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BIOLOGY 205/SECTION 7 DEVELOPMENT- LILJEGREN Lecture 7 Determination and Differentiation 1. DETERMINATION vs. DIFFERENTIATION vs. FATE vs. SPECIFICATION a. We’ve already talked about cells having a certain FATE, that is often specified or modulated by induction events. A level of commitment beyond cell fate is specification. A specified cell has been told what it should be. If it is experimentally isolated, it will follow those instructions; however, its fate can still be influenced by different environments. Beyond cell specification is determination. At this level of commitment, the cell cannot be made to change fates by a different environment, that cell is said to be DETERMINED. A cell is said to be determined if it has made an irreversible (usually) developmental choice among options. A more rigorous definition is that a cell is determined when it has undergone an internal and self-perpetuating change distinguishing itself & its descendents from other cells and committing to a specialized course. This concept differs from DIFFERENTIATION because that refers to the elaboration of the fate of a cell. Differentiation is overt cell specialization: a cell gaining new detectable differences. In other words, a cells knows what it will become (it is determined) before it actually becomes that type of cell (it differentiates). b. Another aspect of determination is that the fate decision becomes selfperpetuating, so that the subsequent progeny of the determined cell usually have the fate (sometimes in modified form) of that cell. 2. Evidence for determination during development. a. Transplant experiments (Spemann 1918): showed changes in cell determination 1. Experiment #1: If take an amphibian embryo at early gastrulation and transplant prospective epidermis (skin) where neural tube would normally form, the transplant tissue forms neural tube and brain. If you reverse the experiment, and put prospective neural plate where skin usually forms, it will form skin. THEREFORE AT THIS STAGE OF DEVELOPMENT BOTH FATES CAN BE SWITCHED BY CHANGING THE ENVIRONMENT. THE CELLS ARE NOT YET DETERMINED. 2. Experiment #2: The same experiment done during a later stage of gastrulation, gives a very different result. At this point prospective epidermis will make epidermis even if it is put where neural plate normally develops. Likewise the prospective neural plate will form neural plate at a epidermal location. THEREFORE AT THIS STAGE BOTH FATES CANNOT BE SWITCHED BY CHANGING ENVIRONMENT, AND THE CELLS ARE DETERMINED. However, when the experiment was done, the cells had not yet differentiated into epidermis or neural plate. b. The ultimate transplantation experiment: CLONING. • Somatic nuclear transfer: replace nucleus of unfertilized egg with that of differentiated somatic cell. • Most famous cloned animal is Dolly, but the cloning process was actually developed in frogs back in the 1950s • How does cloning work? • While occasionally this works at a low frequency so that an entire organism is regenerated, usually this experiment does not succeed! Furthermore, as development proceeds the ability of cells to alter fate progressively decreases. Remember that many clones are abnormal, and/or develop lots of problems. 3. Determination= heritable changes in gene expression. How does it occur? a. Ultimately changes must be heritable (on a cellular level), and must involve changes in gene expression because this regulates machinery available to a cell to carry out specialized tasks. b. With few exceptions, determination occurs as a result of selective gene EXPRESSION without changes in the DNA content or gene arrangement. Selective gene expression appears to result from a combination of (1) information in the cytoplasm of the cell that gets asmmetrically allocated to daughter cells, (2) basechange modifications of the DNA that influence expression of genes (ie. DNA methylation), (3) non base-change modification of the DNA (ie. post-translational modification of histone proteins) . c. The most impressive evidence for cytoplasmic contributions comes from cloning, which demonstrates that the cytoplasm of the zygote can reprogram the nucleus of the differentiated cell back to the starting point. 4. Global mechanisms of regulating gene expression. Heritable changes in the state of the DNA results in determination. a. Methylation. Highly methylated DNA is found in mammals (but NOT in many other animals). Certain enzymes can methylate cytosine, and the methylated DNA is usually not amenable to transcription into RNA. It is thought that the methylated DNA is packaged into larger, tighter chromatin structures that make the DNA inaccessible to RNA polymerase. Methylation is heritable--maintenance methylases methylate "daughter" DNA molecule in the same pattern as "parent" DNA molecule. b. “DNA Imprinting” of egg and sperm DNA is an example of how changes in DNA methylation lead to changes in gene expression. Causes an egg to develop with only the maternal or paternal copy of a gene being active, not both copies. Mechanisms—one parental copy becomes permanently inactivated by DNA methylation. More than 70 genes in mammals known to be imprinted! c. Examples of DNA imprinting that affect fetal growth. (1.) Insulin growth factor 2 (Igf2). Normally this protein stimulates rapid growth and is specifically expressed during fetal development. Only the paternal copy of the corresponding gene is transcribed. (2.) Insulin growth factor 2 receptor (Igf2r). This protein inhibits Igf2 activity and represses fetal growth. Only the maternal copy of the corresponding gene is transcribed. (3.) If Igf2 from dad knocked out (or both copies mutant), mice are born 40% smaller than normal (4.) If Igf2 copy from mom NOT imprinted, get fetal overgrowth. In humans, this is the cause of Beckwith-Weidermann syndrome. (5.) If Igf2r from mom knocked out, fetus is 30% larger than normal and dies late in gestation d. Chromatin structure. DNA in the nucleus is covered with proteins (“string on the beads”. 1. Chromatin=complex of DNA and histone proteins 2. Depending on their packing arrangement, these proteins repress expression of unwanted genes or maintain expression of needed genes. 3. Histone proteins have mechanisms to resume binding after DNA replication. 4. Post-translational modification of histone proteins (methylation, acetylation or phosphorylation) plays a critical role in regulating gene expression. Ie. lack of acetylation contributes to X-inactivation of one X chromosome. In figure, see chromosomes from a fibroblast cell stained with fluorescent antibody to acetylated histone H4, and only one chromosome (X) is not stained, indicating that it has low levels of acetylation. Correlation of acetylation with gene expression. 5. myoD: A master regulatory protein—or is it? a. Experiment#1- Reversing determination Take tissue culture cell line that has properties of fibroblast cells (connective tissue). Grow in azacytidine- this leads to demethylation of DNA Individual cells switch fates; they & descendents can form fat cells, muscle cells, bone cells etc. b. Experiment#2- What genes are demethylated to cause a switch in cell fate? Remember if treat cells with azacytidine it can lead to demethylation of DNA. So in this experiment, fat cells were treated with azacytidine, so they no longer have a fat cell fate. Instead a fraction of them can now go the muscle cell fate route. Myoblasts are committed muscle cell precursors, they are determined but not yet differentiated. Chromatin (DNA packaged in proteins) from these myoblast cells was isolated and cut into pieces, then the different pieces were introduced (transfected) into different untreated fat cells: a fraction of these cells were able to switch fate and become muscle cells. This indicates that those cells that were able to switch fate got a unique piece of chromatin that contained a gene essential for specifying muscle cell fate, which turned out to be myoD. ie. myoblasts contain active genes that control muscle cell fate. Ultimately a single gene was isolated that conferred this property= myoD Resulting theory- myoD is the master regulator of muscle development. If express myoD under the control of a viral promoter and introduce into fat cells, it can convert them to muscle cells. However, just because a molecule can do something in vitro doesn't mean it does the same thing in vivo (in the embryo)! c. Experiment #3- Is myoD expressed at appropriate time to regulate muscle? Use radioactive probes to examine when myoD is expressed in frog embryo. Result- yes, myoD is expressed during muscle differentiation. i.e. It's in right place at right time! d. Experiment #4- What does the myoD protein do? myoD bHLH (basic helix loop helix) protein resembles other "transcription factors" which turn genes on or off. Does myoD bind to the DNA of the right sort of genes? Measure ability of myoD protein to bind to muscle specific genes Result- most muscle specific genes examined have binding sites for myoD e. Experiment #5—Is myoD is the master regulator of muscle development? Eliminate myoD gene from mice- Do they make muscle? Result- oops! They are normal- Make new theory! New theory- myoD and related proteins- myf5, myogenin, etc. cooperate to regulate muscle development. f. Experiment #6—Are myoD and myf5 required for muscle development? Make mice lacking both myoD and myf5 bHLH transcription factors. Result- YES! animals now lack skeletal muscle! Proof of redundancy 6. Cellular memory and the engrailed gene in Drosophila. a. A cell fate choice in Drosophila- posterior vs. anterior in the segment b. c. d. e. 1) Remember, anterior cells make hairs & posterior cells do not, and wingless is one of the signaling molecules that tells cells to be posterior. 2) How is this choice regulated and remembered for rest of life? Early in Drosophila development, a set of transcription factors turn on the engrailed gene specifically in posterior cells. Then these transcription factors are turned off. How does engrailed remain on in some places and off in others? Mechanism #1- cell:cell signaling. 1) Neighboring cells produce and secrete Wingless signal. 2) Engrailed-expressing cells must receive Wingless signal in order to continue to express Engrailed. 3) How do we know this? In embryos lacking Wingless, engrailed is turned on correctly but then turns off. Mechanism #2- autoregulation 1) engrailed encodes transcription factor that binds to DNA near its own gene & keeps it on where it is already on=autoregulation. 2) How do we know this? In mutant embryos with only non-functional Engrailed protein engrailed gene expression comes on correctly and then goes off. Mechanism #3- A specialized set of chromatin-binding proteins 1) engrailed remains on in some cells, off in other for rest of life cycle ≥60 days. 2) If autoregulation explains how it stays on, what explains how it stays off? 3) Theory- several hours after gastrulation a special set of chromatin-binding proteins (Polycomb proteins) bind to the histone-wrapped engrailed gene in cells where it is turned off. These proteins are thought to help wrap up the DNA in a way that keeps it off forever. 4) How do we know this? Scientists removed gene for one of the Polycomb proteins in the adult fruit fly, which caused the engrailed gene to come back on in cells where it had never been on. 5) In contrast, other chromatin-binding proteins (Trithorax proteins) help keep genes on by modifying histone-wrapped genes (nucleosomes) to keep them accessible to transcription factors and RNA polymerase.