8. Regeneration Flashcards
(25 cards)
How does regenerative capacity vary among organisms?
Mammals - some organs e.g. liver
Amphibians and fish - full limbs, tail, fins, lens, CNS, heart and other organs
Planaria - whole anterior and posterior halves of body
Hydra - entire individual from small fragment
Sponges and Ocoels - whole body
What are the conceptual requirements for regeneration?
- Prior to injury, cells and tissues must ‘know’ their own identity within the organism and their position in relation to the other cells = “morphological memory map”
- After injury, the cells and tissues of the organism need to recognise that an injury has occurred
- Organism needs to rapidly respond by closing the wound
- Regenerative response commences and might involve cell proliferation, growth of the tissues, and a re-patterning of the cells to differentiate into the lost structure
- Regeneration must end, having formed correct size and shape of the lost structure, which is both integrated with and in proportion to the rest of the body
How can regeneration mechanisms vary?
Stem-cell mediated regeneration = multipotent stem cell differentiates into many different tissues
Epimorphosis = Cell dedifferentiates into stem cell which can give rise to many different tissues
Compensatory regeneration = Regenerates more of the same tissue e.g. liver
Morphallazis = balance of cell death and cell fate conversions, smaller regenerate at the end
What is involved in regeneration?
Stem cells
Proliferation
Differentiation
Tissue morphogenesis
All fundamental to embryogenesis and homeostasis
Which molecular pathways are reused with some differences?
Immune response = injury comes with possible necrotic cell death, escaping fluids and unprotected tissues. Need to close wound and deploy phagocytic cells to clean up injury.
Induced reprogramming = cells become activated to take on an immature, embryonic-like state before they can use developmental programmed to rebuild tissues. May require novel processes to reprogram adult cells to become embryonic-like.
System integration = formation of new cells that become organised and integrated into existing differentiated tissues e.g. blood vessels and nerve connections
Size recognition and termination = must recognise spatial relationship with surrounding tissues and organism as a whole to ensure regrowth is callibrated to the appropriate scale and is terminated once acheived
Describe regeneration in Hydra?
Radially symmetric body, head and foot
Diploblastic and adult multipotent stem cells
Divide sexually and asexually (budding)
If cut at midgastric level, tail regenerated new head and head regenerates a new tail, each half the size of the original. Doesn’t require cell division. New tissue comes from host. Hypostome produces head inducing signal plus an unknown head inhibitor (doesn’t get secondary axis if transplant into head).
Wnt is head inducer in normal development and regeneration. Wnt3 expressed in tip of hypostome in adult hydra in normal process of budding. Upon amputation of head region, Wnt3 expressed through cut surface, setting up new head organiser.
Wnt is cell surface signalling molecule acting as a cell surface receptor –> signalling cascade –> accumulation of beta catenin –> travel to nucleus –> activate TFs
No proliferation = morphallactic regeneration
Describe regenerations in planarians
3 germ layers
Can reproduce asexually by binary fission which requires neoblasts, a stem cell population scattered throughout animal.
Can dissect and head half will regenerate a tail and vice-versa. Initial wound response and formation of blastema from neoblasts.
Potency addressed by transplantation of single neuroblasts into irradiated animals - some where pluripotent and could rescue animal
Wnt-P1 produced in tail and diffuses anteriorly to form gradient.
Suppresses head formation except at anterior which expressed Wnt inhibitor (Notum).
Act to reset expression of array of positional control genes along AP axis.
Have positional control genes along AP axis and injury-induced wound signaling induces re-patterning of AP expression of notum and Wnt1 at the cut surfaces - no longer inhibiting head in head region so new head can form.
New morphogen patterning redirects positional control gene expression programs that lead to neoblast specialisation and subsequent differentiation
Which cells contribute to embryonic limb formation in chicks/mice?
Limb bud - bilateral bulges at presumptive forelimb and hindlimb locations
Lateral plate mesenchymal cells migrate within limb field and proliferate = limb skeletal and connective tissue precursors
Somite derived mesenchymal cemms (from myotome) migrate into limb field = muscle cell precursors (myoblasts)
Overlying ectodermal tissue provides signals to underlying mesenchymal cells which proliferate creating a limb bud
How does different fgf’s define the position of the limbs?
Fgf10 is expressed at the 4 limb positions in the developing chick lateral plate mesoderm.
Transplanting Fgf10 beads/fibroblasts into chick flank induces 5th ectopic limb.
In somitogenesis, antagonistic relationship between Fgf8 expressed by progenitor zone and RA expressed anteriorly by somites and presomatic mesoderm
Fgf8 also expressed in heart lateral plate mesoderm - acts to inhibit forelimb formation through restricting RA
RA in forelimb field –> Tbx transcription factors –> Fgf10 expression
Which members of the Tbx family determine limb identity? Why not fgf10?
Tbx4 results in leg
Tbx5 results in wing
Fgf10 is expressed in all wings
Fgf bead –> ectopic bead –> some expression of both fore and hindlimb (if it’s in the middle)
What is the evidence that the Apical Ectodermal Ridge (AER) is required for limb growth?
Limb stops developing if AER is removed, more distal structures will be missing.
Positive feedback loop maintains mitosis within limb bus (mesoderm signals to ectoderm with fgf10 and Wnt then ectoderm positively feedbacks to mesoderm using fgf8).
Extra AER induces duplicate limb structure.
Putting leg mesenchyme on wing bud gives leg mesenchyme –> identity of limbs is determined by underlying mesenchyme.
Putting non-limb mesenchyme, AER regresses and limb development ceases –> limb mesenchyme is specialised (has to be intended to develop a limb)
AER replaced by FGF bead means normal wing grows (essential function of AER is source of FGF)
How does the progress zone dictate proximal-distal specification?
Put young PZ on old limb buds –> duplicated radius and ulna
Put old PZ on young limb buds –> no radius or ulna, only digits develop (mesenchyme from the older organism would’ve only been making digits at their timepoint)
Therefore, mesenchyme and not AER confers proximal-distal identity
How is proximal-distal identity controlled by Hox gene expression?
Controls distal axis identity, same hox genes act in forelimb and hindlimb
Pattern in both forelimb and hindlimb = Hox9/10 in most proximal part (Stylopod), Hox11 in middle part (Zeugopod) and Hox12/13 in most distal part (Autopod)
How is proximal-distal identity controlled by opposing morphogen gradients?
2 opposing gradients:
- FGF/Wnt from apical ectodermal ridge specifies distal structures
- RA secreted from flank patterns more proximal structures
Antagonistic relationship on several levels:
- fgf8 and RA oppose each other directly (e.g. RA is direct transcriptional repressor of fgf8)
- fgf8 upregulates RA degradation enzyme cytochrome P450
- fgf8 and RA differentially regulate downstream gene expression
How is AP patterning controlled by the zone of polarising activity (ZPA)?
Transplanting from posterior part of one bud to the anterior part of another –> duplicated digits which were mirror image of each other.
Grafting experiments suggested AP axis specified by block of mesodermal tissue near posterior junction of young limb bud and body wall
Sonic hedgehog mediates polarising activity of ZPA – Shh expressed in posterior region of chick limb bud, precisely where ZPA activity resides
Experiments involving cells expressing Shh mimic ZPA activity
Digit identity controlled by exposure time to Shh (imposing pentadactyl constraint on the limb’s polydactyl potential)
Expression based temporal SHH gradient (pink gets high levels of Shh for a long time, thumb doesn’t)
Shh mutants only get one digit so some digits are shh-independent –> thumb
How is the dorsal-ventral axis controlled by Wnt7a?
Ectoderm dictates DV axis
Wnt7a expressed in dorsal ectoderm of chick and mouse embryos, induces dorsal fate.
Induces expression of TF Lmx1b (Lim1) throughout dorsal mesenchyme.
KO of either Wnt7a or Lmx1b in mouse causes ventralisation
Describe the stages of regeneration in salamanders (axolotl)
- Blood and immune cells flood the amputated area, and a blood clot forms
- Epidermal cells along the edge of the cut migrate over the wound to form wound epidermis (6-12 hours).
- Wound epidermis thickens into apical epidermal cap (AEC) via cell proliferation and migration.
- ECM breakdown and formation of a regeneration blastema containing mitotically ative blastemal cells (resembles limb bud, especially apical epidermal cap) - 5 days
- Outgrowth fuelled by continued proliferation and progressive differentiation of the blastema
Which cells give rise to blastema cells in salamanders?
Differentiated cells of bone, cartilage, fibroblasts, and myocytes – downregulation of genes expressed in differentiated tissues and upregulation of genes characteristic of proliferating cells (e.g. msx1 seen in proliferating progress zone mesenchyme of the embryonic limb)
Blastema cells look morphologically identical
Describe how muscle only gives rise to muscle in salamanders
Embryonic transplantation of genetically marked cells (e.g. somitic mesoderm – myoblasts migrate into limb)
Label found only in muscle –> muscle only gives rise to muscle not cartilage or epidermis
Describe how cartilage only gives rise to cartilage in salamanders
GFP positive limb, transplant to WT, see which cells are GFP positive
Cartilage dedifferentiated –> blastema –> differentiated into cartilage
Label found only in cartilage –> cartilage gives rise to cartilage and not muscle or epidermis
Describe how NS gives rise to NS (using tail as a model not limb) in salamanders.
Transplant SC from GFP axolotl into WT host, then amputate tail distally
Existing CNS is source of cells for the new CNS and not the source of mesodermal and other
tissues
Made cultures from SC giving rise to neurospheres (free-floating clusters of neural stem cells)
Transplantation of single cell in SC, amputation distal to transplant
Outgrowth shows expansion from this single cell
Fully regenerated tail has labelled SC derived from a single cell
GFP label found in all cell types within SC – multipotent NS stem cell
Cells in regenerating limb retain their tissue identity; blastema cells not multipotent; regeneration occurs by region de-differentiation and re-differentiation
Describe how AEC and innervation required for regeneration in salamanders.
AEC stimulates growth of blastema by secreting fgf8.
If limb is denervated before amputation, no regeneration of the limb. Neural conductance is not essential.
NM-ALM (neural modified accessory limb model) assay - decouples nerve source from host animal
Damage to limb nerves during birth of a human infant results in impaired limb growth and size which can be alleviated by surgical repair of the nerves
Describe how positional cues from the epidermis are required for regeneration in salamanders.
If wound is made in epidermis of intact proximal limb and a nerve is diverted to the wound area, an ectopic blastema-like bud will form but not fully regenerated limb – necessary and sufficient for induction
To induce complete limb also need an epidermal graft from the opposite side of the limb (posterior to anterior location) near the wound
Signals from nerves aren’t sufficient along for ectopic limb growth; also need positional cues from an epidermis that are different from the positional cues at the would site itself (positional discontinuities)
Only structures distal to amputation plane are regenerated
Limb blastema is autonomously specified to form limb structures distal to its site of origin = rule of distal transformation
Adult limb contains set of position al value along its proximal distal axis which were set up in embryo
Regenerating limb reads values at amputation site and regenerates positional values more distal to it
Cutting proximal blastema off and replacing it will distal blastema –> all proximal structures
mostly come from proximal tissue (host tissue)
In normal limb development, MEIS and HOX genes expressed in multiple limb progenitor cell types, including cells derived from somitic mesoderm
LPM-derived cells can form recognisable limb segments in absence of muscle formation and
thought they are a dominant cell type directing limb patterning
During regeneration, connective tissue-derived and muscle-derived blastema cells express MEIS, HOXA9 and HOXA13 as found for limb development, but Schwann cells didn’t
From grafting assays – connective tissue cells, but not myogenic cells, guide the proximo-distal outcome of regeneration
Describe the role of RA on the proximal distal axis
Application of RA proximalises (limb regenerates as if it had been amputated at more proximal site)
Dose dependent manner
RA activity gradient is seen along proximal-to-distal axis (highest proximal)
Affecting positional identity in blastema
Regulates expression of Meis TFs
And can influence positional information of cells by affecting cell-cell affinity
