Tutorial Quiz 2 Slides PDF

Tutorial 5: The Discovery of hedgehog and Its Homologs  Morphology of Drosophila larva segments The Drosophila embryo forms repeated morphological units called segments. Each abdominal segment on the ventral side of the larva is characterized by anterior denticles (which point post...

Tutorial 5: The Discovery of hedgehog and Its Homologs  Morphology of Drosophila larva segments The Drosophila embryo forms repeated morphological units called segments. Each abdominal segment on the ventral side of the larva is characterized by anterior denticles (which point posteriorly) and a posterior naked region. Denticles (anterior) Naked (posterior) Denticles A4  The Flies In 1980, Christiane Nüsslein-Volhard and Eric Wieschaus screened thousands of embryos for patterning defects! They set out to identify all of the genes involved in the segmentation process. Everyone else was looking at adult flies! Drosophila embryogenesis  Methodology: Forward Genetics Identifying genes or genotypes that are responsible for a particular phenotype. Adult male flies are treated with a mutagen to generate independent mutations in their gametes. Mutagenized males are crossed to wild-type females to produce many heterozygotes with independent mutations. Breed independent heterozygotes to wild-type flies to produce more heterozygotes with the same mutation. Breed these heterozygotes together to produce homozygotes and screen mutants for abnormal phenotypes. P0 F1 crossed to WT Mutagen = +/+ +/+ +/- +/+ Ethyl methanesulfonate (EMS) F2 = +/- +/- Heterozygous lines +/- +/- F1 = +/- 1000‘s with independent +/- F3 = +/- +/+ +/- mutations +/- ¼ Homozygous mutants +/- +/- -/- If mutant is lethal, examine the larvae  Results of the Forward Genetic Screen Characteristics of hedgehog mutant: Denticle Same number of bands segments Denticle Naked region in each “lawn” segment deleted Denticles duplicated in a mirror image hedgehog is classified as a ‘segment polarity gene’. http://upload.wikimedia.org/wikipedia/en/d/d1/Denticlebands.png WILD TYPE MUTANT Anterior Anterior Posterior Posterior  The Year is 1992 Drosophila hedgehog (hh) was cloned by four independent groups. The sequenced gene could now be used to make a probe to analyze gene expression.  Now the Year is 1993 3 papers were published simultaneously on hedgehog homologs in vertebrates. Andrew McMahon Cliff Tabin Phil Ingham  Homologs, Orthologs, and Paralogs, Oh My! Genes A and B are orthologs. Genes X1 and X2 are paralogs. e.g. Shh in mouse and chicken e.g. Shh, Dhh and Ihh in mouse Genes X1 and X2 are homologs. Genes X1A and X2A are homologs. Genes X2A and X2B are homologs.  Discovery of hedgehog in Mice The first vertebrate hedgehog gene to be found was Desert hedgehog in mice. The other mammalian hedgehog genes identified were Indian hedgehog and Sonic hedgehog. How was this done? Desert hedgehog At this time it had become clear that genes that played important developmental roles in an organism like Drosophila would likely have vertebrate counterparts. Since any hedgehog homologs in vertebrates would share sequence homology with the Sonic hedgehog Drosophila hedgehog gene, the sequence of hedgehog was used to make a probe that could hybridize to unknown vertebrate homologs. Indian hedgehog  Making a Genomic Library A genomic library is a DNA library containing an organism’s complete genome, in the form of small DNA fragments (oligonucleotides). These DNA fragments are inserted into carrier molecules called vectors (such as a viral DNA molecule or plasmid), and packaged into many viral particles. Together, these viral particles contain the entire genome of the organism. Package into phage (in vitro) From Molecular Cell Biology , 2000  Cross-Hybridization Prepared radio- Prepared 1 million labelled probe of phage plaques 700 bp fragment representing of Drosophila hh entire mouse gene genome High stringency Low stringency Low stringency Under low temperature and high salt conditions, cross- Stringent hybridization hybridization can occur with imperfect DNA matching. Why is this important? From M. Alberts et al. Molecular Biology of the Cell, 2002; Figure 8-25.  Screening a Genomic Phage Library DNA cross- hybridization to a labelled probe The fragment can then be sequenced. From Molecular Cell Biology , 2000  Results When they screened the mouse genomic DNA library, the Drosophila hedgehog gene probe hybridized to the Desert hedgehog gene. They were later able to detect two more genes, Indian hedgehog and Sonic hedgehog, using a new probe (based on the sequence of chicken Sonic hedgehog). Amino Acid Sequence Conservation Mouse Dhh Mouse Ihh Chicken Shh Zebrafish Shh Drosophila hh Mouse Shh 61% 63% 84% 68% 48% Mouse Dhh 58% 61% 54% 51% Mouse Ihh 64% 61% 48% Chicken Shh 68% 49% Zebrafish Shh 47%  The Year is Still 1993 Andrew McMahon Cliff Tabin Phil Ingham  Riddle et al. 1993 Study Aim: Look for a hedgehog homologue that may be the ZPA morphogen. Prior Information: Drosophila hedgehog and mouse Desert hedgehog sequences. Highly conserved region Compared the mouse Desert hedgehog sequence and the Drosophila hedgehog sequence to find a conserved region. Found the best conservation between amino acids 161-237 of each protein sequence.  Degenerate PCR To amplify genes from different organisms – these genes are similar but not identical. Primer design is based on amino acid (protein) sequence instead of nucleotide sequence. Use conserved sequence between mouse Dhh and Drosophila hh to probe for homologs in chicken. Goal: Obtain a specific chicken DNA sequence from a (probable) protein sequence. PCR Review: https://www.youtube.com/watch?v=lsSPtYHGl6M  Degenerate PCR design 1. Align two amino acid sequences and look for well-conserved regions. 2. Include 6 – 7 conserved amino acids (equating to ~18-21 base pairs) in the design for forward and reverse primers. Design multiple primers in a “cocktail” to account for every possible nucleotide sequence that could code for those 6 – 7 amino acids. 3. Consider only those amino acid sequences with low degeneracy. Good to include methionine and tryptophan, which are coded only by a single codon (three- letter nucleotide code). Avoid leucine, serine, and arginine, which can each be coded by six different codons. 4. For amino acids coded by 3 to 4 codons, consider using the base inosine (structurally similar to guanine), as it can pair with any of the four bases.  Degenerate PCR Schematic Primer design - Read amino acid: 6 possibilities 1 possibility Create degenerate primer mixture. TG(T/C)ATITA(T/C)ATGCA(T/C)CA(A/G)GA(T/C) ** Where would be a good place to use inosine? ** Why would we want to use it sparingly? From Molecular Cell Biology, Lodish et al.  Degenerate PCR Primer “Cocktail” TG(T/C)ATITA(T/C)ATGCA(T/C)CA(A/G)GA(T/C) = 32 Unique Individual Primers 1. TGTATITATATGCATCAAGAT 17. TGCATITATATGCATCAAGAT 2. TGTATITATATGCATCAAGAC 18. TGCATITATATGCATCAAGAC 3. TGTATITATATGCATCAGGAT 19. TGCATITATATGCATCAGGAT 4. TGTATITATATGCATCAGGAC 20. TGCATITATATGCATCAGGAC 5. TGTATITATATGCACCAAGAT 21. TGCATITATATGCACCAAGAT 6. TGTATITATATGCACCAAGAC 22. TGCATITATATGCACCAAGAC 7. TGTATITATATGCACCAGGAT 23. TGCATITATATGCACCAGGAT 8. TGTATITATATGCACCAGGAC 24. TGCATITATATGCACCAGGAC 9. TGTATITACATGCATCAAGAT 25. TGCATITACATGCATCAAGAT 10. TGTATITACATGCATCAAGAC 26. TGCATITACATGCATCAAGAC 11. TGTATITACATGCATCAGGAT 27. TGCATITACATGCATCAGGAT 12. TGTATITACATGCATCAGGAC 28. TGCATITACATGCATCAGGAC 13. TGTATITACATGCACCAAGAT 29. TGCATITACATGCACCAAGAT 14. TGTATITACATGCACCAAGAC 30. TGCATITACATGCACCAAGAC 15. TGTATITACATGCACCAGGAT 31. TGCATITACATGCACCAGGAT 16. TGTATITACATGCACCAGGAC 32. TGCATITACATGCACCAGGAC  cDNA Library Degenerate PCR used chicken genomic DNA as a template to amplify a 220bp sequence. This sequence was then used as a probe to screen a chicken limb cDNA library in order to find a homologue expressed in the developing limb bud. Cloning with Bacteriophage λ Using Chicken cDNA from the Limb Bacteriophage λ DNA Phage plaques Chicken limb cDNA Transfer to filter Phage DNA absorbed on filter. Add radio-labeled probe (220 bp from degenerate PCR) Recombinant phage with Hybridization occurred chicken cDNA from limb. between the probe, and a fragment containing the Sonic hedgehog gene. From Molecular Cell Biology , 2000  Timeline of Experiments Drosophila hedgehog discovery using a forward genetic screen (Nüsslein-Volhard and Wieschaus). Mouse Dhh discovery using a mouse gDNA library screen (Echelard, and Epstein, and McMahon). Chicken Shh discovery using degenerate PCR and a chick limb cDNA library screen (Riddle and Tabin). Identification of other vertebrate hedgehog homologs using chicken Shh sequence (mouse Shh, mouse Ihh, chicken Dhh, chicken Ihh, and zebrafish homologs). Degenerate PCR Primer Design Practice Question Tutorial 6: The discovery of chick Shh  The year is 1993 Andrew McMahon Cliff Tabin Phil Ingham  Tabin’s search for the ZPA Morphogen. The zone of polarizing activity (ZPA) had been studied since the 1960’s, since it acts as a signalling centre and is the source of a morphogen responsible for patterning the limb. Until this point, the identity of the ZPA morphogen was unknown. Drosophila hedgehog is a secreted morphogen with a patterning function in the body segments of Drosophila (it’s a segment polarity gene). Is there a hedgehog homolog in chicks that could be the ZPA morphogen?  Morphogen “Protein (or chemical) that acts in a concentration-dependent manner to instruct different cell fates across the developing segment.” Transplanted Anterior ZPA III ZPA ZPA Endogenous Posterior ZPA Posterior Anterior Grafting the ZPA from the one limb bud to the anterior region of another limb results in a mirror image duplication of the digits. Quote from S. DiNardo, from Riddle et al., 1993 paper http://www.nature.com/nrm/journal/v7/n1/fig_tab/nrm1830_ft.html  Riddle et al. 1993 Study Aim: Look for a hedgehog homologue that may be the ZPA morphogen. Prior Information: Drosophila hedgehog and mouse Desert hedgehog sequences. Highly conserved region Compared the mouse Desert hedgehog sequence and the Drosophila hedgehog sequence to find a conserved region. Found the best conservation between amino acids 161-237 of each protein sequence.  Degenerate PCR design 1. Align two amino acid sequences and look for well-conserved regions. 2. Include 6 – 7 conserved amino acids (equating to ~18-21 base pairs) in the design for forward and reverse primers. Design multiple primers in a “cocktail” to account for every possible nucleotide sequence that could code for those 6 – 7 amino acids. 3. Consider only those amino acid sequences with low degeneracy. Good to include methionine and tryptophan, which are coded only by a single codon (three- letter nucleotide code). Avoid leucine, serine, and arginine, which can each be coded by six different codons. 4. For amino acids coded by 3 to 4 codons, consider using the base inosine (structurally similar to guanine), as it can pair with any of the four bases.  Degenerate PCR Schematic Primer design - Read amino acid: 6 possibilities 1 possibility Create degenerate primer mixture. TG(T/C)ATITA(T/C)ATGCA(T/C)CA(A/G)GA(T/C) From Molecular Cell Biology, Lodish et al. Cloning with Bacteriophage λ Using Chicken cDNA from the Limb Bacteriophage λ DNA Phage plaques Chicken limb cDNA Transfer to filter Phage DNA absorbed on filter. Add radio-labeled probe (220 bp from degenerate PCR) Recombinant phage with Hybridization occurred chicken cDNA from limb. between the probe, and a fragment containing the Sonic hedgehog gene. From Molecular Cell Biology , 2000  Sonic hedgehog WISH - Where was Shh Expressed? The sequence of chick Shh was now known, cloned, and used to make a WISH probe. Shh was expressed in the posterior of the limb bud, in the zone of polarizing activity (ZPA). Chicken Shh is homologous to Drosophila hh, and the amino acid sequences of the proteins are highly conserved. Because homologs have conserved DNA sequences, they often have similar functions as proteins. A) Close-up view of a stage 20-21 left wing bud (WB). Sonic hedgehog message is found in the These can all give a clue to function, but a LOF or posterior/proximal region of the limb bud (arrow). GOF study must be performed.  Gain-of-Function Study Riddle et al. 1993 used a gain-of-function study to determine whether Sonic hedgehog was responsible for the function of the ZPA. Involves expressing a gene in a location where it is not normally expressed. Resultant morphology provides insight into the role/function of the gene. Methodology Ectopically express Shh in the anterior of the limb bud of chick embryos. Endogenous expression: The normally-occurring expression within a tissue/cell. vs. Ectopic expression: The expression of a gene where it is not normally found. Used a replication competent retroviral vector (RCAS-E).  Replication Competent Retroviral Vector (RCAS-E) Avian retrovirus (RNA virus). Retroviral RCAS-E vector encoding chicken Shh. containing Shh RNA Shh mRNA Shh mRNA Shh cDNA Shh cDNA Constitutive promoter SHH protein Line 15b chicken Figure modified from : embryonic fibroblast (CEF) http://journals.cambridge.org/fulltext_content/E RM/ERM6_13/S1462399404007884sup008.htm Gain-of-Function: Method (Grafting) Insert Shh into RCAS-E vector & infect a specific line (15b) of chick embryo fibroblast (CEF) cells. Infected cells are collected and implanted into the anterior limb bud of stage 19-23 chick embryos. The virus is unable to infect cells of the embryo into which the pellet is grafted. Why is this important for this experiment? Cell migration following grafting was initially tracked using cells expressing alkaline phosphatase (AP). Why would you want to track cell migration? Grafted RCAS-E/AP CEF Cells are Restricted to the Anterior of the Limb Bud The fate of the grafted cells was determined using RCAS-E/alkaline phosphatase grafts (control). Alkaline phosphatase cells are distributed only at the anterior limb (at the origin of the graft). Grafted RCAS-E/AP expressing cells have wild-type digits (II-III-IV). Gain-of-Function: Results Grafts of Shh-expressing CEFs induce mirror image duplications in the limb digit pattern. Same phenotype as ZPA grafts! Extent of duplication depends on the amount and position of the engrafted cells. Grafting other Shh-expressing tissues to the anterior limb bud can also induce digit duplications.  Gain-of-Function: Conclusions SHH is sufficient to polarize the limb bud and induce mirror image digit duplications. Grafts of other Shh-expressing tissues can also induce mirror image digit duplications. SHH-induced mirror image digit duplications are similar to ZPA-induced digit duplications. Shh controls anterior-posterior limb patterning and is the ZPA morphogen. ZPA Graft  Summary of chicken Shh discovery and Functional characterization Drosophila hedgehog discovery using a forward genetic screen (Nüsslein-Volhard and Wieschaus). Chicken Shh discovery using degenerate PCR and a chick limb cDNA library screen (Riddle and Tabin). Chicken Shh functional characterization using a gain of function graft experiment knockout (Riddle and Tabin). Identification of human homolog and link to human disease… STAY TUNED Tutorial 7: Discovery of Human Shh and more Shh phenotypes  Cloning the Human SHH Gene The human SHH cDNA was cloned by degenerate PCR from human genomic DNA in 1995. * used the conserved region of mouse DHH and Drosophila Hh! The PCR product was used to clone the full-length cDNA. They mapped SHH to a region of chromosome 7 by performing PCR on cell lines that contained only portions of human chromosomes (kind of like screening a library!).  Conservation of SHH Homologs High conservation of mouse, chicken and zebrafish SHH proteins, especially the N-terminal portion. - 99% mouse and chicken. - 94% mouse and zebrafish. Function (embryonic patterning) of the proteins is also conserved during development. Probable that the human homolog would have a comparable function. - 99% (N-terminal) amino acid identity Marigo et al., 1995 between mouse and human.  Holoprosencephaly (HPE) Failure of the forebrain to separate into 2 hemispheres. “Holo” = whole “Pro” = front “Encephaly” = disorder of the brain Incidence: 1:16,000 live born; 1:250 embryos. Homozygotes are usually stillborn (lethal). Heterozygous forms range from mild to severe. Prior to 1996, the molecular basis of HPE was unknown.  Range of Holoprosencephaly (HPE) Severe Mild Cyclopia Microcephaly Orbital hypotelorism Proboscis Cleft lip Only 1 front incisor!  Methods 30 Autosomal dominant families (ADHPE) were examined. Roessler et al. PCR amplified SHH exons for single-strand conformation polymorphism (SSCP). SHH implicated as one of at least 4 loci causing HPE. Human SHH maps to the HPE3 critical region on chromosome 7. https://upload.wikimedia.org/wikipedia /commons/2/2f/SHH_location.png  Single Strand Conformation Polymorphism (SSCP) One point mutation can cause major differences in the folded form of single stranded DNA. The strands are the same length, but have different 3D shapes due to differences in folding according to the sterics of each nucleotide sequence. These differences can be detected as differences in electrophoretic mobility. ssDNA (3D) ssDNA (2D) dsDNA Allele 1 Allele 2 https://www.nationaldiagnostics.com/electrophoresis/article/sscp-analysis  Pedigrees of ADHPE with SHH Mutations A. ADHPE 10 B. ADHPE 6 C. ADHPE 15 SSCP analysis = Mutation Carrier D. ADHPE 14 E. ADHPE 2 = Wild-type Restriction digest analysis  Hedgehog Protein Maturation 1 2 3 Arrows = Sites of Mutations 1. A single amino acid change adjacent to the signal sequence. disrupts addition of lipids required for protein function 2. Two different mutations introducing stop codons in the signaling domain. 3. Two mutations changing a highly conserved tryptophan to an arginine or glycine in the signaling domain. These mutations all occurred in the highly-conserved N-terminus portion and result in a non-functional protein! Varjosalo M , Taipale J Genes Dev. 2008;22:2454-2472 ©2008 by Cold Spring Harbor Laboratory Press  SHH Separates the Eye Field Into Two Bilateral Fields by Suppressing Pax6 Pax6 expression is absolutely required for eye development. Shh is expressed along the midline (secreted from prechordal plate mesoderm/notochord). SHH represses Pax6 expression, thereby splitting the eye domain in two. Endogenous Pax6 expression during optic vesicle formation Furukawa T et al. PNAS 1997;94:3088-3093 Arrowheads indicate forming optic vesicles in an E8.5 mouse.  Role of SHH in Proper Eye Development Control Low [Cyclopamine] High [Cyclopamine] = Pax6 expression = SHH activity SHH production by the prechordal plate is necessary for the separation of the eye field into two. When SHH signaling is inhibited (either by a chemical, or by a mutation), the optic vesicle Lamb born from an doesn’t separate, so only a single eye forms. ewe that ate jervine. SHH Loss-of-Function in Humans Note the loss of ‘midline space’ separating the eyes into 2. LOSS of midline space = HYPOtelorism  SHH Gain-of-Function in Humans Note the gain of ‘midline space’. GAIN of midline space = HYPERtelorism  Extreme SHH Gain-of-Function: Janus Cats http://articles.latimes.com/2011/sep/30/news/la-heb-two-faced-cat-diprosopus-20110930 https://www.cnn.com/2020/05/23/us/two-faced-kitten-oregon-trnd/index.html Frank and Louie Biscuits and Gravy (~15 years old) (3 days old)  A molecular model of cosmobia. Brugmann S A et al. Hum. Mol. Genet. 2010;19:1577-1592 © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, http://www.indianpediatrics.net/june2011/images/ra1-1.jpg please email: [email protected]  Limb-Specific Shh Enhancer A sequence present within intron 5 of ZRS the gene Lmbr1 acts as an enhancer of Shh. Lmbr1 Shh The enhancer, called the ZRS ~1 Megabase (ZPA regulatory sequence), is only (= 1000000 bp) active in the posterior of the limbs. ZRS = ZPA Regulatory Sequence = a limb-specific Shh enhancer  Ectopic Expression of Shh in the Anterior of the Limb Causes the Formation of Extra Digits A SINGLE point mutation in the ZRS can cause preaxial polydactyly. The mutation causes expression of Shh in the anterior of the limb bud, resulting in multiple extra digits. The expression is specific to the limb bud (limb-specific enhancer). Maas and Fallon, 2005 This Mutation Can be Found in Cats and Dogs… Hemingway cats Norwegian Lundehund  and Humans, Too! Yang and Kozin, 2009 Lettice et al., 2003  Deletion of the ZRS Deletion of the entire ZRS results in the loss of Shh ZRS expression in the limbs, and degeneration of the distal skeletal elements. Lmbr1 Shh Deletions in the human orthologue of the mouse Lmbr1 ZRS = ZPA Regulatory Sequence gene result in a similar phenotype. Sagai et al., 2005 Ianakiev et al., 2000 The ZRS in Snakes Kvon et al. (2016) ZRS in Snakes Kvon et al. (2016)