The role of AmotL2 in organ formation and tumorigenesis

Tese de mestrado, Oncobiologia, Faculdade de Medicina, Universidade de Lisboa, 2014

Mechano-Chemical Aspects of Organ Formation in Arabidopsis thaliana: The Relationship between Auxin and Pectin

How instructive signals are translated into robust and predictable changes in growth is a central question in developmental biology. Recently, much interest has centered on the feedback between chemical instructions and mechanical changes for pattern formation in development. In plants, the patterned arrangement of aerial organs, or phyllotaxis, is instructed by the phytohormone auxin; however, it still remains to be seen how auxin is linked, at the apex, to the biochemical and mechanical changes of the cell wall required for organ outgrowth. Here, using Atomic Force Microscopy, we demonstrate that auxin reduces tissue rigidity prior to organ outgrowth in the shoot apex of Arabidopsis thaliana, and that the de-methyl-esterification of pectin is necessary for this reduction. We further show that development of functional organs produced by pectin-mediated ectopic wall softening requires auxin signaling. Lastly, we demonstrate that coordinated localization of the auxin transport protein, PIN1, is disrupted in a naked-apex produced by increasing cell wall rigidity. Our data indicates that a feedback loop between the instructive chemical auxin and cell wall mechanics may play a crucial role in phyllotactic patterning.

显花植物器官形成激素调控的分子机理研究

本论文以显花植物水稻和拟南芥为对象，研究植物器官形成激素调控的分子机理。发现拟南芥干细胞决定基因WUS和ABA信号关键调节因子ABI3间存在相互调节关系，它们对质体和根毛发育等具有调控作用；运用细胞学手段对OsAGAP和OsRMC调控根发育的机理进行了研究。这些结果为了解ABA、生长素和JA等激素对分生组织或根等器官的形成的调控机理提供了新资料。 拟南芥WUS基因是一个重要的干细胞决定基因。在茎尖分生组织和花分生组织的动态平衡调节中各存在一个反馈调节环：WUS促进CLV3的表达，而CLV3 反馈抑制WUS表达，它们共同决定干细胞的数目；在WUS+LFY 和AG之间也存在一个类似的反馈环，负责花器官的正常启动和终止。本工作发现在外源ABA 存在下，拟南芥WUS功能获得突变体sef (stem ectopic flowers)子叶黄化过程受到抑制，即sef突变体对ABA 的敏感性降低，而且在sef突变体中ABI3转录水平下调，说明WUS 抑制ABI3的表达。另一方面，在abi3突变体中WUS转录水平下降，启动子分析发现WUS是ABI3所在的B3结构域家族基因的靶基因，酵母单杂交实验表明B3家族蛋白FUS3可以与WUS调控区结合；外源ABA 处理pWUS::GUS植株发现ABA可使WUS异位表达，暗示受ABA信号诱导的B3类转录因子可与WUS调控区结合，从而促进WUS的表达。这些结果支持ABI3/WUS间的反馈调节机理，该调节环可能参与调控拟南芥质体和根毛发育等过程。 双子叶和单子叶植物的发育和器官形成高度依赖生长素极性运输（PAT）， 生长素输入和输出载体的准确定位对于极性运输的正常进行是必要的。在双子叶模式植物拟南芥中，生长素输出载体PIN1的转运和定位受小G蛋白ARF鸟苷酸转换因子GNOM介导。本实验室克隆到一个水稻ARF GAPase激活蛋白OsAGAP， 其超表达引起PAT改变且干扰主根和侧根的发生及发育。前期生理实验显示超表达植株侧根的表型可被膜渗透性生长素NAA恢复，但不能被载体依赖型生长素IAA和2, 4-D恢复。为了解释OsAGAP过表达引起根系发育受到抑制的表型，本工作主要从细胞学角度继续深入分析OsAGAP介导生长素运输的机理。实验发现，OsAGAP超表达植株中AUX1在细胞中分布改变；生长素输出载体PIN1和PIN2的定位不受影响；该基因的超表达使囊泡聚集，形成类似囊泡运输抑制剂BFA处理后的结构；OsAGAP与细胞转运系统中的高尔基体存在部分共定位。以上结果表明OsAGAP调节小G蛋白Arf的活性，参与调控生长素极性运输过程，进而调控根系发育。通过对JA诱导的OsRMC蛋白的膜定位分析及转基因植株根中JA合成水平的测定，为研究此蛋白参与JA信号、调控根系发育提供了直接证据。 此外，论文还对拟南芥STAR2基因的功能进行了一定的初探。

Computer simulation of human iodothyronine deiodinase single-chain antibody

Iodothyronine plays a major role in growth, basic metabolism and organ formation. It has an extremely limited source in the body. In this thesis, we designed iodothyronine(T4) as hapten. Then a single chain antibody displayed on phange was obtained from a human phage displaying a single chain antibody library. The specific genes of E3 was subcloned in P-5E vector. According to its amino acid sequences, we simulate its three dimention structure by computer. It has never been reported in PDB.

C-Kit(+) Cells Isolated from Developing Kidneys Are a Novel Population of Stem Cells with Regenerative Potential

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. STEM Cells 2013;31:1644-1656

The Tomato (Solanum Lycopersicum cv. Micro-Tom) Natural Genetic Variation Rg1 and the DELLA Mutant Procera Control the Competence Necessary to Form Adventitious Roots and Shoots

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Tomato (Solanum Lycopersicum cv. Micro-Tom) Natural Genetic Variation Rg1 and the DELLA Mutant Procera Control the Competence Necessary to Form Adventitious Roots and Shoots

Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively.

Microsurgical and laser ablation analysis of interactions between the zones and layers of the tomato shoot apical meristern

Plants exhibit life-long organogenic and histogenic activity in a specialised organ, the shoot apical meristem. Leaves and flowers are formed within the ring-shaped peripheral zone, which surrounds the central zone, the site of the stem cells. We have undertaken a series of high-precision laser ablation and microsurgical tissue removal experiments to test the functions of different parts of the tomato meristem, and to reveal their interactions. Ablation of the central zone led to ectopic expression of the WUSCHEL gene at the periphery, followed by the establishment of a new meristem centre. After the ablation of the central zone, organ formation continued without a lag. Thus, the central zone does not participate in organogenesis, except as the ultimate source of founder cells. Microsurgical removal of the external L-1 layer induced periclinal cell divisions and terminal differentiation in the subtending layers. In addition, no organs were initiated in areas devoid of L-1, demonstrating an important role of the L-1 in organogenesis. L-1 ablation had only local effects, an observation that is difficult to reconcile with phyllotaxis theories that invoke physical tension operating within the meristem as a whole. Finally, regeneration of L-1 cells was never observed after ablation. This shows that while the zones of the meristem show a remarkable capacity to regenerate after interference, elimination of the L-1 layer is irreparable and causes terminal differentiation.

The auxin influx carrier is essential for correct leaf positioning

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well-documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitor N-1-naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole-3-acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.

Regulation of phyllotaxis

Plant architecture is characterized by a high degree of regularity. Leaves, flowers and floral organs are arranged in regular patterns, a phenomenon referred to as phyllotaxis. Regular phyllotaxis is found in virtually all higher plants, from mosses, over ferns, to gymnosperms and angiosperms. Due to its remarkable precision, its beauty and its accessibility, phyllotaxis has for centuries been the object of admiration and scientific examination. There have been numerous hypotheses to explain the nature of the mechanistic principle behind phyllotaxis, however, not all of them have been amenable to experimental examination. This is due mainly to the delicacy and small size of the shoot apical meristem, where plant organs are formed and the phyllotactic patterns are laid down. Recently, the combination of genetics, molecular tools and micromanipulation has resulted in the identification of auxin as a central player in organ formation and positioning. This paper discusses some aspects of phyllotactic patterns found in nature and summarizes our current understanding of the regulatory mechanism behind phyllotaxis.

Phyllotaxis — a new chapter in an old tale about beauty and magic numbers

Phyllotaxis, the regular arrangement of leaves and flowers around the stem, is one of the most fascinating patterning phenomena in biology. Numerous theoretical models, that are based on biochemical, biophysical and other principles, have been proposed to explain the development of the patterns. Recently, auxin has been identified as the inducer of organ formation. An emerging model for phyllotaxis states that polar auxin transport in the plant apex generates local peaks in auxin concentration that determine the site of organ formation and thereby the different phyllotactic patterns found in nature. The PIN proteins play a primary role in auxin transport. These proteins are localized in a polar fashion, reflecting the directionality of polar auxin transport. Recent evidence shows that most aspects of phyllotaxis can be explained by the expression pattern and the dynamic subcellular localization of PIN1.

Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical–basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical–basal position or the identity of the induced structures.

Cell survival in early embryogenesis requires proper dosage of GCN5 and p300

Histone acetylation is a central event in transcriptional activation. The importance of this modification in mammalian development is highlighted by knockout studies that revealed loss of the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality. Furthermore, early embryogenesis is sensitive to the dosage of p300 and CBP since double p300 +/−CBP+/− heterozygotes die in utero, although either single heterozygote survives. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of HATs interact functionally in vivo, we created mice lacking one or more allele of p300, GCN5 or PCAF. As expected, we found that mice heterozygous for any one of these null alleles are viable. The majority of GCN5 p300 double heterozygotes also survive to adulthood with no apparent abnormalities. However, a portion of these mice die prior to birth. These embryos are developmentally stunted and exhibit increased apoptosis compared to wild type or single GCN5 or p300 heterozygous littermates at E8.5. Tissue specification is unaffected in these embryos but organ formation is compromised. In contrast, no abnormalities were observed in mice harboring mutations in both PCAF and p300 , emphasizing the specificity of HAT functions in mammalian development. ^ Since GCN5 null embryos die early in embryogenesis because of a marked increase in apoptosis, studies of its function and mechanism in late development and in tissue specific differentiation are precluded. Here, we also report the establishment of a GCN5 null embryonic stem cell line and a conditional floxGCN5 mouse line, which will serve as powerful genetic tools to examine in depth the function of GCN5 in mammalian development and in adult tissues. ^

Neuronal type information encoded in the basic-helix–loop–helix domain of proneural genes

The proneural genes encode basic-helix–loop–helix (bHLH) proteins and promote the formation of distinct types of sensory organs. In Drosophila, two sets of proneural genes, atonal (ato) and members of the achaete–scute complex (ASC), are required for the formation of chordotonal (ch) organs and external sensory (es) organs, respectively. We assayed the production of sensory organs in transgenic flies expressing chimeric genes of ato and scute (sc), a member of ASC, and found that the information that specifies ch organs resides in the bHLH domain of ato; chimeras containing the b domain of ato and the HLH domain of sc also induced ch organ formation, but to a lesser extent than those containing the bHLH domain of ato. The b domains of ato and sc differ in seven residues. Mutations of these seven residues in the b domain of ato suggest that most or perhaps all of these residues are required for induction of ch organs. None of these seven residues is predicted to contact DNA directly by computer simulation using the structure of the myogenic factor MyoD as a model, implying that interaction of ato with other cofactors is likely to be involved in neuronal type specification.

Dynamics of Cytokinins in Apical Shoot Meristems of a Day-Neutral Tobacco during Floral Transition and Flower Formation1

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.