SHH Gene and Holoprosencephaly Quiz

Tutorial 5: The Discovery of hedgehog and Its Homologs

• Drosophila embryos form repeated morphological units called segments.
• Each abdominal segment on the ventral side of the larva has anterior denticles (pointing posteriorly) and a posterior naked region.
• In 1980, Christiane Nüsslein-Volhard and Eric Wieschaus screened thousands of embryos to identify genes involved in the segmentation process. They focused on embryonic development, unlike others who studied adult flies.

Methodology: Forward Genetics

• Adult male flies are treated with a mutagen to generate independent mutations in their gametes.
• Mutagenic males are crossed with wild-type females to produce many heterozygotes with independent mutations.
• Independent heterozygotes are bred with wild-type flies to produce more heterozygotes with the same mutation.
• Heterozygotes are bred together to produce homozygotes and screen for mutants with abnormal phenotypes.

Results of the Forward Genetic Screen

• Hedgehog mutants have the same number of segments as normal, but the naked region is deleted and denticles are duplicated in a mirror image.
• Hedgehog is a segment polarity gene.

The Year is 1992

• Drosophila hedgehog (hh) was cloned independently by four groups.
• The sequenced gene enabled the construction of a probe to analyze gene expression.

The Year is 1993

• Three papers were published simultaneously on the hedgehog homologs in vertebrates. Authors include Andrew McMahon, Cliff Tabin, and Phil Ingham.

Homologs, Orthologs, and Paralogs, Oh My!

• Orthologs: genes in different species that evolved from a common ancestral gene and have similar functions.
• Paralogs: genes within a single species that have arisen from a gene duplication event and may have different functions.
• Homologs are genes in different species descended from a common ancestral gene.

Discovery of hedgehog in Mice

• The first vertebrate hedgehog gene discovered was Desert hedgehog in mice.
• Other mammalian hedgehog genes are Indian hedgehog and Sonic hedgehog.
• The sequence of Drosophila hedgehog was used as a probe to identify vertebrate homologs by hybridization.

Making a Genomic Library

• A genomic library is a collection of DNA fragments representing the entire genome of an organism.
• Small DNA fragments are inserted into carrier molecules like viral DNA or plasmids.
• The fragments are packaged into many viral particles, and together these particles contain the entire genome of the organism.

Cross-Hybridization

• High stringency hybridization only allows for the hybridisation of perfectly matched DNA strands.
• Low stringency hybridization allows for the hybridisation of DNA strands with mismatches.
• Low stringency is important when using a probe across species for the identification of similar sequences.

Screening a Genomic Phage Library

• Genomic DNA is cut into smaller fragments.
• Fragments are combined with vector arms and sealed with DNA ligase.
• Fragments, packaged into phage, are screened for the desired sequence by hybridization to a labeled probe.
• The fragment containing complementary sequence can then be sequenced.

Results (Mouse Genomic DNA Library Screening)

• Drosophila hedgehog probe hybridized the Desert hedgehog gene.
• Later, probes based on the sequence of chicken Sonic hedgehog detected further genes, Indian hedgehog and Sonic hedgehog.

Riddle et al. 1993 Study

• The objective was to find a hedgehog homologue, which might be the ZPA morphogen (Zone of Polarizing Activity)
• The study involved comparing mouse Desert hedgehog and Drosophila hedgehog sequences to identify a conserved region.
• The most conserved region was between amino acids 161 and 237.

Degenerate PCR

• Degenerate PCR can be used to amplify genes from different organisms, even when nucleotide sequences are not completely known.
• Primer design is based on conserved amino acid sequences, rather than nucleotide sequences.
• This technique is crucial for identifying homologous genes in unknown organisms.

cDNA Library

• Degenerate PCR was used to create a probe with chicken genomic DNA, enabling the amplification of a 220-bp sequence.
• This sequence was used as a probe to screen a chick limb cDNA library (a collection of cDNA fragments from chick limb tissue).
• This process identified homologs of the homologous genes expressed in developing limb buds.

Cloning with Bacteriophage λ

• Chick cDNA from the limb is used to construct a phage library and screened to isolate a phage containing the desired homologous gene.
• Radio-labelled probes are used in cross-hybridization to screen for the homologous gene.
• The homologous sequence can then be isolated and studied.

Timeline of Experiments

• A chronological account of experiments related to hedgehog gene discovery.
• The experiments started with the Drosophila hedgehog and later extended to mice, chicken and other vertebrates.

Degenerate PCR Primer Design Practice Question

• Practice problems focusing on the design of degenerate PCR primers using known amino acid sequences.

Tutorial 6: The discovery of chick Shh

• Overview of the chick hedgehog's discovery.

Tabin's search for the ZPA Morphogen

• The process of identifying the specific gene responsible for the ZPA signaling center.
• The ZPA, a critical developmental region, was known to produce a signal that determined limb polarity.
• The identity of its morphogen, however, remained unknown before the hedgehog studies.
• The key question was whether, amongst chick homologues of the Drosophila hedgehog gene, was a candidate for the ZPA morphogen.

Morphogen

• A substance that acts in a concentration-dependent manner to direct cell fates during development.

Gain-of-Function Study

• Expressing a gene in a location where it's not normally expressed.
• The resultant changes allow a deeper understanding of the gene's role in development.
• This method aims to determine if Sonic hedgehog is responsible for the ZPA.
• This method used a retroviral vector to express the gene in a new location.

Replication Competent Retroviral Vector

• Description of how retroviral vectors are employed to express target genes, in this case, the Sonic hedgehog gene.
• Retroviral vectors are modified viruses that deliver genes into cells.
• They can be used to express genes in tissues where they aren't usually present.

Gain-of-Function: Method (Grafting)

• Infected chick embryo fibroblasts (CEFs) are implanted in the anterior limb bud zone.
• This process aims to verify if Sonic hedgehog gene is the primary morphogen in limb polarity.
• The method entails using a retroviral vector incorporating the gene to ensure the infected cells can't migrate and thereby infect the whole embryo.

Grafted RCAS-E/AP CEF Cells

• The role of grafted cells in relation to alkaline phosphatase expression within specific regions of the embryo and the effect of this expression on limb development.

Gain-of-Function: Results

• Shh-expressing cells induced mirror image duplication of limb structures.
• This result correlated with ZPA grafting experiments.
• The extent of duplication is dependent on the number and position of transplanted cells.

Gain-of-Function: Conclusions

• SHH is essential for limb bud polarization and inducing mirror image duplications.
• Similarity of SHH-induced and ZPA-induced phenotypes strongly suggests SHH is the ZPA morphogen, controlling anterior-posterior limb patterning.

Summary of chicken SHH discovery and Functional characterization

• Brief overview of the discovery, characterization, and function of chicken Sonic hedgehog (Shh).

Cloning the Human SHH Gene

• The human Sonic hedgehog (SHH) gene was cloned.
• This involved degenerate PCR using the conserved regions of mouse and Drosophilia hh genes.
• The full length cDNA was cloned.
• The SHH gene was mapped to a specific region of human chromosome 7.

Conservation of SHH Homologs

• High conservation of SHH protein, especially the N-terminal portion, between mouse, chicken, and zebrafish.
• The conserved function (embryonic patterning) shows evolutionary implications.
• A high degree of similarity exists in the amino acid sequences of SHH in mammals.

Holoprosencephaly (HPE)

• Forebrain fails to divide into 2 hemispheres.
• The incidence is relatively common.
• Homozygotes are often stillborn.
• Heterozygous forms exhibit varying degrees of severity from mild to severe.
• The molecular basis of HPE was previously unknown.

Range of Holoprosencephaly

• Description of different levels of HPE severity and associated physical features.

Methods (HPE)

• 30 Autosomal dominant families with HPE were examined.
• SHH was suggested as one of several possible loci.
• The SHH gene was found to map to the HPE3 region on chromosome 7.

Single Strand Conformation Polymorphism (SSCP)

• Changes in folding of single-stranded DNA due to point mutations.
• Detection is done by electrophoretic mobility variations.

Pedigrees of ADHPE with SHH Mutations

• This section presents family trees (pedigrees) highlighting cases of Autosomal Dominant Holoprosencephaly (ADHPE) and mutations in SHH.
• These charts visually link the presence of the mutations (and associated symptoms) across generations of a family.

Hedgehog Protein Maturation

• Various sites of mutations affecting hedgehog protein formation and maturation.
• This section presents an overview of mutations causing problems with hedgehog protein maturation.

SHH Separates the Eye Field

• SHH's role in eye formation and development.
• SHH represses Pax6 expression in the eye field, separating it into two bilaterally symmetrical parts.

Role of SHH in Proper Eye Development

• SHH production (from the prechordal plate) is essential for proper eye formation.
• Inhibition of SHH signaling results in a single eye.

SHH Loss-of-Function

• Loss of SHH function results in eye development defects like hypotelorism.

SHH Gain-of-Function

• Gain of SHH function results in eye development defects like hypertelorism.

Extreme SHH Gain-of-Function

• Extreme gain-of-function cases of SHH in cats present as "Janus cats".

Human Molecular Genetics

• A continuum of severity in SHH function relates to the observed phenotypes.

Limb-Specific SHH Enhancer

• The regulation of SHH expression, especially in the zone of limb development, is controlled by the ZRS, which is specific to limb development.

Ectopic Expression of SHH

• Ectopic expression of SHH in the anterior limb bud causes extra digits.
• A single point mutation in the ZRS causes preaxial polydactyly.

This Mutation Can Be Found in Cats and Dogs

• The location of the mutation (and resulting phenotype) is observed in different species, namely cats and dogs.

Deletion of the ZRS

• Deletion of the entire ZRS results in the loss of SHH expression.
• This leads to the absence/defective development of distal skeletal elements in limbs.
• The loss of the ZRS is observed in mammals.

The ZRS in Snakes

• Evolutionary loss of the ZRS in snakes is correlated to the loss of limb-specific enhancer activity.
• The process of evolutionary loss is a form of sequence degradation.

Test your knowledge on the SHH gene, its mutations, and their implications in holoprosencephaly (HPE). This quiz covers various aspects, from the role of SHH in limb development to specific amino acid changes in the hedgehog protein. Dive deep into genetic discoveries and experimental outcomes related to SHH.