Lymphangiogenesis – Development of the Lymphatic Vasculature Flashcards
Q4: Which transcription factor is essential for lymphatic endothelial cell identity?
A: PROX1.
Q5: Which transcription factors regulate PROX1 expression in venous cells?
A: SOX18 and COUP-TFII.
Q6: What is the role of VEGFR3 in lymphangiogenesis?
A: It enables lymphatic progenitors to respond to VEGF-C, guiding their migration from the vein.
🧬 Molecular Players
PROX1: Master transcription factor essential for lymphatic endothelial cell (LEC) identity.
SOX18 and COUP-TFII: Activate PROX1 in the cardinal vein.
VEGFR3: Receptor that enables migration of LECs in response to VEGF-C gradient.
🧪 Key Experiments and Techniques
- Gene Knockout (KO) Approaches
Constitutive KO: PROX1 knocked out in all cells → no lymphatic vessels form.
Conditional KO: PROX1 deletion in endothelial cells using Cre/LoxP under control of the Tie2 promoter → specific loss in vasculature.
- Lineage Tracing
Used to track the origin of cells using fluorescent reporters (e.g., Tomato/GFP switch system).
If cells switch from red (Tomato) to green (GFP) → Cre was active, showing venous origin.
Revealed that most LECs are GFP+, confirming venous origin; a minority remain red, suggesting non-venous origin.
- Inducible Cre-Lox System (CreERT2)
Temporal control using tamoxifen to activate Cre recombinase fused with estrogen receptor (ER).
Helps determine when and where gene functions are needed during development.
cre/loxp system
LoxP = the “cut here” signs 🪧 placed on either side of a gene.
Cre recombinase = the molecular scissors ✂️ that recognizes those signs and cuts out the DNA between them.
Q7: From where do most lymphatic endothelial cells originate?
A: The cardinal vein (venous origin).
Q10: What is the lymph sac (LS)?
A: A primitive lymphatic structure that arises adjacent to the cardinal vein during early development.
Q2: Why is the lymphatic system considered separate from the blood vascular system?
A: It only connects to the blood circulation at one point, functions independently, and serves roles in fluid homeostasis and immune cell trafficking.
Q5: Why is lineage tracing important in developmental biology?
A: It allows researchers to determine the origin and fate of specific cell populations during embryogenesis.
Q4: How does the Cre-LoxP system enable targeted gene manipulation?
A: By using Cre recombinase under a tissue-specific promoter, which excises DNA between loxP sites only in desired cell types.
Q3: What is the significance of the transcription factor PROX1 in vascular development?
A: It reprograms venous endothelial cells into lymphatic endothelial cells, initiating lymphatic identity.
Q9: What marker is used to detect lymphatic endothelial cells in flow cytometry or histology?
A: LYVE1 or Neuropilin-2.
Q10: What does VEGFR3 expression in lymphatic progenitors allow?
A: Responsiveness to VEGF-C, guiding migration out of the vein to form lymph sacs.
Q11: What is the temporal window for lymphatic specification from the cardinal vein?
A: Between 9.5 to 11.5 days post-coitum (dpc).
Q13: A knockout of FOXC1/FOXC2 leads to venous markers being expressed in arteries. What does this suggest?
A: These transcription factors are essential for maintaining arterial identity by repressing the venous program.
Q14: If a lymphatic cell is GFP+ in a lineage tracing experiment using Tie2-Cre, what can be inferred?
A: The lymphatic cell originated from an endothelial cell lineage.
Q15: Why might some lymphatic cells remain Tomato+ (red) in a Cre-GFP reporter system?
A: They may be from a non-endothelial origin, or Cre recombination was inefficient or incomplete.
Q16: How could you experimentally confirm that lymphatic endothelial cells are derived from venous progenitors?
A: Use Tie2-Cre × floxed reporter mice, trace GFP+ cells, and confirm co-expression with lymphatic markers.
Q17: What phenotype would you expect in a Prox1-null mouse embryo?
A: Complete absence of lymphatic vessels and resulting edema due to failed lymphatic development.
Q18: Why would an inducible CreER system be advantageous over constitutive Cre for studying development?
A: It allows precise timing of gene deletion, avoiding early lethality or off-target developmental effects.
Q19: In a lineage tracing study, why is it important to distinguish between green (GFP+), red (Tomato+), and yellow (both)?
A: It helps determine the recombination status and lineage contribution over time, including transitional or mixed populations.
Q20: What is the difference between reverse and forward genetics in the context of lymphangiogenesis?
A: Reverse genetics starts with gene manipulation (e.g., PROX1 knockout) to observe phenotype; forward genetics starts with a phenotype and searches for the underlying gene.
