Lecture 6 (Niche) Flashcards
Describe the targeting of DNA through the Cre/loxP system.
The Cre/loxP system is a method used to artificially control gene expression. The system begins with the Cre gene, short for cyclisation recombination, which encodes a site-specific DNA recombinase. A site-specific recombinase means that the Cre protein can recombine DNA when it locates specific sites in a DNA molecule. These sites are known as locus of X- over P1, or loxP, sequences and are 34 base pairs in length. They are recognised by the Cre recombinase, which recombines the surrounding DNA between loxP sites. This is termed a reciprocal recombination event, and involves the cleaving of double-stranded DNA at loxP sites by the Cre protein. As a result, the DNA between loxP sites is excised and subsequently degraded.
Why is the Cre/loxP-mediated excision of DNA so useful?
Since loxP sequences are 34 base pairs long, there is virtually no chance that they would be randomly found within a genome. Therefore, loxP sequences can be artificially inserted into animal or plant genomes, and used for the precise excision of DNA, without worrying about the potential cleaving of another part of the organism’s genome.
How is a gene regulated using the Cre/loxP system?
A ‘stop’ sequence, consisting of several termination codons, is surrounded by loxP sequences and inserted between the promoter of the target gene and the gene itself. When Cre is present in the cells of that particular organism, it will catalyse recombination between the two loxP sites, thereby removing the stop sequence and enabling gene transcription.
What are the advantages and disadvantages of gene regulation using the Cre/loxP system?
Advantages:
- Versatile; works in almost any cell
- Application to many types of experiment e.g. the labelling of neuronal cells, differentiating them from surrounding glial cells and observing the properties of the promoter
- Combined with tetracycline regulated transcriptional systems leads to a more powerful system
Disadvantages:
- Not all tissue-specific promoters exhibit perfect specificity
- Significant time and costs associated with use of Cre/loxP
Define a stem cell ‘niche’.
A stem cell niche is a set of cells, extracellular matrices, and hormones that surrounds a stem cell and influences its behaviour.
Describe the structure and cellular arrangement of the intestinal epithelium.
The epithelium lining the small intestine is a single cell thick and is composed of three types of differentiated cells. The most abundant epithelial cells, the absorptive enterocytes, transport nutrients essential for survival from the intestinal lumen into the bloodstream. The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals, turning over every five days. The cells of the intestinal epithelium continuously regenerate from a stem cell population located deep in the intestinal wall in pits called crypts. Pulse-chase experiments showed that intestinal stem cells produce precursor cells that proliferate and then differentiate as they ascend the sides of crypts to form the surface layer of the finger-like gut projections called villi, across which intestinal absorption occurs. The time from cell birth in the crypts to the loss of cells (sloughing) at the tip of the villi is only about three to four day, therefore enormous amounts of cells must be produced continually to keep the epithelium intact. The production of new cells is precisely controlled: too little division would eliminate villi and lead to the breakdown of the intestinal surface; too much division would create an excessively large epithelium and may lead to tumourigenesis.
Describe the evidence for the importance of Wnt signalling in the maintenance of the intestinal stem cell population.
Wnts are secreted by mesenchymal cells at the base of the crypts in the wall of the intestine, and are essential for stem cell maintenance. As evidence for the importance of Wnts, over production of active β-catenin in intestinal cells leads to excessive proliferation of the intestinal epithelium. Conversely, blocking the function of β-catenin by interfering with the Wnt-activated TCF transcription factor abolishes the stem cells in the intestine. Thus, Wnt signalling plays a critical role in the intestinal stem cell niche as it does in the skin and other organs. Indeed, mutations that inappropriately activate Wnt signal transduction are a major contributor to the progression of cancer.
Describe the use of Lgr5 as a marker of intestinal stem cells.
By analysing a panel of genes whose expression in the intestine was induced by Wnt signalling, investigators zeroed in on Lgr5, a gene encoding a G protein-coupled receptor, because it was expressed only in a small set of cells at the very base of the crypts. Lgr5 binds a class of secreted hormones termed R-spondins and activates intracellular signalling pathways that potentiate Wnt signalling. Lineage-tracing studies showed that the descendants of these Lgr-expressing cells indeed gave rise to all of the intestinal epithelial cells. This experiment made use of gene-altered mice in which a version of Cre recombinase, an oestrogen receptor-Cre chimera, was placed under the control of the Cre promoter; thus the OR-Cre chimera recombinase was only produced in the few putative Lrg5-expressing cells at the bottom of the crypts. The version of Cre used in the study had been altered so that normally it resides inactive in the cytosol and is transferred into the nucleus only after the addition of an oestrogen analog. There the Cre excises a segment of DNA, activating the expression of a β-galactosidase receptor gene in these cells. Importantly, all the descendants of these cells will also express this enzyme. Immediately after the addition of the oestrogen analog, the only cells expressing β-galactosidase are the stem cells in the crypts. But after a few days, of the descendant epithelial cells expressed β-galactosidase, showing that Lgr5 indeed is a marker of the intestinal stem cells.
Describe a recent experiment on the expression of Lrg5 within intestinal stem cells.
In subsequent studies, single Lrg5-expressing stem cells were isolated from intestinal crypts and cultured on an extracellular matrix containing type IV collagen and laminin; this would ordinarily underlie and support the intestinal epithelia. These cells generated villi-like structures that contained all four differentiated cell types found in the mature intestinal epithelium. Taken together, these experiments establish that expression of the Lgr5 gene defines the intestinal stem cells and shows that these cells are localised at the base of the intestinal crypts, interspersed between the terminally-differentiated Paneth cells.
Describe the function of Paneth cells.
Paneth cells produce several anti-bacterial proteins that protect the intestine from infections; surprising recent evidence shows that Paneth cells also constitute a major part of the niche for the intestinal stem cells. Cultured Paneth cells produce Wnt as well as other hormones, such as EGF and a Delta protein, that are essential for intestinal stem cell maintenance. Co-culturing of intestinal stem cells with Paneth cells markedly improved the formation of intestinal villus-like structures, and genetic manipulations in mice that caused a reduction of Paneth cell numbers concomitantly caused a reduction in intestinal stem cell numbers.
What is bone morphogenetic protein?
Bone morphogenetic protein, or BMP, is an anti-mitogen that instigates cellular differentiation.
What are Gremlin and Noggin?
Gremlin and Noggin are BMP antagonists; their signalling pathway consists of type 1 and type 2 transmembrane receptors and cytosolic SMADs, which regulate the transcription of BMP target genes.
Describe Ephrin B and EphB-mediated regional segregation in the intestinal crypts.
The interaction between Ephrin B ligands and EphB receptors direct cellular localisation and migratory behaviour within the crypt. Cell location dictates cell state by defining exposure levels to secreted molecules and niche interactions. A gradient of EphrinB1/B2 ligands exists within the crypt, with cells at the crypt-villus junction expressing high levels of these molecules, whilst an opposing gradient of EphB2 expression begins at the crypt base. Therefore, the level of Ephrin B ligand and Wnt-induced EphB expression determines cell location.
Interestingly, Paneth cells express EphB3 and no Ephrin B ligands, thereby restricting these cells to the base of the crypt. Crypt-based columnar cells express both EphB3 and EphB2, whilst label-retaining cells express high levels of EphB2, thereby restricting upward migration of these cell types and ensuring that they are localised near their respective niches.
Compare and contrast cells expressing Bmi1 to those expressing Lrg5.
Bmi1 is a Polycomb group repressor that is expressed in quiescent cells that are unresponsive to Wnts and resistant to radiation. These cells can begin to rapidly divide when needed to replace the stem cell population, meaning that Bmi1 is necessary for efficient self-renewing cell division. However, Lrg5-expressing cells are mitotically active and responsive to Wnt signalling, and the population is damaged upon exposure to radiation.
Describe the tissue architecture of the stem cell niche within the testes of the Drosophila.
In the Drosophila testes, two stem cell populations, the germ-line stem cells and the cyst stem cells, intermingle around a cluster of cells called the hub. When the stem cell populations divide, daughters that move away from the hub differentiate and the differentiating germ cells, which begin to undergo transit amplification, become encysted by the differentiating cyst cells. In this niche, the cyst cells produce signals promoting the self-renewal of neighbouring germ cells.
