SAQs Flashcards
In mammalian development, the first cell fate decision produces two cell types from
blastomeres. Name the cell types, and for each, give one gene that is a marker for
that cell type (2 marks)
Trophectoderm. Marker = Cdx2, Gata4/6, Tead4
Inner Cell Mass. Marker = Sox2, Oct3/4, Sall4, Nanog
Name two places (each in a different tissue) in mammalian embryos where
epithelial-to- mesenchymal transformation takes place. (2 marks)
Heart valve formation myogenesis neural crest formation gastrulation neurulation- form neural plate
Give one advantage and one disadvantage for both a genetic and non-genetic means
of lineage labelling in the mouse embryo. (4 marks)
Genetic Adv: Label not diluted by divisions/accurate/inherited by all progeny/ as accurate as the promtor to drive expression
Genetic Dis: Expensive, difficult, time consuming, reliant on promoter activity, complicated
Non-Genetic Adv: Easy, cheap, quick
Non-Genetic Dis: label diluted by cell divisions, not equally inherited, limited accessibility of embryo, accuracy of injection/meand of introduction
Briefly describe how (a) determination and (b) specification of embryonic tissue can
be assessed. (2 marks)
a) Determination: using genetic lineage labelling e.g. green fluorescent protein and observing cell fates from a single lineage
b) Specification of embryonic tissue: using non-genetic dye which is cell specific to follow cell migration
Explain how Cre recombinase with ROSA26 locus transgenics can be used for
lineage analysis. (4 marks)
- Rosa26 is normally ubiquitously expressed
- R26R is a modified Rosa26 containing STOP sequence upstream of the reporter gene (originally lacZ, also now fluorescent proteins now flanked by loxP sites)
- LacZ is not expressed in R26R because of the stop sequence
- In the presence of Cre, stop sequence is removed and lacZ is expressed
- Promoter x-cre mice crossed to R26R can be used to trace lineage of cells where promoter X is active
E.g. Wnt1-Cre is not expressed in neural crest cells, so Wnt1-Cre xR26R marks all neural crest cells and their derivatives
Which receptors bind wnt proteins? (0.5 marks) Name two molecules that can
directly antagonize wnt signalling in the embryo. (1 mark) What happens to betacatenin in the absence of active wnt signalling? (0.5 marks)
Frizzled Receptor
2 molecules: WIF-1, sFRP, Cerberus, dikkopf (Dkk)
beta catenin: destruction complex targets it for ubiquitylation and so degradation by the proteosome
Smad1 is phosphorylated following BMP binding to the BMP receptor. Phosphorylated
Smad1 commonly forms a complex with one of two proteins. What are these
proteins and what is the consequence of the formation of each of these complexes? (2
marks)
SMAD 4 = translocation of complex from cytoplasm to nucleus where target genes are switched on
SMAD 6 = inhibitory so target genes are not switched on
Mouse embryos that are homozygous null mutant for the gene vangl1 develop with
an abnormally wide floor-plate. Say, briefly, why this occurs and what is the
phenotypic consequence. (3 marks)
Occurs due to a neural tube defect where cranial neuropore closure failure occurs because of a broader floor plate;
Vangl1 drives PCP
KO Vangl1 causes Abnormal PCP
this:
disrupts normal actin-mysoin rearragnement,
this prevents constriction of the cytoskeleton in the apical surface of the neural plate cells,
This prevents normal wedging, neural closure and convergence-extension of the neural tube.
Phenotype is craniorachishisis
Describe, briefly, how a graded signal can cause a homogeneous group of cells within
the embryo to become sub-divided into two distinct populations with a sharp boundary between them. (4 marks)
- Graded signal uses different concentrations of a morphogen to induce a rough pattern of 2 domains with different identities e.g. high/low [BMP]
- Mutual repression transcription factors creates strict complementarity of identity
- This is further sharpened by segregation and restriction of intermingling across the border to form 2 distinct compartments rather than a fuzzy border
- cell switching occurs- cells on the incorrect side of the boundary are switched to majority side
What is clonal analysis?
Examination of the position and nature of daughter cells following the labelling of a single cell
Explain how tamoxifen inducible gene knockout works in the mouse (4 marks)
Also worded as: Explain how temporal control of Cre recombinase in vivo in the mouse can be achieved using tamoxifen.
- Transgenic construct encodes a fusion protein of Cre with a mutant E2 receptor ligand binding domain (ERLBD) driven by a promoter (ubiquitous/ tissue specific)
- In the absence of tamoxifen mutant LBD is not activated by natural Oestrogen at physiological levels
- The fusion protein is held in the cytoplasm in a complex with HSP90
- Treatment with tamoxifen releases the HSP90, allowing the Cre to move into the nucleus and excise any floxed sequences
Explain how simple autoregulation can generate regulary cyclic oscillations in gene expression, such as that observed in pre-somitic mesoderm (draw a diagram, if it helps) 4 marks
-Bottom tier: single cell oscilators- so gene expression increases protein synthesis; once protein expression reaches a certain level, this negatively feeds back to inhibit further gene expression for the same protein.
This produces peaks and troughs in gene expression.
Gene X is negatively auto-regulated- repressor x binds in its own transcription.
Genes can be positively or negatively autoregulated
e.g. in somitogenesis the oscillation of Hes7 –> express mRNA–> protein –> which repress the transcription of its own encoding gene via cyclic expression and negative feedback
Notch (forms before delta when inhibiton is lost) delta
what is meant by the terms symmetric and asymmetric cell division as applied to progenitor cells (within the developing cortex, for example)? 2 marks
Symmetric cell division: Proliferative division e.g. of neural epithelial cells to produce 2 identical cells
Asymmetric: Neurogenic division that produces 2 different cells e.g. pre-plate neurons and RGC
Name two different progenitor cell-types that exist within the cortical germinal zones. For each of these give an example of one cell type that they give rise to (0.5 marks each)
Neuroepithelial cells give rise to radial glial cells
Radial glial cells give rise to basal neurones
Neocortical radial glial cells can become intermediate progenitor cells –> basal neurones
Two opposing models have been proposed for proximo-distal patterning of the vertebrate limb. These are the progress and pre-specification models. Describe briefly the experimental evidence that first suggested the progress zone model. How can these findings be reconciled with the pre-specification model? (5 marks)
Progress zone model is maintained as proliferative by FGF8 from AER. As cells leave the progress zone they have a proximo-distal identity. The first cells to leave are proximal, the last to leave are distal. Removal of AER= dorsal truncation. Evidence for the progress zone showed Removing the AER at a later period of development results in less disruption of distal structures than if the AER was removed early in development.
Pre-specification model states cells are specified for each segment in early limb bud; this population of cells expand out as limb bud grows. Cell division is throughout the limbs= able to rescue limb development when the AER is removed by preventing cell death
AER= expression of HoxB13- remove AER, dont get digits
Experimental evidence:
Labeled cells in different position of an early limb bud were restricted to single segments of the limb.
Limbs lacking expression of required FGF4 & FGF8 showed all structures of the limb and not just the proximal parts.
More recently, however, the investigators primarily responsible for both the Progress Zone and Prespecification models have acknowledged that neither of these models accounts adequately for the available experimental data
