MAGING MOLECULAR PROCESSES Flashcards
❓ Q1: Define the following terms and explain their relevance to vascular development:
(a) Vasculogenesis
(b) Angiogenesis
(c) Lymphangiogenesis
✅ Answer:
Vasculogenesis: De novo formation of blood vessels from mesodermal progenitors; establishes initial vascular network.
Angiogenesis: Formation of new blood vessels from existing ones; key in growth and wound healing.
Lymphangiogenesis: Formation of lymphatic vessels from pre-existing lymphatics; critical in immune response and tissue drainage.
❓ Q2: What is a transcription factor (TF), and how do TFs regulate gene expression?
✅ Answer: Transcription factors are proteins that bind specific DNA sequences (e.g., promoters, enhancers) to regulate transcription of genes. They influence the rate of transcription initiation and can recruit co-factors or modify chromatin structure.
🔬 Section B: Methods & Techniques
❓ Q3: Describe the Cre/loxP system. How is it used to study gene function in vascular development?
✅ Answer: Cre recombinase recognizes loxP sites and excises the DNA between them. When driven by a tissue-specific promoter (e.g., VEGF-R3), it allows conditional knockout of genes in lymphatic endothelial cells. It helps assess cell autonomy and lineage tracing in vascular development.
❓ Q4: What is the outcome of a ChIP-seq experiment targeting a transcription factor?
✅ Answer: It provides a genome-wide map of binding sites for the transcription factor, identifying which regulatory DNA regions are directly bound and potentially regulated.
🔎 Section C: Application & Interpretation
❓ Q5: Explain the two kinetic states of transcription factors as revealed by single molecule tracking (SMT). What does each state represent?
✅ Answer:
Short-lived (non-specific) binding (~0.5s): TFs rapidly scan the genome.
Long-lived (specific) binding (~5s): TFs bind high-affinity DNA sites to regulate transcription. These two states reflect the TF’s search strategy.
❓ Q6: You observe transcription bursts in a gene using live-cell imaging. What features of these bursts can vary, and what do they reflect about gene regulation?
✅ Answer: Features include burst frequency, burst size, and duration. They reflect dynamic gene control — e.g., fast bursts = responsive regulation; large bursts = strong gene activation. Burst behavior is influenced by transcription factor binding dynamics and chromatin state.
🧠 Section D: Critical Thinking / Integration
❓ Q7: A TF shows mostly short dwell times on chromatin. What might this indicate about its function?
✅ Answer: It may be broadly surveying the genome (non-specific binding), possibly acting as a general regulator or preparing chromatin for other factors. Few long dwell times suggest low specific activation at current conditions.
❓ Q8: How can combining mRNA live imaging with SMT improve our understanding of transcription regulation?
✅ Answer: It links TF binding behavior (kinetics, dwell time) with actual transcriptional output (burst timing and size), revealing causal relationships between TF dynamics and gene expression.
❓ Q9: Describe how scale affects biological interpretation in vascular development, giving one example each from molecular, cellular, and tissue level.
✅ Answer:
Molecular: PROX1 activates LEC fate via transcriptional regulation.
Cellular: Tip cells migrate using VEGF gradients.
Tissue: Vascular networks form branching patterns and valves. Connecting these levels helps understand how gene changes affect whole organs.
- Describe the principle of the Cre/loxP system
✅ Answer:
The Cre/loxP system is a site-specific recombination technique used to control gene expression in a tissue-specific and/or time-specific way.
Cre recombinase is an enzyme that recognizes loxP sites (short DNA sequences).
When two loxP sites are placed around a gene (this is called “floxing” a gene), Cre can delete or “cut out” the gene between them.
This allows for conditional knockout — you can delete the gene only in certain tissues or at certain times.
- What specific gene promoter has to drive CRE expression to delete (deflox) a gene in the vasculature?
✅ Answer:
To target vascular or lymphatic endothelial cells, Cre should be placed under the control of a lymphatic endothelial cell-specific promoter.
🔹 Example: The VEGF-R3 promoter (also called FLT4)
It is specifically active in lymphatic endothelial cells, so when Cre is under its control, it deletes floxed genes only in those cells.
- What biological question does the Cre/loxP system help answer?
✅ Answer:
It helps answer whether a gene’s effect is cell-autonomous.
That is: Is the gene required in the cell itself to produce a phenotype, or is it acting non-cell-autonomously through other cells?
What does the Cre/loxP system allow us to explore when used with molecular tools like fluorescent reporters?
✅ Answer:
It allows for lineage tracing — defining cell fate trajectories in development.
By combining Cre with a fluorescent reporter cassette (like a floxed-stop GFP), you can permanently label the descendants of specific cells.
This lets you track where cells go, what they become, and how tissues develop.
In vivo models:
Mouse and Zebrafish Two key animal models — mice for mammalian relevance; zebrafish for imaging and rapid genetics.
Forward Genetics Start with a phenotype, identify the gene.
Reverse Genetics Start with a gene, mutate it, and see the phenotype.
LOF (Loss of Function) Knockout (Entire gene is removed or disrupted → no protein made), knock-in (Replace gene with mutant version ), dominant negative (mutant version of the protein that actively interferes with the wild-type (normal) protein), morpholinos (Special synthetic molecules used in zebrafish to block mRNA → prevent translation of specific genes (like temporary KO).
GOF (Gain of Function) Ectopic (Force gene to be expressed where or when it’s not normally active (e.g., turning on VEGF-C in skin to see if lymphatics grow)) expression, rescue experiments (restore normal phenotype).
Gene trap (a reporter into the genome → it “traps” gene expression and lets you see where, when, and what happens when a gene is disrupted.)
ENU screen Chemical mutagenesis followed by complementation testing to find mutations affecting development.
You’re doing an ENU screen, and you end up with multiple mutant animals that all show the same or similar phenotype — like, say, a zebrafish embryo with no blood vessels.
You have two mutants that show the same phenotype (e.g., mutant A and mutant B — both lack blood vessels).
You cross them (mate them).
You look at the offspring (F1 generation) and ask:
Babies are normal (no phenotype) One working copy of gene A from parent B
One working copy of gene B from parent A
➤ So the baby is normal = complementation!
Babies still have the mutant phenotype The baby gets two broken copies of gene A
➤ So the phenotype stays = no complementation
In vitro models:
Used for studying cellular processes in controlled environments.
In vitro EC (Endothelial Cell) lines: Cultured endothelial cells used to study angiogenesis, signaling, etc.
ES cell differentiation: Using embryonic stem cells to model lineage decisions in a dish.
Molecular analysis tools:
ChIP-seq: Identifies genome-wide DNA binding sites of transcription factors.
RNA-seq: Measures transcript levels genome-wide; tells you what genes are expressed and how much.
qPCR: Quantifies expression of specific genes.
In situ hybridization: Shows where specific mRNA is expressed in tissues.
=> You use a labeled complementary RNA probe that binds to the target mRNA → it lights up under the microscope
Spatial map of gene expression (e.g., mRNA in lymphatic endothelium)
Q1: Compare forward and reverse genetics. Give one example of how each might be used in zebrafish.
✅
Answer:
Forward genetics: Mutagenize embryos with ENU, identify phenotypes (e.g., vascular defects), and map the responsible gene.
Reverse genetics: Use morpholinos or CRISPR to knock down a known gene (e.g., VEGF) and study the effect on vessel formation.
Q2: What’s the difference between LOF and GOF experiments? Give one example of each.
✅ Answer:
LOF (Loss of Function): Remove or block a gene to see what breaks (e.g., VEGF KO causes vessel failure).
GOF (Gain of Function): Overexpress a gene to see new effects (e.g., ectopic Notch causes excessive branching).
What biological questions can you answer with:
(a) ChIP-seq?
(b) RNA-seq?
(c) In situ hybridization?
✅ Answer:
(a) ChIP-seq: What genes a transcription factor binds to.
(b) RNA-seq: Which genes are expressed and how much under certain conditions.
(c) In situ: Where a specific mRNA is expressed in a tissue or embryo.
You suspect a TF controls lymphatic identity. What toolset would you use to test this in vivo?
✅ Answer:
Use VEGF-R3-Cre mouse with a floxed TF gene for cell-specific KO.
Combine with a GFP reporter (lineage tracing).
Confirm binding sites using ChIP-seq.
Confirm loss of target gene expression with RNA-seq or qPCR.
Q1: Name three transcription factors involved in LEC specification. Describe the role of each.
✅ Answer:
SOX18: Initiates LEC gene expression.
COUP-TFII: Works with SOX18 to allow LEC fate.
PROX1: Master regulator that locks in lymphatic identity.
A VEGF-C mutant embryo lacks lymphatic sprouting. Which other molecule may be failing to process VEGF-C, and why is this important?
✅ Answer:
CCBE1 is likely defective.
It is required to activate VEGF-C, enabling it to bind VEGF-R3 and drive sprouting.
Explain how LEC valves form and why they are essential.
✅ Answer: Valves form in response to fluid flow and mechanical stress. They are essential for directed lymph flow toward the thoracic duct and prevent backflow. Transcription factors like FOXC2 and GATA2 are key in valve specification.
What is the consequence of knocking out PROX1 in venous endothelial cells?
✅ Answer: Cells fail to maintain lymphatic identity and may revert to blood endothelial cell fate. Lymphatic vessel development is arrested.
Name 3 transcription factors required for lymphatic endothelial cell (LEC) specification and describe their roles.
✅ Answer:
SOX18: Initiates early LEC program.
COUP-TFII: Allows LEC transition from venous ECs.
PROX1: Master regulator; locks in LEC identity.
What stages occur during lymphatic morphogenesis, and what molecular signals define each?
✅ Answer:
Emergence: LECs sprout from veins via VEGF-C; requires SLP-76, Syk (platelet aggregation).
Remodeling: Lymph sacs organize; still VEGF-C driven.
Maturation: Valve formation and stabilization via FOXC2, EphrinB2, Integrin-α9, NFAT1c.
Compare VEGF-R2 and VEGF-R3 in vascular development.
✅ Answer:
VEGF-R2: Blood vessel growth, angiogenesis.
VEGF-R3: Lymphatic-specific; responds to VEGF-C/D; critical for lymphangiogenesis.
What is the role of Integrin-α9 in lymphangiogenesis?
✅ Answer: Required for valve formation in lymphatic vessels; helps align LECs and stabilize flow.
Outline the roles of tip, stalk, and phalanx cells in vessel sprouting.
✅ Answer:
Tip cells: Sense VEGF; lead migration.
Extend filopodia to sense VEGF gradients in the environment
Respond to VEGF-A → VEGFR2, and communicate with stalk cells via Notch signaling
Stalk cells: Proliferate behind tip cells; form vessel shaft.
Receive Notch signals from tip cells to suppress tip-like behavior and promote proliferation
they form the main tube of the new vessel
Phalanx cells: Maintain flow, stabilize mature vessel.
Non-proliferative, low VEGF response; maintain tight junctions and quiescence
mature vessels; maintain barrier function and blood flow
VEGF and anti-angiogenic signals (e.g., TGF-β)
