The tailless ortholog nhr-67 functi

The tailless ortholog nhr-67 functions in the development of the C. elegans ventral uterus

Eliana Verghesea, John Schockena, Sandrine Jacobb, Angela M. Wimera, Rebecca Roycea, Jessica E. Nesmitha, G. Michael Baera, Sheila Clevera, Elizabeth McCaina, Bernard Lakowskib, Bruce Wightmana, ,
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doi:10.1016/j.ydbio.2011.06.007
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Abstract
The development of the C. elegans uterus provides a model for understanding the regulatory pathways that control organogenesis. In C. elegans, the ventral uterus develops through coordinated signaling between the uterine anchor cell (AC) and a ventral uterine (VU) cell. The nhr-67 gene encodes the nematode ortholog of the tailless nuclear receptor gene. Fly and vertebrate tailless genes function in neuronal and ectodermal developmental pathways. We show that nhr-67 functions in multiple steps in the development of the C. elegans uterus. First, it functions in the differentiation of the AC. Second, it functions in reciprocal signaling between the AC and an equipotent VU cell. Third, it is required for a later signaling event between the AC and VU descendants. nhr-67 is required for the expression of both the lag-2/Delta signal in the AC and the lin-12/Notch receptor in all three VU cells and their descendants, suggesting that nhr-67 may be a key regulator of Notch-signaling components. We discuss the implications of these findings for proposed developmental regulatory pathways that include the helix–loop–helix regulator hlh-2/daughterless and transcription factor egl-43/Evi1 in the differentiation of ventral uterine cell types.

Research highlights

► The nhr-67 tailless gene functions in multiple steps of ventral uterus development. ► nhr-67 is required for the Notch-mediated AC–VU signal. ► nhr-67 is a key regulator of lin-12/Notch and the lag-2/Delta. ► nhr-67 functions in differentiation of the anchor cell.

Keywords
Notch; Organogenesis; Nuclear receptors
Introduction
The NR2E1/NR2E2 nuclear receptor transcription factors are conserved among animal phyla where they play major roles in regulating development. The Drosophila gene tailless (tll) functions in embryonic body patterning and the development of neurons ( Daniel et al., 1999, Jürgens et al., 1984 and Pignoni et al., 1990). The vertebrate ortholog of tailless (Tlx) functions in the development of limbic and rhinencephalic brain regions in the mouse and is a key regulator of embryonic and adult neural stem cell development ( Monaghan et al., 1997, Shi et al., 2004 and Yu et al., 1994). The C. elegans genome sequencing project identified nhr-67, which encodes the nematode ortholog of tll ( DeMeo et al., 2008 and Gissendanner et al., 2004). Like most nuclear receptors, nhr-67 encodes a protein with a well-conserved DNA-binding domain and a more weakly conserved ligand-binding domain, although ligands have not been identified for any members of the NR2E1/NR2E2 subclass. RNA-mediated interference (RNAi) experiments revealed that nhr-67(RNAi) animals were slow-growing and displayed cuticle-shedding defects, egg-laying defects (Egl phenotype), and a protruding vulva (Pvl phenotype), suggesting that nhr-67 plays multiple roles in development ( Gissendanner et al., 2004). Fernandes and Sternberg (2007) demonstrated that nhr-67 plays a role in cell fusion events during vulval morphogenesis, Kato and Sternberg (2009) showed that nhr-67 functions in the migration of the male linker cell, and Sarin et al. (2009) identified a role for nhr-67 in neuronal differentiation. In this study, we describe the role of nhr-67 in regulating development of cells that comprise the ventral C. elegans hermaphrodite uterus.

The development of the C. elegans uterus has served as a model for understanding the mechanisms of organogenesis. A series of cell–cell signaling events utilize the Notch pathway to create the adult hermaphrodite uterus, which consists of sixty cells that contain a lumen and connect to the dorsal side of the vulva ( Fig. 1; Newman and Sternberg, 1996 and Newman et al., 1996). The anchor cell (AC) of the ventral uterus plays a pivotal role in coordinating the development of the uterus. The AC is generated from one of two equipotent cells in the immature L2 somatic gonad. The cell that does not become the AC becomes a VU (ventral uterine) cell. The selection of which cell executes each fate depends on birth order and reciprocal signaling between a delta-like signal, LAG-2, and a Notch-like receptor, LIN-12 ( Greenwald et al., 1983, Karp and Greenwald, 2003 and Kimble and Simpson, 1997). The AC expresses the LAG-2 signal and down-regulates expression of LIN-12 receptor, while the VU cell down-regulates expression of LAG-2. When function of either lin-12 or lag-2 is compromised, the VU cell executes an AC fate, resulting in the formation of 2 AC's. Two other somatic gonad cells also serve as VU cells, for a total complement of three VU and one AC in late L2 wild-type animals. While the AC is terminally differentiated in the L2, the three VU cells divide multiple times during the L3 and L4 stages to produce 36 cells that comprise the ventral uterine tissue and part of the uterine–spermathecal junction ( Kimble and Hirsh, 1979).

Summary of ventral uterine development. Larval stages are designated in left ...
Fig. 1.
Summary of ventral uterine development. Larval stages are designated in left column. VU cells are shown as stippled nuclei. The AC is shown in black until it fuses with UTSE. The π cells and their descendants are shown as white nuclei. Cell–cell communication events are designated by solid gray arrows, cellular migrations by broken arrows. The early L3 through late L4 views show the π cells and their descendant cells on one side of the animal only; the total number of π lineage cells is double that shown.
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During the L3 stage, the AC signals using LAG-2 to six adjacent VU descendants via the LIN-12 receptor to execute a single dorsal–ventral division, which is termed the π fate (Fig. 1; Newman et al., 1995, Newman et al., 1996 and Newman et al., 2000). The twelve π cell descendants differentiate into four UV1 cells, which will connect directly to the dorsal side of the vulva, and eight cells that fuse to create a large, thin UTSE syncytial cell that forms the ventral surface of the uterus. The six additional VU descendants that do not receive the LAG-2 AC signal execute a default anterior–posterior division and then divide a second time (ρ fate). Following its final role in orchestrating the development of the uterus, the AC “retires” by fusing with the UTSE syncytium during the L4 stage (Newman et al., 1996).

The similarity among the phenotypes caused by nhr-67 mutations and the phenotypes of mutations in other genes that function in uterine development, combined with a dynamic pattern of nhr-67 expression in AC and VU cells, suggested that nhr-67 might function in a lin-12/Notch pathway to regulate uterine development. To test this possibility, we identified and studied mutations in the nhr-67 coding region and promoter, and explored the regulation of nhr-67 in ventral uterine cells. Our data support a model in which nhr-67 functions upstream of the lin-12/Notch receptor in the VU lineages, upstream of the lag-2/Delta signal in the AC, and in a pathway that includes hlh-2/daughterless and egl-43/Evi1 transcription factors to control ventral uterine development at multiple steps.

Materials and methods
Identification and characterization of nhr-67 promoter mutations

During screens for spontaneous mutations with visible phenotypes in a dog-1(gk10)I background, we isolated a mutation with a partially penetrant Egl and Pvl phenotype superficially similar to the weak sel-12(ar131) presenilin mutation. Genetic mapping experiments placed pf2 on LG IV in an interval between SNP_R13[1] and pkP4085 near dbP7 (data not shown). dog-1 encodes a DNA helicase orthologous to the human BACH1/BRIP1/FANCJ gene implicated in Fanconi anemia and certain cancers ( Cheung et al., 2002 and Youds et al., 2008). Elimination of dog-1 function leads to a high frequency of deletions starting at long G-rich sequences, especially poly-G stretches, that can form multiple G-quadruplexes when the DNA is single stranded ( Cheung et al., 2002, Kruisselbrink et al., 2008 and Zhao et al., 2008). We looked for candidate genes in the region that could explain the Egl phenotype and which contained a G-rich sequence in, or near the gene. For each candidate gene we designed primers flanking the G-rich sequences and tested for the presence of deletions in the pf2 strain ( Table S2). No deletions were identified for the Notch ligands dsl-5 and dsl-6, nor for T01G1.3, but a small deletion was detected in the promoter region of nhr-67 by genomic amplification. A stronger Egl mutation with a larger deletion of the nhr-67 promoter, pf159, was recovered in the same screen.

The pf88 mutation was recovered as a spontaneous mutation arising in the background of a dog-1(gk10) I; sel-12(ty11) spr-3(pf83) X strain. The sel-12(ty11) mutation causes a strong Egl defect and a moderate penetrance Pvl defect ( Cinar et al., 2001 and Gontijo et al., 2009), similar to a weak lin-12(lf) allele. The phenotypic effects of sel-12 mutations are completely suppressed by loss of spr-3 activity which leads to the de-repression of the transcription of the hop-1 presenilin gene ( Lakowski et al., 2003). The pf88 mutation causes a nearly 100% penetrant Egl and Pvl phenotype in the sel-12 spr-3 background, indicating that pf88 is epistatic to spr-3 (data not shown). The pf2, pf88 and pf159 mutations were removed from the presence of all other known mutations in the isolation strains by outcrosses and the presence of the nhr-67 deletion and absence of dog-1(gk10) was confirmed by amplification from genomic DNA using specific oligonucleotides ( Table S2) before phenotypic characteriza

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无尾类nhr-67功能开发中的线虫腹子宫

Eliana verghesea，约翰schockena，桑德琳雅各布，安吉拉M. wimera，丽贝卡roycea，杰西卡E. nesmitha，G.迈克尔baera，希拉clevera mccaina，伊丽莎白，布鲁斯，伯纳德lakowskib，wightmana，
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DOI：10.1016 / j.ydbio。2011.06.007
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摘要
秀丽隐杆线虫的子宫发育的了解，控制器官的调节途径提供了一个模型。线虫，腹子宫通过协调信号子宫锚细胞之间发展（AC）和腹侧子宫（VU）细胞。的nhr-67基因编码的无尾核受体基因的线虫基因。粉煤灰和脊椎动物无尾基因功能的神经元和外胚层发育途径。我们发现，nhr-67功能在多个步骤中的线虫子宫发育。首先，它的功能在AC二分化，它的功能在相互交流及等信号VU细胞之间。第三，这是需要以后的信号事件之间的交流和似曾相识的后裔。nhr-67是两者的滞后/三角信号在交流和LIN-12/Notch受体在所有三VU细胞及其后代的表达水平，提示nhr-67可能是一个关键的调节Notch信号元件。我们将讨论这些研究结果的提出发展调控途径，包括螺旋–环–螺旋调节器hlh-2 / daughterless和转录在腹部子宫的细胞类型的分化因子egl-43 /邪恶的影响。

研究集锦

►的nhr-67无尾基因的功能在多个步骤腹子宫发育。►nhr-67是缺口需要介导的交流–似曾相识的信号。►nhr-67是一个关键的调节LIN-12/Notch和滞后/三角洲。►nhr-67功能在锚细胞分化。

关键词
缺口；器官发生；核受体
介绍
的NR2E1 / nr2e2核受体转录因子是保守的动物门，他们扮演着重要的角色，在规范发展中。果蝇基因无尾（TLL）在胚体形成的功能和神经元的发育（丹尼尔等人。，1999，Jürgens等人。，1984 pignoni等人。，1990）。无尾的脊椎动物的同源基因（TLX）在边缘和嗅脑的大脑区域在小鼠是胚胎和成体神经干细胞发育的关键调节功能（莫纳汉等人的发展。，shi et al., 2004), and yu et al., 1994). the c. elegans genome sequencing project for nhr - 67, which encoded the nematode ortholog of tll, demeo's crew, et al., 2008) and gissendanner et al., 2004). like most nuclear receptors, encoded a protein with nhr or a well conserved dna binding domain and a more weakly conserved ligand binding domain.虽然尚未确定配体的任何成员NR2E1 / nr2e2类。RNA干扰（RNAi）介导的实验表明，nhr-67（RNAi）动物生长缓慢，显示表皮脱落的缺陷，产蛋的缺陷（EGL表型），和一个突出的外阴（PVL表型），这表明nhr-67发展中扮演多重角色（gissendanner等人。，2004）。费尔南德斯和斯腾伯格（2007）表明，nhr-67中起作用的细胞融合的事件在外阴的形态，卡托和斯腾伯格（2009）表明，在男性nhr-67功能连接细胞的迁移，和沙林等。（2009）确定为nhr-67神经元分化的作用。在这项研究中，我们描述了在调节细胞，包括腹线虫雌雄同体的nhr-67子宫发育的作用。

秀丽隐杆线虫的子宫的发展曾为了解形成机制模型。一系列的细胞–细胞信号转导事件通过Notch信号途径打造成年雌雄同体的子宫，其中包括六十个细胞，其中包含一个管腔和连接到外阴背侧（图1；纽曼和斯腾伯格，1996和纽曼等，1996）。腹侧子宫的锚细胞（交流）对子宫的发育起着举足轻重的作用。交流是从一个在未成熟的L2体性腺细胞产生两等效。这并不成为交流成为一个似曾相识的细胞（腹子宫）细胞。其中细胞执行每个命运取决于出生顺序和交互信令三角信号，滞后之间的选择，和Notch受体，LIN-12（Greenwald等人。，1983，卡普和格林沃尔德，2003、金布尔和辛普森，1997）。交流表达的滞后信号及下调LIN-12受体的表达，而武细胞下调表达的滞后。当函数或LIN-12或滞后被攻破，VU细胞进行交流的命运，导致2年代两体性腺细胞交流的形成也是似曾相识的细胞，一个总补体三武一晚L2野生动物交流。而交流是在L2的终末分化，三VU细胞多次分裂在L3和L4阶段产生36个细胞，包括腹子宫组织和子宫–精囊连接部分（Kimble和赫什，1979）。

总结腹子宫发育。幼虫阶段被指定在左。子宫腹侧发育的总结。在左栏中的幼虫阶段被指定。似曾相识的细胞表现为斑点状核。交流是黑色的显示到融合与采用。的π细胞及其后代表现为白核。细胞通讯事件被指定为固体灰箭头，细胞迁移被打破的箭头。早期的L3到L4视图显示π细胞及其后代细胞在动物的一面；π谱系细胞的总数量是双显示。
图选项
L3阶段，采用滞后六相邻似曾相识的后代通过LIN-12受体执行单背–腹侧部交流信号，这被称为π命运（图1，1995；纽曼等人，纽曼等，1996和纽曼等，2000）。十二π细胞的后代分化为四1细胞，这将直接连接到外阴部的背侧，八细胞融合形成一个大的，薄的采用合胞体细胞形成子宫腹侧表面。另外六个似曾相识的后代，没有收到滞后的交流信号执行默认的前–后分然后分一次（ρ命运）。继在策划的子宫发育的最终作用，交流“退休”的融合与采用合胞体L4阶段（Newman等人。，1996）。

通过nhr-67突变和其他基因的突变表型子宫发育，功能引起的表型之间的相似性，结合动态模式的nhr-67表达交流和似曾相识的细胞，表明nhr-67可能在LIN-12/Notch途径调节子宫功能发展。为了检验这种可能性，我们发现，在nhr-67编码区和启动子的突变研究，并探讨了在nhr-67腹部子宫细胞的调节。我们的数据支持这nhr-67功能的LIN-12/Notch受体在武系上游模型，滞后/三角洲上游的信号在交流，在一个通路，包括hlh-2 / daughterless和egl-43／EVI1转录因子在多步腹子宫发育的控制。

材料和方法
识别和表征nhr-67启动子突变

在屏幕上可见的表型在自发突变蛋白（gk10）我的背景，我们分离的突变与部分渗透EGL和PVL型表面上类似的弱SEL-12（ar131）早老素突变。遗传图谱的实验放在一个区间上snp_r13 PF2 [ 1 ]和pkp4085之间的近四dbp7 LG（数据未显示）。蛋白编码的DNA解旋酶的同源人类BACH1 / BRIP1基因牵连/ FANCJ范可尼贫血和某些癌症（Cheung等人。，2002和尤兹等人。，2008）。DOG-1功能消除导致高的缺失开始在长的富G序列的频率，特别是聚乙二醇的延伸，可以形成多个四链体当DNA单链（Cheung et al，2002，kruisselbrink等人。，2008、赵某等人，2008）。我们期待在该地区可以解释EGL表型和含有富G序列候选基因，或基因附近的。对每一个候选基因设计引物侧翼的富G序列和测试在2株缺失存在（表S2）。没有缺失被确定为dsl-5和dsl-6 Notch配体，也t01g1.3，但小的缺失是由基因组扩增在nhr-67启动子区的检测。一个强大的EGL突变具有较大的nhr-67启动子缺失，pf159，是在同一个屏幕中恢复。

的pf88突变恢复作为一种免疫组织化学背景而产生的自发突变（gk10）我；（ty11 SEL-12）spr-3（pf83）X株。该SEL-12（ty11）突变导致强烈的EGL缺陷和适度显PVL缺陷（辛那尔等人贡蒂若等人。，2001。，2009），类似于一个弱LIN-12（LF）等位基因。对SEL-12突变的表型效应的spr-3活动导致的HOP-1早老素基因的转录的抑制损失完全抑制（lakowski等人。，2003）。的pf88突变导致的spr-3 SEL-12背景近100%渗透EGL和PVL的表型，表明pf88上位，spr-3（数据未显示）。2，pf88和pf159突变是从所有其他已知的交配和突变的nhr-67缺失和无蛋白存在的分离株存在删除（gk10）是通过使用特定的寡核苷酸的基因组DNA扩增证实（表S2）在表型特征