Regeneration Flashcards
What is Shmidtea mediterrabea?
Schmidtea mediterranea is a small freshwater flatworm, or planarian, just under a
centimeter long when grown to full size
What is so intruguing about Schmidtea?
For more than a century, planarians such as Schmidtea have intrigued biologists because of their extraordinary capacity for regeneration: a small tissue fragment taken from almost any part of the body will reorganize itself and grow to form a complete new animal.
What happens to Schmidtea when its starved?
his property goes with another: when the animal is starved, it gets smaller and smaller, by
reducing its cell numbers while maintaining essentially normal body proportions.
This behavior is called degrowth, and it can continue until the animal is as little as one-twentieth or even a smaller fraction of its full size. Supplied with food, it will grow back to full size again. Cycles of degrowth and growth can be repeated indefinitely, without impairing survival or fertility.
What is the magic behind the degrowth and regrowth of Schmidtea?
- the magic behind it comes from continual cell turnover
- along with the differentiated cells which do not divide there is a population of small apparently undifferentiated cells which do not divide called neoblasts
- the neoblasts constitute about 20% of the body and are widely distirbuted throughout; by cell division they serve as stem cells for the production of new differentiated cells
- Differenti-ated cells, meanwhile, are continually dying by apoptosis, allowing their corpses to be phagocytosed and digested by neighboring cells. Through this cell cannibalism, the constituents of the dying cells can be efficiently recycled. Cell birth continues in a dynamic balance with cell death and cell cannibalism, no matter whether the animal is fed or starved. In conditions of starvation, the balance is evidently tilted toward cell cannibalism, and in conditions of plenty, toward cell birth.
What is the evidence behind totipotency/pluripotency of neoblasts in Schmidtea?
- high dose of x-rays halts all cell division, puts a stop to cell turnover, and destroys the capacity for regeneration. The result is death after a delay of several weeks. The animal can be rescued, however, by injecting into it a single neoblast isolated from an unirradiated donor
- It follows that at least some neoblasts are totipotent (or at least highly pluripotent) stem cells; that is, cells able to give rise to all (or at least almost all) of the cell types that make up the body of a flatworm, including more neoblasts like themselves.
What is so intruguing about Schmidtea?
For more than a century, planarians such as Schmidtea have intrigued biologists because of their extraordinary capacity for regeneration: a small tissue fragment taken from almost any part of the body will reorganize itself and grow to form a complete new animal.
Outline the regeneration of the limb in axolotl
in the process differentiated cells seem to revert to an embryonic character by first forming on the amputation stump a blastema - a small bud resembling an embryonic limb bud. The blastema the grows and its cells differentiate to form a correctly patterned replacement for the limb that has been lost in what looks like a recapitulation of embryonic limb development
What is blastema?
blastema - dedifferentiated cells at the end of the stump and immigrating progenitor cells and connective tissue cells providing information; generates most of the limb is like an embryonic limb bud but not the same
What is a large contribution in the blastema formation?
a large contribution to the blastema comes from the skeletal muscle cells in the limb stump. These multinucelate cells re-enter the cell cycle dedifferentiate and break up into mononucleated cells which then proliferate within the blastema before eventually redifferenatiating
What can the dedifferentiated cells differentiate into?
contrary to what was believed before lineage tracing using genetic markers the dedifferentiated cells can only differentiate again to the cells that they came from so the muscle derived cells can only give rise to muscles.
Outline the regeneration of the axolotl limb
- Form wound epithelium
- Reorganise ECM; upregulate metalloproteinases (MPPs)
- Dedifferentiate cells within a few mm of the amputation (epigenetic reprogramming allows division)
- Dedifferentiated cells migrate and proliferate under the epithelium
- Blastema forms
- Blastema cells start to redifferentiate to form new limb
What is Kragl method
Kragl method:
Label either embryonic precursors or older in Axolotl limb with stably integrated GFP gene and follow what happens to the cells in the blastema after amputation.
How could you track the development of the blastema to support the hypothesis that it is a mixture of dedifferentiated cells
During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.
What are two kind of regeneration?
- those and other examples are classicaly typed as either physiological (homeostatic) or reparative regeneration
- homeostatic - the regular replacement of cells during homeostasis and ageing; ubiquitous property of vertebrates until the time when the cells can no longer replace themselves and tissues and organs begin to fail
- reparative - regeneration occurs in response to injury
What is the blastema covered with?
The heterogeneous mass of cells is necessarily covered by epidermis (termed the wound epidermis) and thus the definition of a blastema includes this ectoderm-derived component.
What happens in animals that can regenerate?
- in animals that posses regenerative capabilities local amputation initiates the first transformation from mature tissue into a transient undifferentiated proliferative state (blastema) that is followed by the second transformation where morphogenesis and re-differentiation replace the missing structures
- the first transformation is a specialised wound healing response that ends with a formation of a blastema
How can you distinguish a regenerative and a non-regenerative response?
- this can be distinguished from a non-regeneratve response where re-epithelialization is followed by reconstitution of a mature basal membrane, wound contraction and the deposition of a densley layerd firbous scar tissue that defines regeneration incompetence
- during a regenerative response where the first transformation ends with the formation of a small mass of undifferentiated proliferating cells the second transformation involves proliferative expansionof the cell population, patterning and ultimately the orderly differentiation of the cells into the multitude of cell types that make up the tissues of the replacement structure
What is the difference between blastema and cancer?
While the persistence of active cycling cells can be used to identify the blastema, this alone hardly differentiates a blastema from a tumour. Therefore, the presence of proliferating cells must be used in combination with other factors. Given that uncontrolled growth is a characteristic of tumor cells and controlled growth is a characteristic of blastemal cells, comparative profiling of cycling tumor cells and blastemal cells may identify a panel of markers specific to cycling blastemal cells. Until these markers are found, however, one must look to other factors to uniquely define a blastema.
What are the conclusions from the ear regeneration studies?
As a comparative system between regenerating and non-regenerating species, the ear punch assay will reveal whether specific molecules or pathways can serve as early indicators for blastema formation and which factors alone or in combination are required to maintain blastema morphogenesis.
How can you identify the genes responsible for regeneration?
they performed parallel expression profile time courses of healing lateral wounds versus amputated limbs in axolotl. The comparison between wound healing and regeneration allowed them to identify amputation specific genes
What phases of gene expression did they identify in during regeneration?
- by clustering the expression profiles of the samples they could detect three distinguishable phases of gene expression
- early wound healing
- transition phase
- establishment of the limb development program
What experimental method did they use to detect genes in regeneration?
To validate the quantitative aspect of the gene expression data we performed qPCR analysis on seven representative targets
When do differences between regeneration and healing emerge?
- wound healing diverges from reegeneration at 24h
- cellular stress often starting at 24h and sustained
How did they discover molecular funelling?
Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud–like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.
How did they discover molecular funelling?
Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud–like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.
What did Rivera and Morris discover?
Connective tissue (CT) cells are labeled via the inducible Cre-loxP system.Connective tissue cells
and their derivatives are isolated throughout post-injurylimb regeneration
and profiled via single-cell RNA-sequencing (scRNA-seq). These experiments demonstrated that mature, heterogeneous connective tissue cells “funnel” via a de-differentiatedblastema
progenitor before transitioning to an embryonic limb bud-like state. From this state, the heterogeneity of mature connective tissue is re-established in the regenerated limb.
What role does [p53 play in the regeneration process
The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation.
What happens if the levels of p53 don’t drop during regeneration?
- Stabilization of the p53 level at the time of blastema formation, when it normally decreases, led to an impairment of the regeneration process
- This finding suggests that down-regulation of p53 activity is required for blastema formation.
