The Mouse as a Model for Mammalian Development

Transcription

1 BIOLOGY 205/SECTION 7 Lecture 10 DEVELOPMENT- LILJEGREN The Mouse as a Model for Mammalian Development Since we are mammals, our development is considered the system for which other organisms are "models". 1. Special difficulties of studying mammalian development. a. Development occurs inside mother; post-implantation development cannot be studied in vitro. can only observe embryos up to implantantation in a dish but not beyond. b. Need to form a placenta complicates many early morphological events. c. Life cycles are long (even mice take weeks vs. days for fruit flies and worms). d. Mice relatively expensive to maintain in the lab compared to flies or worms. e. Experimental manipulation of human development not possible due to ethical constraints. 2. A molecular/genetic approach to mammalian development. a. Mouse is the model for mammalian development. For a mammal, has a relatively short life cycle & is relatively small & easy to maintain. Numerous mutations isolated that alter morphology, development, or even behavior. Ie. in Asia where mice were kept as pets and bred for specific traits, mutants were found that twirled around in circles. b s: DNA cloning & sequencing developed. Genes encoding many abundant proteins were identified (but most encoded specialized proteins of differentiated cells, eg. hemoglobin, keratin, muscle actin, myosin, NOT key regulators of development). c. Last 20 years- return to a genetic approach. Forward genetics too hard and expensive in mice (screening for mutants would mean raising 10,000 s of mice 10s of millions of dollars). d. Alternative genetic approaches have paid off: i. Isolation of homologs of Drosophila developmental genes in mammals & realization that previously-cloned mouse genes are homologs of Drosophila genes ii. Isolation of genes encoding classical mouse mutations. (contributed by mouse breeders, spontaneously occuring mutations found in people s labs) iii. The ability to create mutations in cloned genes and reintroduce them into the mouse (gene knockouts) has revolutionized the study of development in mice. This is a reverse genetic approach, you start with a gene of interest and then examine loss-offunction mutants to determine its function. iv. Creating transgenic mice that express particular genes where they aren t normally expressed (ie. gain-of-function) also provides valuable information. 3. Creating "knockouts" (making mutations in cloned mouse genes) or gain-offunction mice. a. By far the most exciting recent advantage of the mouse is the ability to manipulate the DNA of the germline in a controlled way. This allows us to

2 add or subtract specific genes and study the effects on both development and disease mechanisms. How? b. For years, the only way to make mutations was hard way: Treat mice with mutagens- look at very large numbers of progeny for new mutants. This was very expensive given numbers of mice required. And there wasn t a way to add a gene in transgenic mice. However, in the late 1980 s mouse molecular biologists dreams came true. The realization of these dreams took two big steps: i. Find cells that you can grow in culture (in a dish) but that retain potential to become part of an embryo ii. Develop a method of altering a single gene in cultured cells. c. Solution to step #1: Embryonic stem cells. Scientists found can grow blastocyst cells (from inner cell mass) in culture and make mouse of them. Can transplant embryos at blastocyst stage into female mice by making mother think she s pregnant with hormones. This leads to production of chimeric mice embryos. Using this discovery we can add a gene and make transgenic mice. ES cells are TOTIPOTENT cells are able to generate all cell types. d. Solution to step #2: Gene targeting in ES cells. Mario Cappechi and Oliver Smithies (who s here at UNC; check out the Daily Tar Heel article about him posted on the supplemental information link for our class website) developed technology to alter ES cells so that one of the two gene copies has a piece of DNA inserted (interfering with gene function). These cells would be heterozygous. e. Here s a simplified version of the plan using BMP7 as an example of a gene to knockout in a mouse. BMP7 is a secreted ligand related to Vg1 (TGF-beta superfamily). 1. Make a version of gene with a selectable marker (usually a drug resistance gene) inserted in middle. This has 2 outcomes: i. it prevents the protein encoded by the gene from being made ii. it allows selection for this DNA by growing cells in the drug 2. Put modified gene into embryonic stem cell line & select for integration of selectable marker by homologous recombination. Now cells have one mutant copy of gene and one good copy. 3. Select for ES cells heterozygous for KO gene by screening with selectable marker (neomycin drug). 4. Inject ES cells heterozygous for KO gene into blastocyst-stage embryos from donor mouse. 5. Inject chimeric blastocysts into host mouse to generate chimeric mice. Some cells hopefully become part of embryo s germline. 6. Cross chimeric mice to wildtype mice to get mice heterozygous for mutation. 7. Cross heterozygous mice to get mice homozygous for mutation (1/4 of progeny)- hope for mutant phenotype. Total Time estimate- 15+ months, if it works, but if it does, fame and fortune (well at least a better shot at a job!) f. Exciting result: if you knockout the mouse BMP7 gene, you get mice without eyes and the kidney is severly atrophied. g. Nightmare scenario after all that work- no phenotype. Example: myod 4. Can use this technology to create disease models in mice- ie. cystic fibrosis. e. Cystic fibrosis is the most common lethal genetic disease in U.S: 30,000 patients in the US, 10 million symptomless (heterozygous) carriers

3 f. CF patients produce an abnormally thick, sticky mucus that clogs the lungs and leads to life-threatening lung infections. These thick secretions also obstruct the pancreas, preventing digestive enzymes from reaching the intestines to help break down and absorb food. With treatment patients usually live into their mid-30s. g. Defect- missing chloride ion channel encoded by CFTR gene. (Remember this protein is in the same transmembrane channel protein family as the White protein in flies that pumps pigment precursors.) By blocking transport of chloride, the mucus lining the lung dries up and becomes abnormally thick. h. Smithies lab pioneered studies of cystic fibrosis model by knocking out the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene in mice. i. Mice with CFTR mutations die soon after birth with defects similar to those in people. They can now design and test new therapies using these mutant mice. 5. Studying the function of mouse homologs of Drosophila developmental genes. The relative ease of studying development in Drosophila lead to the rapid identification of numerous genes involved in regulating development. Perhaps the greatest surprise that came with the ability to manipulate genes in mice was that many genes controlling development in Drosophila also regulate similar developmental processes in mammals. e. Example #1- The homeotic gene complexes. 1. We've seen how transcription factors of Drosophila homeotic gene complexes control fruit fly segment identity. Imagine the surprise of biologists when they learned that very similar gene complexes are present in humans and other mammals. How do we know that the Hox genes are the vertebrate homologs of fly homeotic genes? i. The mouse Hox genes were discovered using a homeobox DNA probe from fly. ii. In fly, genes organized so that latter genes in a cluster are expressed most posterior, and in mouse the same organization exists! In vertebrates, also see temporal regulation. Ie. latter genes in a cluster are expressed during later stages of development. iii. Once these genes were discovered, the mouse scientists wanted to know how they affected vertebrate development, so they made deletions of individual genes by gene targeting in ES cells. The first results were hard to interpret because they deleted genes expressed in the head region (like genes 1-4 in a group). It was clear that head was affected but HOW was confusing. One reason for confusion is that in some cases deleting one gene did not produce a dramatic phenotype! iv. But finally when they deleted Hox c8, a clear result was obtained. KO Mice made L1 (lumbar vertebrae 1, no rib) into more anterior T13 (thoracic vertebrae 13, which has a rib). They had made mice with an extra rib!! And just like in flies, where deleting Ubx makes T3 into T2, here a posterior structure was changed to one more anterior. Once they found other Anterior-Posterior landmarks in the vertebrae, they found other examples where deletion of a Hox gene caused a homeotic transformation. v. Analysis of Hox gene function in vertebrates more complicated than in flies since 4 clusters. Scientists realized

4 that each PARALOG GROUP (ie. Hoxa10, Hoxc10, and Hoxd10) has multiple members that often compensate for each other. Overlap was subsequently shown by the fact that deleting all members of a paralog group often gave a more severe phenotype than any single or double mutants. f. Example #2- The wingless/wnt-1 genes. 1. Recall that in flies, the wingless gene encodes the signal required to set up posterior (hairless) cell fate within each segment 2. Mammalian homolog=wnt-1. Identified as cause of mammary tumors in mice. 3. wnt-1 is normally expressed in mouse embryo in certain regions of developing nervous system. 4. The wnt-1 knockout made- mutant mice lack a large region of their mid and hind-brains. The part missing includes the region expressing wnt-1 and guess what- the adjacent posterior region that normally expresses the mouse homolog of engrailed! g. Example #3- Pax genes. 1. The Pax genes are a family of transcription factors important for early fruit fly development. We ve already briefly talked about the pax6 gene being important for fly eye development. Pax proteins share a common DNA-binding domain called the "paired box". 2. Pax genes were also found in mice, and map to chromosomal locations near mouse mutations affecting embryonic development. It is now clear that mutations in Pax genes cause these mutant phenotypes. Moreover, in 2 cases, human diseases are known that are similar to these mouse mutations. It has now been shown that each of these diseases result from mutations in human Pax genes i. Mutations in mouse Pax-3 are responsible for the classic mouse mutant Splotch, which has neural tube defects. Mutations in Human Pax-3 cause Waardenburg syndrome I- and also result in neural crest defects. (we ll talk about this disorder again at the end of the lecture) ii. Mutations in mouse Pax-6 gene are responsible for the classic small eye mutant. Mutations in Human Pax-6 are responsible for the disease called aniridia (no iris). When a fly homolog of Pax-6 cloned, it was found to encode the gene mutated in fly eyeless mutants. Amazingly, the same gene directs eye development in flies, mice, humans, and even squid! 6. Classical mouse genetics revisited- Steel and White-spotting. A pair of mutations found many years ago provide an interesting story about how different migrations are controlled during development. e. There is a dominant mutation called White spotting. Heterozygous mice have a big white spot on their belly. They are also slightly anemic and have reduced fertility. Humans are also sometimes piebald (term usually used to describe horses that have large white spots) and have lack of pigment in their midline. f. Homozygous mutant mice die during development. What is their problem? No blood cells, germ cells, or melanocytes (derived from neural crest cells) (homozygous mice are totally white). g. All of these cells undergo MIGRATION as part of their development. For example, blood cells migrate from the fetal liver to the bone marrow, and

5 melanocytes are a type of neural crest cell that migrate from the top of the neural tube down the body to all sites in the skin where pigmented cells are found. h. Around the same time, scientists found another mutation called Steel, because the mice had gray or steel-colored hair instead of black. The Steel heterozygotes also had some white hair. 1. Homozygous mutant embryos were also embryonic lethal (and white), and the same cells were not migrating as in White-spotting mutant mice! 2. The scientists predicted that ONE MUTATION WAS IN A SIGNAL AND ONE MUTATION WAS IN THE RECEPTOR FOR THAT SIGNAL. But which was which? 3. they did some mosaic experiments (similar to the Drosophila delta/notch experiment) in which they put blood cells (precursors) from Steel homozygous embryos into a wild-type host. The mutant blood cells migrated to the bone marrow. 4. they did the same experiment with blood cells from White-spotting homozygous embryos and found that these mutant blood cells did not migrate to the bone marrow. Therefore, they could not be rescued by the environment being wild-type. i. Based on these results, they concluded that White-spotting was expressed in these migrating blood cells, and was CELL-AUTONOMOUS, whereas Steel was not expressed in these cells but acted on them ie. was NON-CELL-AUTONOMOUS About 20 years later, the genes for White-spotting (also called White) and Steel were finally identified. j. Others had independently isolated a gene called c-kit, that if expressed at wrong time & place led directly to cancer. c-kit is a transmembrane receptor tyrosine kinase, and the gene encoding c-kit is the same gene mutated in White-spotting. k. Other scientists isolated an extracellular protein that acts as a "growth factor" This growth factor, which allows blood cells to grow in tissue culture turns out to be the protein mutated in Steel mutants. Therefore, the Steel mutation is in a protein called stem cell factor, which is the ligand or signal that binds to the c-kit receptor. Steel was found to be expressed in cells associated with the migratory pathways that cells expressing White-spotting travel. l. Human Waardenburg syndrome II-- results from mutation in MITF (microphthalmia transcription factor), a transcription factor at the end of White-spotting signal transduction pathway. m. Waardenburg syndrome and piebaldism are related disorders.