Pbx-Directed Control of Cellular Behaviors that Drive Midface Morphogenesis

Pbx 定向控制驱动中面部形态发生的细胞行为

• 批准号: 10620238
• 负责人: Licia Selleri
• 金额: $ 57.88万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10620238
• 关键词: Apoptosis; Apoptotic; Bioinformatics; Biological Assay; Bromodeoxyuridine; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Proliferation; Cells; Cephalic; Cleft Palate; Cleft lip with or without cleft palate; Congenital Abnormality; Cryoultramicrotomy; Development; Diagnostic; Effector Cell; Embryo; Epithelial Cells; Epithelium; Exhibits; Face; Fostering; Frontonasal Prominence; Genes; Genetic; Genetic Transcription; Grant; Histone H3; Human; Image; Immunofluorescence Immunologic; Impairment; In Situ Hybridization; Individual; Lip structure; Live Birth; Maxillary Prominence; Mediating; Mesenchyme; Microdissection; Molecular; Molecular Profiling; Morphogenesis; Mus; Neural Crest; Pathogenesis; Pathway interactions; Population; Pregnancy; Premaxillary palate; Process; Publishing; Quantitative Reverse Transcriptase PCR; Reporting; Research; Societies; Stains; Testing; Time; Tissues; Transcript; Work; cdc Genes; cell behavior; comparison control; craniofacial; epithelial to mesenchymal transition; experimental study; in vivo; inhibitor; mouse model; mutant; orofacial cleft; palate repair; prenatal; progenitor; programs; repair strategy; single-cell RNA sequencing; spatiotemporal; tissue repair; transcription factor; transcriptome; wound healing

PROJECT SUMMARY Human cleft lip with or without cleft palate (CL/P), the most common craniofacial birth defect, is caused by im- paired fusion of the facial prominences. During normal morphogenesis, the frontonasal and maxillary promi- nences fuse at a three-way seam, the lambdoidal junction (l), to form the upper lip/primary palate. If the epithe- lium that covers the prominences persists at the l, orofacial clefting ensues, as we previously reported in mouse embryos deficient for PBX transcription factors (TFs). We described that: 1) prominence fusion requires coordi- nation of two distinct cellular behaviors, apoptosis and epithelial-to-mesenchymal transition (EMT), respectively, in two discrete subpopulations within the l epithelium; 2) these two subpopulations are characterized by different molecular signatures; and 3) mice deficient for PBX TFs with CL/P lack both of these epithelial subpopulations. Our preliminary evidence in the mouse indicates that: I) the λ epithelium is heterogeneous and comprises 6 main cell subpopulations prior to prominence fusion, as demonstrated by single cell RNA sequencing; II) one of the subpopulations, that we termed “l fusion effectors”, is enriched for genes that have been associated with human or mouse orofacial clefting, for genes encoding pro-apoptotic factors, and for inhibitors of cell cycle progression; III) “l fusion effector” cells are located at the tip of the prominences; and IV) the proportion of “l fusion effectors” is perturbed in compound Pbx1/2 mutant embryos with CL/P compared to controls. Based on these results, we posit that the “l fusion effector” cluster comprises cells that are prime executors of prominence fusion; is halted in the cell cycle, a prerequisite for subsequent cellular changes, like apoptosis and EMT, to achieve fusion; and is quantitatively and/or qualitatively perturbed in mouse models of CL/P. We will test this hypothesis via the following Specific Aims: 1) Determine the spatiotemporal dynamics of the epithelial “l fusion effector” subpopulation throughout midface prominence fusion. We will establish whether the “l fusion effector” clus- ter is transient or if it persists after fusion, as well as track the progenitors and descendants of “l fusion effector” cells in vivo across space and developmental time. 2) Establish the molecular mechanisms underlying cell cycle arrest in “l fusion effector” cells during upper lip/primary palate fusion. We will uncover in vivo cell cycle dynamics of “l fusion effector” cells and assess whether PBX-dependent regulation of cell cycle genes mediates cell cycle control in this cluster. 3) Determine whether the perturbations of the “λ fusion effector” cluster in Pbx1/2 mutants are recapitulated in p63- or Bmpr1a-deficient mice with CL/P. We will establish whether the cellular and transcriptional changes of the l epithelium resulting in CL/P in Pbx1/2 mutants are recapitulated in all three mouse models, or if they are distinct. This research will lead to discover new genes for prenatal diagnostics of CL/P and open strategies for CL/P repair through reactivation of developmental programs that are defective in orofacial clefting. Broadly, this work will foster mechanistic studies testing whether other fusion processes are mediated by cell cycle arrest.

项目摘要 人类唇裂伴或不伴腭裂（CL/P）是最常见的颅面出生缺陷，其原因是先天性唇腭裂。 面部表情的成对融合在正常的形态发生过程中，额鼻和上颌前突， 鼻突在三向接缝处融合，即鼻突连接处（L），以形成上唇/主腭。如果表皮- 正如我们以前在小鼠中报道的那样， 胚胎缺乏PBX转录因子（TF）。我们描述了：1）日珥融合需要协调- 两种不同的细胞行为，细胞凋亡和上皮间质转化（EMT）的国家，分别， 在L上皮内的两个离散的亚群中; 2）这两个亚群的特征在于不同的 分子标记;和3）具有CL/P的PBX TF缺陷的小鼠缺乏这两种上皮亚群。 我们在小鼠中的初步证据表明：I）λ上皮是异质的，包括6个主要的上皮细胞， 突出融合前的细胞亚群，如通过单细胞RNA测序所证明的; II）突出融合前的细胞亚群之一， 我们称之为“L融合效应子”的亚群富集了与人类基因组相关的基因， 或小鼠口面裂，用于编码促凋亡因子的基因，和用于细胞周期进程的抑制剂; III）“l融合效应物”细胞位于双链末端;和 与对照相比，在具有CL/P的复合Pbx 1/2突变体胚胎中受到干扰。基于这些结果，我们 假设“l融合效应物”簇包含作为突出融合的主要执行者的细胞; 在细胞周期中，是后续细胞变化（如凋亡和EMT）实现融合的先决条件; 在CL/P小鼠模型中定量和/或定性干扰。我们将通过 以下具体目标：1）确定上皮“l融合效应器”的时空动态 整个面中部突出融合的亚群。我们将确定“融合效应器”是否- ter是瞬时的，或者如果它在融合后持续存在，以及跟踪“l融合效应子”的祖细胞和后代。 跨空间和发育时间的体内细胞。2)建立细胞的分子机制 在上唇/原腭融合期间“L融合效应”细胞的周期停滞。我们将发现体内细胞 “I融合效应”细胞的周期动力学，并评估细胞周期基因的PBX依赖性调节是否 在这个簇中调节细胞周期控制。3)确定“λ融合效应器”的扰动是否 Pbx 1/2突变体的簇在p63或Bmpr 1a缺陷的CL/P小鼠中重现。 在Pbx 1/2突变体中导致CL/P的l上皮细胞的细胞和转录变化是否 在所有三种小鼠模型中重现，或者如果它们是不同的。这项研究将导致发现新的基因， CL/P的产前诊断和通过重新激活发育程序进行CL/P修复的开放策略 在口面裂方面有缺陷从广义上讲，这项工作将促进机制研究，测试其他 融合过程由细胞周期停滞介导。

项目成果

Toward Microsurgical Correction of Cleft Lip Ex Utero through Restoration of Craniofacial Developmental Programs.

通过恢复颅面发育程序来实现宫外唇裂的显微外科矫正。

• DOI: 10.1097/prs.0000000000003417
• 发表时间: 2017
• 期刊: Plastic and reconstructive surgery
• 影响因子: 3.6
• 作者: Dong,Xue;Landford,WilminaN;Hart,James;Risolino,Maurizio;Kaymakcalan,Omer;Jin,Julia;Toyoda,Yoshiko;Ferretti,Elisabetta;Selleri,Licia;Spector,JasonA
• 通讯作者: Spector,JasonA

Pbx loss in cranial neural crest, unlike in epithelium, results in cleft palate only and a broader midface.

• DOI: 10.1111/joa.12821
• 发表时间: 2018-08
• 期刊: Journal of anatomy
• 影响因子: 2.4
• 作者: Welsh IC;Hart J;Brown JM;Hansen K;Rocha Marques M;Aho RJ;Grishina I;Hurtado R;Herzlinger D;Ferretti E;Garcia-Garcia MJ;Selleri L
• 通讯作者: Selleri L