---start--- 1. list and briefly describe 3 levels of organization involved in skeletal development/function 2. identify one abnormality for each level and state pathogenesis 3. why is it sometimes better not to make a protein at all than to make a partially functional one Osteochondrodysplasias the handout contains all the key figures and notes except the disease phenotypes. slide: rat skeletons there are a huge number of skeletal disorders that occur spontaneously, or are inherited. they share similar characteristiccs in all vertebrate species but are best characterized in humans (funding) but we can apply info to the other species. we know this b/c the data mostly comes from mice. ultimate goal - try to come up with therapies or preventatives for these diseases. before doing that we have to understand the biochemical bases and pathogenetic mechanisms responsible for the phenotypes we see. review of skeletal development (not in handout; should know from other classes): endochondral bone developement (all neck down bones except clavicle) mesenchymal cells migrate-->become chondrocytes which form cartilage model---> endochondral ossification occurs and forms bone and marrow intramembranous bone developement: skull, clavicle mesenchymal cells ---->bone, compact bone with no marrow endochondral ossification involves the hyaline cartilage model and at some point, central chondrocytes mature and hypertrophy; blood vessels invade, marrow cavity forms, and bone deposits on top of cartilage. starts in central area and radiates out, gradually replacing hypertrophic cartilage with bone and marrow, and creating growth plates which allow for longitudinal growth until maturity. intramembranous: mesenchymal cells change to bone cells, start to make matrix. cells around them start changing, doing same thing. happens in circumferenteral rings. shape and size changes occur via remodelling. so realize and appreciate, this is intricately orchestrated. there are multiple steps to go wrong, at each level. osteochondrodysplasias: developmental disorders of cartilage and bone in humans we know about 150+ of them very broad phenotypic spectrum of abnormalities from very mild (osteoarthritis) to very severe or lethal. genetic basis for most of the diseases unknown at this time. astonishingly, causes of many of them have been found in the past couple of years. primarily this is due to advances in transgenic technology. Dr. Richardson thinks the horse is the ideal model for studying osteoarthritis. She says mouse is good for most other disorders :) the reason is b/c they are small, gestation is short, they have big litters, there are many strains of mice, many inbred uniform strains, genetics are well defined in mice; most importantly transgenic technology was developed for mice (Dr. Brinster made one of the first ones in 1980) so, skeletal development involves a large sequence of intricate events. defects in any one step can cause disease. when you think of skeletal development, divide it into different levels. there are three. 1. positional information 2. proliferation and formation of skeletal part 3. function of skeletal cells level 1: global specification of skeletal pattern. occurs very early in embryonic life. no differentiation of cartilage or bone cells yet. shortly after gastrulation. notochord and a zone in limb bud called ZPA zone of polarizing activity make a morphogen, an inducing factor, which induces expression of many genes, which specify the pattern of the skeleton. someone got a nobel prize for finding thi morphogen. the nature of the morphogen has been hotly debated - thought it was retinoic acid but 3-4 yrs ago they found it was actually Sonic hedgehog. (SHH) (really). as soon as SHH is turned on, it activates a series of genes. some of the genes are homeobox genes - HOX and PAX in particular. homeobox genes - these encode for proteins that are transcription factors that bind DNA and activate transcription of downstream genes. these all share common structure. two domains - variable region (through which these molecules interact with neighboring molecules) and homeodomain or homeobox, which is extremely conserved. homeobox has HLH motif (helix/loop/helix) which binds DNA. through that binding, downstream genes are activated. the homeobox genes are expressed such that they subdivide embryo into morphogenetic fields. these genes subdivide body into regions that become different tissues, organs. the specification of positional information preceeds organ structure formation. cells respond to these genes by becoming an arm, or leg, or whatever. HOX and PAX genes - since these specify positional info, mutation probably results in something mising. homeotic mutations: transformation of one body part into another part classic example - described in fruit fly - antennopedia mutation. one mutation in a homeobox results in antennae being legs. that's a homeotic transformation. HOX genes: homeodomain containing genes that divide embryo along anterior to posterior axis. activated by SHH, directly specify positional fate of cells. in mammals, genes are in 4 clusters on 4 chromosomes. genes in each cluster are related. all are expressed in different but overlapping domains. arranged such that the order of expression along anterior to posterior axis is important. so the HOX genes are lined up in order...HOX1 to HOX13. all the HOX1 are the most 3' genes, expressed in most anterior part of body. genes closer to 5' are expressed in posterior part of body. so they are on the chromosome in the order of expression, anterior to posterior. in mammals there are 4 chromosomes, A, B,C, and D, all having similar arrangement of HOX genes. all HOX1s are similar to each other, not as similar to HOX2. get it? HOX1 on A is like HOXB2, C2, etc. not as similar to HOXA5. all the HOX1s have overlapping expression in head area, and so forth. mutations in these genes - first example involves HOX3 genes. these affect axial area. HOX3 mutations: cervical/cranial junction in mouse. bone i red, cartilage is blue. we see parts of skull and cervical vertebrae. in center is anterior arch. this top is normal. on right is abnormal. HOXD3 is knocked out. w see skull bones are different shaped. atlas is missing anterior arch. . atlas is incorporated into skull bone. the atlas is fused to skull - has become more anteriorized. axis is thicker than normal. very subtle here. this is what happens with one gene inactivated. if two genes are inactivated - here D3 AND A3 are knocked out. the A3 knockout has very subtle changes, as with D3 knockout. but when HOXa3 and d3 are knocked out, atlas is completely gone. so cluster of HOX3 genes are needed to specify formation of atlas bone. also, these genes have overlapping functions. if you remove one, other genes can compensate. if you remove two, you can't compensate. know this example: HOX11 genes - a11 and d11. these are expressed in radius and ulna. AADD is normal. aaDD (A knockout) have misshapen radius and ulna 0- slightly thick. AAdd have a slight fusion of radius and ulna - again very subtle. but aadd - no a11 or d11 present - complete absence of radius and ulna. so these genes specify positioning information required for formation of radius and ulna. inactivating one gene can be compensated for. losing more than one gene loses the whole pattern. it's better not to have a gene, than to have a whole partially functional gene. know this: mutation in HOXD13 gene: - syndactyly or polydactyly occurs. phenotypes: (note - all these people are heterozygotes having one normal and one mutant gene) -five fingers present, but 4th has two nails. the fourth digit is duplicated internally. -syndactyly - fusion of 3,4,5 with only one knuckle externally. xray shows carpal like bones - 7 of them - where metacarpals should be. -only three toes on foot - normal first metatarsal, second is small, and 3,4,and 5 are fused. so you get fused or extra digits. this mutation is insertion of polyA stretch in the variable region. mutation in variable domain causes this partially functional molecule. still has homeobox domain intact - can bind and activate downstream genes - but it can't appropriately interact with surrounding molecules to get normal cues. so you get altered pattern. you can use this to answer a question. if you completely inactivate the gene, other genes usually acan compensate. however, a partially fucntional molecule will cause abnormal function and inappropriate interaction with other things, producing weird phenotype. another example of another homeobox gene, not to write down: PAX genes: homeobox genes containing a pair-rule domain. they subdivide axial body along dorsal to ventral axis (instead of AP) also activated by SHH, specify fate of vertebral column. 9 genes identified. PAX1 and 9 identified in mice as spontaneous mutations: PAX1 is undulated tail, 9 is kinky tail. these are slightly curved, or twisted tails. note also they have hunched appearance - b/c vertebral column is abnormal. PAX1 mutants - vertebral column abnormal, patterning of ribs altered - some fold back dorsally. PAX9 mutants - subtle changes in vertebrae, dramatic changes in ribs, most have lost dorsoventral identity and are waving back and forth. PAX1 and 9 lost: no ribs at all form. so these genes specify positional information for formation of ribs. you could see scoliosis in a pet caused by a pax mutation. summary: level 1 SHH is inducible signal that activates homeobox genes the genes set up morphogenetic fields or gadients providing positional info mutations result in transformation of one part to another or absence of a part mechanism is inability to activate downstream genes required to initiate morphogenesis. example: mutationn of hox11 a and d which involve radius and ulna, also mutation of hoxd13 in humans in variable region. when you do not have a product made you can often compensate, but when abnormal molecule is made it will function in an inappropriate way. Level 2: local control of skeletal growth and shape. this happens when cells are already positioned, know if they will be bone or cartilage, and what they will be. but now they have to proliferate and form size and shape of the part. two examples of mutations - intramembranous - craniosynostosis - skull looks cloverlike due to premature fusion of sutures b/w plates. a number of genes can mutate to cause this - we'll discuss MSX2, a transcription factor containing a homeobox. it's expressed in sutures of skull. mechanism of this gene isn't known, but somehow it interacts w/surrounding tissues to mediate interactions b/w bone cells, brain, and sutures. as brain grows, skull has to expand to accomodate it. mutations in MSX2 cause premature fusion of calvariae at the sutures via inappropriate and accelerated cell division of calvarial osteoblasts. when the gene is mutated, the cell cycle of these cells is disrupted. looking at mouse skulls- mutant skull has no open sutures, it's all fused. histologically we can see that normal animals have thin bone plates with nonmineralized CT in between. in the mutant there are larger bone plates, overlapping eachother, with no gap in between. that's one mutation affecting the cell cycle. slide: baby with achondroplasia phenotype - most common form of dwarfism - disproportionate - head and trunk normal, limbs shortened. many dog breeds are mutants like this - dachshunds, corgis, basset hounds. they probably have mutations in these genes: fibroblast growth factors, FGFs. these are signalling molecules that bind a receptor and activate tyrosine kinase signal transduction cascade. this activates cell division. these FGFs regulate cell cycle, influence maturation fate. they are involved in all stages of skeletal development. mutations inn FGF or FGF receptors can alter chondrocyte proliferation or maturation, making really long or really short limbs. we've found mutations in the receptor that result in craniosynostosis, too. anyway. achondroplasia results from mutations in FGF3 receptor. rads: limb bones are deformed and thickened, b/c cell cycle of chondrocytes is altered. histology: instead of normal gradient of resting to hypertrophic chondrocytes above the growth plate, we have a disorganized mess - cell cycles are altered, all these cells are dividing abnormally, there's a big mass of tissue andit isn't properly replaced by bone and marrow. summary: local control of skeletal growth and shape involves local regulatoin of growth and differentiation via growth factors, transcription factors, and cytokines. mutations here result in disproportionate growth of specific componenets. inability to regulate cell proliferation is the pathogenetic mechanism. Level 3 - function and homeostasis of skeletal matrix cells are already differentiated and have formed the parts. now, to function correctly, cells must make matrix. slide: baby bat stained for red bone and blue cartilage. these stains are coloring the ECM, not cell. that's cool :) cartilage - cells surrounded by vast ECM bone - individual cells surrounded by even more matrix - hypocellular compared to cartilage. it's mineralized matrix b/c there is so much matrix, any defect in ECM components affect structure and function. if you consider the vertebrate- think of a scaffold of ECM molecules, with cells living in it, interacting with it. it not only provides support and structure, but instructional cues to the cells. two examples of mutations: 1. mechanical structure is altered 2. behavior is altered collagen - either fibrillar or non fibrillar type I (fibrillar) and type X (nonfibrillar) type I is most abundant. mutations affect structural stability. is major protein of ECM and is most predominant protein in body - in bone, teeth, tendon, blood vessels. is made of products from two genes. has alpha1 and alpha2 chains, which associate at carboxyl domain and fold into alpha-helical molecules. they do this b/c primary structure is such at every third AA is a glycine. so all glycine residues end up in central core,making stable and rigid molecules, which form extensive lateral associations forming the fibrils, which provide tensile strength and serve as a template. mutations in type I collagen cause osteogenesis imperfecta: -characterized by extreme brittleness of bone -other signs: poor or altered mineralization, osteoporosis, joint laxity, blood vessel rupture, blue sclera -huge variation in phenotype, depending on mutation - lethal to barely noticable cause: mutation in either type I collagen gene slide: kid with moderate OI. he only has onne point mutation. one glycine residue is substituted out. he's pretty deformed - barrel chest, limb deformities rads: bone in legs very deformed, with multiple fractures, poor mineralization. ribs are beaded - those are old fractures. also multiple existing fractures. another moderate OI case - woman in her 30s. tremendous scoliosis. limbs completely bent. incompletely mineralized. OI has been studied for several decades and to date there are over 450 mutations identified in alph1 or alpha2 type I collagen genes. you can predict phenotype based on site of mutation. any mutation outside the triple helical domain is less severe. most within the triple helix are lethal or very severe - usualy lethal. mechanism - dominant interference. - results from the expression of partially functional collagen chains that compete with normal chains for binding at the carboxyl domain. partially functional molecules will associate with normal ones, but won't trimerize correctly - resulting in all of them being degraded and having dramatic reduction in amount of collagen. or, if they aren't all degraded,they form mutant trimers that persist, and interfere with endogenous collagen function. they can't form proper fibrils so you lose the whole structure - huge loss in stability of bone. type X collagen - made only by hypertrophic chondrocytes at junction with bone. mutant type X collagen gene seems to be related to spondylometaphyseal dysplasia. this collagen forms networks, which are present in pericellular matrix of hypertrophic chondrocytes - area right around the cell is where you see this collagen. mutant mice look ok up to weaning. then they get perinatal lethal phenotype with hunching, kinking of neck, and waasting. this is very rapid. fine one day, sick the next day. can't survive over 24 hrs. this is in 25% of them. 75% of them get varying forms of dwarfism. may be 1/3 normal size or almost normal. relatively normal lifespan, but get nonhealing skin ulcers and very aggressive lymphosarcomas. the defect is a problem with endochondral ossification. you can see the defects in long bones the best - first, the growth plate is compressed in the hypertrophic chondrocyte zone - the only place where type X collagen is expressed. also, the transition to bone is altered- number and size of trabecular bone is very reduced. but shockingly, the bone marrow is also different. there is much less myeloid and more erythroid development. there is WBC precursor depletion. also depletion of entire hematopoietic compartment. so there is really a marrow aplasia or dysplasia. why? hypothesis: endochondral ossification may establish the marrow stromal microenvironment prerequisite for blood cell differentiation. experiment: study of LNs, spleens, thymuses. couldn't find LNs in these mice. thymus and spleen are small, very hypoplastic. spleens also discolored. that's due to the fact that the red pulp is missing. histologically - thymus - mutant has very little cortex. cortex should contain immature T cells from the bone marrow. this cortex is severely hypocellular. similar thing in spleen - mutants have very little red pulp, very small lymphatic nodules, B cells are reduced by about 50%. so marrow isn't really releasing B or T cells into circulation. human disease - 4 possible candidate diseases - within a week they found a mutation in type X collagen in a human disease. a mild form of dwarfism. they rae about 4 feet tall, with limb deformities. this is pretty mild compared to mice - b/c mutation is in a different domain in the molecule. in animals, they deleted central triple helical part and got dominant interference. in the people, the mutations are in the carboxyl domain which means the mutants can not bind and form trimers. so the dominant interference doesn't occur. heterozygotes have 50% less type X collagen. the mice have a persistent abnormal molecule and are more severely affected. Alaskan malamute dwarf - they have chondrodysplasia linked to hemolytic anemia, could be related. so we're trying to find out links b/w skeletal development and hematopoiesis. one lab study showed that altering a cell cycle of chondrocyte could cause chronic myelogenous leukemia... summary of level 3 - inovlves function and homeostasis of ECM. alterations in any ECM part alter structure and function of tissue zone and subsequent cell behavior - as seen with mutation in type I collagen altering mechanical propery, or type X mutation latering cell behavior (hematopoiesis) mechanism: production of abnormal molecule which either interferes with normal function or alters the environment. ---end---