-9, neither Mitf + nor Dct + cells were observed (data not shown). Thus, Mitf + cells in the neural crest were severely reduced in these Mitf mutations and lacked Dct expression which effectively precluded their further in vivo examination (Opdecamp et al., 1997). This observation is consistent with the fact that mutant mice are devoid of differentiated neural crestderived melanocytes in skin, hair follicles, choroid, Harderian gland and inner ear. Interestingly, mutant embryos still showed a few Kit + cells in locations where migrating neural crest cells were expected (Fig. 4D–F), suggesting that unlike Dct expression, Kit expression is not absolutely dependent on functional Mitf. This observation is consistent

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Fig. 4. (A,B,C) Mitf and Kit protein double-label immunofluorescence of wild type embryos at the indicated time points and anatomical levels. The majority of Mitf + cells (red or yellow, nuclear fluorescence) are also positive for Kit (green, cytoplasmic fluorescence). (C) Note that in the sacral area, cells in both the dorso-lateral (arrow) pathway and close to the neural tube (arrowhead) are double positive. (D,E,F) Sections of embryos homozygous for Mitfmi-ew. Note rare Kit + cells lacking nuclear Mitf signal. Magnification bar: 50 mm.

with earlier in vitro and in vivo findings in the trunk region (Opdecamp et al., 1997). In addition to the above-mentioned locations of Mitf + cells in early embryos, starting at E9.5, weak but specific Mitf expression was also found in the ventricular walls of wild type but not Mitfvga-9 embryos. Also, after E13.5 in wild type embryos, there were individual, weakly Mitf-positive cells around the dorsal aorta, the heart atrium, the atrioventricular bulbar cushion, the cervical muscles, Meckel’s cartilage and in hindlimb mesenchyme, particularly around the muscle primordium and ossifying bone (data not shown). However, Mitf expression was progressively lost

in these cells and no pigmented cells appeared in these locations. In mutant embryos, independent of the allele, such cells were undetectable. Although it is conceivable that these individual cells were derived from the neural crest, their precise origin and the role Mitf plays in them remain to be determined. 2.3. The role of Mitf in eye development 2.3.1. Mitf expression in the developing wild type eye The earliest embryonic Mitf expression was observed in the developing eye. Already, beginning at the 24 somite

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its proximal part (arrow, Fig. 7A for the 25–26 somite stage). At the 29–30 somite stage (Fig. 7B), the optic placode was induced and the optic vesicle has developed into the optic cup with a clear separation into an inner layer which will become the retina (Fig. 7B, arrowhead) and an outer layer which will form the RPE (Fig. 7B, arrow). At this stage, Mitf expression was prominent in this outer layer and absent from all other eye structures. At E13.5, the monolayer of the RPE was still positive for Mitf and additional Mitf + cells were seen behind the optic cup and in the surface ectoderm overlaying the developing eye (Fig. 7C, arrows). These latter cells were likely neural crest-derived and may develop into choroidal and anterior iris pigment cells and pigment cells of the periorbital skin (see below). At E16.5, Mitf was still expressed in the RPE and individual cells in the adjacent mesenchyme (Fig. 7D, arrows). However, with increasing time, Mitf expression became less prominent and at birth was largely undetectable, except in hair bulb cells in the overlying skin (Fig. 7E, arrows); labeling of adjacent sections with Dct showed positivity of these hair bulb cells but also of the iris and the RPE (Fig. 7F). Thus, in the RPE and iris, Dct expression may continue beyond birth even though Mitf has ceased to be expressed at detectable levels.

Fig. 5. Mitf expression in the otic vesicle area. (A) 29–30 somite stage. Note Mitf + cells in a region corresponding to the ventro-medial pathway close to the otic vesicle (ov). (B) At E13.5, Mitf + cells are found in close association with the otic vesicle. (C,D) Adjacent sections through the otic vesicle at E16.5. (C) shows Mitf + cells in a restricted area in the future cochlear duct, and (D) shows the same area labeled for Dct. Magnification bar: 140 mm for (A); 110 mm for (B); 200 mm for (C,D).

stage (E9.5), i.e. before the expression of Dct, the neuroepithelium of the optic vesicle was weakly positive for Mitf at

2.3.2. Effect of Mitf mutations on the RPE Our earlier analysis of Mitf expression in Mitf null mutant eyes suggested that RPE cells remain anatomically identifiable even at E13.5 (Hodgkinson et al., 1993). Examination of the eyes of Mitfmi-ew homozygotes now showed that this was the case even at birth. In fact, parts of the newborn Mitfmi-ew RPE still expressed Mitf in addition to Dct (arrowheads, Fig. 7G,H) while expression of these genes was absent in hair bulb cells (arrows, Fig. 7G,H, compare with E,F) as expected if neural crest-derived cells were missing in the hair bulbs. We now extended these studies to earlier time points and used the above-mentioned Mitf alleles (Mitfvga-9, Mitfmi and Mitfmi-ew) and an additional allele,

Fig. 6. Incorporation of Mitf + cells into the skin. (A–D) Albino embryos or mice. (A) At El2.5, trunk Mitf + cells are found both beneath and within the surface ectoderm. (B) At E16.5, Mitf + cells are in the dermis and epidermis. (C) Newborn skin shows Mitf + cells in hair follicles where Mitf-positivity persists beyond birth (D). (E) shows a cross-section through a whisker hair bulb of a four week-old pigmented mouse, stained by immunofluorescence for Mitf protein. Black color represents melanin pigment, green color immunofluorescent signal. Note the fluorescence in pigmented and unpigmented cells. Magnification bar: 28 mm for (A,B); 37 mm for (C); 50 mm for (D); 26 mm for (E).

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Fig. 7. Mitf expression in the developing eye of albino A2G embryos and newborn mice. (A) Budding optic vesicle at the 25–26 somite stage. Note the Mitf in situ hybridization signal in the proximal parts of the neuroepithelium of the vesicle (arrow). (B) Mitf-positivity in the presumptive RPE (arrow) at the 29–30 somite stage, while the presumptive retina (arrowhead) is negative. (C) At E13.5, the future RPE is positive and individual, presumably neural crest-derived Mitf + cells (arrows) are also seen behind the RPE. (D) Similar appearance at E16.5, with arrows pointing to Mitf + cells presumably derived from the neural crest. (E,F) Adjacent sections of a newborn eye. Note the absence of Mitf-labeling in RPE and iris but the presence of Mitf + cells in hair follicles (arrows). In (F), hair follicle cells are positive for Dct (arrows), as is the iris and the thin layer of the RPE (arrowhead). (G,H) Adjacent sections of a newborn Mitfmi-ew/mi-ei eye. Note the absence of Mitf (G) and Dct (H) labeling in hair follicles (arrows) but positivity for Mitf and Dct of the RPE-derived (back) layer of the iris (arrowhead). Magnification bar: 34 mm for (A); 37 mm for (B); 115 mm for (C); 200 mm for (D); 128 mm for (E-H). mi-ew

Mitf , which encodes a predominantly nuclear protein with an ile → asn substitution in the DNA-binding basic domain. This latter protein is unique in that it mediates partial interallelic complementation of other alleles and in one of its isoforms retains in vitro DNA-binding activity,

provided it is dimerized not with Mitf but with one of the related proteins Tfeb, Tfe3 or Tfec (Hemesath et al., 1994; Steingrı´msson et al., 1994). Immunocytochemical analyses of wild type and mutant RPEs (E14.5) are shown in Fig. 8. In wild type, Mitf was a predominantly nuclear protein (Fig. 8A). In Mitfvga-9, as expected from a null allele, Mitf protein did not accumulate appreciably in the RPE (Fig. 8B). In MitfMi-wh, the protein was predominantly nuclear, and in Mitfmi and Mitfmi-ew, the respective proteins were distributed throughout the cells, consistent with previous in vitro results (Takebayashi et al., 1996). More importantly, however, these immunocytochemical analyses also revealed that the mutant RPE cells were not only present but were actually more numerous than in wild type. In addition, particularly in Mitfvga-9, Mitfmi and Mitfmi-ew, the RPE was thickened, with the cells assuming a more columnar shape compared with the cuboidal or flat shape observed in wild type. These results nicely confirmed a previous report, 30 years ago, that showed that Mitfmi and MitfMi-wh RPEs display a higher number of mitotic cells than wild type (Packer, 1967), and a more recent study on Mitfmi-vit (Tang et al., 1996). Thus, in contrast to neural crest-derived melanocytes which are few in number and rapidly become undetectable, mutant RPE cells hyperproliferate and survive, though they assume abnormal shapes. The fact that mutant RPE cells persisted throughout development allowed us to test what effects Mitf mutations might have on the expression of melanocyte-specific genes. The analysis of Dct, Tyrp1 and Tyr expression in wild type and the four different mutant embryos (E14.5) is shown in Fig. 9. Of the three melanogenic enzyme genes, Dct was the least and Tyrp1 and Tyr expression were the most reduced in all four mutants. In fact, Dct expression was barely affected both in Mitfvga-9, which shows no detectable Mitf protein (Fig. 8B), and MitfMi-wh, which had the mutant protein predominantly in the nucleus and showed only mild thickening of the RPE (Fig. 8C). This latter mutant also still allowed for weak Tyrp1 and Tyr expression. In contrast, in Mitfmi and Mitfmi-ew embryos, which display prominent thickening of the RPE (Fig. 8D,E), Tyrp1 and Tyr expression were undetectable (Fig. 9). Thus, even though Dct, Tyrp1 and Tyr all have E-box motifs in their promoters, the in vivo effects of Mitf mutations on expression of these genes are different. A differential regulation of Tyr and Tyrp1 versus Dct has also been suggested on the basis of in vitro co-transfection experiments (Yasumoto et al., 1997). 2.4. Absence of expression of the Mitf-relatives Tfeb and Tfe3 in the neural crest and neuroepithelium As mentioned above, the MitfMi-wh protein, much as wild type Mitf protein, is capable of forming DNA-binding heterodimers with the related bHLH-Zip proteins Tfeb, Tfe3 and Tfec, and it was suggested that these heterodimerization partners might play a role for Mitf to exert its function (Hemesath et al., 1994). However, in none of the Mitf +

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Fig. 8. Immunolabeling of Mitf protein in wild type RPE and RPE of homozygous Mitf mutant embryos at E14.5. (A) Wild type (albino strain A2G). Note the monolayer of RPE cells with nuclear labeling. (B) Mitfvga-9. RPE is thickened but Mitf-labeling is undetectable. (C) MitfMi-wh. Note nuclear labeling in RPE that displays more cells than wild type. These cells are more columnar and the nuclei in some areas arranged in more than one layer. (D) Mitfmi. A thickened RPE shows prominent Mitf-labeling both in nuclei and cytoplasm. (E) Mitfmi-ew. Similar thickened RPE with nuclear and cytoplasmic staining. Magnification bar: 25 mm.

Fig. 9. Effect of Mitf mutations on gene expression in the RPE. Serial sections through E14.5 eyes of wild type and the homozygous Mitf mutant embryos as indicated on the top were probed by in situ hybridization with an Mitf, Dct, Tyrp1 or Tyr probe (as indicated on the left-hand side). Note that the RPEs of all mutants are present but express different levels of the indicated genes. Also note that only wild type shows presence of neural crest-derived cells expressing these genes (arrows). See text for details. Magnification bar: 200 mm.

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Fig. 10. Summary of the expression pattern of wild type Mitf. The top row of pictures shows a schematic representation of the expression pattern in eye and neural crest at the indicated developmental stages as obtained by non-radioactive in situ hybridization. The lower part of the figure lists the Mitf-positive stages for the neuroepithelium and the different parts of the neural crest and their derivatives. As mentioned in the text, there are Mitf + cells in additional anatomical areas, including in the heart, whose origin has not yet been determined. NC, neural crest.

cells did we observe any appreciable expression of Tfeb or Tfe3 at stages when the Mitf mutant phenotype becomes manifest (data not shown). Thus, it is unlikely that these related proteins are relevant for Mitf to function during development and we predict that knock-out mice in which these related genes are deleted will not show a neural crest or eye phenotype similar to Mitf mutant mice. 2.5. Summary and conclusion The results from these studies are summarized in Fig. 10. The RPE represents the first place of Mitf expression, followed by expression in the neural crest in a rostro-caudal sequence. The periods during which wild type neural crest cells and their derivatives are positive for Mitf, and the types of their positive derivatives, are shown in the lower half of Fig. 10. Our results show that Mitf mutations severely affect

Mitf + cells in the neural crest. These cells are rare and lack Dct expression. In contrast, the initial steps of RPE development are not affected but RPE cells show abnormal proliferation, shape, and Tyrp1 and Tyr expression but they largely retain Dct expression. This clearly establishes that melanocytes of different origin, though dependent on the same Mitf transcription factor gene for their development, respond differently to mutations in this gene and suggests that Mitf plays different roles in these two types of pigment cell.

3. Experimental procedures 3.1. Mice and harvest of embryos Mice of the strains C57BL/6 (pigmented) and A2G

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(albino) were used as Mitf wild type controls. For in situ hybridizations, A2G embryos were used predominantly since we observed that the presence of melanin in pigmented mouse embryos may result in non-specific labeling. Embryos homozygous for Mitf mutant alleles were obtained either by homozygous × homozygous mating or by heterozygous × heterozygous mating and identification of homozygous embryos by absence of eye pigmentation. The alleles used were: Mitfvga-9 (background: mixed C57BL/6/ C3H) (Tachibana et al., 1992; Hodgkinson et al., 1993); Mitfmi-ew (background: C57BL/6) (Hertwig, 1942); MitfMiwh (background: C57BL/6) (Grobman and Charles, 1947); and Mitfmi-ew (background: Naw) (Miner, 1968). The noon on which a vaginal plug was found upon mating was designated embryonic day 0.5 (E0.5). At the indicated time points, embryos were harvested and placed into minimal essential medium, containing 5% fetal bovine serum before further processing. For accurate staging of embryos younger than E11.5, the number of somites were counted. All animal procedures were approved by the institutional review board. 3.2. Riboprobes Riboprobes were made by in vitro transcription of appropriate plasmids with either T3 or T7 RNA polymerase and incorporation of digoxigenin-labeled UTP or 35S a-labeled UTP. The template for the Mitf probe, MC1, was described previously (Hodgkinson et al., 1993), as was that for the mouse Dct probe (Steel et al., 1992; Opdecamp et al., 1997). For the mouse Tfe3 probe, a 1.1 kb fragment corresponding to the 3′-UTR of the Tfe3 cDNA (Roman et al., 1991) kindly provided by K. Calame was subcloned in pBS. Mouse TfeB cDNA clones were obtained by screening a lgt10 library derived from adult mouse heart (Clonetech) using human cDNA as a probe. Positive clones were subcloned into pBluescript KS- and further characterized. One clone containing a 1.9 kb insert was sequenced and identified as mouse TfeB by sequence comparison with the human TfeB sequence. A fragment coding for the carboxyl 184 amino acids (excluding the conserved bHLH-Zip region) and 373 bases of the 3′-UTR was further subcloned and used to generate antisense riboprobes. For all probes, corresponding sense probes were generated and used as controls. 3.3. In situ hybridization Non-radioactive in situ hybridization was performed essentially as described previously (Hodgkinson et al.; 1993; Opdecamp et al., 1997). For double-labeling in situ hybridization, a mixture of digoxigenin-labeled and 35Slabeled probes was applied. Digoxigenin-labeling was first visualized in the standard manner, followed by coating of the glass slides with Ilford K-5 D emulsion (Polysciences Inc.). After appropriate times of exposure, the hybridization signals were visualized by standard development and fixation procedures. The slides were viewed and photographed

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using a Polyvar microscope set for differential interference contrast (DIC) mode to view the digoxigenin-labeled cells and for dark field to view the grains. Both images were then scanned and the dark field image was converted into pseudocolors and overlayed over the DIC image. 3.4. Immunofluorescence and immunocytochemistry Double indirect immunofluorescence of cryostat sections using a rabbit anti-mouse Mitf serum was performed as described (Opdecamp et al., 1997). For immunocytochemistry, cryostat sections were postfixed in 4.0% formaldehyde for 10 min, treated with 10% normal goat serum and then exposed to a 1:2000 dilution of the rabbit anti-Mitf antibody. The presence of the antibody was revealed by the avidin-biotin complex method (Vectastain).

Acknowledgements We thank Drs. Ian Jackson and David Fisher for plasmids, Dr. Vincent Hearing for Dct antibodies, Dr. Nitin Gogate for initial help with in situ hybridization and Drs. Eirı´kur Steingrı´msson, William Pavan, Monique Dubois-Dalcq and Lynn Hudson for valuable comments on the manuscript. During the course of this work, C.C. was a HHMI-NIH Research Scholar.

References Asher, J.H., Jr. 1968. A partial biochemical and morphological description of the action of the gene Wh causing anophthalmia in the Syrian hamster, Mesocricetus auratus, 1968. MA Thesis, California State College at Long Beach, USA, p. 360. Bentley, N.J., Eisen, T., Goding, C.R., 1994. Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator. Mol. Cell. Biol. 14, 7996– 8006. Boissy, R.E., Moellmann, G.E., Lenzer, A.B., 1987. Morphology of melanocytes in hair bulbs and eyes of vitiligo mice. Am. J. Pathol. 127, 380–388. Boissy, R.E., Beato, K.E., Nordlund, J.J., 1991. Dilated rough endoplasmic reticulum and premature death in melanocytes cultured from the vitiligo mouse. Am. J. Pathol. 138, 1511–1525. Cable, J., Huszar, D., Jaenisch, R., Steel, K.P., 1994. Effects of mutations at the W locus (c-kit) on inner ear pigmentation and function in the mouse. Pigment Cell Res. 7, 17–32. Chabot, B., Stephenson, D.A., Chapman, V.M., Besmer, B., Bernstein, A., 1988. The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335, 88–89. Copeland, N., Gilbert, D.J., Cho, B.C., Donovan, P.J., Jenkins, N.A., Cosman, D., Anderson, D., Lyman, S.D., Williams, D.E., 1990. Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles. Cell 63, 175–183. Geissler, E.N., Ryan, M.A., Housman, D.E., 1988. The dominant-white spotting (W) locus of the mouse encodes the c-kit proto-oncogene. Cell 55, 185–192. Green, M.C., 1989. Catalog of mutant genes and polymorphic loci. In: Lyon, M.F., Searle, A.G. (Eds.), Genetic Variants and Strains of the Laboratory Mouse. Oxford University Press, New York, pp. 12–403.

166

A. Nakayama et al. / Mechanisms of Development 70 (1998) 155–166

Grobman, A.B., Charles, D.R., 1947. Mutant white mice. A new dominant autosomal mutant affecting coat color in Mus musculus. J. Hered. 38, 381–384. Hemesath, T.J., Steingrı´msson, E., McGill, G., Hansen, M.J., Vaught, J., Hodgkinson, C.A., Arnheiter, H., Copeland, N.G., Jenkins, N.A., Fisher, D.E., 1994. microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family. Genes Dev. 8, 2770–2780. Hertwig, P., 1942. Neue Mutationen und Kopplungsgruppen bei der Hausmaus. Z. Indukt. Abstammungs-Vererbungsl. 80, 220–246. Hodgkinson, C.A., Moore, K.J., Nakayama, A., Steingrı´msson, E., Copeland, N.G., Jenkins, N.A., Arnheiter, H., 1993. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell 74, 395– 404. Hughes, M.J., Lingrel, J.B., Krakowsky, J.M., Anderson, K.P., 1993. A helix-loop-helix transcription factor-like gene is located at the mi locus. J. Biol. Chem. 268, 20687–20690. Isozaki, K., Tsujimura, T., Nomura, S., Morii, E., Koshimizu, U., Nishimune, Y., Kitamura, Y., 1994. Cell type-specific deficiency of ckit gene expression in mutant mice of mi/mi genotype. Am. J. Pathol. 145, 827–836. Keshet, E., Lyman, S.D., Williams, D.E., Anderson, D.M., Jenkins, N.A., Copeland, N.G., Parada, L.F., 1991. Embryonic RNA expression patterns of the c-kit receptor and its cognate ligand suggest multiple functional roles in mouse development. EMBO J. 10, 2425–2435. Knapp, B.H., Polivanov, S., 1958. Anophthalmic albino: a new mutation in the Syrian hamster. Am. Naturalist 92, 317–318. Lerner, A.B., Shiohara, T., Boissy, R.E., Jacobson, K.A., Lamoreux, L.M., Moellmann, G.E., 1986. A mouse model for vitiligo. J. Invest. Dermatol. 87, 299–304. Matsui, Y., Zsebo, K.M., Hogan, B.L., 1990. Embryonic expression of a haematopoietic growth factor encoded by the S1 locus and the ligand for c-kit. Nature 347, 667–669. Miner, G., 1968. Mouse News Lett. 38, 25. Mintz, B., 1967. Gene control of mammalian pigmentary differentiation, I. Clonal origin of melanocytes. Proc. Natl. Acad. Sci. USA 58, 344–351. Moore, K.J., 1995. Insight into the microphthalmia gene. Trends Genet. 11, 442–448. Motohashi, H., Hozawa, K., Oshima, T., Takeuchi, T., Takasaka, T., 1994. Dysgenesis of melanocytes and cochlear dysfunction in mutant microphthalmia (mi) mice. Hear. Res. 80, 10–20. Mouse Genome Database (MGD), 1997. Mouse Genome Informatics, Jackson Laboratory, Bar Harbor, Maine, World Wide Web (URL: http:/ /www.informatics jax.org/). Moutier, R., Ostrowski, K., Lamendin, H., 1989. Microphthalmia: a new recessive mutation in the Norway rat. J. Hered. 80, 76–78. Nir, I., Ransom, N., Smith, S.B., 1995. Ultrastructural features of retinal dystrophy in mutant vitiligo mice. Exp. Eye Res. 61, 363–377. Online Mendelian Inheritance in Man, OMIM (TM), 1997. Center for Medical Genetics, Johns Hopkins University, Baltimore, MD and National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD. World Wide Web URL http:/ / www.ncbi.nlm.nih.gov/Omim/. Opdecamp, K., Nakayama, A., Nguyen, M.-T.T., Hodgkinson, C.A., Pavan, W.J., Arnheiter, H., 1997. Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor. Development 124, 2377–2386. Packer, S.O., 1967. The eye and skeletal effects of two mutant alleles at the microphthalmia locus of Mus musculus. J. Exp. Zool. 165, 21– 46.

Reid, K., Nishikawa, S.-I., Bartlett, P.F., Murphy, M., 1995. Steel factor directs melanocyte development in vitro through selective regulation of the number of c-kit + progenitors. Dev. Biol. 169, 568–579. Roman, C., Cohn, L., Calame, K., 1991. A dominant negative form of transcription activator mTFE3 created by differential splicing. Science 254, 94–97. Sato, S., Roberts, K., Gambino, G., Cook, A., Kouzarides, T., Goding, C.R., 1997. CBP/p300 as a co-factor for the microphthalmia transcription factor. Oncogene 14, 3083–3092. Spritz, R.A., Hearing, V.J. Jr., 1994. Genetic disorders of pigmentation. Adv. Hum. Genet. 22, 1–45. Steel, K.P., Barkway, C., 1989. Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear. Development 107, 453–463. Steel, K.P., Davidson, D.R., Jackson, I.J., 1992. TRP-2/DT a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development 115, 1111–1119. Steingrı´msson, E., Moore, K.J., Lynn Lamoreux, M., Ferre´-D’Amare´, A.R., Burley, S.K., Sanders Zimring, D.C., Skow, L.C., Hodgkinson, C.A., Arnheiter, H., Copeland, N.G., Jenkins, N.A., 1994. Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences. Nature Genet. 8, 256–263. Tachibana, M., Hara, Y., Vyas, D., Hodgkinson, C., Fex, J., Grundfast, K., Arnheiter, H., 1992. Cochlear disorder associated with melanocyte anomaly in mice with a transgenic insertional mutation. Mol. Cell. Neurosci. 3, 433–445. Takebayashi, K., Chida, K., Tsukamoto, I., Morii, E., Munakata, H., Arnheiter, H., Kuroki, T., Kitamura, Y., Nomura, S., 1996. The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nuclear localization potential. Mol. Cell. Biol. 16, 1203–1211. Tang, M., Ruiz, M., Kosaras, B., Sidman, R.L., 1996. Increased cell genesis in retinal pigment epithelium of perinataI vitiligo mutant mice. Invest. Ophthalmol. Vis. Sci. 37, 1116–1124. Tassabehji, M., Newton, V.E., Read, A.P., 1994. MITF gene mutations in patients with Type 2 Waardenburg Syndrome. Nature Genet. 8, 251– 255. Yasumoto, K.-I., Yokoyama, K., Shibata, K., Tomita, Y., Shibahara, S., 1994. Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene. Mol. Cell. Biol. 14, 8058–8070. Yasumoto, K.-I., Yokoyama, K., Takahashi, K., Tomita, Y., Shibahara, S., 1997. Functional analysis of microphthalmia-associated transcription factor in pigment cell-specific transcription of the human tyrosinase family genes. J. Biol. Chem. 272, 503–509. Yavuzer, U., Keenan, E., Lowings, P., Vachtenheim, J., Currie, G., Goding, C.R., 1995. The microphthalmia gene product interacts with the retinoblastoma protein in vitro and is a target for deregulation of melanocyte-specific transcription. Oncogene 10, 123–134. Yokoyama, K., Yasumoto, K.-I., Suzuki, H., Shibahara, S., 1994. Cloning of the human DOPAchrome tautomerase/tyrosinase-related protein 2 gene and identification of two regulatory regions required for its pigment cell-specific expression. J. Biol. Chem. 43, 27080–27087. Zsebo, K., Williams, D.A., Geissler, E.N., Broudy, V.C., Martin, F.H., Atkins, H.L., Hsu, R.-Y., Birkett, N.C., Okino, K.H., Murdock, D.C., Jacobsen, F.W., Langley, K.E., Smith, K.A., Takeishi, T., Cattanach, B., Galli, S.J., Suggs, S.V., 1990. Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63, 213–224.