human embryology week 6

human embryology week 6

1. Neural induction

   • Process of forming the neural plate from the ectoderm
   • Midline ectodermal cells are induced to become “neural plate” by a signal from the underlying axial mesoderm (= notochord)
     • The node is the anterior end of the primitive streak
       • Cells that pass through the node form the notochord = axial mesoderm (underneath the ectoderm
   • Ectoderm that receives inducing signal becomes neural plate
     • Axial mesoderm cells signal to ectoderm
       • Ectoderm without this signal becomes surface ectoderm
         • Neural plate ectoderm becomes thickened
           • Surface ectoderm remains thinner
   • Molecular basis of neural induction
     • Involves a series of repressive interactions
       • BMP4 initially throughout ectoderm : inhibits neural induction
         • Noggin, follistatin, chordin secreted by node/notochord: bind to and inhibit BMP4
   • Pathways involves in neural induction:
     • BMP2 binds to serine-threonine type I / type II receptor complex  phosphorylation
     •  phosphorylation of R-Smad (Smad1, 5, or 8)  forms complex with Smad4
     • translocates to nucleus to activate (non-neural) or repress (neural) target genes.
       • Noggin (Nog) from axial mesoderm binds to BMP: prevents activation of receptor.
         • FGF and Wnt signaling lead to the inhibition of bmp4 transcription.

2. Primary Neuration

   • Process of converting the neural plate into the neural tube = Neural tube closure
     • Closure continues cranially (into brain) and caudally (into spinal cord): “zippering” action
       • Neural tube closure starts in the middle of the embryo (the future cervical region)
   • Neuroectoderm and surface ectoderm initially continuous
     • As neuroectoderm fuses, surface ectoderm also fuses
       • Neuroectoderm and surface ectoderm detach
   • Neural tube closure initiates ~ midway along the A-P axis (future cervical region).
     • Starting point is called Closure 1
       • Continues cranially (into the hindbrain)
         • caudally (into the spinal cord)
           • in a “zippering” fashion

3. Neural tube closure in mouse

   • Closure initiates at closure 1 site and then continues both cranially (into the hindbrain) and caudally (into the spine) Embryonic day (E) 8.5 in the mouse (~22-23 days in humans)
   • A second site of initiation of closure occurs at about the forebrain-midbrain boundary: closure 2 Embryonic day (E) 9.5 in the mouse (~24-25 days in humans) From Closure 2 the NT closes caudally (into the midbrain) and rostrally (‘towards the face’ i.e. into the forebrain)
   • A third site of initiation of closure occurs at the most rostral end of the neural plate (front of the forebrain) : closure 3 Closure 2 and 3 occur at almost the same time: E9.5 in mouse (~24-25 days in humans) From closure 3 the NT closes caudally, into the forebrain
   • Between closure 2 and closure 3 is a region of temporarily open neural plate – Anterior neuropore  Closed by continuation of closure - rostrally into the forebrain from closure 2 and caudally into the forebrain from closure 3

4. Forward genetic approach

   • Positional Cloning
   • Step 1: Map the gene to a chromosome
     • Step 1: Map the gene to a chromosome
       • Find out which chromosome carries the mutant gene
         • Heterozygous mutant animals are first crossed to another (wild-type) strain
           • Mutant and wild-type chromosomes are from different strains
             • Analyse inheritance of the phenotype in relation to co-inheritance of DNA markers from the different strains
               • Intercross heterozygous mice to generate homozygous mutants
   • Step 2: Refine the genetic mapping to a small part of a chromosome
     • Step 2: Fine genetic mapping
       • Find out which part of that chromosome carries the mutant gene: Relies on occurrence of recombination
         • Meiotic recombination
           • Meiotic crossover generates recombinant chromosomes: allow sub-chromosomal position to be identified
           • Use recombinants to identify the part of the chromosome carrying the mutation: a region always carrying the ‘red’ DNA markers in the mutant offspring
       • Analysing many recombinants will define the smallest region that must carry the mutation = the critical interval. All the genes in the critical region are positional candidates
   • Step 3: Identify the genes in the critical region
     • Step 3: Identify the genes in the critical region
       • All the genes in the critical region are positional candidates
   • Step 4: Look for a genetic disruption
     • Step 4: Screen candidates for mutations
       • Prioritise list of genes: (known function, expression data…)
       • Screen the genes for a mutation! Direct sequencing of exons and splice junctions
       • May not be a coding sequence mutation…. Use RT-PCR to assess for splicing defects and expression changes
       • Verify : additional evidence, functional data
   • Step 5: Find further supporting evidence

5. DNA markers

   • Microsatellites are short runs (<100bp) of simple repeat sequences (= Short Tandem Repeats). Number of repeat units varies in different alleles.
   • SNPs are sites where there is a single base difference between alleles

Images and files in the map