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In vivo analysis of cell division during vertebrate developmentKieserman, Esther Kathleen 19 October 2009 (has links)
In this work, we identified and characterized developmentally regulated aspects to
cell division in the Xenopus laevis. We found that cells in the early neural plate divide in
an oriented manner. This orientation is established by Cdc42 controlled maintenance of
stable interactions between the spindle and the cell cortex. This role of Cdc42 is
developmentally regulated and cells dividing later in a related tissue, the tail epidermis,
are not under this control. Moreover, we find that the cell divisions in the early neural
plate are further specialized in their mechanisms of cell division. Cells in the early neural
plate exhibit exaggerated anaphase-B movements, a delayed onset of cytokinesis, low
density of midzone microtubules and a rapid cytokinetic furrow ingression as compared
to the late tail epidermis, another ectodermally derived tissue. These modifications to the
mechanism of cell division appear to be because of a reduced level of PRC1, a
microtubule bundling protein, and thus modifications to the central spindle structure.
Finally, we find that cytokinetic mechanisms may be functionally related to the process
of ciliogenesis. We find proteins known to localize to the central spindle localized to the
rootlet of the basal body of cilia in multiciliated cells of the mucociliary epidermis. This localization may be related to vesicle transport during both these processes. This work
reveals unexpected plasticity to fundamental mechanisms of cell division. / text
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Contribution of Lipophilic Secondary Metabolites to the Toxicity of Strains of Freshwater Cyanobacterial Harmful Algal Blooms, Identified Using the Zebrafish (Danio rerio) Embyo as a Model for Vertebrate DevelopmentJaja-Chimedza, Asha D 21 March 2014 (has links)
Cyanobacteria (“blue-green algae”) are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called “harmful algal blooms”, particularly in freshwater systems, a number of these metabolites have been associated - as “toxins”, or commonly “cyanotoxins” - with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates.
We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.
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Wnt5a Signaling Independently of the Planar Cell Polarity Pathway Resulting in Convergent Extension and Neural Tube Closure During Vertebrate DevelopmentBarrott, Jared James 14 August 2008 (has links) (PDF)
Vertebrate development is regulated by cellular communication by mechanisms of cell fate and cell behavior. These crucial mechanisms are regulated by cellular signaling and in the case of cell fate, cellular signaling results in transcription of developmentally important genes. Communication between cells can also result in regulation of cell behavior by acting on cytoskeletal elements rather than nuclear factors. One of the cellular signals that regulate both cell fate and cell behavior is the family of Wnt signaling molecules. Wnt5a is one of 19 Wnt molecules and has been previously demonstrated to play critical roles in many important processes in embryonic development as well tumor suppression. Despite many studies that lend credence to a pathway that regulates cell behavior for Wnt5a rather than cell fate, the identity of the pathway(s) Wnt5a impinges upon remains unclear. Despite the possibility of Wnt5a signaling through multiple pathways, here, focus is given to the non-canonical Wnt signaling pathway, a pathway that regulates cell behavior, also known as the Wnt/Planar Cell Polarity (PCP) pathway. The involvement of Wnt5a in the Wnt/PCP pathway was demonstrated with a genetic approach: crossing Wnt5a heterozygous mice with mice heterozygous for a component of the Wnt/PCP pathway to uncover genetic interactions in vivo. Hence, Wnt5a X Looptail (Lp) (Wnt/PCP) heterozygous crosses have been performed. Double heterozygotes for this intercross did not exhibit a decrease in viable progeny as compared to the decreased numbers of Lp heterozygotes. These observations demonstrated a lack of genetic interaction between Wnt5a and the PCP pathway. Wnt5a mutants possess phenotypes associated with deficits in the Wnt/PCP pathway, namely convergent extension (CE) defects and neural tube closure defects. However, upon further investigation of the increased penetrance of craniorachischisis in Wnt5a-/-;Lp+/-, Wnt5a mutants do not display the characteristic broadening of the neural floor plate commonly associated with Lp-/-. This supports that Wnt5a and PCP signaling are parallel pathways that have converged to regulate different aspects of CE and neural tube closure. Despite the complexity of Wnt5a and its potential involvement in multiple pathways, dissection of this will explain the broad range of phenotypes observed.
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Global Analysis Of Transcriptional Control Driving Zebrafish GastrulationSimon Wilkins Unknown Date (has links)
Gastrulation, literally “formation of the gut” is ultimately an inadequate term to describe one of the most dynamic periods during vertebrate developmental biology. During gastrulation coordinated cell movements reshape the non-descript blastula into the structured gastrula and simultaneously specify the three germ layers: endoderm, mesoderm and ectoderm. The morphogenetic movements of gastrulation are highly conserved between species, but the links between their genetic and biomechanical regulation are poorly understood. The zebrafish embryo – externally hatched, optically clear and amenable to genetic manipulation – is an ideal vertebrate model in which to study both morphogenetic movements and their genetic control. This thesis provides a detailed analysis of the zebrafish Mix-type homeobox transcription factor, Mtx2, both in terms of its role in gastrulation and the molecular mechanisms regulated by Mtx2. This approach involved detailed examination of the Mtx2 loss-of-function phenotype and, subsequently, use of this phenotype as the basis for a microarray screen to identify and investigate Mtx2-dependent genes. One specific Mtx2-dependent gene, katanin-like 1 was investigated further by loss-of-function studies. Prior to this study the mtx2 gene was identified by homology, within its homeodomain, to other Mix-family transcription factors, but both its function and transcriptional targets remained unknown. Using a morpholino knockdown approach, this thesis demonstrates that Mtx2 is essential for vegetal movement (epiboly), but not specification, of the embryonic germ layers and extra-embryonic tissues during zebrafish gastrulation. The recruitment of filamentous actin (F-actin) to a punctate band at the blastoderm margin, was previously shown to be responsible for progression of epiboly. However, formation of this structure is demonstrated to be Mtx2-dependent. Microarray expression profiling of the Mtx2 loss-of-function phenotype was performed to screen for novel genes with roles in gastrulation. This approach identified Mtx2-dependent genes with roles in cytoskeletal dynamics, cell-cell adhesion and endocytosis and vesicular trafficking – processes known to be involved in morphogenetic movements. Many Mtx2-dependent genes are co-expressed with mtx2 in the extra-embryonic yolk syncytial layer (YSL), the teleost functional equivalent of mammalian visceral endoderm. The subset of Mtx2-dependent genes co-expressed with mtx2 and that contain Mtx2-binding sites within their 2kb proximal promoter represent the genes with the greatest likelihood of being direct Mtx2 transcriptional targets. A novel homologue of the microtubule severing protein Katanin, known as katanin-like 1 (katnal1) met all these conditions. Morpholino knockdown of Katnal1 demonstrates that like Mtx2, Katnal1 is essential for gastrulation in zebrafish. A cloned Katnal1mCherry fusion construct was observed to associate with microtubules, and demonstrated bi-directional trafficking around transfected mammalian cells. Analysis of the microtubule network in wild-type and morpholino injected zebrafish embryos demonstrated that remodelling of the extensive microtubule network found in the YSL and yolk cytoplasmic layer (YCL) is Katnal1-dependent. Nuclear division within the YSL and F-actin recruitment to the blastoderm margin are also Katnal1-dependent. This thesis therefore demonstrates, for the first time directly, the multiple, specific roles played by the microtubule network of the YSL/YCL. Katnal1 is highly conserved from Drosophila to mammals and is dynamically expressed during mouse gastrulation. The Mtx2 binding motif in the katnal1 2kb proximal promoter can be bound by both Mtx2 and its putative mouse homologue Mixl1. This suggests that katnal1 may also be a direct target of Mtx2. At the technical level, these results demonstrate the validity of screening for novel developmentally important genes using a zebrafish microarray-based approach, the potential of such an approach to, ab initio, identify a candidate list of transcription factor targets and confirm the utility of the zebrafish as a developmental model. At the biological level, this work collectively suggests that Mtx2 is a central regulator of the morphogenetic movement of epiboly and that Katnal1-dependent microtubule remodelling drives multiple aspects of gastrulation, potentially from Drosophila through to humans.
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Xenopus Laevis TGF-ß: Cloning And Characterization Of The Signaling ReceptorsMohan, D Saravana 01 1900 (has links)
The amphibian species Xenopus laevis, along with mouse and chicken is a very important model system, used widely to dissect the molecular intricacies of various aspects of vertebrate development. Study with Xenopus has clear advantages in terms of various technical considerations including the ease of handling early stage of embryos and due to the remarkable documentation of several early molecular events during development. The concept of inductive interactions between various cell types during early development was first revealed by the studies performed in Xenopus, and among the various factors proposed for mesoderm induction, the members of transforming growth factor-β (TGF- β) superfamily have been considered to be the most probable candidates. About forty different members of the TGF-β superfamily have been cloned and characterized from various organisms. The superfamily members like activins and BMPs have been studied extensively with respect to their functional role during development. While BMPs were assigned as candidates for inducing ventral mesoderm, activins oppose the role of BMPs by inducing dorsal mesoderm. Studies that helped in delineating their roles were performed using three approaches that utilized the ligands, receptors or down stream signaling components (Smads). All the three components were studied with respect to their endogenous expression pattern and effects of ectopic expressions of the wild type or dominant negative mutants. These approaches led to the accumulation of evidences supporting the importance of these signaling molecules. All the above mentioned studies were only possible due to the cloning and characterization of cDNAs of the various proteins involved in the signaling pathway including the ligands. TGF-β2 and 5 are the two isoforms of TGF-β cloned from the amphibian system. We have earlier cloned and characterized the promoter for TGF-β5 gene, which suggested possible regulation of this factor by tissue specific transcription factors. Messenger RNA in situ hybridization analysis to study the TGF-β5-expression pattern during Xenopus development, showed spatial and temporal expression pattern. The expression was confined to specific regions that include notochord, somites, and tail bud among others,
in the various stages analyzed. This suggested a possible role for TGF-β5 in organogenesis during the amphibian development. To better understand the role of TGF-β in Xenopus development, studies to examine the specific receptor expression pattern for this growth factor is very essential. With the lack of any reports on cloning of TGF-β receptors from this system, the aim of the present study was to isolate and characterize the receptors for TGF-β from Xenopus laevis. PCR cloning using degenerate primers based on the conserved kinase domains of this class of receptors, coupled to library screenings enabled the identification of two novel receptor cDNAs of the TGF-β receptor superfamily. Characterization of the isolated cDNAs suggested that one of them codes for a type II receptor for TGF-β. Further the cDNAs were found to be ubiquitously expressed during development, as judged by RT-PCR analysis. The cloned cDNAs can now be employed as tools, to study the expression pattern by means of mRNA in situ hybridization, on the various developmental stage embryos and to perform studies using antisense and dominant negative mRNA injection experiments in vivo. Such studies will greatly assist in delineating the role of TGF-β ligands and receptors during amphibian development.
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