Carolines' Module

Carolines' Module

1. Planarian Experiments: This guy called T.H.Morgan experimented on planarians, a type of flatworm, and discovered the idea of MORPHOGEN GRADIENTS. Planarians regenerate quite often and successfully regenerate a new head and tail using the morphogen gradient theory. Neoblasts, undifferentiated stem cells, are used for regeneration. Different transcription factors are activated in the neoblasts, activation is dependent on the morphogen gradients, to ensure polarity is maintained so you get one head and one tail and not both of either one.

   • Lewis Wolpert's French Flag Model: This model is based on the French flag with the 3 sections; blue, white and red. Each section has a different threshold for the morphogen to respond to the morphogen. Genes found in the blue section have a high threshold so a large concentration of the morphogen (ligand) is needed for the blue section genes to respond and be expressed. Genes found in the red section have a low threshold so a low concentration of the morphogen (ligand) is needed for the red section genes to respond and be expressed. Genes in the white section have an average threshold to the morphogen.
     • The French flag model works but how is this morphogen gradient set up? This gradient can be set up in different ways but the French flag model holds true no matter what way the gradient was set up.
   • e.g. 1 of morphogen gradient: You can see the affect morphogen gradients have on planarian regeneration. When the Wnt pathway is turned OFF -> bi polar head. When Wnt pathway is turned ON -> bi polar tail. Wnt P-1 is wound responsive and later on found posteriorly. Wnt P-2 is only found in posterior facing wounds, so on the head segment. Another signalling factor, Notum, was only found on anterior facing wound, so on the tail segment. Research showed that Notum inhibits wnt but it also depends on wnt to produce it. As you can see from the diagram, Beta-catenin produces Notum so there is a Beta-catenin gradient in the planarian. Beta-catenin is high at the tail, so notum can be expressed and wnt inhibited. Beta-catenin is low at the head so no notum is expressed so wnt pathway is turned on. This balanced expression allows the formation of one head and one tail.
   • e.g. 2 of morphogen gradient: Morphogen gradients are vital for the proper positioning of motor neurons in the spinal cord. Motor neurons form ventrally and sensory neurons form dorsally. The neural tube comes from the neural plate which comes from the mesoderm. Notochord produces a floor plate to form. If there an extra notochord there will be an extra floor plate. Extra floor plates produce more floor plates. Notochord produce motor neurons by releasing Shh (sonic hedgehog). Shh is expressed in the notochord first then in the floor plate. This causes the motor neurons to move away. If the notochord and floor plate is removed then there are no motor neurons. If you remove the notochord but keep the floor plate then motor neurons are still seen because the floor plate releases Shh as well. How is that Shh produces motor neurons? Because Shh inhibits class I of transcription factors in the neural tube and expresses class II of transcription factors.
     • Class II transcription factors give rise to motor neurons.

2. ·

   • forelimb vs hindlimb. For forelimb formation Hox 4 and 5 are received which causes the expression of TBX5. TBX5 is a member of the T box gene family which act as transcription factors. For hindlimb formation Hoxc8, 9 or 10 are expressed which causes the expression of transcription factor PITX1 which goes on to act as a transcription factor for HLEA and HLEB which causes the expression of TBX4. Mutations in either of these transcription factors results in all sorts of developmental disorders. Ectopic injection of TBX can cause the limb to transform limb type.

3. Cell fate: This is a good analogy to describe how cell fate gets determined. If an embryonic stem cell is at the top of the hill, the way it rolls down the hill is determined by what transcription factors force the stem cell to roll a particular way. Embryonic stem cells have all the transcription factors turned on so can become anything but as the cell fate gets more restricted more transcription factors get turned off and on. This pattern of what transcription factors are turned on and off are inherited to the daughter cells which is called cell memory.

   • transdifferentiation can happen if there is an overexpression of transcription factors. You can induce transformation in the lab and these cells are called iPS cells.

4. iPS cells: iPS cells are similar to embryonic stem cells, they have the ability to become any kind of cells depending on what culture conditions you grow the cells in. They have so much potential, they can be used for regenerative therapy, helping restore function of mutated stem cells OR growing new organs eliminating the need for organ transplants. They are useful in research too, providing a constant source of cells to study in vitro and helping understand the disease process in particular genetic mutations. However there are cons to using iPS cells. If using the retroviral insertion method that makes it a permanent genetic modification (could use mRNA to encode OSKM instead). Another problem is myc is an oncogene so it could promote cancer. Another problem is that cells are cultured using 'xeno' media. Lastly using a patient's own cells that have mutations could be slow and costly to derive, a potential solution to this is to use banks of safe iPSC.

   • Ways to produce iPC

5. Limb Development: the pentadactyl limb - it's observed in so many different animals like humans, horse, whale, bird etc - and 3 basic components: [1] Stylopod [2] Zeugopod [3] Autopod. Limbs develop from limb buds which is basically loose mesoderm/mesenchymal cells wrapped in ectodermal epithelium. Limb buds start off as small bud.

   • AER [Apical Ectodermal Ridge] is the thickened part of the ectodermal epithelium and it acts like a cap for the limb bud. It releases FGF (fibroblast growth factor) which causes the mesoderm cells to grow so the limb develops. AER divides the dorsal and ventral side so it controls the growth of the limb along the proximal to distal axis so that means it grows outward. AER is only there when the limb is growing - so it's TRANSIENT and disappears after the limb has fully developed. If AER is removed too early, before the limb has developed then the limb will stop growing. If an extra AER is added, this process is called grafting, then an extra limb bud will form. AER signals FGF 8 specifically, that is the FGF required for limb growth. This is confirmed by the experiment where FGF8 was ectopically (meaning made outside of AER and in the lab, was injected into the limb bud and growth occured as normal.
     • FGF - fibroblast growth factor is in charge of cell growth. It's a ligand, once bound to its receptor causes signalling cascade and turns on the expression of the target genes. Target genes are the ones that aid in cell growth. In the limb development process many different types of FGF are involved. AER expresses FGF8 and FGF10 are found in the mesoderm/mesenchyme. There are many mutations found in the FGF receptor which results in all types of syndromes but all of these syndromes don't have proper limb development because of the mutation in the FGF receptor so the ligand, FGF, can't bind properly.
     • The AER forms where the dorsal ectoderm meets the ventral ectoderm. The dorsal and ventral ectoderm both express their own transcription factors to define the dorsal ectoderm and ventral ectoderm.
       • The dorsal ectoderm expresses Wnt7a which interacts with ZPA, maintaining the ZPA and AER interaction. Wnt7a also helps express Lmx1b.
       • The ventral side expressed En-1 which represses the effects of Wnt7a making sure the ventral side stays ventral and doesn't get changed to a dorsal side.
   • ZPA [Zone of Polarising Activity]: is the organiser of the developing limb which organises the growth along the anterior/posterior axis and organises the proper segmentation of the limbs. Anatomically, it's a part of the mesoderm/mesenchyme. If you add an ZPA in the limb bud it causes a mirror image of the limb to form. This suggests ZPA releases a morphogen in the same manner as the French flag model, this explains why there is a mirror image as graphically you would get a parabola forming. Shh is the morphogen here because ZPA is known to express it. This was also expressed by an experiment where ectopic Shh was produced by a retroviral infection and the same effect of the mirror image digits was seen.
     • Shh mutations. Mutations in Shh are lethal meaning that embryo won't develop and will die during pregnancy. However mutations in genes that act as transcription factors to Shh aren't lethal but impact limb development greatly.
       • e.g. 1 Acheiropodia: this developmental disorder results in a loss of distal limbs. Mutation comes from a single base pair deletion of LMBR1 which is a long range transcription factor of Shh, about 1kb upstream.
       • e.g. 2 Preaxial polydactyl (meaning a duplicated digit 1) is caused by a point mutation in ZRS, which is a long range control of Shh expression.
       • e.g. 3 when one digit forms. This comes from a mutation in MFCS1 which is a type of ZRS mentioned above. The mutation is specifically a deletion.
       • e.g. 4 Hemimelic toes in mice, meaning having extra toes, comes from a single base pair change in ZRS. Wildtype has a G but Hx mutants have an A.
     • Shh works on the French flag model, this is how digit segmentation works. Genes close to ZPA have a high threshold for Shh to be expressed and the further away mesoderm cells are from ZPA then they require a low amount of Shh for their target genes to be turned on. Time of exposure to Shh plays a role in digit segmentation as well as the French flag model.
       • Self organisation of digit in the autpod. As previously discussed Shh, expressed at the ZPA, is in charge of segmentation of the digits but this French flag model cannot explain why it segments into 5 digits specifically which is why the TURING REACTION DIFFUSION MECHANISM was thought of. This mechanism cannot explain why there is individual variation within the digit which is why together both models explain digit segmentation perfectly. In limb development Wnt and BMP are the activators and Sox9 is the inhibitor.
     • Gli3: is a key factor in autopod development. There is a disorder called Greig Cephalopolysyndactyly where the digits aren't segmented properly, this indicates it plays a role in Shh expression. Without Shh Gli 3 is in a repressor form so it represses Shh expression and dHand. When Shh is present, Gli3 doesn't go to its repressed form allowing the expression of Shh and dHand.
   • Progress Zone: This is the final part of the limb bud. This is where the mesoderm/mesenchymal cells grow. It's important the right mesdoderm cells are found in the progress zone. For example if you want a leg limb then you need leg mesoderm cells in the progress zone. If it's arm mesoderm in the progress zone instead then the limb won't develop. The mesoderm cells in the progress zone are diving and undifferentiated, they grow by receiving FGF signals from the AER. The progress zone also sends a signal called Gremlin which is a survival signal. This survival signal makes sure the AER stays while the limb is developing. Once the limb has finished developing, the progress zone will stop sending Gremlin so once AER stops receiving survival signal it will dissapear.
   • Some organisms don't have limbs and there is a genetic basis for this. With comparative genomics, scientists realized among species with limbs the genome is highly conserved. Snakes, who don't have limbs, have an area missing (the ZRS gene), specifically the Ets1 binding site in the ZRS gene.

6. Hox genes and the tetrapod. Hox genes are really important. They are a family of genes that are transcription factors. Hox genes dictate the order of the body so they are ones that say head is here, abdomen here and legs/tail here. Within the limb they also determine if it's a forelimb or hindlimb and organize the 3 sections of the limb. There are 4 clusters of the Hox gene, HOX A, HOX B, HOX C AND HOX D. Within each cluster there are 13 genes. All HOX genes are required as each gene is in charge of a particular part of the limb/body so if there is a loss of function of one of the hox genes then the body and/or limb won't form properly.

   • Hoxa11 mutation and pentadactyl. Pentadactlyl limbs arose from an event where Hoxa11 didn't get expressed as highly as it ususally does and expression of Hoxa13 got upregulated so only 5 digits formed.
   • In tetrapods (fish, birds, amphibians) there is a reverse expression of Hox genes allowing the formation of the autopod. The ancestral line had an early expression of HoxD genes but in tetrapods HoxD is expressed later in limb development allowing the formation of the autopod.

7. Mammalian Regeneration. A mice model that was meant for studying autoimmune diseases also had a high regenerative ability. They studied this MRL mouse and found that it had 20 changes in the genome on multiple chromosomes. Especially in p21! This meant that the cells stayed stuck in G1/S phase in the cell cycle allowing the cells to grow more.

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