Lecture summaries.

SES #	TOPICS	LECTURE SUMMARIES
1	Introduction
2	Blastema formation	Reading 1 Frogs can regenerate limbs within a window of time during embryonic development. Yokoyama and coworkers provide evidence that Wnt signaling, one of the main developmental signaling systems, is required for limb regeneration in developing frogs. To do this, the authors make use of transgenic lines in which a secreted inhibitor of Wnt signaling, Dkk1, is induced by heat-shock. Using this system, they propose that Wnt signaling is required for the initiation of regeneration but not subsequent steps. Are there limitations to this type of strategy for studying gene function? Reading 2 Zebrafish can regenerate several tissues, including the caudal fin. In a forward genetic screen, Whitehead et al. identify a mutant, dob, that fails to regenerate the tail fin, characterize this defect, and propose that mutations in fgf20 are responsible for the dob phenotype. What functional assays and line of reasoning do the authors use to argue that the dob mutant is specifically defective at initiating regeneration? The second part of the paper deals with cloning the gene responsible for the dob phenotype. Think back to your genetics class. What exactly is the evidence that dob is caused by a mutation in fgf20 ? Are you convinced? What other limitations are there to the forward genetic approach in the study of an adult trait?
3	Progenitor cells I, stem cells	Reading 1 What is the source of cellular material used to construction regenerating tissues? Planarian flatworms are capable of regenerating nearly any missing parts, and this activity is impaired by irradiation of animals, which causes depletion of a cell type termed neoblasts (as well as causing other effects as well). Neoblasts are small, contain relatively little cytoplasm, and subsequent work demonstrated that this cell population encompasses the dividing cells of the animal. In this classic paper, Baguna and coworkers perform experiments to determine whether transplantation of neoblasts is sufficient to restore regenerative abilities and homeostatic maintenance of tissues to irradiated planarians. How do the authors enrich for neoblasts? How do they label neoblasts—are there any concerns about this method? How can researchers provide confidence that measured differences in quantities reflect actual differences? Reading 2 What molecular processes regulate neoblasts? Reddien and coworkers present an analysis of the function of a gene, smedwi-2, which is expressed specifically within subpopulations of neoblasts. Inhibition of smedwi-2 by RNA interference results in regeneration and homeostatic tissue maintenance failure. These phenotypes that are almost identical in outward appearance to those caused by irradiation, which eliminates neoblasts. Does this mean that smedwi-2 is required for neoblast maintenance? Often in the study of developmental genetics, perturbation of a gene can produce multiple effects, and an inevitable concern is specificity of the phenotype. One way to solve this problem is to try to identify the first thing that goes wrong. These authors characterize the cellular defects caused by inhibition of smedwi-2 and find that the proximal defect is not a failure to maintain neoblasts, but rather a failure for them to differentiate or for their differentiated progeny to persist.
4	Progenitor cells II, dedifferentiation	Reading 1 Are stem cells ultimately the source of most new tissue in regeneration, or can differentiated cells be the source of new material?  In regeneration of the newt limb, evidence has been presented for both cases.  In this paper, the authors investigate the fate of individual, labeled differentiated myotubes, and conclude that myotubes are capable of giving rise to non-myotube cells during regeneration.  Myotubes are multinucleated and arise during development by fusion of numerous progenitors.  How did the authors label the myotubes?  How can the authors detect dedifferentiation? Reading 2 In mammals, muscle homeostasis is thought to involve stem cells called "satellite cells." These authors identify a cell within the newt limb that shares simlarity to mammalian satellite cells and argue that these cells can contribute to differentiated muscle. What enabled identification of newt satellite cells? What is the evidence that newt satellite cells function as stem cells within the animal? How can the results of this paper and the last one be reconciled?
5	Morphallaxis	Reading 1 Freshwater hydra can regenerate whole body parts from nearly any type of injury. Hydra are also observed to regenerate via "morphallaxis" or by large changes to the pre-existing tissues. A small fragment removed from the middle of a normal hydra regenerates all missing parts to become a fully-functional, but small regenerated hydra, and the original size can be obtained again slowly after feeding. Although cell division occurs during regeneration in hydra, these authors show that cell division is not necessary for hydra regeneration. Therefore, pre-existing tissues are capable of adopting new positional or functional fates independently of cell division in hydra. Reading 2 Planarians regenerate by making some entirely new tissues ("epimorphosis") and also by a complex and poorly understood process of rearranging and/or altering "pre-existing" tissues present immediately after amputation ("morphallaxis"). How can accomplish tissue remodeling when it cannot take in nutrients, as is the case during regeneration? Autophagy is a cellular process of cell cannibalization that can recycle resources during stress. These authors identify a gene involved in autophagy and find that inhibition of this gene by RNAi results in abnormally small blastemas and in some cases lysis during regeneration. The authors argue that autophagy plays an important role in tissue remodeling during planarian morphallaxis.
6	Positional information I, regeneration polarity	Reading 1 How do regenerating animals specify the identity of missing structures? The property of specification of structures at endpoints of the primary axis during regeneration is known as "regeneration polarity." Freshwater hydra are capable of regenerating nearly any missing part, and are organized along an oral-aboral axis, with tentacles protruding from the oral end. These authors argue that perturbation canonical Wnt signaling, a highly conserved signaling pathway, alters regeneration polarity in Hydra. Specifically, administration of a chemical inhibitor of the protein GSK-3beta causes formation of ectotopic tentacles. How do the authors conclude that the inhibitor acts on GSK3-beta in hydra? What do the transplantation assays demonstrate? Reading 2 Freshwater planarians are also capable of regenerating nearly any type of missing parts. They also have the property of regeneration polarity because animals can appropriately regenerate a head at anterior-facing wounds or a tail at posterior-facing wounds. These authors show that components of Wnt signaling regulate regeneration polarity in planarians. Inhibition of Smed-beta-catenin-1 by RNAi caused regeneration of a head rather than a tail at posterior-facing wounds. Conversely, inhibition of Smed-APC-1 caused regeneration of a tail rather than a head at anterior wounds. How can the authors conclude that Smed-beta-catenin-1 acts downstream of Smed-APC-1 in the regeneration polarity decision? Based on the phenotypes, where would beta-catenin activity be highest or lowest? Planarians diverged from Hydra more than 500 million years ago. What is the significance of a shared involvement of Wnt signaling pathways in control of regeneration polarity? What are alternative explanations?
7	Positional information II, limb regeneration	Reading 1 In different regenerating systems, positional information is needed to specify the identity of structures along an axis. In the newt limb, amputation of regions at any point along the proximo-distal axis result in regeneration to restore only what is missing. These authors develop an assay for proximo-distal blastema identity by transplantation of blastema from a given region onto the blastema-stump interface of another limb. Remarkably, during the course of regeneration, the donor blastema displaces to associate with the host blastema at the location on the P-D axis from which it was derived ("affinophoresis"). Retinoic acid is a substance used in cell signaling and is involved in a large number of developmental processes and ectopic retinoic acid treatment caused limb blastemas to adopt proximal fates in a previous publication. In this paper, the authors find that grafting retinoic acid-treated donor blastemas onto untreated host limbs caused a failure of the donor to displace from a proximal position. Additionally, grafting untreated donor blastemas onto retinoic acid-treated host limbs caused the donor to displace only to very proximal regions. Therefore, retinoic acid proximalizes both blastema identity and host-graft differential displacement. Are there mechanisms that could account for this observation other than cell-cell affinity? What are some of the predictions of the authors' model of differential affinity, and how could they be tested? What is the evidence that retinoic acid controls proximo-distal identity in vivo? Reading 2 How is proximo-distal identity controlled during regeneration? These authors identify a gene, Prod1/CD59 that is transcriptionally induced by retinoic acid treatment. In tissue culture, proximal tissue explants engulf distal tissue explants, and administration of neutralizing Prod1/CD59 antibodies impairs this process. How did they accomplish their screen? Of the genes that were up or down regulated, how did they choose Prod1/CD59 to study? Where is Prod1 expressed? How do the authors argue that treatment with Prod1/CD59 antibodies specifically inhibits the activity of Prod1/CD59?
8	Positional information III, intercalary regeneration	Reading 1 Recall that intercalary regeneration is the growth of intervening tissues which occurs after surgically juxtaposing two tissues with different positional identities. As we mentioned in class the last few weeks, pattern formation can be understood as a distinction "between the events by which cells are assigned positional values according to their physical locations...and the subsequent responses of the cells." In this paper, French et al. synthesize a large number of experimental observations regarding the outcome of intercalary regeneration in newt and cockroach limbs and drosophila imaginal discs and put forth a comprehensive model to explain this data. This rather simple "polar coordinate" or "clockface" model of intercalary regeneration offers an explanation for a strikingly wide variety of seemingly unconnected observations made on patterning in intercalary regeneration. This is a very long paper, so first read for understanding the model as it applies to the three case studies, and then as your time allows consider the evidence the authors give for the model. As time allows in class, we will discuss these two aspects of the paper and try to consider whether there are other models that could explain the data. Reading 2 Drosophila imaginal discs are ~2D disc-like structures in the larva that grow outward into appendages during metamorphosis. Classical experiments show that in general, surgical fragmentation of an imaginal disc leads reproducibly to one part regenerating the missing tissues while the other part duplicates its own structures rather than filling in missing ones. In this paper, Gibson and Schubiger show that the secreted signaling protein hedgehog is required for anterior to posterior repatterning that occurs during intercalary regeneration in imaginal discs. Specifically, they show that in hedgehog loss-of-function mutants, a leg imaginal disc regenerates rather than undergoing its normal duplication after wounding.
9	Organ regeneration	Reading 1 Zebrafish heart tissue can regenerate following injury. In this paper, Lepilina et al. show that heart regeneration proceeds by formation of a blastema in which cardiac progenitor cells differentiate to become new cardiac muscle cells, followed by expansion and migration of an epicardial (cells surrounding the heart) population of cells to cover the wound. Some of these epicardial cells undergo an epithelial-mesenchymal transition and contribute to vasculature during regeneration, a process shown to be dependent on FGF signaling. Reading 2 Liver regeneration proceeds by proliferation of hepatocytes until the original size of the liver is restored. Therefore, injury somehow induces proliferation in what remains of the existing liver. In this classic paper, the authors test the hypothesis that proliferation is induced by humoral agents in the blood. To do this, they fuse the circulatory system of uninjured and injured rats and observe an increase in DNA synthesis in the liver of the uninjured rats.
10	Adult stem cells	Reading 1 Classic experiments demonstrated the existence of hematopoietic stem cells (HSCs) which can self-renew, and also differentiate to become all types of cells in the blood (this is why a bone marrow transplantation works in human patients). But can HSCs differentiate into non-blood types of cells, and do they do this in a normal animal? This paper was one of many papers published in 1999-2001 providing evidence that HSCs can differentiate into non-blood cell types. Reading 2 This paper refutes the previous one by providing evidence that HSCs do not populate non-blood cell lineages. Think about the evidence in each paper. Based on the evidence, which model is correct? Is there a way the data can be reconciled into one model? What other experiments could be done to provide support for either model?
11	Homeostasis I, progenitor cells	Reading 1 The pancreas maintains itself throughout adulthood. What is the source of new pancreatic beta cells: undifferentiated stem cells or some other kind of differentiated cell? The authors employ a system for inducible, permanent labeling of pancreatic beta cells and any possible progeny and show that pre-existing pancreatic beta cells, rather than undifferentiated stem cells, are the major source of new pancreatic beta cells in adult mice. Reading 2 Many tissues must undergo self-renewal in order to maintain themselves throughout adulthood. These authors demonstrate that like the mammalian gut, the fly gut undergoes self-renewal during adulthood. The authors identify a putative intestinal stem cell population based on incorporation of BrdU, cell size and expression of markers, and then use a genetic labeling strategy to show that these cells are indeed the progenitors of differentiated cells in the gut. Furthermore, they show that the differentiation of the stem cells may be regulated by the Notch signaling pathway.
12	Homeostasis II, stem cell self-renewal and stem cell niche	Reading 1 Stem cells must be capable of both self-renewal and differentiation. Molofsky et al. find that the polycomb transcriptional repressor Bmi-1 is required for self-renewal but not differentiation of neuronal stem cells in the mouse. Polycomb group proteins alter chromatin structure to prevent the association of transcription factors with DNA, and are known to regulate key developmental regulators in flies and mammals, such as Hox genes. Molofsky et al. provide evidence that one of Bmi-1's targets important for maintaining self-renewal is the cell cycle inhibitor p16Ink4A (which inhibits cyclinD/cdk4 complexes to arrest cells in G1). Reading 2 It has been observed in many systems that the stem cells of a tissue divide very slowly. Therefore, one method of identifying stem cells is to pulse label with BrdU or some other labeled nucleotide, and then chase for a long period of time. In this type of experiment, slowly dividing cells are seen as "label-retaining cells." In addition, the self-renewing capability of some types of stem cells requires cell-cell interactions with specialized differentiated cells around them, termed the stem cell "niche" or "microenvironment". In this paper, Tumbar et al. use a modified lineage tracing strategy to identify a stem cell niche within the mouse skin and show that these cells likely rapidly divide after leaving the niche and then contribute to differentiated skin in homeostasis and regeneration.
13	Regenerative medicine	Reading 1 Embryonic stem cells in mammals can grow indefinitely in culture and also differentiate into any type of tissue. The ability to generate pluripotent stem cells from differentiated cells would have numerous medical implications, including the creation of patient-specific stem cells, or the ability to study any tissue from disease patients. It was known that transfer of the nucleus of a differentiated cell into an enucleated oocyte could cause the differentiated nucleus to dedifferentiate to an embryonic stem cell-like state, but at a low efficiency, and the molecules involved in this process were unknown. In this breakthrough paper, Takahashi and Yamanaka identify four factors as sufficient for inducing fibroblast cells to acquire embryonic stem cell-like properties in the mouse (this type of strategy was subsequently performed in human cells as well). How did the authors perform their screen? What characteristics of the screen seemed to be critical in order to use it to identify the four factors? How did the authors assay for pluripotency? What kinds of applications can you imagine that this technology would allow for medicine or research? Reading 2 Individuals with Marfan's syndrome exhibit muscle deterioration and an inability to produce muscle mass, and this disorder is caused by a deficiency in the extracellular matrix protein fibrillin-1. Although fibrillin-1 deficiency was originally believed to result in a structural defect, some reports had shown that TGF-beta signaling was overactivated in some Marfan's syndrome patients. These authors show that inhibition of TGF-beta signaling retores muscle structure and repair using a mouse model of Marfan's syndrome in which fibrillin-1 is genetically inactivated. Thus, inhibition of TGF-beta signaling might be useful as a therapy for patients with Marfan's syndrome.
14	Oral presentations	Each student will give an oral presentation about an article selected from the List of Papers on the assignments page.