Lecture 7 - 8 Flashcards Preview

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Flashcards in Lecture 7 - 8 Deck (20)
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1

In which places in the adult brain can new neurogenesis be seen

The subependymal/subventricular zone of the lateral ventricles and the subgranular zone of the dendate gyrus of the hippocampus

2

Where do the new neurons that migrate to the olfactory placode come from

The subependymal/subventricular zone

3

Where are the new neurons associated with the acquisition of memory born from

The subgranular zone of the dendate gyrus of the hippocampus

4

Describe the niche in which adult neural stem cells are found

In a niche containing a variety of different cells including astrocytes transit amplifying progenitors ependymal cells neurons and blood vessels

5

Outline the origin of these adult neural stem cells

The adult Neural stem cells originated from radial glial-like cells which themselves are derived from neuroepithelial cells

6

Describe how the adult neural stem cells form

Neuroepithelial symmetrically divide to extend the neural tube. At later point one of the daughters of the dividing neuroepithelial becomes a radial glial cell. Through asymmetric division of this radial glial one daughter remains and one moves along the projection and differentiates. The retained daughter of the radial glia acts as this adult neural stem cell

7

Radial glia first give rise to the glia and then to neurons T or F

F – vice versa

8

Why is it expected that there is a hypothalamic stem-like cell

The needs of the body at different times of the year are fundamentally different hence the ability to produce new neurons to regulate the core body functions would be beneficial this would be provided by retaining a stem-like population

9

A cross section through the mouse median eminence of the hypothalamus revealed a radial-glial-like cell thought to be a stem-like cell what is the name given to these types of cells and what is their appearance

Tanycytes – cell bodies lie at the ventricular zone and possess long basal processes which extend towards the marginal zone

10

What must a neuronal cell be able to do in order to be considered multipotent

Give rise to neurons and glia

11

Neurogenesis in the adult hypothalamus can be stimulated by a high-fat diet how was this determined

Injection of the thymine analogue BrdU which integrates into the DNA at S phase. This enabled the visualisation of this de novo proliferation and neurogenesis in mice on high fat diets

12

Where specifically was neurogenesis seen in the mice on high fat diets and what is the significance of this

Neurogenesis was seen in the arcuate and ventromedial nuclei these contain the neurons responsible for the central regulation of energy and metabolism (POMC and neuropeptide-Y neurons)

13

Describe how to make a tissue-specific knockout

Create a mouse in which loxP sites have been introduced either side of a target gene this is now known as a floxed allele. Create another mouse in which cre-recombinase is put under the control of a promoter that directs expression of genes only in the target cell(s)/tissue(s). In this gene cre-recombinase coding sequence replaces coding sequence of the gene with the upstream promoter conserved. Cre-recombinase is the enzyme that works on the loxP sites. Cross the cre-recombinase mouse with the floxed allele containing mouse. The progeny produced from the cross will have normal functioning target gene except in the heart. This is because these cells are co-expressing the floxed target gene and the cre-recombinase enzyme which will effectively cut-out the target gene hence preventing its expression

14

Give an example of a conditional approach that will allow you to see where a gene is expressed and lineage trace the progeny

Put the cre-recombinase enzyme under control of promoter for wnt1 expression where wnt1 is expressed in the neural crest and roof plate cells. Create a second mouse where LacZ (or any reporter gene) is downstream of a STOP codon. This means that the reporter gene won’t be expressed unless the stop codon has been removed. However flank the stop codon with loxP sites. Breed the two mice and add X-Gal to the developing embryos. Staining will be seen in cells co-expressing cre-recombinase and floxed stop codon and hence will have the stop codon cut out and lacZ expressed. This expression of lacZ will be maintained in all the progeny of these cells and hence can be traced in the lineage of these cells

15

How can we use conditional approaches to create temporal reporters to study gene function

CreERT2 encodes a Cre-recombinase fused to a mutant oestrogen ligand-binding domain (ERT2). This ERT2 domain requires the presence of tamoxifen for its activity. This means that Cre is only activated when tamoxifen is injected into the live mice. You can then insert the creERT2 coding region downstream of a promoter expressed in a target tissue (i.e. a stem cell gene promoter). Cross this mouse with a mouse containing a floxed target gene. You can then knockout this target gene at a specific point in development by injecting the live mouse with tamoxifen

16

How was it determined that the hypothalamic tanycytes were a radial glia-like population and capable of giving rise to multiple different cells

The GLAST gene was identified to be expressed in radial glia. The creERT2 coding sequence was then inserted downstream of the GLAST promoter sequence in one mouse line. These mice were crossed with mice containing a floxed STOP sequence upstream of a reporter gene such as LacZ or GFP. The progeny of these mice could then be injected with tamoxifen and a specific time and then the cells expressing the reporter genes would be only those that are radial glia cells. Indeed the tanycytes of the mice hypothalamus were the only one expressing the reporter gene. The mice were then allowed to develop longer to see if the progeny of the tanycytes could also be seen to express the reporter. The tanycytes were seen to have given rise to both neuronal and glial cells. Hence the population of tanycytes are radial glia able to self-renew and give rise to other populations of radial glia as well as neurons and astrocytes

17

What is the major limitation of genetic lineage tracing in determining the multipotency of cells

This technique images a population of cells and thus it cannot be determined if a single cell alone can give rise to this variety of cell fates

18

How was it that the fluorescently labelled tanycytes of the mouse hypothalamus were ultimately determined as being multipotent

Complementary in vitro studies show that tanycytes exhibit neural stem cell properties. FACS sorting was used to separate out the fluorescent cells from the rest of the median eminence cells created from the conditional tissue specific reporter mouse. Then a set of serial dilutions were carried out to obtain single cells which were then plated and cultured. These single cells continued to divide to produce a neurosphere derived from that single cell. The ball of cells (neurosphere) were then placed in differentiating conditions and markers for various different hypothalamic cells (Neuropeptide-Y POMC DA GABA Somatostatin GnRH etc.) were stained for. This showed that these cells were indeed capable of giving rise to the different types of hypothalamic neurons

19

Using BrdU staining it was determined that under normal caged conditions proliferation and neurogenesis/gliogenesis is very low in mice what can be inferred about this neurogenesis pathway in adults

The neurogenesis pathway is most like in some way associated with stress

20

What was seen as a result of implantation of corticosterone soaked beads into the region of the hypothalamus where the tancytes are found and why

Implantation of a corticosterone soaked bead to see if this stimulates proliferation in the hypothalamic tanycytes. This provides more evidence the adult neurogenesis is associated with stress