BIOM 3550 Study Notes (Lec 17-26) PDF

Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Lecture 17 – Hoxology FRUITFLY DEVELOPMENT: Almost all the initial work on homeotic mutations in fruitfly (can see profound work early) Centrolecithal, first ~10 cell divisions = meroblastic Early development → nuclear d...

Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Lecture 17 – Hoxology FRUITFLY DEVELOPMENT: Almost all the initial work on homeotic mutations in fruitfly (can see profound work early) Centrolecithal, first ~10 cell divisions = meroblastic Early development → nuclear division without cellular cleavage ○ Track nuclei, migrate to periphery ○ Partitioning ~11-12 division, but still syncytial at bottom Pair-rule genes expressed first before cellularization Holoblastic cleavage = ~12-14 cell divisions = Hox gene expression ○ Genes playing roles to define segments/regions and their identities Cytoplasmic polarity by Maternal Effect genes → Hunchback protein gradient → Gap genes → Pair-rule genes → Segment Polarity Genes + Segment Selector (Hox) Genes which give specific addresses to the cells within segments ANTIBODIES + REPORTER CONSTRUCTS: Kaufmann predicted that patterning of Drosophila = most parsimonious/simple decided by series of binary divisions ○ Have morphogen gradient → get anterior + posterior → then anterior-anterior, posterior-anterior, anterior-posterior, posterior-posterior → etc. Walter Gehring found that antibodies presented in the top panel in discrete bands → directly matched what Kaufmann suggesting ○ Therefore, hierarchical arrangement of genes forming discrete bands Promotor Expression Assay: multiple stainings of genes in vivo 1. Take promotor of various genes 2. Hook it up to reporter gene => where/when gene expressed? 3. Different colours for different genes ○ In real-time, in living embryo => can see how mutation affects genes (and other effects too) FRUITFLY STEREOTYPICAL PATTERN/SEGMENTS: T2 holds the wing & limb = biramous segment (both derived from imaginal disc + both are appendages) T3 holds pair of halteres & limbs = biramous segment ○ Halteres = vestigial wings (rear pair of wings that shrunk down into balancing organ) Head develops from fusion of ~6 segments that all carry imaginal discs for limbs (except for top one)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Shrunk down to become mouth-feeding parts ○ One segment where limbs have migrated up to the top to become the antennae (NOT biramous)’ HOMEOTIC MUTATION – HOMEOSIS: Homeosis: misaddressing of segment so it behaves like another Example 1: T3 develops pair of wings instead of halteres (T3 behaving as if its T2) ○ Atavism = evolutionary throwback (structure more primitive → fly ancestors had 2 pairs of wings) Example 2: Antennapedia (ant) gene normally expressed in thoracic region, when ectopically expressed in head = antennae look like legs ○ Imaginal disc that gives rise to the antennae receives information as if it were thoracic SEGMENTATION: = Metamerism (metameres = reiterative segments) Cells organized into groups => groups can act independently (change in address, behaviour not contingent on what its neighbours doing) Genes act to regulate segment formation, address, and differentiation (head vs trunk?) ○ Selector Genes involved are TFs: Homeobox genes (these genes can have other domains, not just homeobox) ➔ = One category of TFs – subdivided into families, Hox genes are subgroup (TFs → homeobox genes → Hox genes) Encode proteins that bind DNA and regulate other genes Characterized by 3 helical domains ➔ 3rd helix binds major groove of target DNA sequence Turn target genes on or off (some are activators, some are repressors) DIMERIZATION OF HOMEOBOX GENES: Hox gene has dimerization associated with its homeodomain that allows it to interact with other homeobox genes (e.g., Ubx = ultrabithorax, Exd = extradentricle) ○ If dimerized, targets will have binding domains for both Ubx & Exd Spacing between these 2 domains in the target DNA sequence = important  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 The combinations for hetero/homodimerization are extensive and are going to regulate how easily the gene products interact with DNA sequences of different target genes Exd → ortholog in humans = Pbx ○ Will bind to many different Hox genes (can heterodimerize with others) ○ Has its own specific AA sequence in the 3rd helix, binds its own specific DNA consensus sequence HOX CLUSTER: Selector genes (HOM, Ant, or Hox complex) Orthologs = same category of gene, highly conserved, providing important function (e.g., pb in fly, a2/b2 in mammals = orthologs) ○ More closely related to each other than those genes on the same cluster Paralogs = genes beside each other, on same cluster (e.g., a2 paralogs = a1 + a3) Hox genes in FLY = spatially colinear (3’ anterior, 5’ posterior) ○ Some expression domains overlap ○ Tend to express hazy bands that over minutes become very sharp/defined (ancestral/original way they were expressed) ○ Dorsal/Ventral patterning conserved in regards to Hox genes Hox genes in MAMMALS = spatially AND temporally colinear (3’ anterior first, then 5’ posterior) ○ Gene duplications from 7 to 13 genes/paralogs Sequences of genes suggest long history of duplications ○ Cluster duplications from 1 to 4 Not all clusters have all genes → but all 4 clusters combined have all genes  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 HOX GENE PROPERTIES: Hox genes have regulatory domains/elements specific to themselves, but also other Hox genes ○ a1 has promotor/enhancer region specific to a1, but also has remote enhancers that may play role in regulating a2-a4, etc. = shared regulatory behaviour (why these genes have remained unchanged as clusters for such a long time) Hox protein products autoregulate (e.g., a2 motor enhancer has binding site for a2 protein) and sometimes regulate neighbours Hox genes have other binding motifs for other classes of TFs ⇒ ex. RARE = Retinoic Acid Response Element ○ Sensitive to receptors that bind vitamin A More earlier/anterior (3’) genes = more vitamin A sensitivity → can inhibit head development (no formation of mid-hind brain junction) Homeotic transformation Hox Genes – Somite Fate & Vertebrae HOX GENE OVERLAPPING EXPRESSION – MAMMALS: In mammals, Hox genes express in a nested hierarchy of overlapping domains Early genes = anterior ○ Exquisitely sharp expression domain from the get go (laying down Hensen’s node) ○ Expression domain expands posteriorly (but fades out – NOT sharp) Expression domains overlap and different combinations of genes expressed ⇒ genes are specifying identity of respective segments in a combinatorial way ○ = combinatorial Hox code ○ E.g., most anterior cervical (occipital) somites patterned by Hox a1+a2, but when you get more thoracic get a1-a6 = different combinations of Hox genes expressing in the cells at those particular anterior/posterior levels Assemble all 39 paralogous groups of Hox genes & look at expression patterns ○ Unique combination of genes spaced at ~2 somites → after re-segmentation = 1 vertebral body SOMITE FATE: 1) Occiptal Somites (contribute to cranial vault) ○ 4 pairs ⇒ occipital bones of skull  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 2) Cervical Somites ○ 7 pairs ⇒ neck 3) Thoracic Somites ○ 7 pairs ⇒ ribs attached at sternebrae (sternum) ○ 6 pairs ⇒ rib ends free floating 4) Lumbar Somites ○ 5-6 pairs ⇒ lower back 5) Sacral Somites ○ 4 pairs ⇒ pelvic region 6) Tail Somites (humans have NONE) ○ Variable ⇒ tail Each somite acted upon by Hox genes, given specific identity ○ Cells in those structures will differentiate in manner appropriate to location = different fates for the different somites ○ Therefore, mutations in Hox genes or change in evolutionary patterning alter fate of somites (what # and type of vertebrae it will become) Gain & Loss of Function EXPERIMENTS: Prediction => knockout in fly should be same in mouse (get homeotic mutation) 2 approaches: 1) Ectopic Expression => Gain of Function Mutant ○ Ectopically expressed gene driven by promotor its usually not ○ E.g., express posterior gene in head/more anteriorly = dominates (rule of posterior prevalence) 2) “Knockout” => Loss of Function Mutant ○ E.g., removal of posterior gene results in anteriorization in domain normally expressed (remain under sway of former/anterior gene expressed) ○ Posteriorizing cue absent (does not occur) EXAMPLES: Ectopic anterior expression of a posterior Hox gene = posteriorization: ○ Cervical (more posterior) gene expressed into occipital (more anterior) region = NO cranial vault + rear head bones, instead get extra cervical vertebrae Deletion of a Hox gene → lost posterior cue = anteriorization:  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Ex.1: Anterior cue that made vertebrae 13 thoracic gets reiterated → next somites behave as if they’re T13 = 14th thoracic vertebrae Prior agenda continues to sway ○ Ex.2: Normally, 4th sacral vertebrae not attached to the upper tree => if anteriorization, attached b/c behaving as if S3 Atavism Atavism: recurrence in an organism of a trait or character typical of an ancestral form (evolutionary throwback) ○ Due to changes in Hox patterns / removal of Hox gene activity Ex. 1: middle ear conductive bone morphology more typical of reptilian forms Ex. 2: interdigitating ribs at sternum – Hox a4 (Horan et al., 1994) ○ Normally => ribs form, first 6-7 meet at midline, get sternum formation ○ Abnormally => ribs interdigitate Older specimens have 11 cervical vertebrae (instead of 7) + lack cranial vault: ancestral Hox act on all somites? (e.g., mouse cervical genes in occipital regions = Atavistic approximation of dinosaurs) ○ Slight tweaks to Hox gene expression (when + where) may have played role in evolution of skeleton today ○ Delay onset of the posterior cues in Hox genes when somites being specified = leave room for the earlier somites formed to do something new Delayed onset of Hox gene patterning of somites is what permits the occipital somites to form cranial vault instead of cervical vertebrae More recent skeletons have 7 vertebrae (more evolved dinosaurs): ○ Likely Hox turned on later to permit occipital somites to contribute to occipital bones (enlarging cranial vault to accommodate brain) Delayed onset of Hox gene activity = cranial vault Chick vs. Mouse Hox Gene Expression Have same genes, just changes in where/when expressed ○ Timing/regulation important in changes in morphologies  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Hox genes expressed when average ancestry is old → found at period of time particular to phylotype Tweaks in timing determine what organism will form (chick or whale or human… etc.) Both have 4 pairs of occipital somites Chick = longer neck ○ Overtime, Hox gene expression domains delayed = more cervical vertebrae Hox 5/6 expression = later in chicks (marks transition from cervical to thoracic vertebrae) Both Hox 5/6 and Hox 9/10 boundaries are where appendages arise in chick + mouse ○ Hox 5/6 => limb/wing bud forms ○ Hox 9/10 => hind limb forms Lecture 18 – Problems with Hox Code Model Hox Code model => combinatorial spatial model, set agenda for development of segments ○ 39 homologs in humans that act in a combinatorial way, to act upon discrete segments (of ~2 somites) along the dorsal axis PROBLEMS: 1) Hox protein target specificity ○ If they all recognize a similar DNA sequence motif (TAAT) how can they each specify a different vertebral identity (i.e., activate different genes at different times in each place)? Homeodomains so similar, recognize similar motif TAAT… how do they have specificity? 2) Phenotypes not always as expected ○ If combinatorial Hox code specifies discrete addresses (positional identity), then mutation should yield a phenotype immediately within the normal domain of Hox expression Homeotic transformations NOT what would predict Problems: Target Specificity 3rd helix of homeodomain binds to and affects major groove of target gene regulatory sequence (TAAT)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Does flanking sequence of target gene motif play a role in modulating access? Are there aspects around the TAAT sequence that regulate specificity? ○ Or does rest of Hox protein affect shape in subtle way to affect specificity of action? Are there aspects of homeodomain outside the 3rd helix that regulate the specificity? Combination of elements of DNA target site & Hox protein regulate specificity: 1) Shape of Hox protein (locally/globally) 2) Conformation of DNA to which the Hox protein binds Are there partner proteins that play a role in guiding specificity? ○ Hox genes form homo/heterodimers (with other Hox and homeobox – Exd/Pbx – genes) Specificity lent from: 1) Spacing between the Hox & Pbx binding sites 2) Conformation of dimer itself Subtle changes in AA sequence of protein partners = role in spacing + intimacy of contact ○ Sometimes with dimers, only ONE partner binds = changes shape of “key” that fits into DNA motif “lock” HOX PROTEIN STRUCTURE/PARTNERS VARY: Hox proteins dimerize with => each other, Exd/Pbx (Exd = Pbx in humans – have 3 copies), MEIS 1) Hexapeptide (pentapeptide region) binds partner protein ○ Regulates ability of partner proteins (e.g., Pbx) to interact with Hox proteins = amplification of # of protein combinations 2) Linker region endows this binding site with its partner specificity 3) Distance between homeodomain and Exd/Pbx/MEIS binding region (hexapeptide) varies ○ Distance between homeodomain + hexapeptide increases as you go from Hox 1-4, etc. Explains how Hox 1 & 2 interact with different downstream target genes than Hox 3, 4, 5, 6 (shape of complex different = the specificity of targets they interact with different)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Testing Different Structural Attributes SOME TESTS: 1. Reporter Gene Promoter Assays (tests 1000s of kb of target gene regulatory region) 2. DNase Footprinting Assays (tests 100s of bp of target gene regulatory region) 3. Gene Mobility Shift Assays (tests 40-60 bp of target gene regulatory region) 4. Gene Disruption Animal Model (“knockout” mice) 1. REPORTER GENE CONSTRUCTS: 1) Put reporter plasmid (e.g., LacZ, Luciferase) and a Hox gene expression plasmid into cell line ○ Transfect reporter construct (or mutated Hox protein) into cells, incubate 2) Lyse cells, provide substrate (e.g., for LacZ/Luciferase) 3) Analyse colour/light output – do alterations to target sequence change output? (quantitative measure of how much the Hox protein has interacted/turned on reporter) ○ Use the reporter in cell/tissue culture system to generate colour/photometric change that you can measure with machine ○ Alter different parts of target sequence (experimentally mutated) Start big, work small => e.g., knockout 5’ most 1000 bp… still active? Localize where critical sequence is with which Hox protein interacts (i.e., TAAT site, flanking sequence adjacent protein binding motifs) ➔ Have local sequence, can do site-directed mutagenesis to change TAAT site or sequences immediately on either side of it => see effects of missed sequence 4) What changes to Hox gene alter output? ○ Can do Gel Mobility Assay after Example: Luciferase Assay ○ Examples of Mutations to Hox Expression Plasmid: Alter sequence encoding 3rd helix (binding specificity) Alter sequence encoding partner-interacting domains Alter sequence encoding NH-terminal activation domain  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Examples of Mutations to Target Promotor Sequence: Deletion mutants to see WHICH TAAT sites are important Mutate TAAT site Mutate TAAT flanking sequences Mutate dimerizing partner (co-transfect) DNA-binding motif Drawbacks: tested cells should NOT have the Hox protein of interest, its partners, or other homeodomain encoding proteins → interact with target and skew things 2. DNAse FOOTPRINTING ASSAY: 1) Add target DNA sequence (usually 200-300 bp, lifted out of promotor sequence) and TF (Hox) => do they bind TFs? 2) Incubate at physiological conditions ○ Target sequence incubated with lysate from cells expressing specific Hox (e.g., Hox a2) ○ Allow the Hox TF to bind to target DNA sequence (e.g., TAAT) 3) Add DNase for a short while ○ Eat up the DNA 4) Enzyme will generate random lengths of DNA, EXCEPT where protected by Hox protein/TF = DNase footprint (protected) CO-OPERATIVITY: Can have multiple binding sites for Hox protein in promotor sequence => happens when several sites (TAAT) really close together Co-operativity: binding at one site activates/promotes binding at another site ○ Once one Hox protein bound to sequence, easier for another to bind = binding co-operativity ○ Facilitates activation of target gene (logarithmic activation) Isn’t just which Hox gene is on, but how much is on/expressed (concentration… how much protein is available?) ○ Which, where, how long, which partners, how much Beachy’s Experiment: ○ Cryptic sites in the DNA construct target → Ant (antennapaedia) promotor/enhancer had been cloned into plasmid ○ In the cloning process, site where cloned had TAAT-like part  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 When co-operativity happening, made it possible for Hox protein to bind to even that site 3. GENE MOBILITY SHIFT ASSAY (GMSA): Target sequences 40-60 nt long 1) Design a sequence + its complementary strand 2) Label using P32 (radiolabelled) 3) Incubate in a tube labelled oligonucleotide with cell lysate that has Hox protein ○ Can alter sequence of oligonucleotide OR Hox gene that encodes TF (e.g., Hox a2) ○ If Hox TF binds to sequence = complex of radiolabelled DNA bound to protein 4) Mix in various concentrations, run out on gel ○ i) Dark band at bottom = unbound probe/oligonucleotide (not bound by TF → small, mobile, goes through gel quickly) ○ ii) Band near top = probe bound by TF (bigger, slower, has different charge) ○ iii) Band even closer to top = complex, probe bound by TF dimer (2 TFs bound together to DNA, even bigger + slower) 5) Can test specificity of interaction by adding unlabelled oligonucleotide (cold probe) to see if it competes for binding with available protein/TF 6) Can also add antibody to Hox (e.g., Hox a2) ⇒ get Supershift ○ iv) Band closest to top = Hox protein bound to huge antibody specific to it, complex (antibody+Hox) binds to probe (even slower) Can confirm its the protein you wanted put into system (Hox a2) and not some other protein  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 4. “KNOCKOUT” MICE – GENE DISRUPTION ANIMAL MODEL: 1) Cell lines derived from inner cell mass (ES cell lines) → grow in culture 2) Electroporate altered gene construct into cells = produce neoresistance enzyme, NOT Hox protein ○ Replace exon containing start codon with small gene for neomycin resistance = Hox gene missing first exon, with stop codon at end 3) At very low frequency, this DNA construct will align with homologous sequence in the genome of these cells = crossing-over event ○ Homologous sequences switch out, replace normal sequence with mutant 4) Have 1-2 ES cells that are mutant ○ Neo resistance encoding construct has integrated at some random place OR integrated into homologous site 5) Treat the cultures with lethal dose of antibiotic => only cells with neo resistance gene will survive (cells that carry mutated Hox) ○ Have to sort out which have replaced endogenous Hox vs. which haven’t Challenges = discriminating between mutant cell lines that have integrated/recombined into homologous site (replaced gene of interest) and b/n those that have integrated randomly ➔ Have other markers (e.g., Tk – thymidine kinase) that help discriminate (in construct, outside neomycin) ➔ Positive & negative selection (neo resistance, but NO Tk resistance)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 6) Expand population, inject into blastula stage embryo => will join inner cell mass + contribute to embryo ○ Hope some mutant cell progeny contribute to germ line 7) At end = knockout mice that carries mutant gene in some of its cells (perhaps in germ cells) SUMMARY: 1. Hox binding is mediated by the 3rd helix of the homeodomain to the major groove of DNA at a consensus site (TAAT) 2. The N-terminal arm of Hox reaches out and makes contact with the minor groove 3. The pentapeptide motif binds partner TFs such as the homeobox gene Exd/Pbx or MEIS 4. AA sequence flanking the DNA binding domain (e.g., the 3rd helix) matters 5. The binding motif on DNA is contextually accessible: ○ Flanking sequence matters ○ Other DNA binding motifs, and their relative spacing matters 6. The presence and amount of TF matters to: ○ Form homodimers ○ Form heterodimers 7. Concentrations of Hox protein matter => CO-OPERATIVITY Problems: Unexpected Mutant Phenotypes (Anomalies) Mutant phenotypes not always as predicted => expect transformations at anterior border of expression (know where gene is normally expressed, but when mutated get effects at different location?) 1. Transformations do not always occur at Hox expression boundary 2. Next gene does not seem to specify its usual somite/vertebral identity, but to specify generically one somite more posterior (order of somites proceeds smoothly without interruption) 3. Mutant phenotypes sometimes cover broad regions (not sharp), and don’t perturb just at the boundary 4. Mutant phenotypes sometimes present in reiterative manner 5. Deletion of entire cluster has surprisingly little effect (in terms of anterior-posterior and dorsal-ventral axis) None of these exceptions is consistent with the notion of the Hox Code specifying precise spatial mapping identity (e.g., a particular Hox gene specifies somite contributing to vertebra T4, etc.)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ANOMALIES: Hox genes normally exhibit sharp anterior boundaries of expression ○ According to the Hox Code model, this is where positional (posteriorizing) information should be installed and phenotypes should change ○ Expect anomaly to occur here if knocked out… BUT: E.g., Hox gene expressed at C4 contributing to cervical vertebrae 4 → but when mutated, see change/anomaly at cervical to thoracic transition See reiterative anomalies, effects all the way down Irrespective of normal Hox boundary, phenotypes map to borders of body transition (cervical to thoracic, thoracic to lumber, etc.) ○ Whole body plans gets shifted down => therefore, Hox genes AREN’T giving unique spatial identification, but giving more generic addressing (e.g., whatever you are now, be one segment more posterior) SOME EXPLANATIONS TO ANOMALIES: 1. Transformations do not always occur at Hox expression boundary ○ Explanations? Somite re-segmentation could fuzzy the phenotype Functional redundancy of Hox genes could compensate and delay manifestation of phenotype 2. Next gene does not seem to specify its usual somite/vertebral identity, but to specify generically one somite more posterior (order of somites proceeds smoothly without interruption) ○ Explanation? In the leap to “catch up” and cover for the missing previous posterior cue, some of the morphological attributes are smoothened The rest of the anomalies are incompatible with a Hox Code-Position model ○ Especially cannot explain why, when the entire Hox C cluster is deleted = there is little effect on spine Problems with the Neo-Cassette Homologous KO targeting vector introduces an artifact ○ Neo-cassette => if used to knockout one Hox gene, also affects activity of neighbour genes (therefore, insertion of neo-cassette interrupted behaviour of chromatin in areas that were somewhat more remote) Neo resistance selection marker acts as a genetic insulator – activity of adjacent genes no longer coordinated… this is a product of:  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Hox genes sharing regulatory elements, therefore stay clustered (if knockout one gene may be interrupting shared regulatory sequences) ○ Chromatin domain integrity being violated and not behaving properly (architecture is abnormal, epigenetic factors altered) => affect how other genes behaving ○ Timing of gene activation is thrown off, and since there is normally cross-talk => this too is disrupted Spatial and temporal (e.g., Hox 1 turns on, then 2, then…) colinearity affected Solution? ○ Cre-Lox removal of selectable marker HOX CLUSTERS & ANOMALIES: Hox genes NOT providing discrete spatial identification, but generic temporal cues (e.g., be one segment more posterior) ○ = Clusters unwinding + dividing temporal cues in coordinated fashion Therefore, by introducing neo-cassette = affect the timing of the unpacking of that particular cluster ➔ Ex. 1: one cluster not in synchrony with others = reiterative anomalies ➔ Ex. 2: remove entire cluster, but the other 3 still working fine temporally = NO anomaly Conclusions Hox clusters are operating to posteriorize in a generic manner, not positionally codified TIMING of gene activation is critical, not WHICH specific gene is active ○ Example: in flies => patterning/homeodomain encoding genes (gooseberry and paired) Knocking out gooseberry = neural effects  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Turned on paired using gooseberry promotor → when/where paired normally expressed, had gooseberry instead = rescued phenotype ➔ One could functionally rescue the other if turned on in place of the mutant ◆ Generic quality → as long as a homeobox gene (doesn’t matter which) is turned on at the right place at the right time = rescue ○ Example from other studies: in mice (Wolfgang Wurst) Engrailed = a homeobox repressor TF → causes big neural problems when mutated in fly When knocked out in mouse, almost NO discernable effect (subtle brain defect and learning disability) There is a 2nd Engrailed gene in mouse → maybe one is covering the other? ➔ En1 and En2 (share only 55% AA similarity) turn on at slightly different times during brain development ➔ If En2 is expressed in place of En1 in a En1-/- background = phenotype is rescued ◆ Took En1 promotor to drive expression of En2 (En1 mutants mouse rescue b/c of En2) ◆ Therefore, doesn’t matter which Hox gene being expressed as long as one expressed at right place + time = functional rescue Lecture 19 – Developmental Clocks & Head Organizer WHY DID HOX TRANSFORMATIONS OCCUR AT ZONES OF MAJOR BODY TRANSITION? Transformations often occurred at transition between cervical to thorax, thorax to lumbar, etc. Why? Theme of 6 or 7 somites/segments between anomalies in mammals (presomitic mesoderm into somites ⇒ clocking systems) ○ 6-7 prosomeres (forebrain + midbrain) ○ 7 rhombomeres (hindbrain segments) ○ 7 cervical somites ○ 7 thoracic somites with ribs meeting at sternum 6 thoracic somites with ribs free floating at ends ○ 5-6 lumbar somites ○ 5 sacral somites  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ 4 coccyx somites or more (in tailed animals) WHY? DEVELOPMENTAL CLOCKS! 3 Clock Systems: 1) Segmentation Clock 2) Cell Cycle Clock 3) Hox Activation Clock Segmentation Clock (Somitogenesis Clock/Wavefront Model) Related to timing of cell cycle + unwinding of Hox clock => involves oscillator + moving wavefront 1) Hensen’s Node migrates posterior-ward along dorsal midline/groove => leaves behind presomitic mesoderm ○ Hensen’s Node = source of retinoic acid (RA) = “posteriorizing cue” 2) Posterior primitive streak is source of FGF/Wnt signal (both secreted) 3) 2 gradients are antiparallel and form a determinative wavefront that moves with Hensen’s Node (moving posterior) ○ Both RA + FGF/Wnt diffusing => anti-parallel gradients (one anterior, one posterior) 4) An oscillating molecular signalling clock affects pre-somitic cells at the determinative wavefront => presomitic mesoderm partition + pinch off to form somite WAVEFRONT: 1) Cells are subjected to molecular oscillator while Hensen’s Node passes down the dorsal axis ○ Molecular oscillator from posterior primitive streak (wave of genetic activity)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 2) Some point behind Hensen’s Node, competing RA and FGF/Wnt gradients open a window of opportunity for somitic cells (determinative wavefront) to be receptive to the oscillator 3) Presomitic mesoderm responds by pinching off to form a new segment ○ Molecular cues (oscillator) pulsing UP → going on/off at the same time wavefront is moving DOWN/posterior = presomitic mesoderm → somite when oscillator & wavefront intersect (get segmentation of presomitic mesoderm at overlap) OSCILLATOR: Involves limited oscillatory network ⇒ Notch/Delta Pathway 1) Notch = cell surface receptor ○ NotchICD = intracellular domain 2) Notch activation involves: ○ Notch glycosylation ○ Interaction with partner (e.g., Delta) on neighbouring cell 3) Delta binds to Notch => induces NotchICD to break off 4) NotchICD is trafficked through cytosol + nuclear membrane => into nucleus 5) NotchICD interacts with other TFs (e.g., CSL) => interact with promotor/enhancer region => transcription of target genes activated: ○ 1) Lunatic Fringe (acetylglucosaminyltransferase) Secreted Inhibits glycosylation (necessary for interaction between Notch & Delta) ➔ = Inhibits activating ability of Notch by Delta (autoregulatory loop) ○ 2) Hes7 (hairy enhancer split = TF) Helix TF Suppresses activation of Lunatic Fringe OSCILLATOR AT WORK: Secreted Inhibitor of Notch = Lunatic Fringe Regulatory sequence (promoter) from Lunatic Fringe to drive expression of a marker, GFP – green fluorescent protein ○ Reporter = GFP, hooked to promotor enhancer of Lunatic Fringe ○ Where Lunatic Fringe expressed, so is GFP  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 = can see the sequential and transient waves of the marker working up from the posterior to meet the presomitic mesoderm at the determinative wavefront OSCILLATOR IS QUITE ROBUST – REDUNDANCY: Notch/Delta is only one side of the oscillator, there is another side to the cycle that works on the off-beat – Axin/Wnt ○ Axin/Wnt = reverse side of Notch/Delta (when Notch/Delta = ON, Axin/Wnt = OFF → vice versa) FGF also oscillates → so essentially 3-part redundancy: 1) Notch/Delta 2) Axin/Wnt 3) FGF Segmentation timing and therefore somite length + number affected by: ○ Radical changes in cell cycle, temperature, etc. E.g., Local heating/cooling of embryo changes rate of cell cycle The cells that segment = are in the correct phase of the oscillatory cycle and at the determinative wavefront Larget Body Segment Zones? SO HOW DO WE EXPLAIN LARGER BODY SEGMENT ZONES? Claudio Stern (Columbia University New York City) Chick Embryos 1) Inject fluorescent marker = fluoresceinated dextran (intracellular carbohydrate with chains → too big, can’t leave cells) ○ Fine glass needle filled with marker, inject into Hensen’s node 2) Only daughter cells will remain marked (b/c only way to get dextran is by inheriting) 3) Follow fate of daughter cells ○ Lineage tracking experiment ○ See where the marker ends up in notochord/somites CELL CYCLE DYNAMICS VARIES IN AXIAL VS. PARAXIAL MESODERM: Somites => periodicity every 6-7 Notochord => periodicity every 1.5-2 somite equivalent distance  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Axial mesodermal cells divide more quickly than presomitic cells = lineage tracking marker (dextran) seen more periodically in notochord than in somites Is a somite–notochordal concordance required for major changes to body morphology to occur? ○ Both notochord + somite in the same phase of cell cycle → is every ~6-7 somites Segmentation of the Brain 1. Rhombencephalon (Hindbrain): respiratory rhythm, motor activity, sleep, and wakefulness 2. Mesencephalon (Midbrain): vision, hearing, motor control, sleep and wakefulness, arousal/alertness, and temperature regulation 3. Prosencephalon (Forebrain) becomes the: a. Telencephalon: cerebral cortex and subcortical structures (including the hippocampus, basal ganglia, and olfactory bulb) b. Diencephalon: thalamus, hypothalamus, posterior pituitary gland, and the pineal gland 1. RHOMBENCEPHALON: Rhombencephalon displays transient segmentation into rhombomeres ○ When hindbrain developing, breaks into 7 discrete segments (anatomical segments disappear) Is ONLY part of brain that lies ABOVE notochord ○ Notochord only till isthmus (mid+forebrain DON’T have notochord) Gives rise to Cranial Ganglia Is ONLY part of brain in/near which Hox genes are active Cells within become clonally restricted & don’t cross rhombomere boundaries ○ E.g., r3 progeny cannot be sent elsewhere (sharp boundaries)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 LINEAGE RESTRICTION OF RHOMBOMERES: 1) Inject lineage tracking marker (dextran) into the neural ridges early = BEFORE rhombomeres formed ○ = Have some degree of plasticity in the cells derived — could contribute to rhombomere 1/2/etc. 2) Inject cells at time rhombomeres forming ○ = become lineage restricted to just that one rhombomere Therefore, temporal order to clonal restriction ○ At the transition from pre- to post-rhombomere segmentation that Hox genes are expressing HOX EXPRESSION IN RHOMBENCEPHALON: Hox genes expressing in discrete domain ○ Each rhombomere has unique combination of Hox gene expression (unique address) 2. FORE & MIDBRAIN SEGMENTS? Neuromeres: ○ Meier & Jacobson SEM of developing mouse brain in the early 1980s ○ Saw and published evidence of transient “neuromeres” of fore- and mid-brain ○ NOT widely accepted until recently Thought it was just an artifact of fixation Prosomeres: ○ Puelles & Rubenstein => cataloguing expression patterns of homeobox genes as they were activated during brain development (in mouse) Break fore+midbrain into 6 discrete areas categorized by unique combinations of gene expression ○ NOT morphological segments (being segmented at GENETIC level) ○ Expression domains define them Domains overlap and combine to define discrete boundary-delimited areas  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ➔ Diffuse expression that becomes progressively more refined 6 distinct regions Lecture 20 – Other Head Organizers & Chordoneural Hinge (Tail) Is There a Separate Head Organizer? RECALL MODEL FOR D/V & MESODERM PATTERNING IN FROG: High Wnt + TGF- (Nodal) signalling => Nieuwkoop Center => Siamois => Chordin, Noggin => Spemann Organizer (dorsal mesoderm) More ventrally = TGF- signalling (e.g., Veg1, BMPs) only => ventral mesoderm NEURULA, NEURAL PLATE, NEURAL CREST: 1) Neural Plate forms along the dorsal midline ○ Wnt/Chordin underneath the neural plate 2) Neural Ridges meet at the midline 3) Neural Tube sinks, ectoderm covers the top If the Hox genes AREN’T expressing in mid+forebrain, what is organizing the segments? SEPARATE HEAD ORGANIZER? Behringer’s mouse mutant → Lim1 = Lhx1 ○ Homeobox gene expressed in organizer ○ Later expresses in notochord ○ Mutants lack any head forward of the hindbrain When Lim1/Lhx1 knocked out => forms normally body, but everything AFTER hindbrain is absent ➔ Why area that is not supposed to be impacted by Hox (mid+forebrain) is being the most impacted?  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Subsequent studies showed that Lhx1 is necessary to gastrulation movement & cell adhesion Notochord, Isthmus – Head Organizer? HOW CAN NOTOCHORD ORGANIZE HEAD? DOES IT? Effect is probably indirect and acting on isthmus = midbrain/hindbrain junction – a constriction ○ Notochord plays role in organizing stuff in FRONT of it (end of notochord, at isthmus) Serves as boundary between two regions (sharp expression domain) Is site of expression boundary of important prosomeric genes that also have big brain phenotypes ○ 1) Gbx2 (gastrulation brain 2) in hindbrain ○ 2) Otx2 in midbrain Also site of FGF expression ISTHMUS: Mid/Hindbrain Boundary (MHB) => sharply delineated ○ Ectopic expression of Gbx2 up into midbrain represses Otx2 expression there => generates posterior neural structures ○ Ectopic expression of Otx2 down into hindbrain represses Gbx2 there => induces anterior-like neural structures Gbx2 and FGF mutually INDUCE Otx2 and FGF mutually REPRESS Otx2 induces Wnt which induces FGF which in turn stimulates Gbx2 ○ Otx2 → Wnt → FGF → Gbx2 Overexpress Gbx2 = turns down Otx2 expression (and vice versa → reciprocal restrictive behaviour) Anterior Cell Tiers – Head Organizers in Zebrafish? ARE THERE OTHER REGIONS IMPORTANT TO HEAD DEVELOPMENT? Monte Westerfield’s lab → a father of zebrafish developmental studies  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Deleted tiers (suck out with needle) of anterior ectoderm cells at mid gastrula Past the yolk syncytial layer => cells organized in discrete tiers ANTERIOR TIERS CRITICAL TO BRAIN DEVELOPMENT: Some specific tiers of cells are critical to anterior neural development ○ Deleting tiers 1 & 6/7 cells = head defects (something acting in these tiers to organize head) DELETION OF TIERS ALTERS BRAIN (MOLECULAR) MARKERS: 1) emx1 = homeobox gene 2) dlx2 = homebox gene ○ emx1 + dlx2 involved in prosomere model (e.g, tier 1 removed = patterning not occurring b/c emx1+dlx2 missing) 3) shh (sonic hedgehog) = secreted factor ○ Expressed on neural floor plate ○ Helps promote apoptosis (role in modelling) ○ In this case, shh overexpressed = more cells dying off TRANSPLANT STUDY: Anterior-most ectoderm (induced to anterior neural fate) = potent organizing activity ○ What if transplanted ectopically? => inject with fluorescent marker: 1) Tier 1 cells have limited inductive ability ○ NECESSARY BUT INSUFFICIENT (did NOT induce a whole head, but had induced brain markers emx1+dlx2) 2) Tier 3 (control) do NOT induce brain markers ANTERIOR PLAYS AN ORGANIZING ROLE, ALONG WITH OTHER DOMAINS: 1. Anterior neural plays an organizing role 2. Elicits expression of brain marker genes 3. Is not sufficient to recapitulate total organization ○ Hence the term: “necessary but insufficient” SUMMARY OF 4 ORGANIZING PLAYERS: All of them induce some of the brain markers, but can’t develop head alone  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 1) Isthmus 2) Ventral Floor Plate 3) Anterior Neural Ridge (anterior tiers of cells) 4) Zona Limitans Intrathalamica Tail Organizer – Chordoneural Hinge DURING NEURULATION: Both head AND tail → neural ridges are still open at far ends (suture up gradually as embryo elongates) ○ = loop of neural fold at top + bottom that still haven’t closed = therefore, different organizing activity ○ Lots of evolutionary divisions CHORDONEURAL HINGE: In fish + amniotes => Hensen’s Node continues to have some organizing activity, but movement slowed down at tail end ○ Down there… directive cell movement involving chordal (notochord) mesoderm = Chordoneural Hinge Extending down further and further (slower) Distinct bud/growth cell with discrete organizing activity TAIL ORGANIZING ACTIVITY: Explant LATE dorsal lip organizer to ectopic sites (in frog, fish, chicks) stimulates addition of tail somites => predominantly posterior notochord 1) At this stage – Chordoneural Hinge => notochord + open neural plate merge, cells convergent extending + extruding 2) Proliferation of extrusion stimulates activity of BMP & Eve 3) BMP & Eve provide the stimulus => more outward extrusion of cells ○ BMP (bone morphogenic protein) is a secreted growth factor from the TGF family ○ Eve (even skipped) is a homeobox encoding gene (remember pair-rule genes in fly) that acts as a repressor of Hox & Wnts 4) Expression is stimulated by physical cell migration TAIL BUD ORGANIZER = TRANSPLANTABLE & INDUCES LOCAL ACTIVITY: Excise and transplant tail bud organizer (chordoneural hinge) to ectopic site = growth of tail ○ Degree of BMP + convergent extension regulates HOW LONG tail is  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Why Don’t We Have a Tail? TBXT GENE: Tbxt = T-box TF (large family) Tbxt found in jellyfish to humans (highly conserved old gene = important function) Originally found/used as pan-mesoderm in riboprobe in situ hybridizations → mesoderm marker in frog – later restricts to dorsal lip & notochord (chordoneural hinge) ○ Tbxt first expressed in mesoderm => in frog + mice starts expressing in dorsal lip + ALL mesoderm, but later restricts to only axial mesoderm/notochord Human homolog of brachyury (means “short tail”) and T-mutant (mice) In mice: ○ Tbxt expresses in ICM, then mesoderm, then Hensen’s node & notochord ○ Naturally occurring (mild) Tbxt mutation causes kinked or shortened tails ○ Severe Tbxt mutation leads to embryonic lethality early in development => notochord defects, leading to neural patterning defects Defects happen in early neurulation Notochord abnormal = doesn’t give Shh, Noggin, Chordin signals to get neural fate HOMINOIDS = ALU SEQUENCE INSERTION = NO TAIL: Alu sequence (GC rich) interspersed all throughout genome ○ Alu (SINE – short interspersed elements) are primate-specific retroviral remnants => 11% of our genome! Tbxt activity in chordoneural hinge linked to presence/absence of tail (hominoids have extra DNA inserted into Tbxt gene = absence of tail)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 1) Presence of tail in non-hominoids => normally have ONE Alu element in Tbxt gene between exons 5/6 2) Absence of tail in hominoids => have SECOND Alu element in Tbxt gene between exons 6/7 ○ Get internal looping due to complementarity between 2 Alu sequences => results in exon 6 being spliced out = get different protein = NO tail REMOVE EXON 6 IN MICE = NO TAIL: Mutant mouse without exon 6 = NO tail ○ Good evidence of involvement by polypeptide sequence encoded by exon 6 in giving function to Tbxt during the elongation of chordoneural hinge INSERTION OF REPEAT SEQUENCES = SHORTENED/NO TAIL: Replace Alu sequence with RCS2 (reversed sequence that permits same sort of complementarity) ○ => induce internal complentarity + looping => exon 6 spliced out => different lengths of tail depending on degree of internal complementarity (how effective was it in many cells in that area?) CONCLUSION? Tbxt function in chordoneural hinge area is important to tail as well as general mesoderm development ○ Tbxt has activating roles: Has target genes in chordoneural hinge Playing role in helping mesoderm to continue its proliferation, convergent extension, and segmentation to form additional vertebrae necessary to form a limb When exon 6 is NOT translated, Tbxt does not support progression of the chorodoneural hinge (dorsal lip and notochord) Hominoid evolution (tail loss) resulted from insertion of Alu sequence, internal transcript complementarity, and alternative splicing  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Lecture 21 – Limb Buds Limb Origins Experimental Developmental Biology begins with studies on limb buds 250 years ago – letters from Spallanzani to Bonnet (salamander limb regeneration) Studies on limbs suggest there is a field of competent cells on the flank that is somewhat larger than what actually contributes to the limb ○ Before even limb bud formed => area of flank tissue competent to respond to cues/signals “Area around bull’s eye target” ○ Extirpation experiments: Remove tissue from area where limb bud forms => tissue around can regenerate what’s missing to create functional limb Therefore, zone that can contribute to formation much larger than what actually becomes limb CONSERVATION OF LIMB EMERGENCE BOUNDARIES: Where do limb buds arise? => always emerge at typical junction of Hox genes: ○ Hox C6 and HoxC9 expression domains delimit emergence of forelimb bud (fin, limb, wing) at the anterior & posterior margins respectively Anteriorly, Hox C6 = fore/wing limb bud (+ transition to thoracic vertebrae) Posteriorly, Hox C9 = hindlimb Limb Bud Development BUG ELONGATES & CHONDROGENIC PATTERNING BEGINS: 1) Early Limb Bud ○ Migration, proliferation 2) Emerging Limb Bud ○ Proliferation (bud/mound of cells accumulates just under epithelilum) ○ Mound becomes specialized into Apical Ectodermal Ridge (AER)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Initiation of Zone of Polarizing Activity (ZPA) ZPA = where armpit ultimately is (area of cells that includes both integument/ectoderm + mesenchyme underneath) 3) Elongating Limb Bud ○ Proliferation ○ ZPA forms ○ Aggregation begins 4) Palette / Early Chondrogenic ○ End of limb flattens (limb grows outwards like tube, begins to flatten – more paddle-like where fingers form) ○ Proximal chondrogenesis (chondrogenesis = beginning of cartilage formation) Right under AER (growth zone) → as the limb forming, cells proliferating that are suspended in gel-like matrix (HA), not much cell-contact (embryonic-like cells) ➔ The cells left behind lose HA matrix, form aggregates (cluster) ➔ Begin to chondrify to form nodules that form cartilage 5) Chondrogenic ○ Interdigit apoptosis ○ Distal chondrogenesis LIMB DEVELOPMENT IN AMNIOTES: AER = fine ridge of skin ectodermal cells that are bounding the external periphery of the palette LIMB BUD GROWS FROM SEVERAL SOURCES: Cells migrate from: ○ 1. Lateral mesoderm (e.g., from Hensen’s Node – some cells migrate to flank and start proliferating) ○ 2. Somitic mesoderm ○ 3. Mesonephric mesenchyme (comes from part of developing kidney) Glomeruli/kidney forming goes through 3 phases of building: 1) Pronephros → falls apart, then next built  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 2) Mesonephros → falls apart, then next built ○ When it disintegrates, those mesodermally derived will migrate to limb buds (and genital ridges where gonads form) 3) Metanephros → becomes kidney ○ 4. Neural crest ○ => Hence MESENCHYME Cell proliferation under ectoderm: ○ Mound of cells forming Apical Ectodermal Ridge (AER) AER secreting cues (growth factors) into mesenchyme that are necessary to support rapid cell proliferation & undifferentiated state ➔ Mesodermal cells collecting in HoxC boundary area & proliferating a lot CHICK VS. MOUSE LIMB BUD DEVELOPMENT: Similar phases of development: 1) Shh secreted in ZPA (early on in limb bud development) 2) Also early on, AER secreting growth factors (in FGF family → e.g., FGF8) Both important in early growth phase of limb development Roles of Hox, AER, FGF, Mesoderm, & Pitx1 in Limb Development 1. HOX GENE EXPRESSION BOUNDARIES DETERMINE LOCATION ON DORSAL AXIS: Along dorsal axis => nested hierarchy of Hox gene overlapping expression domains Hox C6 initiates activation of T-box genes => turns on Tbx5 Hox9 turns on Pitx1 (homebox gene related to Bicoid) => Pitx1 turns on Tbx4 (Hox9 +Tbx5? → Pitx1 → Tbx4) ○ Pitx1 expressed in pituitary gland Rathke’s pouch (precursor to pituitary gland) = anterior-most structure IN EMBRYO that forms in us ➔ Is formed by invagination of roof of mouth T-box genes turn on FGFs in mesenchyme => FGFs stimulate AER which also secretes FGF (secretion from AER critical to supporting proliferation) ○ T-box genes → FGF → AER → FGF  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 2. ROLE OF AER & FGF: AER sustains undifferentiated and proliferative state of cells immediately underneath ectoderm by secreting FGF If you remove AER = remove source of FGF = subsequent proliferation/differentiation ceases ○ If AER removed early on = only most proximal structures will differentiate ○ If AER removed later = more distal structures forming Need AER all the way through development to support enough growth for differentiation of digits AER is source of FGF: ○ A common approach to assess morphogenic signals in the limb has been to soak beads in a substance (FGF, Shh, RA), and to implant them ○ E.g., mimic AER activity by installing beads soaked in FGF => rescue limb development to some extent => FGF mediates outgrowth and permits patterning to occur (through secretion of FGF = AER supporting proliferation + undifferentiated state) Enlarge AER by Graft or Mutant = overexpression of FGF = supernumerary digits ○ Talpid3 Mutant (first found in chick) => elongated AER Can be mimicked by grafting extra AER ○ Encodes a receptor for Shh ○ Since knocked out in mouse 3. ROLE OF MESODERM IN LIMB BUD: Age of mesenchyme determines proximo-distal differentiation pattern/potency ○ E.g., mix/match experiments: 1) Take limb region + remove it 2) Put on some (old/new) mesenchyme & (old/new) ectodermal cap ○ ⇒ old mesenchyme underneath ectoderm = ONLY distal  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ ⇒ young mesenchyme = proximal then distal forming Age of AER is NOT important to proximo-distal pattern Mesenchyme Determines Fore- vs. Hind-Limb Identity: ○ Fore- vs. hind-limb is determined by Tbx 4 & Tbx5 (TF family that binds DNA through a T-box) Tbx5 (activated by Hox C6) determines forelimb ➔ Tbx5 activates Pitx1 Tbx4 (activated by Hox C9/Pitx1) determines hindlimb ○ Flank mesenchyme is NOT competent Mesenchyme does still require ectoderm from limb bud area ➔ If you install mesenchyme anywhere else along flank = NO limb Area where Hox 6 to 9 boundary are → renders the ectoderm in that area competent to form AER 4. PITX1 IN HINDLIMB DEVELOPMENT: Pitx1 activates Tbx4 in hindlimb ○ HoxC9 → Pitx1 → Tbx4 (hindlimb) Ectopic expression in chick accomplished by using a viral vector to ectopically express Pitx1 in forelimb 1) Pitx1 activates Tbx4 in wing bud 2) Wing bud forms more like leg/hindlimb (clawed toes + scales rather than feather germ) Zone of Polarizing Activity (ZPA) ZPA = ectodermal + mesenchymal ZPA gives polarizing, posterior cue ○ Morphogen is probably Shh ○ Historically, Retinoic Acid (RA) thought to play a role since it mimics ZPA Beads immersed in either Shh or RA can mimic a ZPA ○ Morphogen = dose-dependent (fewer cells/morphogens = less posterior induction) Can create mirror image symmetric structure 1) Graft ZPA into anterior margin = induce posterior traits 2) Mirror duplication of digits → posterior digit forming posteriorly AND anteriorly  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Retinoic Acid (RA) CLASSIC RETINOIC ACID STUDIES: Vitamin A disturbs limb development if overexpressed Summerbell → RA beds into chick limb buds 1) Used beads to test (soaked in Shh/RA) 2) Implant beads in posterior or anterior margin → see what happens ○ Both Shh and RA could independently mimic ZPA ○ E.g., if put anteriorly, like grafted ZPA Supernumerary digits => posteriorization of anterior flank ➔ *Only do this in the area that limb buds grow* (therefore, specific zone of cells competent to respond to these diffusible signals/morphogens) Other studies: ○ ZPA ○ Shh ○ Not flank mesenchyme RA & THE RECEPTORS: 1) RA passes through membrane (easily b/c highly lipophilic) 2) Pass through nuclear membrane of envelope (transportation network to get inside) 3) Retinoic Acid Receptors (RARS, RXRS, RORS, etc.) bind RA ○ RARs = subclass of steroid family receptors 4) Act upon DNA motif (RAREs) to alter gene expression ○ RARE – Retinoic Acid Response Element CRABPs bind to and remove RA from circulation ○ CRABPs = Cytoplasmic Retinoic Acid Binding Protein (act as sponge to prevent RA from getting into nucleus) RA & CRABP: Gregor Eichele → chick limb buds: 1) Removed and assayed anterior 2/3 and compared to posterior 1/3 2) Homogenized from hundreds of limb buds  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 3) HPLC Chromatography 4) More RA near ZPA (posterior) than elsewhere Malcolm Maden: 1) Purified CRABP 2) Reared antibody 3) Did assays on limb buds 4) CRAPB richer near anterior than posterior region Therefore, RA (more posterior – ZPA) + CRABP (more anterior) expressed assymmetrically => anti-parallel gradients ○ When taken together, the amount of RA available to bind to the RARs is enhanced posteriorly Putting It All Together Axial Hox Gene Expression: 1) Anteriorly, Hox C6 (between cervical & thoracic) a) → Tbx5 b) → FGFs (mesenchyme – from several sources) c) → AER → FGF 2) Posteriorly, Hox 9 (between lumbar & sacral) a) → Tbx4, Hand2 (TF in E-box family, activates Shh) b) → Shh/RA c) → ZPA AER & ZPA = mutually dependent ○ Remove AER => ZPA signal dies out ○ Remove ZPA => AER starts to die out  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 The longer the limb grows, the further removed AER cells become from the ZPA (= stop proliferating and start differentiating) ○ As AER cells proliferating → bud = larger + longer = AER further away from ZPA Further diffusion distance + more growth = both AER/ZPA die out (b/c need each other) => practical limit to limb growth THERE IS AN ANTERIOR SIGNAL = GLI 1: Gli 1 (TF) acts to repress ZPA ○ If removed, get posteriorized limb BMP = mesenchymal specific growth factor How Did the Snake Lose Its Legs? HOX GENES & LIMB DEVELOPMENT: 5’ members of the Hox cluster => late genes, play a role in limb development ○ These LATE-expressing Hox genes activate Shh T-box genes turn on Hox cluster → Hox genes act upon Shh (from python studies – used to have hindlimbs, but disappeared over evolutionary time) PYTHON & ANOLE LIZARD: Similarities between python snake & anole lizard Pythons have the remnants of hindlimb structure ○ Pelvic girdle, femur, claw = vestigial structures MANY LIMB GENE EXPRESSION PATTERNS REMAIN SIMILAR (CONSERVED): Took markers of limb development to see how expressed in python & lizard Found that python has limb bud, Shh expression (very little in ZPA), similar Gli-3 pattern of expression, and FGF-8 in AER ○ => BUT begins to fall apart (AER deteriorates) => Limb development arrests (AER starts to form, but Shh expression dies out = no ZPA = no support of AER) Hox genes expressed the same in between them In python → Hand2 hindlimb-specific gene dies out (like Shh) CRISPR OUT THE SHORT ZRS OF Shh IN MOUSE: Shh critical to ZPA => in python, lacking its discrete sequence (ZRS) ZRS in promotor enhancer region of Shh (regulates Shh activity, has binding elements for Hand2 + Hox genes)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Whats happens if removed in mouse? => hind limb development aborted (“serpentized” b/c no Shh = no ZPA = no AER support = no proliferation support) Small tweaks to timing + boundaries of gene expression (e.g., change in activity of single gene) = radically change morphology of outcome Lecture 22 – Limbs, Evolution, & Hoxology LIMB SCAFFOLDS: 1) Cells proliferate in bud ○ AER => proliferative/undifferentiated state plays role in limb bud outgrowth 2) Drop behind progress zone ○ As the limbs begins to grow, AER separates in diffusion distance to the ZPA => deterioration => limb begins to flatten into palette stage 3) Begin to aggregate (matrix degrades from gelatinous, hyalin-rich substrate – keeps cells separated) ○ Cell-to-cell distance breaks down 4) Collagens secreted ○ Collagen = precursor to cartilage = precursor to bone 5) Chondrogenesis (cartilage formation) 6) Bone replaces cartilage (ossification) METAPTERYGIAL ARCH: When cellular aggregations (cartilage) form, they can do so in 3 ways: 1. De novo condensation (by themselves) 2. Bud off 3. Branching In tetrapods, condensation pattern always follows a stereotypical pattern ○ No matter what organism in tetrapods = the order + pattern of cartilage condensations are always the same ○ Always occurred in same order (humerus → radius → ulna → digits forming in arch-like progression) ⇒ Metapterygial arch = the bauplan of limb development  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Homology = conserved features from human forelimb to whale fin (pattern of morphogenesis highly conserved) Fin to Limb Transition FIN TO LIMB TRANSITION: Progression from linear (fins = NO arch) to arched development of bony elements (one after another – tetrapods) ○ How did this evolve? SHUBIN, COATES ET AL. – FOSSIL RECORD ON ELLESMERE ISLAND: Ellesmere Island => deposits of rock equal to when creatures climbing out of ocean to land (fin to limb transition) ○ Zebrafish => linear progression ○ Tiktaalik => found radius + ulna – beginning of arch forming ○ Acanthostega => half in/out of water, developed more digits (extensive AER) ⇒ Now, tetrapods have less digits (don’t need more) Hox Gene Regulation HOX MUTANT MICE DEMONSTRATED LIMB DEFECTS: 1) Knock out a member of Hox 9 orthologs = missing elements of stylopod/shoulder  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 2) Knock out … Hox 10 = missing humerus 3) Knock out … Hox 11 = … radius + ulna 4) Knock out … Hox 12 = … carpals 5) Knock out … Hox 13 = … metacarpals What areas within cluster regulate this? ○ => regulatory activity of Hox directed by areas/sequences OUTSIDE the cluster HOX GENE REGULATION: 3 phases of Hox gene regulation: 1) Early, anterior Hox expression largely regulated withIN cluster (unwinding of cluster chromatin) Later expression regulated by 2 domains: 2) Control of limb, genital tract expression by 3’ remote enhancer (telomeric T-DOM) 3) Control of posterior axis genes by 5’ remote enhancer (centromeric C-DOM) C-DOM & T-DOM = Topologically Associated Domains (TADs) ○ Dynamic chromatin folds on itself = brings remote domains of chromatin into contact with Hox genes Early Hox cluster => all the genes being locally controlled => being subdivided into discrete domains by virtue of topology ➔ There are insulator sequences between these domains ◆ Bind specific protein ◆ Act as an insulator to define the boundary of how much folding can go (provide constraints within which topological changes can play a role) LATE CONTROL = T-DOM & C-DOM: Telomeric T-DOM (3’) = acts on earlier (proximal) Hox genes Centromeric C-DOM (5’) = late regulatory domain, acting upon distal-most Hox genes (late regulation in digits) These C/T enhancers work by folding into close proximity to Hox genes  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Proteins bind to these enhancers → when topology permits → come into close association with Hox genes to activate them Topologically Associated Domains (TADS) Heat Map: ○ Lots of red = enhancers curl around + make contact (with transcriptional protein attached) to affect some change on Hox cluster Hox Cluster: ○ Spacers/insulators that allow separation between C-domain enhancers coming into contact vs. T-domain enhancers coming into contact ○ Different members within Hox cluster belong together by: close proximity with each other & with remote binding proteins to turn them on/off BOUNDARIES DEFINE CHROMATIN INTERACTION DOMAINS/POSSIBILITIES:  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 T-DOM: ○ Remote 3’ enhancer → separated from Hox by 3’ domain desert (gene-sparse stretch of chromatin) 1) When 3’ enhancer bind their sequence… 2) => Topological change that permits that enhancer region & attached protein to come to close contact with Hox genes (9/10/11) 3) => Activates those Hox genes ○ Bringing in remote activity to turn on genes in a time- + space-specific manner 4) These Hox genes will turn on, in around posterior edge ○ Domains of Hox genes (overlapping in nested hierarchies) turned on one after another in posterior domain => Hox 9/10/11 turn on Shh = ZPA active C-DOM: ○ Later in development = secondary activation of Hox genes by 5’ enhancer (spaced by 5’ domain desert) Turns on later genes for a 2nd time Hox Deployment HOX DEPLOYMENT EXPLAINS METAPTERYGIAL ARCH: 2 phases of Hox Deployment (early & late phase of Hox gene activation/expression): 1) Early → look in mouse/chick/fish = pretty much the same (discrete domains to identify different parts of limbs) 2) Late → in fish, LACK 2nd period of Hox gene activation = NO metapterygial arch Phylotype (metapterygial arch) regulated by zootype (Hox genes) ○ Redeployment of Hox genes allows for patterning of metapterygial arch HOX DEPLOYMENT VARIATION EXPLAINS LIMB MORPHOLOGIES: HoxD13 = last to be activated, most distal/posterior domain Hox D13 Mutations: ○ Gain of function → synpolydactyly (fusion of digits) or brachydactyly (fewer, sometimes broader digits) ○ Loss of function → polydactyly (extra digital bony elements) Gli 1/3 = also play role in restricting posteriorization by combining inhibitory cues in the anterior region ○ If Gli 3 suppressed = anteriorizing cue lost = get extra digits  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Gonad Development Hox genes also expressed in segment-specific way in formation of genital tract ○ Combinatorial pattern of Hox genes plays role in gender differentiation IN FEMALES: Precursor to female structures = Mullerian duct ○ HoxA9 => ampullae + fallopian tubes ○ HoxA10 => uterus ○ HoxA11 => cervix ○ HoxA13 => contributes to formation of upper vagina IN MALES: ED => HoxC4 Epididymis => HoxC4, HoxC9, HoxA9 + Meis2 (partner of Hox genes) Vas Deferens => HoxA9 + Meis2, HoxA11+D10+D13 HANDS & GONADS – SEXUAL DIMORPHISM: IVF clinics => noticed people with fertility problems were slightly more likely to have asymmetric hand size (left vs. right) Index vs. Ring finger length ratio (2D:4D) was slightly more likely to be off  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Normally, Index vs. Ring finger length: In females = tend to be roughly equal In males = index finger shorter ○ Effect in genital tract = therefore, probably effect in hands (b/c Hox genes involved in both) Hypothesis = Testosterone:Estrogen levels in utero affects Hox gene expression ○ MORE Androgen (Testosterone) and Estrogen Receptors (AR & ER) in 4th digit than in 2nd Some reported links to sexual behaviour/preferences, psychology, autism, left-handedness, etc. Lecture 23 – Gender Determination The Indifferent Stage Aristotle = first to comment upon it Many organisms have a single orifice for excretory and reproductive functions (e.g., cloaca excretes feces/urine, but also gametes) ○ Birds ○ Fish ○ Some amphibians Early in development, we ourselves present only a cloaca-like structure that only later gives rise to separating openings for feces, urine, reproduction ○ Later the anal + vaginal outlets begin to separate Aristotle and Lucretius (a Roman philosopher) believed males were more evolved and fully developed than females ○ Since females retained cloaca-like opening, while males grew a phallus as the opening "sutured up” Cloaca = primitive vagina (females represented arrested form of development) ○ Reflected societal roles of the time, where women were denied rights like voting and were treated as property EXTERNAL APPEARANCE OF INDIFFERENT STAGE: Developing external genitalia At indifferent stage: ○ Genital opening separated from anus ○ On either side of it have genital swellings (labial/scrotal swellings)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 In males: lips/swellings suture up, glands begins to elaborate, and phallus to elongate ○ Scar down the phallus, around the scrotum (scar runs up between scrotum + phallus) = remnant of suturing In females: the labial swellings give rise to the labium majora + labium minora (the lips surrounding the vagina) ○ No elaboration of glands beyond what was already present Gilbert: ○ Adam’s rib from which Eve was derived => mistranslated ○ Rib was actually central support member (penile bone) When God took a bone out, was removing penile bone ○ Primates are only mammals that LACK a penile bone → erection is caused solely by hydrodynamic pressure manipulation INTERNAL PLUMBING OF INDIFFERENT STAGE: Within the abdominal cavity = 2 sets of plumbing in parallel: ○ 1) Wolffian ducts → give rise to male reproductive tract Wolffian ducts are part of intermediary structure in the course of kidney development (mesonephric duct) ○ 2) Mullerian ducts → give rise to female reproductive tract One or the other survives (the other withers away + degenerates depending upon process of sex determination) Gonad Development FIRST STEPS TO GONAD DEVELOPMENT: 1) Primordial Germ Cells (PGCs) migrate to genital/gonadal ridge from allantois (external sac) via hindgut ○ PGCs = give rise to spermatogonia/oogonia ○ Come from outside of body (allantoic sac), migrate in through the hindgut to the genital ridge (allantois → hindgut → genital ridge) 2) Mesonephric mesenchyme migrates to genital ridge  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ At the time kidney developing, intermediary structure forms called mesonephros → contributes cells (mesonephric mesenchyme) that also migrate to ridge 3) Cells proliferate and assemble into sex cords Examining PGCs & Genital Ridges in Mouse: ○ Take a mouse => open up its gut & remove all the viscera ○ As looking down into the abdominal cavity, see ridge covering where spine is at the bottom of the cavity => at either side of that, 2 accessory ridges (genital/gonadal ridges) ○ If do this to embryo early enough => see small clear popcorn-like kernels of cells (PGCs) Not yet embedded in either testes or ovaries => free Have broad distribution along the genital ridge (some organisms, reach all the way up to head region) FIRST STEPS (COMMON IN BOTH GENDERS): 1) Nephric/kidney system undergoes change over span of its development => 3 different nephric systems: 1. Pronephros = primitive system ○ Glomerular-like structures that represent the connection/folding between bloodstream + what will become kidney glomeruli ○ Found in very primitive organisms ○ Only appears transiently during our development When it forms, begins to degenerate/dismantle and give rise to 2nd structure 2. Mesonephros / Mesonephric Ducts ○ Present in primitive organisms ○ Dismantles as the 3rd structure assembles 3. Metanephros => becomes the mature Kidney 2) As the mesonephric system begins to dismantle => its mesenchyme migrates to the genital/gonadal ridge ○ Cells are redeployed + migrate + proliferate (contribute to mesenchyme of limb bud + gonads) 3) Mesonephric mesenchyme cells assemble into rows (columns) called the sex cords ○ In females: organization to limited extent ○ In males: more pronounced organization that consolidates  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Why develop all 3 structures, when 2 degenerate? ○ Ernst Haeckel: “ontogeny recapitulates phylogeny” = embryogenesis goes through the same steps that were developed throughout evolution (development is recapitulating evolution) An organism’s development will take it through each of the adult stages of its evolutionary history/phylogeny Why? Metabolically expensive to build structures that then have to be taken apart ○ 1) Perhaps a reflection of layers of genetic networks/modules that lie on top of one another (e.g., can’t progress to the pair-rule genes unless you have gone through the gap genes first) Deploy one suite of genes that set the circumstances for the next suite of genes to become active, and so on ○ 2) OR… may be that the early embryo has an accumulation of cells that are packed within a certain density (this mass of cells in 3 dimensions = something like a tuning fork) Different sizes + densities of cell packing are going to resonate with morphogenic fields in different ways Different reaction-diffusion systems (e.g., Turing’s reaction-diffusion model) resonating with different numbers + packing densities of cells → would predispose to the formation of one tubule set, then another, then finally a 3rd INDIFFERENT STAGE IN 3D: Ducts (Wolffian + Mullerian) go out through what will become the umbilical cord, which connects to the allantoic sac or vitelline duct ○ PGCs migrate in through allantois and distribute along the genital ridges Mesonephric ducts feeding off much larger tubule (Wolffian duct) ○ When mesonephros deteriorates, only the Wolffian duct remains which serves as primordial tissue for development of male reproductive organs Arrows denote migration of dismantled mesonephric cells to the genital ridges, which will come to play different roles in males or females  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Decision Time – Female or Male? MALES: 1) PGCs migrate to genital ridge 2) Mesonephric mesenchyme migrates to genital ridge 3) Mesenchyme proliferates and forms substantial sex cords ○ Sex-cords = well ordered → consolidate 4) Cells of genital ridge (contribute slightly to cortex) become: thick interstitial tissue, Leydig cells = future source of testosterone 5) Sex cords eventually form into Sertoli cells of seminiferous tubules = future source of MIH/MIS (Mullerian Inhibiting Hormone/Substance) ○ Sex cords are elaborating => will form long files that will roll up to form seminiferous tubules ○ Sertoli cells help nurse spermatogonial cells through their maturation process to form spermatozoa, which are extruded into the lumen of the seminiferous tubules 6) MIS/MIH inhibits Mullerian development = Mullerian ducts degenerate 7) Elaboration of the interstitial tissue between the seminiferous tubules where the Leydig cells reside => start secreting testosterone ○ Testosterone absolutely neccesary for the survival of Wolffian duct tissues 8) Wolffian ducts to differentiate into: epididymis (segmented), seminal vesicle, & vas deferens FEMALES: 1) PGCs migrate to genital ridge 2) Mesonephric mesenchyme migrates to genital ridge ○ Cells embed/organize into medulla of gonads 3) Mesenchyme abortively form sex cords (doesn’t proliferate much) ○ Starts to form sex cords, but DON’T persist 4) Cells of genital ridge assemble into cortex/cortical cells => proliferate to become granulosa cells (support development of the oocytes) ○ Periphery of gonads formed by cortex/cortical cells of genital ridge 5) In the absence of testosterone => Wolffian ducts degenerate ○ No support for development of Leydig cells = NO testosterone = degeneration of Wolffian ducts 6) Mullerian ducts differentiate into: fallopian tubes, uterus, cervix, & upper vagina ○ Sex cord degeneration + formation of granulosa cells = NO Sertoli cells = absence of MIH/MIS = Mullerian ducts persist  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Experiments – What Directs Gender Determination? HORMONES? FROG EMBRYO EXPERIMENT: 1) Taking amphibian (frog) eggs => culturing them in petri dishes 2) Adding estrogen/testosterone to water ○ Add estrogen: ALL embryos developed to look like anatomical females ○ Add testosterone: ALL embryos developed to look like anatomical males (irrespective of what genotype is – XX or XY or ZW or ZZ) Some frogs determine gender like birds (ZW heterogametic females, ZZ males) But some, like spotted leopard frogs are like us (XX females, XY males) PARABIOSIS: Frog Embryo (Rana pipiens – XX, XY frog) 1) Scratch ectoderm off developing embryos ○ 2 scratched embyros together in petri dish (plasticine on bottom, make well) 2) Press them together while healing 3) = Conjoined twins ○ Mixing genders: XX + XY combinations always develop anatomically MALE ➔ Something circulating in bloodstream → hormone that is driving differentiation of gender to males Genetic Gender Expected Frequency Observed Phenotype XY XY 25% Both Male XX XX 25% Both Female XX XY 25% Both “Male” XY XX 25% Both “Male” = 75% male SIMILAR CHIMERAS IN MICE: 1) Chimera => genetic manipulation of stem cells ○ Mix/press them into blastula-stage embryos (some cells would contribute to germ-line) Can do this with entire relatively intact blastula-stage embryos 2) Strip them of the membrane that surrounds them  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 3) Press them together => they form 1 big ball from 2 separate early-stage embryos 4) Same results = 75% male outcomes ○ Masculinized genetic females are NOT fertile Anatomically male embryos => only XY cells would contribute to germ line (XX are constitutionally prohibited from becoming sperm) Similar to Freemartin cattle (mixed-gender twins with shared placenta) ○ Natural phenomenon – rare, but happens ○ Testosterone in male will circulate and disrupt gender differentiation of female twin (will not develop normally) WHAT CONTROLS GENDER? Just hormones? or something else? ○ Problems with hormone studies: estrogen + testosterone don’t start circulating until well AFTER the mesonephric mesenchyme has been recruited So what is it that’s getting this gendered difference in hormone production? Human Chromosomal Anomalies – SRY STUDIES IN MOUSE/HUMANS – CHROMOSOMAL STATUS VS. ANATOMICAL PHENOTYPE: XY missing short arm of Y chromosome => Female ○ Despite being XY, anatomically female XY missing long arm of Y chromosome => Male ○ Developed normally as males short arm Y XX translocation => Male ○ Translocation had shifted part of the Y chromosome to one of the X chromosomes If X chromosome had acquired the short part of Y = anatomically male long arm Y XX translocation => Female XO (no Y chromosome) => Female SHORT ARM OF THE Y CHROMOSOME CONTROLS GENDER: Therefore, something on the Y chromosome that confers gendered status, that drives the “ball” in masculinizing direction:  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Comparing different short Y arm mutants —> narrowed to 6 different genes What is the factor produced by the small arm on the Y chromosome? ○ SRY (sex region Y) => a high mobility group class of TF (includes SOX genes, of which SOX9 is a major player in gonad determination) Belongs to same family as Hox9 NOT homeobox gene, NOT leucine zipper or steroid zinc finger domain Have specific DNA-binding domain that separates them from other families ○ Expresses on the ridge in males Early in development around the time mesonephric mesenchyme is being recruited ○ Loss of function mutants fail to support male gonadal development (= feminization) ○ Ectopic expression in females causes male gonadal development (= masculinization) The Genetic Hierarchy of Action 1) SRY → turns on SOX9 ○ SOX9 = also member of high mobility group TFs (also involved in limb development) 2) SOX9 → Steroidogenic Factor (SF) → Leydig → Testosterone ○ SF → SOX9 (auto-activates) 3) SOX9 → FGF → Sertoli → MIH/MIS ○ FGF encourages proliferation that leads to the expansion of sex cords → which leads to Sertoli cells → and the production of MIH/MIS Males vs. Females IN MALES: 1) High SRY = high SOX9 = high SF = high FGF 2) High mesonephric recruitment & proliferation => sex cords grow 3) LOW cortical recruitment => Leydig cells develop  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Cortical cells recruited from the genital ridge begin to differentiate into interstitial tissue, including Leydig cells 4) High SF = Leydig cells produce testosterone = Wolffian ducts persist 5) High FGF = High Sertoli & MIH/MIS = Mullerian ducts degenerate ○ FGF supports proliferation/recruitment of mesonephric mesenchyme & elaboration of sex cords => give rise to Sertoli cells 6) Wolffian ducts form the: testes, epididymis, seminal vesicle, vas deferens 7) Anal and “cloacal” openings separate 8) Labia-scrotal folds suture => Scrotum develops => Glans tops a growing Phallus IN FEMALES: 1) No SRY (b/c no Y chromosome) 2) LOW mesonephric recruitment => sex cords dissolve 3) Higher cortical recruitment => granulosa cells develop 4) NO SRY, SOX9, SF = NO Leydig cells = NO testosterone = Wolffian ducts degenerate 5) NO SOX9, FGF = NO Sertoli cells = NO MIH/MIS = Mullerian ducts persist 6) Mullerian ducts contribute to: ovaries, fallopian tubes, uterus, cervix, upper vagina ○ PGCs migrate to gential ridge → genital ridge cells (cortical cells) surround them → become embedded → ovary beginning to form 7) Anal and “cloacal” openings separate 8) External features elaborate => outer labial folds (labia majora), inner folds (labia minora), clitoris Female SRY? – Dax1 & XIST Both are encoded or reside on the X chromosome DAX1: Steroid nuclear receptor family (like SF) Gene resides on X Chromosome – XY have one copy, XX have 2 ○ Males have 1 copy, females have 2 XY males with autosomal translocation of Dax1 can be dose-dependent sex reversed NO functional DNA binding domain Binds to and competitively removes SF from action => inhibits Testosterone ○ Acts as a cofactor → probably binds to/heterodimerizes with SF, removing it from action  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 XIST: XIST does NOT make protein X chromosomes = large + contain many genes, while Y chromosome = very small + carries few genes XIST = X inactivation gene → removes one of the X chromosomes from activity ○ XX females must inactivate one X chromosome (randomly at blastula stage) to attain gene dosage compensation → if both active = embryonic lethal b/c so many genes ○ XIST expressed from both X at first, but then more from one than the other One of the X chromosomes expresses more XIST – begins to coat the surface of the X chromosome (coated with XIST RNA) XIST expression from the other X chromosome begins to fade out ○ XIST RNA gloms onto X and serves to nucleate epigenetic repression => Barr body RNA = nucleation signal for epigenetic processes to close down/compress/inactivate the X chromosome ➔ Results in a Barr body (radically compressed X chromosome, very little can be expressed) There is leaky expression from this inactivated X chromosome (DAX1 may be one of the genes that is expressed from this Barr body) Calico Cats: ○ Calico cats have patchy coat colours (white, grey, and orange) due to X-linked pigment genes Grey+orange colours determined by pigment genes on the X chromosome ○ During the early blastula stage of development, one X chromosome is randomly inactivated in each cell/blastomere In some areas, the paternal X chromosome (from the father) is inactivated In other areas, the maternal X chromosome (from the mother) is inactivated ○ This random X inactivation leads to patches of cells with different pigment expressions (clones go on to populate body), which form the distinctive calico pattern ○ Since X inactivation involves two X chromosomes, most calico cats are female (XX)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 Rare male calico cats (XXY) exist if they have an extra X chromosome XIST EXPRESSED IN FEMALE NUCLEUS: Did riboprobe in situ hybridizations to illustrate how the XIST locus operates Female nucleus → 2 X chromosomes: ○ DNA stained with intercalary dye — propidium iodide (commonly used to disclose where DNA is located) ○ Within this nucleus, chromosomes not entirely heterochromatic (not a metaphase spread) Probe made from labelled cDNA for XIST (labelled with yellow fluorescent dye) ○ Since female, 2 X chromosomes = 2 copies of XIST gene Also localized with riboprobe in situ hybridizations, RNA transcript that has been delivered by these genes (both loci) ○ Accreting around only ONE of those X chromosomes (marking that chromosome for compression by epigenetic proteins) If XIST non-functional = both X chromosomes active = embryonic lethal Examples of Disorders/Syndromes Related to Gender 1. ANDROGEN INSENSITIVITY SYNDROME: Lack functional testosterone receptor => XY individuals make testosterone, but cells cannot perceive it (don’t receive the signal) Cells of the Wolffian ducts are not receiving the support they require = degenerate (NO Wolffian AND Mullerian ducts) ○ 1) High SRY, high SOX9, high SF, high FGF ○ 2) High mesonephric recruitment and proliferation => sex cords grow ○ 3) Low cortical recruitment, Leydig cells develop ○ 4) High SF = Leydig cells produce testosterone ○ 5) Testosterone NOT sensed = Wolffians degenerate = NO testes, epididymis, seminal vesicle, vas deferens ○ 6) High FGF = high Sertoli and MIH/MIS = Müllerians degenerate Will have labia minora + labia majora forming, but Mullerian ducts didn’t form = NO fallopian tubes, uterus, cervix, upper end of vagina  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ 7) Anal and “cloacal” openings separate ○ 8) External = feminine, but blind end vagina Anatomically FEMALE, genotype is XY —> not meiotically predisposed to making oocytes STERILE b/c there hasn’t been neccesary hormonal support for production of gametes 2. TURNER SYNDROME – XO INDIVIDUALS: XO = 1 X chromosome, but NO Y or 2nd X chromosome ○ Lack Y chromosome = NO SRY expressed ○ Infertile b/c missing a sex chromosome, meiosis cannot proceed normally (can’t produce gametes in normal fashion) External Phenotype = FEMALE 3. KLINEFELTER SYNDROME – (X)XXY INDIVIDUALS: Male (XY) individual with extra X chromosome ○ Have Y chromosome = have SRY ○ Have multiple X chromosomes = multiple copies of XIST = multiple Barr bodies Gene dosage effects controlled to some extent, BUT… Infertile b/c Barr bodies are a little bit leaky = gamete maturation will be abnormal (also b/c there is an abnormal number of chromosomes) External Phenotype = MALE 4. SWYER SYNDROME – XY INDIVIDUALS BUT NO SRY: XY, but SRY mutated to the point of disfunction (b/c of an expansion of trinucleotide repeats – just like Huntington’s disease, etc. => progressive inactivation of SRY)  Lecture 17 => 18 => 19 => 20 => 21 => 22 => 23 => 24 => 25 => 26 ○ Infertile b/c neither ovarian + testicular tissues form – due to lack of support (have streak gonad instead) External Phenotype = FEMALE Alternative Modes of Sex Determination 1. FLY: Sex chromosome to Autosome (X:A ratio) determines anatomical differentiation ○1.0 = female ○0.5 = male Although XY => Y only contains fertility factors – NOT involved in gender development per se ○ Y chromosome just helps when sperm are specified to help them mature XY = only 1 sex chromosome + 2 autosomes → ratio = ½ = 0.5 2. NEMATODE WORM – C. ELEGANS: Ratio of X:A determine whether an individual will be hermaphroditic or male: 1) Hermaphrodite ○ 5 pairs of autosomes, 2 X chromosomes ○ Can self fertilize 2) Male (more rare) ○ 5 pairs of autosomes, 1 X chromosome Half of the sperm they produce carry X, half carry null 0 ○ Must mate 3. FISH: Some, like salmonids, are XY (males heterogametic) or ZW (females heterogametic – many birds/reptiles) determined Others, by social status, maturity, and size => reef fishes (e.g., clownfish) are sequential hermaphrodites ○ 1) Protoandrous → start male, but transform to females later in life Testes change shape + function + histological characteristics & take on the attributes of ovaries => mate with smaller males ○ 2) Protogynous → start female, but transform to male later  Lect