Functional genomic analysis of neural crest development

神经脊细胞是脊椎动物特有的胚胎结构，它产生了包括产生色素的黑素细胞在内的多个谱系。色素沉着，定义为色素在皮肤、头发和眼睛中的放置以供着色，是独特的，因为色素沉着的位置、数量和类型在调节色素产生细胞的途径中提供了遗传异质性的视觉表现。这种遗传异质性的范围从正常到病理性色素沉着表型。临床上，正常的人类色素沉着包括各种皮肤和头发的颜色以及点状色素沉着，如黑色素细胞痣(痣)或雀斑(雀斑)，而异常的人类色素沉着表现为显著减少或增加的色素水平，分别称为色素减少和色素过度沉积。对色素沉着的分子遗传学的阐明揭示了对黑素细胞发育和功能至关重要的基因。此外，许多色素沉着障碍在黑素细胞以外的细胞中表现出额外的缺陷，并且对这些疾病中的遗传侮辱的鉴定揭示了多向性，其中不同细胞类型的不同功能需要单个基因。因此，揭开容易看到的色素沉着障碍的遗传学已经确定了黑素细胞和较不明显的细胞类型/组织之间的分子相似之处，源于共同的发育起源和/或共同的遗传调控途径。我们利用各种遗传和基因组方法来发现人类色素沉着障碍的病因学，通常侧重于这样一个事实，即破坏色素沉着的发育突变对于理解控制相关疾病的异常途径具有指导意义。 黑素细胞分化：编码转录因子TFAP2A的基因突变导致模式生物中的色素异常和人类的过早白发。然而，TFAP2A及其冗余作用的类似物的多效性功能使得TFAP2类活性对黑素细胞分化的确切贡献尚不清楚。确定这一贡献可能有助于解释为什么与良性痣相比，晚期黑色素瘤TFAP2A的表达减少。为了确定在黑素细胞中依赖于TFAP2A表达的基因，我们描述了斑马鱼组织和缺乏TFAP2A的小鼠黑素细胞，发现支持色素表型的一小部分基因的表达是依赖于TFAP2A的，包括DCT、MC1R、MLPH和PMEL。然后，我们在小鼠和人类的黑素细胞中进行了TFAP2A芯片序列分析，发现更大的色素沉着基因子集与TFAP2A结合的活性调节元件有关。这些元素也经常被MITF结合，MITF被认为是黑素细胞发育的“主要调节器”。例如，TRPM1的启动子同时与TFAP2A和MITF结合，我们发现最小的TRPM1启动子的活性在TFAP2A结合位点缺失后失去。然而，Trpm1的表达不依赖于TFAP2A，这意味着额外的TFAP2类似物冗余地驱动黑素细胞分化，这与先前在斑马鱼上的结果一致。TFAP2a和Tfap2b在小鼠黑素细胞中都有表达，我们的研究表明，在Wnt1-Cre介导的神经脊TFAP2a和Tfap2b缺失的小鼠胚胎几乎完全缺乏黑素细胞，但保留了神经脊来源的感觉神经节。这些结果表明，与MITF类似的TFAP2对黑素细胞谱系的诱导也是必要的。最后，我们在斑马鱼中观察到TFAP2A和MITFA之间的遗传相互作用，但发现人工上调TFAP2A的表达并不会增加MITFA亚型或功能丧失突变体中的黑色素水平。总而言之，这些结果表明，TFAP2类似物与MITF等谱系特异性转录因子一起工作，直接调节黑素细胞末端分化的效应因子。此外，他们还表明，TFAP2A活性和MITF活性一样，有可能调节黑色素瘤细胞的表型。 黑素细胞状态：缺氧和HIF1信号直接调控肿瘤进展、侵袭和转移的组织特异性基因反应。通过整合HIF1基因敲除和低氧诱导的基因表达变化，本研究确定了黑素细胞特异性的、HIF1依赖/低氧响应的基因表达特征。将这些基因表达变化与HIF1芯片序列分析相结合，确定了黑素细胞中81个HIF1直接靶基因。通过Logistic回归(P值=0.0013)和ROC曲线分析(AUC=0.826，P值和t；0.0001)，HIF1的10个直接靶标GAPDH、PKM、PPAT、DARS、DTWD1SEH1L、ZNF292、RLF、AGTRAP和GPC6的表达水平与黑色素瘤无病状态的缩短显著相关。这个HIF1调节的图谱定义了黑素细胞在低氧条件下的特异性反应，并证明了HIF1作为侵袭性细胞状态守门人在调节细胞代谢、染色质和转录调节、血管形成和侵袭方面的作用。

Neural crest cells are an embryonic structure unique to vertebrates that gives rise to multiple lineages including the pigment-producing melanocytes. Pigmentation, defined as the placement of pigment in skin, hair, and eyes for coloration, is distinctive because the location, amount, and type of pigmentation provides a visual manifestation of genetic heterogeneity in pathways regulating the pigment-producing cells. The scope of this genetic heterogeneity ranges from normal to pathological pigmentation phenotypes. Clinically, normal human pigmentation encompasses a variety of skin and hair color as well as punctate pigmentation such as melanocytic nevi (moles) or ephelides (freckles), while abnormal human pigmentation exhibits markedly reduced or increased pigment levels, known as hypopigmentation and hyperpigmentation, respectively. Elucidation of the molecular genetics underlying pigmentation has revealed genes important for melanocyte development and function. Furthermore, many pigmentation disorders show additional defects in cells other than melanocytes, and identification of the genetic insults in these disorders has revealed pleiotropism, where a single gene is required for various functions in different cell types. Thus, unraveling the genetics of easily visualized pigmentation disorders has identified molecular similarities between melanocytes and less visible cell types/tissues, arising from a common developmental origin and/or shared genetic regulatory pathways. We utilize a variety of genetic and genomic approaches to discover the etiology of human pigmentation disorders, often focusing on the fact that the developmental mutations disrupting pigmentation are instructive for understanding abnormal pathways governing related disorders. Melanocyte differentiation: Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the "master regulator" of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells. Melanocyte cell state: Hypoxia and HIF1 signaling direct tissue-specific gene responses regulating tumor progression, invasion, and metastasis. By integrating HIF1 knockdown and hypoxia-induced gene expression changes, this study identifies a melanocyte-specific, HIF1-dependent/hypoxia-responsive gene expression signature. Integration of these gene expression changes with HIF1 ChIP-Seq analysis identifies 81 HIF1 direct target genes in melanocytes. The expression levels for 10 of the HIF1 direct targets - GAPDH, PKM, PPAT, DARS, DTWD1, SEH1L, ZNF292, RLF, AGTRAP, and GPC6 - are significantly correlated with reduced time of disease-free status in melanoma by logistic regression (P-value=0.0013) and ROC curve analysis (AUC=0.826, P-value<0.0001). This HIF1-regulated profile defines a melanocyte-specific response under hypoxia, and demonstrates the role of HIF1 as an invasive cell state gatekeeper in regulating cellular metabolism, chromatin and transcriptional regulation, vascularization, and invasion.