Melanoblast Development Research Articles

Abstract LB233: Single-cell transcriptional analysis of embryonic melanoblasts pinpoints embryonic cell states that underlie targeted therapy and immune checkpoint blockade resistance

Abstract Melanoma is a malignant neoplasia of pigment-producing melanocytes notable for its ability to metastasize early during primary tumor growth. Melanomas display typically high mutation burden—a hallmark associated with immune checkpoint blockade therapy (ICBT) sensitivity—but can still evade ICBT. These melanoma phenotypes likely relate to embryonic neural crest cells, a population of cells from which melanocytes arise. For instance, BRAF inhibition-evading melanoma cells adopt neural crest-like states. However, the exact embryonic lineage underlying BRAFi resistance remains unknown as does its relationship with any neural crest-like states that underlie ICBT resistance. To discover such states, we used a mouse fluorescent reporter model of melanoblast development in which melanocyte-specific GFP is driven by the dopachrome tautomerase promoter (iDct-GFP). Embryonic day (E) 11.5 and 15.5 melanoblasts were isolated and sequenced using single-cell transcriptomics (scRNA-seq). These time-points enriched for cells migrating from the dermis to epidermis (at E11.5) or for cells colonizing the hair follicle (at E15.5). These characteristics could help metastatic progression, namely migration across the basement membrane in early invasive melanomas and colonization of metastatic sites. We classified these embryonic melanocytes and their precursors into multiple clusters which in turn were grouped into four key lineages: Neural Progenitor, Schwann Cell Progenitor (SCP), Mesencyhmal (Mes) and Melanocytes (Mel). To detect the presence of these states in tumors, we derived developmental gene modules (DGMs): sets of genes that were preferentially co-expressed in each embryonic cell state. DGMs stratified TCGA samples; SCP-high and Mes-high subtypes were associated with worse survival compared to Mel-high tumors. Moreover, SCP-high tumors had lower T cell infiltration, and were associated with higher NGFR expression, a known marker of ICBT resistance. High expression of SCP DGMs was associated with poor response to anti-PD1 ICBT. Targeting factors underlying the SCP state might therefore sensitize tumours to ICBT. Mes DGMs on the other hand were associated with targeted therapy resistance. Cross-referencing DGMs with published RNA-seq data from melanoma cell lines suggest that SCP and Mes DGMs are induced by pro-inflammatory cytokines secreted by immune cells in the tumor microenvironment. Together, these observations suggest that the SCP state is implicated in the phenomenon of inflammation-induced de-differentiation, a process by which melanomas are able to evade ICBT. Our identification of the SCP and Mes states, along with a deeper understanding of their transcriptional programs and signaling pathways, may reveal clinically actionable targets to disrupt immuno- or targeted-therapy resistance. Citation Format: Vishaka Gopalan, Chun Wai Wong, Chi-Ping Day, Eva Perez-Guijarro, Maxwell P. Lee, Yuhong Jiang, Howard H. Yang, Maira Alves-Constantino, Antonella Sassano, Cari Smith, Mark Simpson, Sung Chin, Jessica Ebersole, Charli Gruen, Emily Wu, Sridhar Hannenhalli, Adam Hurlstone, Glenn Merlino, Kerrie Marie. Single-cell transcriptional analysis of embryonic melanoblasts pinpoints embryonic cell states that underlie targeted therapy and immune checkpoint blockade resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB233.

Epidermal Expression of Melanocyte Related Proteins in Normal Skin and in Vitiligo

Melanocytes are specialized neural crest-derived cells that are responsible for skin pigmentation. In vitiligo, loss of functioning epidermal melanocytes results in loss of pigment. Based on published studies, many transcription factors including microphtalmia-associated transcription factor, SRY (Sex Determining Region Y)-Box 10 are responsible for differentiation of melanocytes from neural crest cells as well as development of melanoblasts). However, the pathogenesis of loss of pigmentation in skin still unclear. We studied the expression of several melanocyte-related proteins and markers in vitiligo samples and comparing them to normally pigmented skin samples. This may provide a more insight into the pathogenesis of vitiligo which is a still controversy. We assessed the expression of six antibodies including S100, Melan-A (to detect fully formed, functioning melanocytes), CD117 (a specific relevant tyrosinase inducer), MITF and SOX-10 (important related transcription factors), and BCL-2 (stem cell markers) in 32 skin samples. We demonstrated significant decline in the expression of all examined immunomarkers in the skin of vitiligo when compared to normal pigmented skin. Our data may speculate that vitiligo pathogenesis does not involve only destruction of functioning active melanocytes, but also, other steps of cellular stimulation of induction of melanocytes and melanogensis are significantly affected as well.

Stabilization of β-catenin promotes melanocyte specification at the expense of the Schwann cell lineage.

ABSTRACTThe canonical Wnt/β-catenin pathway governs a multitude of developmental processes in various cell lineages, including the melanocyte lineage. Indeed, β-catenin regulates transcription of Mitf-M, the master regulator of this lineage. The first wave of melanocytes to colonize the skin is directly derived from neural crest cells, whereas the second wave of melanocytes is derived from Schwann cell precursors (SCPs). We investigated the influence of β-catenin in the development of melanocytes of the first and second waves by generating mice expressing a constitutively active form of β-catenin in cells expressing tyrosinase. Constitutive activation of β-catenin did not affect the development of truncal melanoblasts but led to marked hyperpigmentation of the paws. By activating β-catenin at various stages of development (E8.5-E11.5), we showed that the activation of β-catenin in bipotent SCPs favored melanoblast specification at the expense of Schwann cells in the limbs within a specific temporal window. Furthermore, in vitro hyperactivation of the Wnt/β-catenin pathway, which is required for melanocyte development, induces activation of Mitf-M, in turn repressing FoxD3 expression. In conclusion, β-catenin overexpression promotes SCP cell fate decisions towards the melanocyte lineage.

Selection signatures in goats reveal copy number variants underlying breed-defining coat color phenotypes.

Domestication and human selection have formed diverse goat breeds with characteristic phenotypes. This process correlated with the fixation of causative genetic variants controlling breed-specific traits within regions of reduced genetic diversity, so called selection signatures or selective sweeps. Using whole genome sequencing of DNA pools (pool-seq) from 20 genetically diverse modern goat breeds and bezoars, we identified 2,239 putative selection signatures. In two Pakistani goat breeds, Pak Angora and Barbari, we found selection signatures in a region harboring KIT, a gene involved in melanoblast development, migration, and survival. The search for candidate causative variants responsible for these selective sweeps revealed two different copy number variants (CNVs) downstream of KIT that were exclusively present in white Pak Angora and white-spotted Barbari goats. Several Swiss goat breeds selected for specific coat colors showed selection signatures at the ASIP locus encoding the agouti signaling protein. Analysis of these selective sweeps revealed four different CNVs associated with the white or tan (AWt), Swiss markings (Asm), badgerface (Ab), and the newly proposed peacock (Apc) allele. RNA-seq analyses on skin samples from goats with the different CNV alleles suggest that the identified structural variants lead to an altered expression of ASIP between eumelanistic and pheomelanistic body areas. Our study yields novel insights into the genetic control of pigmentation by identifying six functionally relevant CNVs. It illustrates how structural changes of the genome have contributed to phenotypic evolution in domestic goats.

Melanoblast development coincides with the late emerging cells from the dorsal neural tube in turtle Trachemys scripta

Ectothermal reptiles have internal pigmentation, which is not seen in endothermal birds and mammals. Here we show that the development of the dorsal neural tube-derived melanoblasts in turtle Trachemys scripta is regulated by similar mechanisms as in other amniotes, but significantly later in development, during the second phase of turtle trunk neural crest emigration. The development of melanoblasts coincided with a morphological change in the dorsal neural tube between stages mature G15 and G16. The melanoblasts delaminated and gathered in the carapacial staging area above the neural tube at G16, and differentiated into pigment-forming melanocytes during in vitro culture. The Mitf-positive melanoblasts were not restricted to the dorsolateral pathway as in birds and mammals but were also present medially through the somites similarly to ectothermal anamniotes. This matched a lack of environmental barrier dorsal and lateral to neural tube and the somites that is normally formed by PNA-binding proteins that block entry to medial pathways. PNA-binding proteins may also participate in the patterning of the carapacial pigmentation as both the migratory neural crest cells and pigment localized only to PNA-free areas.

Genome-wide association analysis identifies potential regulatory genes for eumelanin pigmentation in chicken plumage.

Plumage color in chicken is determined by the proportion of eumelanin and pheomelanin pigmentation. As the main ingredient in plumage melanin, eumelanin plays a key role in the dark black, brown and grey coloration. However, very few studies have been performed to identify the related genes and mutations on a genome-wide scale. Herein, a resource family consisting of one backcross population and two F2 cross populations between a black roster and Yukou Brown I parent stockbreed was constructed for identification of genes related to eumelanin pigmentation. Chickens with eumelanin in their plumage were classified as the case group, and the rest were considered the control group. A genome-wide association study of this phenotype and genotypes using Affymetrix 600K HD SNP arrays in this F2 family revealed 13 significantly associated SNPs and in 10 separate genes on chromosomes 1, 2, 3 and 5. Based on previous studies in model species, we inferred that genes, including NUAK family kinase 1 (NUAK1) and sonic hedgehog (SHH), may play roles in the development of neural crest cells or melanoblasts during the embryonic period, which may also affect the eumelanin pigmentation. Our results facilitate the understanding of the genetic basis of eumelanin pigmentation in chicken plumage.

Pigmented Lesions of the Nervous System and the Neural Crest: Lessons From Embryology.

Neurosurgeons encounter a number of pigmented tumors of the central nervous system in a variety of locations, including primary central nervous system melanoma, blue nevus of the spinal cord, and melanotic schwannoma. When examined through the lens of embryology, pigmented lesions share a unifying connection: They occur in structures that are neural crest cell derivatives. Here, we review the important progress made in the embryology of neural crest cells, present 3 cases of pigmented tumors of the nervous system, and discuss these clinical entities in the context of the development of melanoblasts. Pigmented lesions of the nervous system arise along neural crest cell migration routes and from neural crest-derived precursors. Awareness of the evolutionary clues of vertebrate pigmentation by the neurosurgical and neuro-oncological community at large is valuable for identifying pathogenic or therapeutic targets and for designing future research on nervous system pigmented lesions. When encountering such a lesion, clinicians should be aware of the embryological basis to direct additional evaluation, including genetic testing, and to work with the scientific community in better understanding these lesions and their relationship to neural crest developmental biology.

FOXD3 Promotes PAX3 Expression in Melanoma Cells.

Several key transcription factors regulate cell growth, survival, and differentiation during neural crest and melanoblast development in the embryo, and these same pathways may be reactivated in tumors arising from the progenitors of these cells. The transcription factors PAX3 and FOXD3 have essential roles in melanoblasts and melanoma. In this study, we define a regulatory pathway where FOXD3 promotes the expression of PAX3. Both factors are expressed in melanoma cells and there is a positive correlation between the transcript levels of PAX3 and FOXD3. The PAX3 gene contains two FOX binding motifs within highly conserved enhancer regulatory elements that are essential for neural crest development. FOXD3 binds to both of these motifs in vitro but only one of these sites is preferentially utilized in melanoma cells. Overexpression of FOXD3 upregulates PAX3 levels while inhibition of FOXD3 function does not alter PAX3 protein levels, supporting that FOXD3 is sufficient but not necessary to drive PAX3 expression in melanoma cells. Here, we identify a molecular pathway where FOXD3 upregulates PAX3 expression and therefore contributes to melanoma progression.

White spotting phenotype induced by targeted REST disruption during neural crest specification to a melanocyte cell lineage.

Neural crest cells (NCCs) emerge from the dorsal region of the neural tube of vertebrate embryos and have the pluripotency to differentiate into both neuronal and non-neuronal lineages including melanocytes. Rest, also known as NRSF (neuro-restrictive silencer factor), is a regulator of neuronal development and function and suggested to be involved in the lineage specification of NCCs. However, further investigations of Rest gene functions in vivo have been hampered by the fact that Rest null mice show early embryonic lethality. To investigate the function of Rest in NCC development, we recently established NCC-specific Rest conditional knockout (CKO) mice and observed their neonatal death. Here, we have established viable heterozygous NCC-specific Rest CKO mice to analyze the function of Rest in an NCC-derived melanocyte cell lineage and found that the white spotting phenotype was associated with the reduction in the number of melanoblasts in the embryonic skin. The Rest deletion induced after the specification to melanocytes did not reduce the number of melanoblasts; therefore, the expression of REST during the early neural crest specification stage was necessary for the normal development of melanoblasts to cover all of the skin.

Druggable targets in pediatric neurocutaneous melanocytosis: Molecular and drug sensitivity studies in xenograft and ex vivo tumor cell culture to identify agents for therapy.

Neurocutaneous melanocytosis (NCM) is a rare congenital disorder that presents with pigmented cell lesions of the brain or leptomeninges in children with large or multiple congenital melanocytic nevi. Although the exact pathological processes involved are currently unclear, NCM appears to arise from an abnormal development of melanoblasts or melanocyte precursors. Currently, it has an extremely poor prognosis due to rapid disease progression and lack of effective treatment modalities. In this study, we report on an experimental approach to examining NCM cells by establishing subcutaneous tumors in nude mice, which can be further expanded for conducting molecular and drug sensitivity experiments. Analysis of the NRAS gene-coding sequences of an established NCM cell line (YP-MEL) and NCM patient cells revealed heterogeneity in NRAS Q61K that activated mutation and possibly consequential differential sensitivity to MEK inhibition. Gene expression studies were performed to compare the molecular profiles of NCM cells with normal skin fibroblasts. In vitro cytotoxicity screens of libraries of targeted small-molecule inhibitors revealed prospective agents for further evaluation. Our studies provide an experimental platform for the generation of NCM cells for preclinical studies and the production of molecular and in vitro data with which to identify druggable targets for the treatment.

A microarray study of altered gene expression during melanoblasts migration in normal pigmented White Leghorn and hyperpigmented mutant Silky Fowl

Melanoblasts originating from neural crest cells can migrate through the mesenchyme of the developed embryo and give rise to melanocytes. Unlike the melanocytes that are confined to the integument in other vertebrates, melanocytes in Silky Fowl can reach the ventral regions of the embryos owing to differences in gene expression in the process of melanoblasts migration. In this study, we used microarray profiling to identify differences in gene expression between White Leghorn and Silky Fowl. Differential expression of 2517 microarray probes (P 2) was observed in Silky Fowl compared to White Leghorn. After filtration by cluster analysis, functional annotation and pathway analysis, eight differentially expressed genes were identified to be closely related to the development of melanocytes. Moreover, differences in expression of immune genes were also detected between Silky Fowl and White Leghorn. The differentially expressed genes associated with melanocyte development were verified by q-PCR, and results were highly consistent with the microarray data. The genes with significantly altered expression involved in melanoblast migration and development suggested that different microenvironments resulted in the abnormal melanoblast migration in Silky Fowl, although there were no big differences in melanoblast development between these two breeds. The candidate genes discovered in this study are beneficial to understand the molecular mechanism of hyperpigmentation in Silky Fowl.

Hereditary hypomelanocytoses: the role of PAX3, SOX10, MITF, SNAI2, KIT, EDN3 and EDNRB genes

Hypo- and hyperpigmentation disorders are the most severe dermatological diseases observed in patients from all over the world. These disorders can be divided into melanoses connected with disorders of melanocyte function and melanocytoses connected with melanocyte development. The article presents some hereditary hypomelanocytoses, which are caused by abnormal melanoblast development, migration and proliferation as well as by abnormal melanocyte viability and proliferation. These disorders are represented by Waardenburg syndrome, piebaldism and Tietz syndrome, and are caused by different mutations of various or the same genes. The types of mutations comprise missense and nonsense mutations, frameshifts (in-frame insertions or deletions), truncating variations, splice alterations and non-stop mutations. It has been demonstrated that mutations of the same gene may cause different hypopigmentation syndromes that may have similar phenotypes. For example, mutations of the MITF gene cause Waardenburg syndrome type 2A as well as Tietz syndrome. It has also been demonstrated that mutations of different genes may cause an identical syndrome. For example, mutations of MITF, SNAI2 and SOX10 genes are observed in Waardenburg syndrome type II and mutations of EDNRB, EDN3 and SOX10 genes are responsible for Waardenburg syndrome type IV. In turn, mutation of the KIT gene and/or heterozygous deletion of the SNAI2 gene result in piebaldism disease. The knowledge of the exact mechanisms of pigmentary disorders may be useful in the development of new therapeutic approaches to their treatment.

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.

Irradiance, but not fluence, plays a crucial role in UVB‐induced immature pigment cell development: new insights for efficient UVB phototherapy

Light exposure modulates development of living organisms. In the field of medicine, light has frequently been used for regenerative purposes. Excimer light (308nm) has demonstrated superior efficacy in treating vitiligo, a condition requiring development of melanoblasts and a model for studying nerve cell regeneration, as compared to narrow-band ultraviolet B (NBUVB; 311nm). Using mouse-derived melanoblast cells to examine the pro-differentiation effects of these two light sources, we demonstrated that at equivalent fluence, excimer light induces melanoblast differentiation, while NBUVB failed to so. Mechanistically, activation of aryl hydrocarbon receptor pathway and nuclear translocation of epidermal growth factor receptor are involved in pro-differentiation effects of excimer light. Reduction in irradiance by filter abrogated the effects of excimer light in melanoblasts, even when equivalent fluence was delivered by the same light source. As ultraviolet B (UVB) irradiation is closely associated pigment cell development, future therapy employing UVB for pigmentation purposes should incorporate irradiance as a crucial specification.

The EJC component Magoh regulates proliferation and expansion of neural crest-derived melanocytes

Melanoblasts are a population of neural crest-derived cells that generate the pigment-producing cells of our body. Defective melanoblast development and function underlies many disorders including Waardenburg syndrome and melanoma. Understanding the genetic regulation of melanoblast development will help elucidate the etiology of these and other neurocristopathies. Here we demonstrate that Magoh, a component of the exon junction complex, is required for normal melanoblast development. Magoh haploinsufficient mice are hypopigmented and exhibit robust genetic interactions with the transcription factor, Sox10. These phenotypes are caused by a marked reduction in melanoblast number beginning at mid-embryogenesis. Strikingly, while Magoh haploinsufficiency severely reduces epidermal melanoblasts, it does not significantly affect the number of dermal melanoblasts. These data indicate Magoh impacts melanoblast development by disproportionately affecting expansion of epidermal melanoblast populations. We probed the cellular basis for melanoblast reduction and discovered that Magoh mutant melanoblasts do not undergo increased apoptosis, but instead are arrested in mitosis. Mitotic arrest is evident in both Magoh haploinsufficient embryos and in Magoh siRNA treated melanoma cell lines. Together our findings indicate that Magoh-regulated proliferation of melanoblasts in the dermis may be critical for production of epidermally-bound melanoblasts. Our results point to a central role for Magoh in melanocyte development.

Postnatal lineage mapping of follicular melanocytes with the Tyr::CreERT2 transgene

One of the main advantages of using inducible and conditional transgenes to study pigment cell biology is that they allow for genetic manipulation within melanocytes after roles in general neural crest or melanoblast development have been fulfilled. Specifically, we focus here on the ability of the Tyr::CreER(T) (2) transgenic line to alter genes within follicular melanocytes postnatally. Using the Gt(ROSA)26Sor(tm1sor) reporter allele, we present in detail the expression and activity of Tyr::CreER(T) (2) when induced during hair morphogenesis and adult hair cycling. We find that despite similarities in expression pattern to endogenous TYR, Tyr::CreER(T) (2) is effective at targeting both undifferentiated and differentiated melanocytes within the hair follicle. We also find that Tyr::CreER(T) (2) provides the highest levels of recombination when induced during the early phases of hair growth. In conclusion, the descriptions provided here will guide future analyses of gene function within the melanocyte system of the hair follicle when using this Tyr::CreER(T) (2) transgene.

Emerging Roles of ADAMTSs in Angiogenesis and Cancer

A Disintegrin-like And Metalloproteinase with ThromboSpondin motifs—ADAMTSs—are a multi-domain, secreted, extracellular zinc metalloproteinase family with 19 members in humans. These extracellular metalloproteinases are known to cleave a wide range of substrates in the extracellular matrix. They have been implicated in various physiological processes, such as extracellular matrix turnover, melanoblast development, interdigital web regression, blood coagulation, ovulation, etc. ADAMTSs are also critical in pathological processes such as arthritis, atherosclerosis, cancer, angiogenesis, wound healing, etc. In the past few years, there has been an explosion of reports concerning the role of ADAMTS family members in angiogenesis and cancer. To date, 10 out of the 19 members have been demonstrated to be involved in regulating angiogenesis and/or cancer. The mechanism involved in their regulation of angiogenesis or cancer differs among different members. Both angiogenesis-dependent and -independent regulation of cancer have been reported. This review summarizes our current understanding on the roles of ADAMTS in angiogenesis and cancer and highlights their implications in cancer therapeutic development.

Modeling melanoblast development.

Melanoblasts are a particular type of cell that displays extensive cellular proliferation during development to contribute to the skin. There are only a few melanoblast founders, initially located just dorsal to the neural tube, and they sequentially colonize the dermis, epidermis, and hair follicles. In each compartment, melanoblasts are exposed to a wide variety of developmental cues that regulate their expansion. The colonization of the dermis and epidermis by melanoblasts involves substantial proliferation to generate thousands of cells or more from a few founders within a week of development. This review addresses the cellular and molecular events occurring during melanoblast development. We focus on intrinsic and extrinsic factors that control melanoblast proliferation. We also present a robust mathematical model for estimating the doubling-time of dermal and epidermal melanoblasts for all coat color phenotypes from black to white.

Impaired development of melanoblasts in the black‐eyed white Mitfmi‐bw mouse, a model for auditory–pigmentary disorders

Microphthalmia-associated transcription factor (Mitf) is a regulator for differentiation of melanoblasts that are derived from the neural crest. The mouse homozygous for the black-eyed white (Mitf(mi-bw)) allele is characterized by the white coat color and deafness, with black eye that is associated with the lack of melanocytes in skin and inner ear. The Mitf(mi-bw) mutation is an insertion of the LINE1 retrotransposable element into intron 3 of the Mitf gene that causes the selective deficiency of the melanocyte-specific Mitf isoform, Mitf-M. Here, we show the expression of Mitf-M mRNA in the trunk region of the homozygous Mitf(mi-bw)(bw) mouse at embryonic days (E) 11.5 and E12.5, but Mitf-M mRNA is undetectable at E13.5. In addition, using bw mouse that carries the lacZ transgene under the control of a melanoblast-specific promoter, we show that the number of migrating melanoblasts in bw embryos was less than 10% of that in control embryos at E11.5 and E12.5, and melanoblasts disappear by E13.5. The loss of melanoblasts in bw embryos was probably caused by apoptosis. Finally, forced expression of Mitf-M in the cultured neural tube of bw embryos ensured the differentiation of melanoblasts. Therefore, the correct dose of Mitf-M is required for the normal development of melanoblasts.

Multiple Roles of Integrin-Linked Kinase in Epidermal Development, Maturation and Pigmentation Revealed by Molecular Profiling

Integrin-linked kinase (ILK) is an important scaffold protein that mediates a variety of cellular responses to integrin stimulation by extracellular matrix proteins. Mice with epidermis-restricted inactivation of the Ilk gene exhibit pleiotropic phenotypic defects, including impaired hair follicle morphogenesis, reduced epidermal adhesion to the basement membrane, compromised epidermal integrity, as well as wasting and failure to thrive leading to perinatal death. To better understand the underlying molecular mechanisms that cause such a broad range of alterations, we investigated the impact of Ilk gene inactivation on the epidermis transcriptome. Microarray analysis showed over 700 differentially regulated mRNAs encoding proteins involved in multiple aspects of epidermal function, including keratinocyte differentiation and barrier formation, inflammation, regeneration after injury, and fundamental epidermal developmental pathways. These studies also revealed potential effects on genes not previously implicated in ILK functions, including those important for melanocyte and melanoblast development and function, regulation of cytoskeletal dynamics, and homeobox genes. This study shows that ILK is a critical regulator of multiple aspects of epidermal function and homeostasis, and reveals the previously unreported involvement of ILK not only in epidermal differentiation and barrier formation, but also in melanocyte genesis and function.