BIO 448 Exam 3: vertebrate regeneration

read and comprehend

• Stem cells are undifferentiated​

• Have the ability to become many different cell types ​

• Undergo a process of self-renewal​

Embryonic stem cells (ES) cells:​

Found at the blastula stage in an embryo. ES cells give rise to all the cells that are part of the organism. They are pluripotent. ​

Adult stem cells: ​

Present during development and continue through adulthood. ​

​

Give rise to specific cell types . ​

​

These are multipotent. ​

1) Bone Marrow ​

2) Intestinal Tissue ​

3) Lower layers of the skin ​

epithelium​

4) Skeletal Muscle ​

5) Hepatic stem cells (liver) ​

• metamorphosis

• regenerative

• Limbs and Tail and Spine​

What is an axolotl?​

• They are an adorable salamander.​

• They are critically endangered in the wild from habit loss in Mexico​

• They are a neotenic species, in which _____ is delayed (sometimes permanently). They have external gills, 4 limbs and a tail​

• They are highly ____ and can be grown relatively easily in lab. Not clear why axolotls (and other salamanders/newts can regenerate so well!)​

• Regenerates:______

Limb Regeneration:​

1. Formation of the wound epithelium ​

2. Nerve Innervation ​

3. Formation of an apical epithelial cap and blastema tissues​

4. De-differentiation and migration of cells​

5. Proliferation of cells and similar patterning to limb bud formation! ​

Cell types in limb: muscle, skin, bone, neurons, vascular system (blood vessels, etc.) ​

No scar tissue that forms (which differs from humans!) ​

• nAG

1. Formation of the wound epithelium ​

• After amputation, there is little blood loss ​

• The surrounding epidermis cells migrate to cover the wound surface, within 6-12 hours. ​

• Unlike humans, there is no scar that forms. ​

VERY different from humans ​

2. Nerve Innervation ​

• After the wounds heal, nerves are recruited to the site of injury.​

• How do we know nerves are required for limb regeneration? ​

• Nerve innervation is important and required for regeneration ​

• Spoiler alert: Still not clear! ​

• In newts, a growth factor protein identified as ___ was identified that can rescue the regeneration phenotype when nerves are cut.​

• What is nAG? ​NAG (growth factor) which rescues the defect of nerve not being able to innervate ​

​

nAG:​

• Newt Anterior-Gradient Protein​

• No clear homologs in humans  ​

• In fact, it is not clear that Axolotls have nAG.​

• In conclusion, nerves are important in vertebrate limb regeneration, but it is not clear why.

• After the wounds heal, nerves are recruited to the site of injury.​

• Signaling between the nerves and the wound epithelium helps recruit cells to the wound site and aids in the formation of the apical epithelial cap. ​

• The apical epithelial cap will become a major signaling center in establishing the proximal-distal axis of the limb. ​

Limb Regeneration:​

1. Formation of an apical epithelial cap and blastema tissues​

2. De-differentiation, proliferation and migration of cells​

​- When the apical epithelial cap forms, signals are established that induces dedifferentiation of cells surrounding the wound site. ​

Limb Regeneration:​

1. Dedifferentiation occurs when differentiated cells become progenitor cells. Progenitors cells are more mitotically active than differentiated cells and can migrate! ​

​Cells that were differentiated... dedifferentiate and migrate to the blastema ​

Different from what is happening in planaria (neoblast stem cells migrate) ​

​

read and comprehend

Look at this blastema picture. What structure does this look similar to?​

• There are functional similarities between the Apical Epithelial Cap (AEC) and the AER (Apical Epidermal Ridge) & the Dedifferentiation zone and the Progress Zone ​

What factors might you think are involved in Blastema Formation? Some evidenced suggests that RA and FGF signaling functions during blastema formation, but overall, its not clear. ​

​

1. Do cartilage cells make muscle after amputation? ​

2. Immunostaining to look at muscle cell fate markers; used GFP to label cartilage (They stained cartilage with GFP; stained MHCI with immunostain (muscle)) - did a transplant from cartilage ​

3. Found no Pax7 signals; regenerated in the skeleton; Cartilage cells do not make muscle cells ​

Figure 2:

Answer the following questions about the figure:​

1. What was the question the researchers were addressing​

2. What technique did they perform to address the question​

3. Where were the major conclusions​

4. Do you have any questions about the figure? ​

Transplant in limb that was already developed

1. They were trying to test if muscle makes cartilage or epidermis (does muscle cell become other cell fates during regeneration) ​

2. Transplanted GFP tagged presomitic mesoderm and single cell PCR (single cell PCR, GFP tagging and transplant analysis, immunoflourence (figure C) ​

3. Major conclusions: muscle does not make cartilage or epidermis. It stays muscle ​

Transplant in embryo (stage: just after gastrulation; beginning of neurlation – line dictates the primitive streak) ​

Bc of fate mapping, they know it’s the tissue that gives rise to the somites... and transplants it in a similar location ​

Some cells expressing Pax7 are also expressing GFP – (figure C) this suggests that some GFP cells are expressing Pax7 (muscle)… some of the blastema cells are making muscle ​

D: looked at specific markers for muscle, dermis, skeleton, and schwann cells – showed that GFP cells were not giving rise to any of the types except for muscle ​

NOT giving rise to tissue that it did not used to be

Figure 3:

Answer the following questions about the figure:​

1. What was the question the researchers were addressing​

2. What technique did they perform to address the question​

3. Where were the major conclusions​

4. Do you have any questions about the figure? ​

Experiment that was performed: sequencing the genes before and during/after regeneration to see what was being turned on (next gen sequencing) - which genes are upregulated and which are downregulated (gives us genes to test!) ​

What was one of the major findings: found genes involved in ECM, cell-cell adhesion (cahderins), and immune-system related processes (the immune-system related genes were downregulated)

Axolotls can also recover from spinal cord injuries. But how? ​

Describe the experiment that wasperformed?​

​

​

What was one of the major findings?​

neoblast

de-differentiation

Planaria use ____ model ​

Axolotl use _____ model ​