1. REGENERATION II

2. Regeneration in Hydra: Morphallaxis and Epimorphosis
Hydra is a genus of cnidarians found in freshwater. Most are only 0.5 cm long with a tubular body.
There is a “head” at its distal end consisting of a conical hypostome (the mouth) surrounded by a ring of tentacles beneath it which catch food.
There is a “foot” at the proximal end which is also known as the basal disc and helps the animal stick to surfaces under water.
They are diploblastic animals having only two germ layers ectoderm and endoderm.
Though they do not have a true mesoderm, they have secretory cells, stinging cells (nematocytes), gametes and neurons that are not part of the two epithelial layers.

3. Hydra can reproduce sexually but do so only under adverse conditions
Hydra can reproduce sexually but do so only under adverse conditions. They usually reproduce by budding off a new individual.
A hydra’s body is not stable. The cells of the body column are constantly dividing and are displaced to the extremities of the column from which they are shed. Thus the hydra’s body is always regenerating
The cellular replacement happens from three cell types. The ectodermal and endodermal cells are constantly dividing to produce more epithelia. The third cell type is a multipotent interstitial stem cell found in the ectodermal layer which generates the gametesm neurons secretory cells etc.
The three cell types are all that is needed to form a new hydra.

4. Experimental embryology or one might say experimental biology began with Abraham Tremblay’s of hydra regeneration in 1741
If the hydra is cut into as many as 40 pieces, “there are reborn as many complete animals similar to the first”. Each piece would regenerate a head at its original apical end a foot at its original basal end.
Every portion of the hydra’s body along the apical-basal axis has the potential to form a head and a foot.
However, the polarity of the organism is co-ordinated by a series of morphogenetic gradients that permit the head to form only at one place and the basal disc to form only at another.

5. Evidence for such gradients was obtained from the grafting experiments done by Ethel Browne in the early 1900s
When hypostome tissue from one hydra is transplanted to the middle of another hydra, the transplanted tissue forms a new apico-basal axis with the hypostome extending outwards.

6. When a basal disc is transplanted to the middle of another hydra, a new apico-basal axis forms but with the opposite polarity with the basal disc extending outwards.

7. When tissues from both ends are transplanted simultaneously into the middle of another hydra, either no new axis forms or the new axis has little polarity.
These experiments have been interpreted as follows:
There is a head activation gradient (highest at the hypostome)
There is a foot activation gradient (highest at the basal disc)
The head activation gradient can be measured by implanting rings of tissue from various levels of a donor hydra into a particular region of the host trunk. The higher the level of the head activator in the donor tissue the greater the percentage of implants that will induce the formation of new heads.

8. Formation of secondary axes following transplantation of head regions into the trunk of a hydra
Ethel Browne noted that the hypostome acted like an “organizer” of the hydra.
Broun and Bode (2002) confirmed this by demonstrating that:
When transplanted the hypostome can induce host tissue to form a secondary body axis.
The hypostome produces both head activation and inhibition signals.
The hypostome is the only “self-differentiating” region of the hydra.
It contains a head inhibiting signal which supresses the formation of new organizing centers.
Bode also found that even transient contact with the hypostome region was sufficient to induce a new axis from a host hydra.
Hypostome tissue grafted onto a trunk induces the hosts own trunk tissue to become tentacles and head
Subhypostomal donor tissue grafted onto a trunk self-differentiates into a head and upper trunk.

9. The major head inducer of the hypostome organizer is a set of Wnt proteins acting through the canonical β-catenin pathway
Wnt expression during hydra budding.
These Wnt proteins are seen in the apical end of the early bud defining the hypostome region as the bud eleongates.
If GSK3 is inhibited throughout the body axis ectopic tentacles form at all levels and each piece of the trunk has the ability to stimulate the outgrowth of new buds.

10. Morphallaxis and Epimorphosis
When a hydra is cut, the Wnt pathway is activated in the lower portion (in the portion that will form the new head).
If the cut is made close to the head (just below the hypostome), Wnt3 in the epithelial cells will cause the remodelling of existing cells to form a new head; hence this is morphallactic regeneration.
However, if the hydra is cut at mid-section, the cells derived from the interstitial cell (neurons, nematocytes etc.) undergo apoptosis immediately below the cut site. Before dying these cells produce a burst of Wnt3. This Wnt3 activates β-catenin in the interstitial cells causing a wave of cell proliferation in these cells as well as remodelling of epithelial cells. So regeneration is thus epimorphic.

11. The head inhibition gradients
Grafting experiments provide evidence for a head inhibitor gradient:
A. Subhypostomal tissue does not generate a new head when placed close to an existing head.
B. Subhypostomal tissue generates a head if the existing host head is removed.
C. Subhypostomal tissue generates a new head when placed far away from an existing host head.
The host head appears to make an inhibitor that prevents the grafted tissue from forming a secondary axis and new head.
The identity of the head inhibitor remains unknown. It appears to be labile with a half-life of only 2-3 hours.
It is thought that the head activator (Wnts) and head inhibitor are both made in the hypostome, but the head inhibitor gradient falls of more rapidly than that of the head activator.
The place where the head activator in uninhibited by head inhibitor becomes the budding zone.

12. What prevents the bottom third of the Hydra from forming a head?
Head formation at the base appears to be prevented by the production of another substance a foot activator
Bud location as a function of head and foot inhibition gradients. In young adult hydras the gradients of head and foot inhibitors appear to block bud formation. However, as the hydra grows the sources of these labile substances move apart creating a region about 2/3rds down the trunk where the levels of both inhibitors are minimal. This is where buds form.
Several small peptides have been found to activate foot formation.
The specification of cells as they migrate from the basal region through the body column may be mediated by the gradient of a tyrosine kinase Shinguard.

13. Prometheus’s punishment
Compensatory regeneration in the mammalian liver
Today the standard assay for liver regeneration is a partial hepatectomy, where some lobes of the liver are removed. Although the removed lobe does not grow back the remaining lobes enlarge to compensate for the loss of the missing tissue.
Such compensatory regeneration-the division of differentiated cells to recover the structure and function of an injured organ has been observed in the mammalian liver and the zebrafish heart.
Prometheus’s punishment
A Greek legend

14. The human liver regenerates by proliferation of existing cells
The human liver regenerates by proliferation of existing cells. The liver cells do not fully dedifferentiate when they enter the cell cycle nor is there a regeneration blastema formed.
Mammalian liver regeneration has two lines of defence:
The normal, mature adult hepatocytes which are normally not dividing are instructed to join the cell cycle and proliferate till they have compensated for the missing part
There is a population of hepatic progenitor cells that are normally quiescent but activated when the injury is severe and the adult hepatocytes cannot regenerate well (due to senescence, alcohol or disease).

15. Normal mammalian liver regeneration
The five types of cells in the adult liver namely the hepatocytes, duct cells, fat-storing cells, endothelial cells and Kupffer macrophages all begin to divide to produce more of themselves.
Each cell type retains its cellular identity and the liver retains the ability to perform all its functions e.g. bile synthesis, albumin production, toxin degradation etc.
Several pathways may initiate liver cell proliferation the end result of which is to downregulate but not totally supress the genes required for the differentiated functions of the cell while activating the genes that make the cell proliferate.

16. How is injury to the liver sensed in order to trigger regeneration?
The removal or injury to the liver is sensed through the blood stream as some liver-specific factors are lost while others such as bile acids and gut lipopolysaccharides increase.
These lipopolysaccharides induce some non-hepatocytes to secrete some paracrine factors that induce the hepatocytes to enter the cell cycle. The Kupffer cells secrete IL-6 and TNFα while stellate cells secrete Hepatocyte growth factor (HGF) and TGF-β. The specialized blood vessels of the liver also produce HGF and Wnt2.

17. Hepatocytes still connected to each other in the epithelium cannot respond to HGF. How is regeneration triggered?
The hepatocytes activate cMet (the receptor for HGF) within an hour of partial hepatectomy.
The trauma of partial hepatectomy may activate metalloproteinases that digest the ECM and permit the hepatocytes to separate and proliferate.
Metalloproteinases may also cleave HGF to its active form.
Together the factors produced by the Kupffer cells, endothelial cells and stellate cells allow the hepatocytes to divide by preventing apoptosis, activating cyclins D and E and repressing cyclin inhibitors such as p27.

18. How does the liver stop growing when it reaches the appropriate size?
Not known.
One clue comes from parabiosis experiments. Partial hepatectomy in one parabiosed rat will cause the other’s liver to enlarge.
Some factors in the blood appear to establish liver size.
Huang and colleagues (2006) propose these factors are bile acids.
Partial hepatectomy stimulates the release of bile acids in blood. This activates a transcription factor Fxr in hepatocytes and promotes their proliferation.

19. The second line of regenerative ability in the mammalian liver
If the hepatocytes are unable to regenerate the liver sufficiently within a certain amount of time, the second line is activated.
Oval cells, a small progenitor cell population that can produce hepatocytes and bile duct cells start dividing.
Oval cells appear to be kept in reserve and are used only after hepatocytes have attempted to heal the liver.

20. Questions remaining:
What are the molecular mechanisms by which these regeneration promoting factors interact?
How is the liver first told to begin regenerating?
How is the liver told to stop regenerating when it reaches appropriate size?