BIOM3550-Final (2) PDF

L17: Hoxology Recall: fruit fly development Nuclear division without cellular cleavage Done in fruitflites Can see something happening in the early stage Looking at an embryo dividing on basis of centrolecithal, meroblastic cleavage for the first 10 or so cleava...

L17: Hoxology Recall: fruit fly development Nuclear division without cellular cleavage Done in fruitflites Can see something happening in the early stage Looking at an embryo dividing on basis of centrolecithal, meroblastic cleavage for the first 10 or so cleavages Can track nuclei and see where they are going Stage 11-12, beginning to partition 12 first cells that are homoblastically cleaving - pole plasm Stage 13 they begin to completely enclose, all cells are partitioned Maternal effect, gap genes and some early pair end genes are all expressing before the cells are cellularized Second polarity, they are expressing in fully enclosed cells When the hox genes start expressing is 13-14 cell division  Cytoplasmic polarity being induced by maternal genes in a one cell organism Pair end - fully partitioned, multicellular cells. This is where hox genes and selector genes start to give specific addresses to cells within those areas Done by reporter constructs/antibodies Another scientist also predicted that antibodies presented in the top panel in discrete bands that directly match what Kaufman was predicting Hierarchical arrangement of genes forming progressively more and more discrete and well defined genes  Take promoter from various genes and can hook it up to a reporter gene. Where's that gene expressed? Fly segments talking about stereotypical patterns withs structures that directly map larva or discrete areas in the fly T2 holds the wing and a limb - derived from imaginal disc, both are appendages, biramous segment T3 holds the halteres - segment that is biramous, has both a pair of wings and limbs that are emerging Each segment has discrete identity and specific attributes that are possible to discern Head is developed from fusion of somewhere in the neighborhood of 6 segments - all segments except the top one carry imaginal discs for limbs. For the most part, shrunk down to become the small feeding mouthparts One segment where it migrates to the top and become antenna - not biramous Homeosis talking about misaddressing of one segment so it behaves like another Homeotic mutation: Homeosis  A mutation induced T3 to develop a pair of wings instead of halteres Atavism: evolutionary throwback. Structure that forms that is more formative of a primitive than the more modern evolved state so its the reappearance of a trait that was present in an ancestral species but lost in its modern descendants Flies ancestor had two pairs of wings, with this mutation T3 is behaving as T2 and growing wings instead of halteres - good example of an atavism Timing and placing of homeotic selector genes plays a role in evolution Homeotic mutation: Homeosis (antennapedia) Homeotic mutation: causes one body part to develop as a structure normally found on another segment Antennapedia gene is a homeotic selector gene from the Hox family Antennapedia is normally expressed in thoracic region where it promotes the formation of legs, but it is is expressing up in the head - ectopic expression A: is head of the fly, the head develops antenna on either side of the midline B: antenna looks a lot more like legs than antenna Homeotic transformation Segmentation (metamerism) Metamerism: The division of the body into repetitive units called segments or metameres Cells organized into groups Groups can act independently (if you change the address of one it can behave as that) Genes act to regulate segment formation Genes act to regulate segment address  Genes act to regulate segment differentiation (head vs trunk) Selector genes involved are transcription factors Selector genes: a subset of transcription factors that determine the identity and function of specific segments Homeobox genes (TF factors can include leucine zippers, paired box genes, zinc finger domains) and TF factors that have a subset of TF that are homeobox genes Homeobox genes are one category of TF that are themselves sub divided into sub families. Some belong to PAX, AREX, HOX. HOX is a subcategory of homeobox genes which is a subcategory of TF. Also does not mean it is only coding a homeobox. Homeodomain is characterized by 3 helices that bind the major groove of DNA. some homeobox are activators, some are repressors - Hox genes are usually activators Encode proteins that bind DNA and regulate other genes Characterized by 3 helical domains Helix 3 binds major groove of target DNA sequence Turn target genes off or on Dimerization domain associated with homeodomain allowing it to interact with other homeobox domain Combinations of heterodimerization or homodimerization will regulate how easily the key fits the lock How easily the genes products interact with DNA sequences of target genes Selector genes (HOM, Ant, or Hox complex) Hox genes in fly are spatially collinear Spatial collinearity: refers to the arrangement of Hox genes on the chromosome in a sequence that corresponds to their expression domains along the body axis. Arranged in 3’ to 5’ direction that correlates with their expression domain Some of these expression domains overlap These genes when they express, express in fuzzy bands that over the span of a few minutes to hours become very sharp Our pattern of expression is different Pattern of loose expression that becomes progressively more refined Orthologs in mammals  Gene duplication that increased the number from 7 to 13 genes and increased clusters from 1 to 4 Not all clusters have all the genes Hox orthologs: genes on other clusters. Would be b2 for a2. Genes in different species that evolved from a common ancestral gene and retain similar functions. Hox paralogs - genes inside. Genes within the same species or genome that arose through duplication and may diverge in function. A4, b4, c4, d4 are more closely related than a5, b5 for example Orthologs very high conserved sequences - providing a strong function Many genes share regulatory domains - in part why they have remained so unchanged as clusters over time Protein products will autoregulate but also can regulate some of their neighbors Highly conserved response elements, DNA regulatory motifs or other TFs RARE - sensitive to receptors that bind vitamin A. vitamin A sensitivity - early genes tend to be more sensitive Product of duplication 13 theoretical paralogs, each cluster is missing a couple Looking at 4 clusters combined, at least one cluster is expressing one of those genes Might be evolutionary room to perform new structures  Dorsal ventral patterning in flies is conserved with respect to hox genes 3’ genes like labial, hox A1 are always expressed near the head end 5’ like hoxB13 expression more posteriorly In mammals not just spatial, but also temporal colinearity 3’ is early expressing anterior, and 5’ is posterior and late expressing Hox genes: In mammal, genes are express in a nested hierarchy of overlapping domains The early expressing genes express anterior and expression domain extends a lot posteriorly Unlike fly which start fuzzy, in mammals there is sharp anterior domain from the gecko As hensen's node is moving down, hox A1 is expressing a little bit less as it goes down, then hox 2 turns on, sharp anterior expression domains, hox 3 turns on node is further down Expression domains many of them overlap - depending on if you're looking at cervical, thoracic - there's a different combination of genes overlapping Genes specifying identity in a combinatory way If you assemble all 39 hox genes, the unique combination of genes seem to be spaced at roughly 2 slides Somite fate Occipital somites 4 pairs Occipital bones of skull Cervical somites 7 pairs Neck Thoracic somites 7 pairs Ribs attached at sternebrae 6 pairs Rib ends free floating Lumbar somites 5-6 pairs Lower back Sacral somites 4 pairs Pelvic region Tail somites Variable Tail Each somite will be acted upon by hox genes and given a specific identity End result, end up with different fates for different somites Have 4 somite pairs that will contribute to occipital bones If hox genes are playing a role in specifying, then you would expect mutation in hox genes to alter the fate of whether or not a somite develops into a cervical somite (and what pair) Mutations or evolution to change the number and type  Hox genes first characterized, gene knockout technology was coming into broad use If we knock out in fly and get homeotic mutation same should happen in mouse Ectopically the second copy of the gene would be driven by a promoter that would express where it wouldn't normally express - ex: take a posterior gene and express it into the head. With fly, if you do that you would expect posterior prevalence rule (master of everything and should override the anterior program). When you ectopically do this, it posteriolizes the structures If you knockout a gene? One of the posteriolizing cues will be absent. Everytime the gene turns on it is a later and more posterior cue. Anterior combination genes hold its way. So what would have been regulated by that mutation gene, is remaining under the sway of the previous. Results in anteriorization of the information in the domain it is normally expressed in Ectopic express = gain of function Knockout = loss of function Gain and loss of function explained WT hox genes - hox olive, teal, green, yellow turn on successfully over the span of development. Specifying more and more posterior domains If there is a loss of function - knockout hox green, hox teal will continue to provide that information and that domain will perform under sway of hox teal, giving it more posterior like characteristics Hox yellow in head (usually anterior) will make a structure that is posterior Deletion of a hox gene -> lost posterior cue=anteriorization This loss means that the affected body segments or regions cannot properly develop posterior traits  Instead, they "default" to a more anterior identity because anterior Hox genes (which specify head or middle body structures) remain active. Anterior cue appropriate to what made 13 thoracic gets reiterated The next somites behave as if they are T13 Have a 14th thoraxis vertebrate Removal of posteriorization gene has caused anteriorization of structures they normally would have covered Ectopic anterior expression of a posterior hox gene -> posteriorization Take posterior gene and express anterior Ectopic expression refers to the abnormal activation of a gene in a location where it is not normally expressed If a posterior Hox gene is expressed in the anterior (front) part of the body, this disrupts the normal developmental pattern. The posterior Hox gene can override the anterior identity signals and impose posterior characteristics on structures that would normally develop as anterior In this example, Express a cervical gene to occipital somite - cranial bones disappear and get something that vaguely looks like it Posterior genes posteriorilzie occipital somites so instead of forming occipital genes Hox Atavism An atavism is a recurrence in an organism of a trait or character typical of an ancestral form - an evolutionary throwback Middle ear conductive bone morphology more typical of reptilian forms Normally ribs form and first 6-7 meet at midline, aggregation of cells to form sternum Ribs instead of growing out at midline, they interdigitate, ends are between each other Older specimens have 11 cervical vertebrae: ancestral Hox act on all somites? Older dinosaurs have 4 more cervical vertebrae (11) Lack a cranial vault One of the implications of these hox mutation studies - slight tweaks to when and where they express might have played a role in evolution of structures If you delay onset of posteriolizing cues in hox genes, when somites are being specified leave room for the early somite form to do something new Delayed onset of hox genes patterning of somites is what permits the occipital somites to form cranial vault instead of cervical vertebrae More recent skeletons have 7 vertebrae Likely Hox turned on later to permit occipital somites to contribute to occipital bones (enlarging cranial vault to accommodate brain)  Larger cranial vault As evolution occurs and brain enlarges permitted by cranial vault Cervical vertebrae have ribs growing out More or less have same genes, what changes is when and where they are expressed Chick vs mouse - both have 4 pairs of occipital somites but average chick has a longer neck Hox gene expression domains are delayed in chick compared to mice In chick, more somites in the neck adopt a cervical identity before transitioning to thoracic identity = greater number of cervical vertebrae in chicks contributing to longer neck Hox 5 and 6 is cervical to thoracic for us same as in chicks, but chicks delay the activation of hox 6 so there are more cervical vertebrae Boundary between hox 5,6 and 9,10 - at these levels the appendages (limb buds) arise. In us and mouse, boundary of 5 and 6 is transition from cervical and thoracic but where forelimb buds develop - in chicks delayed Thoracic to lumbar - 9,10 hindbrain will form In chicks the boundary, this transition occurs further down the axis than in mice, which also contributes to differences in proportions  Nested hierarchy of overlapping domains - gives unique patterns Genes found particular to phylotype Tweaks in timing at early stages whether it develops to whale, chick…. Phylotype: vin Baer, Hackel L18: Problems with the Hox code model - specificity Problems Hox protein target specificity If they all recognize a similar DNA sequence motif (TAAT) how can they each specify a different vertebral identity (ie; activate different genes at different times in each place)? 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. Target specificity Third helix of homeodomain binds to and affects major groove of target gene regulatory sequence (TAAT) Does the rest of Hox protein affect shape in a subtle way to affect specificity of action? Does flanking sequence of target gene motif play a role in modulating access? Are there partner proteins that play a role in guiding specificity? Hox genes form homo and hetero dimers Homeobox genes have their own DNA binding motifs, the spacing between the hox binding site and pbx binding site would lend some specificity as well as the conformation of the dimer itself When you have a homo or hetero dimer it changes the shape of the key that fits into the DNA binding lock Hox protein structure/partners Hox proteins dimerize with: Each other Exd/Pbx MEIS Hexapeptide (AKA pentapeptide region) binds partner protein - regulates the ability of these partner proteins to interact with the hox proteins Linker endows this binding site with its partner specificity Distance between homeodomain and Exd/Pbx/MEIS binding region varies Homo and hetero dimers Amplification of number of protein combinations that you can conceptualize 39 hox genes expressing differently in combination with Pbx 1,2,3…, MEIS 1,2,3…. Different DNA target sites is becoming larger and larger Some sites will be very attractive and some less with a gradient in between As you go from hox 1 to 2 to 3… the distance between hexapeptide and homeodomain increases. Shape and specificity will be different Testing different structural 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 Put reporter plasmid and a Hox gene expression plasmid into cell line Lyse cells, provide substrate Analyze color/light output – do alterations to target sequence change output? What changes to the Hox gene alter output? Potential target gene e.g. actin - have promoter and hook it up to a reporter e.g. lacZ, going to use the reporter in the cell system to generate a colorimetric/photometric change that you can measure with the machine. Alter different parts of target sequence (start big work small) knock out e.g. 5’ most 1000 bp then 1500bp then 2000bp and see if you still had activity of promoter gene - localize where critical gene is, where hox interacts. When you can narrow it down you can do site directed mutagenesis to change the site and see what the effect is Can also change sequence of hox gene you are using Reporter constructs usually help on 1000s of bp but can narrow it down Example: Luciferase assay Have gene for hox gene of interest, let cells grow, lyse them, apply them with luciferase, have all these difference constructs - hox variants Drawback of this approach: cells in which you are testing, you hope are naked of the hox protein that you are interested in testing and not some other homeodomain protein that occurs naturally that will interest. Also hoping that some partners that interact with hox aren't interacting 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 Examples of Mutations to Target Promoter Sequence Deletion mutants to see WHICH TAAT sites are important Mutate TAAT site Mutate TAAT flanking sequences Mutate dimerizing partner DNA binding motif Dnase footprinting assay Add target DNA sequence and transcription factor (Hox) Incubate at physiological conditions Add DNase for a short while Enzyme will generate random lengths of digested DNA EXCEPT where protected by Hox Works by: have a sequence of DNA usually on the order of 200-300 bp that you have lifted out from promoter sequence. Have target sequence and incubate it with a lysate  from cells in which you have been expressing Hox A2, allow hox A2 protein to bind to TAT motif if it is present and then add Dnase. Dnase will start eating up the DNA except where it is covered by and protected by the protein When you add Dnase and don't let it chew for long, arrest the degradation very quickly. Some highly degraded, some not. As it's chewing back, it ends up on a gel with a ladder. Have a sequence, binding site for POI, degraded it, looking for confirmation that it binds there Sometimes get surprises where you have multiple binding sites for hox protein in this promoter sequence, usually where this phenomenon occurs is when there are several of these sites very close together Cooperativity Binding cooperativity - if you had multiple sites and proteins bound to TATC sequences, the reporter or target gene would be turned on a little bit more which each transcription  factor that binds. But if were acting to unwound the chromatin to make it more accessible, would be cooperativity Worked so well that there were cryptic sites in DNA construct that he was using as a target Availability of multiple copies of a protein and interaction with multiple target sites facilitates activation in the gene It is not just which hox gene is on in a given cell but how much that hox gene is on Gene mobility shift assay (GMSA) Good for target sequences of 40-60 nucleotides Would have identified computationally or on basis of reporter assays, areas that are of real significance Design a complementary strand to set off - label using p32 these fragments Incubate your labeled oligonucleotide with cell lysate in which you have expressed protein. Incubate in a tube near to physiological concentration of salts. If hox A2 binds to this particular sequence - complement. Radioactive DNA bound to protein Can alter either the sequence of oligonucleotide or can alter the gene that encodes the TF like Hox A2 Would mix in varying concentrations and run on a gel, saran wrap, x-ray film over top, dark band at bottom which is unbound oligonucleotides that has not been bound by a TF If bound by a protein, bigger and different charge and migrates through the gel slower GMSA continued  Add cold unlabeled oligonucleotides to test specificity to see if it can compete Can add antibody to Hox A2 to see if you get a supershift - now see not just unincorporated probe, have hox protein bound to a huge antibody specific to it - can reassure that what you're looking at is indeed Hox A2 Knockout mice Cell lines derived from inner cell mass that are used - can grow them in culture Would incorporate a gene construct to disrupt the target gene for a TF like Hox A2 Would alter the gene construct, replace exon that contains start codon with a small gene for neomycin resistance So have a Hox A2 gene that is normal in all respects but ex: missing its first exon that is replaced with neomycin resistance - so it Hox A2 cannot be produced If it were expressed like Hox A2, would portray neomycin resistance and would not be produced in Hox A2 protein At low concentration, DNA construct would align and get a crossing over - end result is homologous sequences will switch out - replace normal sequence with mutant sequence Have one or two ES cells that are mutant, maybe neomycin resistance encoding construct has integrated some random place, or where you'd hope  Can treat these cultures with a lethal dose of antibiotic - and only those cells with neomycin resistance gene will survive, all others wiped out Have a collection of cells that you know carry the Hox A2 that has been mutated to express neomycin resistance instead Have to sort out which has replaced the exogenous hox A2 vs which have a random integration somewhere else Populate or expand population and inject into a blastula stage embryo, they will join the inner cell mass and contribute to the embryo that derives Don't know which cell/tissue type, mutant will contribute to, but hope some progeny will contribute to germ line Knockout mice Challenge: discriminating between mutant cells lines that have incorporated and those who have incorporated randomly Have other markers that help you discriminate Have a knockout mouse and can look to see what it is missing 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 transcription factors such as the homeobox gene exd/Pbx or MEIS 4. Amino acid sequence flanking the DNA binding domain 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 transcription factor matters to: - Form homodimer - Form heterodimers 7. Concentrations of Hox protein matter – cooperativity Problems Mutant Phenotypes Not Always as Predicted: Expect transformations at anterior border of expression If you know where genes usually express themselves, why do they sometimes get effects in different places? 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, and don’t perturb just at the boundary 4. Mutant phenotypes sometimes present in re-iterative manner 5. Deletion of entire cluster has surprisingly little effect - None of these exceptions is consistent with the notion of the Hox code specifying precise spatial mapping identity (ie a particular Hox gene specifies somite contributing to vertebra T4 etc.). Anomalies Hox genes normally exhibit sharp anterior boundaries of expression Anomalies tend to occur not in middle, but pushed down to a junction to areas of morphology transition Phenotypes that cluster around areas of not necessarily where you'd expect on basis of expression pattern of the gene Hox genes giving whatever you are now, one segment more posterior According to the Hox code model, this is where new positional (posteriorizing) information should be installed and phenotypes should change Irrespective of normal Hox boundary, phenotypes map to borders of body transition (cervical to thoracic, thoracic to lumbar etc.) Some explanation 1. Transformations do not always occur at Hox expression boundary Explanations? Somite resegmentation 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, and especially cannot explain why, when the entire Hox C cluster is deleted, there is little effect on the spine. Problems with neo-cassette Homologous KO targeting vector introduces and artifact Neo resistance selection marker acts as a genetic insulator – activity of adjacent genes no longer coordinated. This is a product of: Genes sharing regulatory elements Chromatin domain integrity being violated and not behaving properly (architecture is abnormal, epigenetic factors altered) Timing of gene activation is thrown off, and since there is normally cross talk… this too is disrupted Solution? Cre-Lox removal of selectable marker  Conclusion: what the hox genes are doing is not just providing spatial identification but temporally generic cues to be one segment more posterior. Clusters are dividing temporal cues in a coordinated fashion If you introduce neomycin cassette marker, affect timing of that particular cluster 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 from other Studies Engrailed – a homeobox repressor transcription factor Causes big neural problems when mutated in fly When knocked out in mouse, almost no discernible effect (subtle brain defect and learning disability) There is a second Engrailed gene in mouse En1 and En2 turn on at slightly different times during brain development En1 and En2 share only 55% amino acid similarity If En2 is expressed in place of En1 in a En1-/- background – phenotype is rescued L19: Body structure, segmentation clock and Hox Reprise Why did Hox transformations occur at zones of major body transition? Simple and complex signals Transformations often occurred at transition between cervical to thorax, thorax to lumbar et. Why? Theme of 6 or 7 somites/segments between anomalies in mammals Periodic segments like tagmata 6-7 Prosomeres fore and midbrain (more next lecture) 7 Rhombomeres (hindbrain segments - more next lecture) 7 Cervical somites 7 Thoracic somites with ribs meeting at sternum 6 Thoracic somites with ribs free floating at ends 5-6 Somites Lumbar 5 Somites Sacral 4 Somites Coccyx or more in tailed animals Why? Clocks! Three Clock Systems – Segmentation Clock (redundant clock, linked to cell cycle and hox clock) , Cell Cycle Clock, Hox Activation Clock Segmentation Clock (Somitogenesis Clock/Wavefront Model) Involves oscillator and moving wavefront of activity Hensen’s node migrates posterior-ward Hensen’s node is source of Retinoic Acid “posteriorizing Cue” - as node moves down, plays role in progression of somite clock Retinoic acid at anterior, FGF/Wnt posterior Posterior primitive streak is source of FGF/Wnt signal - both are secreted Two gradients are antiparallel and form a determinative wavefront that moves with Hensen’s node - where the gradients intersect also moves posteriorly (at the wavefront) An oscillating molecular signaling clock affects pre-somitic cells at the determinative wavefront - something happens at wavefront at oscillating signals Wavefront Cells are subjected to molecular oscillator while Hensen’s node passes down the dorsal axis Some point behind Hensen’s node, competing RA and FGF/Wnt gradients open a window of opportunity for somitic cells to be receptive to the oscillator Presomitic mesoderm responds by pinching off to form a new segment  Segmentation wavefront ward and stops at the wavefront each time, new segments formed Determination wavefront - provides synchronized window of opportunity for oscillator to act Hensen's node continues to move down The oscillator Creates boundaries Notch pathway Notch B cell surface receptor. Has external domain, transmembrane domain and intracellular notch domain Notch has to be glycosylated and interact with partner or adjacent cells to be active Adjacent cells has delta which stimulates notch to release its ICD into nuclear membrane Can interact with TFs Certain TFs need to be activated by notch icds CSL is an example Oscillator continued Notch ICD interacts with CSL complex in promoter region Notch activation Hes7 which is a helix TF Hes7 inhibits lunatic fringe and is activated by notch On switch Also activates lunatic fringe. It inhibits glycosylation. Secreted factor. When secreted reduces activity of notch by preventing glycosylation Notch activates it, feedback inhibition Off switch The 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 (reverse side)– Axin/Wnt. FGF also oscillates – so essentially 3-part redundancy. Radical changes in cell cycle, temperature etc., affect segmentation timing and therefore somite length and number - functional redundancy Can mess with oscillator by messing with cell cycle Cells that are in the correct phase of the oscillatory cycle and at the determinative wavefront, these cells are the ones that segment. Clock wavefront model acts as specific subpopulations of cells at certain stages So how do we explain larger body segment zones? Inject fluorescent marker fluoresceinated dextran (intracellular) - studied lineage of chicks Only daughter cells will remain marked Follow fate of daughter cells  Fine glass needle Filled with dextran Too big to leave cells Can see when and where dexron is trafficked Only way cells get dextron in them is by inheriting from mother cells Cell cycle dynamics varies in axial versus paraxial mesoderm Dividing more quickly than somite cells Is a somite - notochordal concordance required for major changes to body morphology to occur? Where you have both notochord and somites in the same phase of cell cycle is every 6-7 somitic cells Might be why tagmata are separated by this measurement 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: Telencephalon: cerebral cortex, ands subcortical structures, including the hippocampus, basal ganglia, and olfactory bulb Diencephalon: thalamus, hypothalamus, posterior portion of the pituitary gland, and the pineal gland Rhombencephalon When hindbrain develops, breaks into 7 discrete segments called rhombomeres Rhombencephalon displays transient segmentation into rhombomeres Is only part of brain that lies above notochord Gives rise to Cranial Ganglia Is only part of brain in/near which Hox genes are active Cells within (with rhombomeres) become clonally restricted, and don’t cross rhombomere boundaries - share boundaries Notochord has, at its anterior end, has the isthmus Lineage restriction of rhombomeres Taking lineage tracking molecules and putting them into somites before becoming rhombomeres. They are plastic, not lineage restricted Once rhombomeres, they become lineage restricted Hox expression in rhombencephalon Hox genes are present in the hindbrain Discrete domains Hierarchy of expression Each segment has a unique address Fore and midbrain segments Neuromeres: Meier and Jacobson SEM of developing mouse brain in the early 1980s Saw and published evidence of transient “neuromeres” of fore- and midbrain Not widely accepted until recently Produced a series of studies where they thought they saw transient structures in the mid and forebrain segments Naysayers say it was an artifact of fixation, from adding things like ethanol to dead tissue, not too helpful of an argument Prosomeres Puelles and Rubenstein Not morphological segments Expression domains define them Domains overlap and combine to define discrete boundary delimited areas 6 distinct regions Segmentation at least happening on a genetic level Fore and midbrain elements that act in segments Started cataloging expression patterns of homeobox genes as they were being expressed in mice L20: Introduction to other organizers Recall model for D/V and mesoderm patterning in frog Nieuwkoop center - high Wnt signaling+TGF-Beta (Nodal) signaling -> induces Spemann organizer/dorsal mesoderm More ventrally - TGF-Beta signaling only -> ventral mesoderm Neurula, neural plate, neural crest Neural plate forms along the dorsal midline Neural ridges meet at the midline Neural tube sinks, ectoderm covers the top If hox genes are not expressed in mid and forebrain, is there a separate organizer? Is there a separate head organizer? Behringer’s Mouse mutant (Lim1 = Lhx1) Homeobox gene expressed in organizer -> in Spemenn or Hensens node Later expresses in notochord Mutants lack any head forward of the hindbrain  Subsequent studies showed that Lhx1 is necessary to gastrulation movement and cell adhesion Why the areas not affected by hox genes being most affected was a question of concern How can notochord organize the head? Does it? Effect is probably indirect and acting on isthmus (midbrain/hindbrain junction – a constriction) Serves as boundary between two regions Is site of expression boundary of important prosomeric genes that also have big brain phenotypes Gbx2 (gastrulation brain 2) in hindbrain, Otx2 in midbrain Also site of FGF expression Imp segmentation boundary Isthmus Otx2 and GbX2 really sharply separated here Isthmus may play an organizer role Made mice with overexpressed G6X or Otx Overexpressed Otx turns down Gbx and the other way around Isthmus continued 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 Mechanisms are in place for sharp boundaries Are there other regions important to head development Monte westerfields lab (a father of zebrafish developmental studies) Deleted tiers on anterior ectoderm cells at mid gastrula Suck out cells with a needle Cells are organized into discrete tiers Have tiers before the head Believed tier cells play an organizing role Anterior tiers critical to brain development Some specific tiers of cells are critical to anterior neural development Tiers 1, 6 and 7 affected the development of brain Deletion of tiers alters brain markers  Emx1 and dix2 are homeobox genes Shh is sonic hedgehog, a secreted factor Induce predictable absences cell expression when specific are removed Gorgeous transplant study Anterior most ectoderm (induced to anterior neural fate) have potent organizing activity What if transplanted? Tier 1 cells have limited inclusive ability Tier 3 (control) do not Took tier 1 cells and transplanted them ectopically. Do they organize extra main structures in this site? Found that transplants induce some prosomere molecules Tier 3 cells did not induce these cells Cannot support the formation of hindbrain with just tier 1 cells 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” Cannot organize brain structures on their own when transplanted ectopically Summary of 4 organizing players: Isthmus Ventral floor plate Anterior neural ridge Zona limitans intrathalamica Tail organizer (chord neural hinge) Late dorsal lip organizer simulates addition of tail somites - predominantly posterior notochord Bmp and even skipped (eve) provide the stimulus Bmp is a secreted growth factor from the TGF family Eve is a homeobox encoding gene (remember pair rule genes in fly) that acts as a repressor of Hox and Wnts Expression is stimulated by physical cell migration Both head and tail during neurulation - neural ridges are still open at the far ends (head and tail). They will suture up gradually as the embryo elongates. Impart why there is slightly different organizing activity going on there  Hensen's node zipping up neural groove, neural plate forming but in fish and amniotes that the hensen's node/dorsal lip continues to have organizing activities. In both cases, movement of tissue slows down at tail side Presomitic mesoderm extending down further and further - slower. Acts as a separate bud with discrete organizing activity If you explant dorsal lip in frog/fish/chick to ectopic sites, it produces more posterior structures depending on how old it is. The later the transplant, the less anterior it is to the extent that by the time you get to the stage looking at chordoneural hinge - notochord, open neural plate merge - have cells extruded by convergent extension. This proliferation seems to act to stimulate the activity of BMP Tail bud organizer is transplantable and induces local activity Figure A: Open neural tube, not suturing on middle line. Notochord and axial mesoderm on the bottom along with neural plate Excise bottom transplant it to B, get a tail Degree to which BMP is stimulated is going to regulate how long the tail is Why don't we have a tail - TBXT gene A t-box transcription factor (large family) - first discovered. Expressed in mesoderm in a huge array of animals Have DNA binding domain=t-box Tbxt found in jellyfish through to humans When it's expressed early in development, it is in mesoderm Originally found/used as pan-mesoderm in riboprobe in situ hybridizations - mesoderm marker in frog – later restricts to dorsal lip and notochord Doing linkage analysis, found errors/mutations in TBXT Human homolog of brachyury and T-mutant (mice) – brachyury means “short tail” In frog and mice starts expressing in dorsal lip and in all mesoderm, but then restricts to axial mesoderm Mice expressed earlier than in frogs In mice, it expresses in ICM, then mesoderm, then node and notochord  In mice, naturally occurring (mild) mutation causes kinked or shortened tails In mice, severe mutation leads to embryonic lethality early in development – notochord defects, leading to neural patterning defects Defects occur after gastrulation is over Notochord is abnormal and doesn't give normal signals of noggin to transform overlying ectoderm to neural End of neurulation have chordoneural hinge, predominantly axial/paraxial mesoderm Also express in mesoderm of in chordoneural hinge mesoderm Alu sequence insertion Alu (SINE - short interspersed elements) are primate specific retroviral remnants - 11% of our genome Correlation between activity of TBXT in choroneural hinge linked to the presence or absence of a tail All tailed primates have a particular structure to T box T gene different than those primates with no tail Have an extra bit of DNA in the gene SINE - numerous Subclass of SINE which predominated in primates = Alu sequences Can find them interspersed distributed all throughout the genome In most Tbox T genes, Alu element that is interjected in between exons 4 and 5 and exon 6 In the case of hominid, second Alu sequence between exon 6 and 7 When gene is being transcribed, mRNA prespliced has Alu sequences either side of exon 6 and permit exon 6 to be spliced out Internal looping that occurs probably as a consequence of complementarity With extra Alu, have looping, exon 6 gets looped out, protein is different in tail vs tail in primates Remove exon in mice - no tail What would happen if you use a mouse model and remove exon 6? So it would lack that particular amino acid sequence? Trying to mimic functional effect of Alu sequences Generate mutant mouse = tailless Good evidence Insertion of repeat sequences has same effect RCS2 is a reversed sequence that permits the same sort of complementarity as the Alu insert Replace Alu sequence with some other bit of sequence? Used RCS2 that was reversed (possibility for internal complementarity), going from one direction on one strand vice versa, would be internal complementarity, and this would induce looping and excision of exon 5 from transcript and as a consequence exon 6 would be absent from the amino acid sequence  Mice with this defect showed a range of morphologies depending on how effective this internal complementarity was ranging from tailless to tails that were short and kinked Conclusions TBXT function in the chordoneural hinge area is important to tail as well as general mesoderm development. Helps mesoderm continue its convergent extension, proliferation When exon 6 is not translated, the transcription factor does not support progression of the hinge (dorsal lip and notochord). Hominid evolution (tail loss) resulted from insertion of Alu sequence, internal transcript complementarity, and alternative splicing. L21: Limb development Growing out on a limb Limb origins Aside: 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 that actually contribute to the limb. Very early in development, an area of flank tissue that is confident to respond to cues. Flank is a target - center will revert to become the limb Take area where bud will form, take tissue away, the zone that has the confidence to respond is much larger than what will give rise to the limb Conservation of limb emergence boundaries Where do limb buds arrive? Fore limbs and hind limbs emerge at junctions of Hox genes HoxC6 and HoxC9 expression domains delimit emergence of forelimb bud (fin, limb, wing) at the anterior and posterior margins respectively Hox6 is transition of cervical to thoracic, where limb bud will emerge HoxC9 - defining boundary where you get formation of a hind limb Mouse, occipital  All in common, wherever Hoxc6 is expression where you get transition to thoracic and emergence of limb bud Bud elongate and chondrogenic patterning begins As the bud grows, the cells proliferate, it grows outwards and then begins to flatten - called pallet Where the digits will form, programmed cell death between the digits Chondrogenesis (process by which cartilage is formed and developed in the body) - in the limb as it's forming, cells that are proliferating like crazy tend to be suspended in a gel like matrix (rich in hyaluronic acid), cells don't have a lot of contact with another. Look like embryonic cells - highly proliferated. Occurs right under AER (growth zone), cells left behind lose water rich gel like matrix and form aggregation. Differentiation and beginning of cartilage formation Limb development in amniotes Zone of polarizing activity Early in bud stage, AER and ZPA forming A lot of cells migrating in limb buds Have ectoderm and mesenchyme (mixture of cells) Cells coming from lateral mesoderm Limb bud grows from several cell sources Cells Migrate from: Lateral mesoderm Somitic mesoderm Mesonephric mesenchyme Neural crest Hence mesenchyme Proliferation that occurs requires AER. AER secreting into mesenchyme signals to promote rapid proliferation Cells proliferation under ectoderm Forms Apical Ectodermal Ridge (AER) Mesonephric mesenchyme Kidney forming - goes through 3 phases of building Pronephros buds - cells that contribute to this, fall apart and mesonephros formed, falls apart then mesonephros builds which becomes the kidney Sources of mesoderm  Ectoderm in blue with mound of cells forming apical ectodermal ridge - secreting growth factors Cells migrating from somites, lateral plate, disintegrating mesonephric Collecting in Hox6 boundary network and proliferation like crazy Limb bud development Shh plays an important role, and seems to be secreted in ZPA. Intrinsic role in ZPA Shh turned on early during limb bud development as well as growth factors secreted by AER (fall into FGF family) FGF8 secreted by AER, some involved FGF4 and 2 Hox gene expression boundaries determine location on dorsal axis  Where HoxC6 expressing, initiate or stimulate activation of T box genes turns on t box 5 HoxC9 turns on PitX1- homeobox gene, characterized based on expression in pituitary gland Tbox genes activate FGFs in mesenchyme FGFs stimulate in part the AER AER starts secreting FGF, that secretion is critical to supporting proliferation Role of AER *** If you remove the AER, and remove source of FGF, subsequent proliferation ceases AER sustains und If you remove it later, get distal structures forming Need AER all the way through development to support enough growth for differentiation for digits to form Sustains proliferation states of cells immediately underneath by secreting growth factor FGF AER is a source of FGF FGF and the AER Can mimic AER activity by installing beads that have been soaked with 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 Naive ectoderm + beads, can rescue limb development to some extent AER through secretion of FGF is supporting proliferation and can mimic it by implanting a source of FGF FGF mediates outgrowth and permits patterning to occur Enlarge AER by graft or mutant Mutants in chick that have an abnormally large AER - get extra digits forming Limbs are abnormal and have more than the normal number of digits Talpid3 involves shh Role of mesoderm in limb bud Age of mesenchyme determines proximo-distal differentiation pattern/potency Age of AER is not important to proximo-distal pattern Can do mix and match experiments, take a limb region, remove it and put on some mesenchyme and an ectodermal cap (old or new cap, old or new mesenchyme). If you replace the ectoderm with young ectoderm or old ectoderm = NO difference, will provide source of FGF, patterning normal Take different ages of mesenchyme and put it under ectoderm changes everything = changes how much will form, if you put old under ectoderm = just dorsal, if you put young = dorsal and proximal Mesenchyme is responsible for determining this differentiation. Hind limb in t box 4 = hind limb, fore limb expressed in t box 5 Mesenchyme determines fore - vs hind limb identity Fore- versus hind- limb is determined by Tbx 4 and Tbx5 Transcription factor family that binds DNA through a T-box Flank mesenchyme is not competent Tbx5 determines forelimb Tbx4 determines Hindlimb Tbx5 activates Pitx1 (homeobox gene related to bicoid) Area where HoxC6 and 9 boundaries are that renders ectoderm in that area, generic flank tissue won't do it Pitx1 in hindlimb development Pitx1 activates Tbx4 Ectopic expression in chick accomplished by using a viral vector to ectopically express Pitx1 in forelimb Pitx1 activates Tbx4 in wing bud Wing bud forms more like leg (clawed toes and scales rather than feather germ) Made a viral construct that would express Pitx1 - would infect this into cells. Those cells, if infected, would express Pitx1. Instead of forming a limb that grew feather germs and elongate digits, get scales forming and limbs that look more like feet - no feather germs, scales instead. = good evidence to help delineate pathway Zone of polarizing activity ZPA gives polarizing, posterior cue Morphogen is probably Shh Historically, RA thought to play a role since it mimics ZPA Beads immersed in either Shh or RA can mimic a ZPA Can create mirror image symmetric structures If you take ZPA and graft it into the anterior margin you would induce posterior traits Mirror image duplication of digits, posterior digit forming posterior and anterior Explant of graft of ZPA activity in anterior margin creating a morphogenetic gradient, get duplication Dose dependent - have fewer cells or morphogens then you get less induction of posterior structures Shh mutants in fly would produce mirror image duplicate wings Experiments done in chick embryos with vitamin A - vitamin A disturbed limb development if you applied it in pharmacologically significant doses. Led to experiments if vitamin A is playing a role? Had reads, soak it in a medium containing shh or in vitamin A - implant beads in posterior or anterior margin to see what happens. Found that both shh and/or retinoic acid could mimic the polarizing activity. If you put in anterior zone, it would be as if you grafted ZPA activity Experiment: expressed shh in cells, spun cells down, took out pellet, implanted those, shh cells would also mimic ZPA activity Classical retinoic acid studies Summerbell (RA beds into chick limb buds) Only do this in areas limb buds normally grow - zone of cells competent Usable signal Other studies: ZPA Shh Not flank mesenchyme Recall: Ra and the receptors Lipophilic RA passes through membrane CRABPs bind to and remove RA from circulation RARs etc. bind RA and act upon RAREs to alter gene expression Zinc finger domain factors - subclass is steroid family receptors - receptors that will have their ligand, thyroid, estrogen, testosterone hormone Subclass of steroid class that are retinoic acid receptors  In cytoplasm, have steroid class receptors that will bind retinoic acid. Pass the membrane of nuclear envelope, they can bind to their motif Many different vitamin A receptors that bind to different receptors Have transportation network, get receptors into the nucleus where they can interact with DNA motif In cytoplasm, have CRABPs, seems to act as a sponge to absorb acid to get to the nucleus where it can interact with retinoic acid receptors RA and CRABP Gregor Eichele Chick limb buds Removed and assayed anterior 2/3 and compared to posterior ⅓, would solubilize them and analyze them by HPLC Homogenized from hundreds of limb buds HPLC More RA near ZPA than elsewhere Look for how much retinoic acid in these cells Difference in anterior vs posterior cells? Was a difference but not a huge difference. ⅔ less vitamin A anteriorly than posteriorly Malcolm Maden Purified CRABP Reared antibody Tended to be expressed more anteriorly than posteriorly Retinoic acid was distributed asymmetric Did assays on limb buds CRABP richer near anterior than posterior margin  If you have a gradient of retinoic acid high in ZPA, but CRABP in anti parallel gradient, the effectiveness is to absorb retinoic acid and enhance gradient CRABP is rich anteriorly, RA is slightly richer posteriorly. When taken together, the amount of RA available to mix is enhanced posteriorly Putting it all together Remove AER, ZPA signal dies out Remove ZPA, AER starts to die out HoxC6 activating TBX5 which activates FGFs - proliferation signal that acts to stimulate AER AER cranks out FGFs which supports continued proliferation of cells In posterior margin, sonic hedgehog is expressing If you remove shh or ZPA, the AER dies If you remove AER, the ZPA dies Mutually dependent May explain the longer the limb grows the further removed the cells become from the ZPA, the more likely they are to stop proliferating and start differentiation Imposes a growth limit FGFs and mesenchyme is what stimulates growth of ridge which secretes FGFs - maintain proliferated, undifferentiated states of cells underneath the ectoderm In proximity of AER, remain proliferating and differentiating If you remove one or the other the other will fail - AER and ZPA Hoxc9 boundary is where the hind limb bud begins to form - acts through TBX4  Hand2 - transcription factor of ebox family and plays a role in activation of sonic hedgehog There is an anterior signal There is an anterior signal as well not all just ZPA Gil1 a lot more anterior - seems to be acting to repress ZPA Hox genes and limb development 5’ members of the Hox cluster - late genes, play a role in limb development (More to follow – after we talk about gender differentiation) These late expressing Hox genes activate Shh. T Box genes turn on the hox cluster Hox genes act upon shh - some of the proof comes from studies on python. Known from fossil record that limbs have disappeared over the span of evolutionary time How the snake lost its legs  Vestigial pelvic girdle, claw and femur Remnants of a hind limb Many limb gene expression patterns remain the similar (conserved) Took markers of limb development - like shh, gli1, gli3 - looked to see how expressed in lizard hind limb development and python hind limb development Looked at early markers, seemed to have shh expressed at least in the ZPA Gli3 has a similar pattern of expression in anterior end of things  Fgf8 begins to form an AER, expressed in AER but in stage 3 of development it begins to fall apart and the AER deteriorates Link between ZPA and AER - AER is starting to form but ZPA with expression to shh starts but poops out then bud development arrests Many limb gene expression patterns remain the similar (conserved) Hox genes - genes expressed during early limb development and late in digit formation expressed the same in python and in lizard Hand 2 starts like shh, but poops out What is it that differentiates lizards and us from pythons? Power of computational genomics. Took the sequences around many of these genes known to be playing a role in limb development. In shh, gene critical to ZPA - in python was lacking a discrete sequence CRISPR out the short of ZRS ZRS in promoter enhancer region WT mice - zone that plays a role that regulates shh activity In python, this is absent If we remove this small area of promoter enhancement in the mouse? Get a mouse that develops serpentinized - begins to form a hind limb and ends up that hind limb is important because no shh, no ZPA, no support of AER, no support of proliferation and differentiation = no hind limb development ZRS - binding elements for Hand 2 and for hox genes Change in regulation of a single gene. All the other genes to make a limb, all cellular capacity are there, but the change in the activity of a single gene is sufficient L22: Limbs, Evolution and Hoxology: Reprise Limb scaffolds Cells proliferate in bud Drop behind progress zone Begin to aggregate (matrix degrades from gelatinous, hyaline-rich substrate) Collagens secreted Chondrogenesis Bone replaces cartilage (ossification) Aggregation of cells and consequence off degradation of matrix that keeps cells separated When cell cell distance breaks down and they get mushed together Metapterygial arch When cellular aggregations form, they can do so in three ways: 1. De novo condensation - by themselves, if cells aggregate 2. Bud off 3. Branching In tetrapods, condensation pattern always follows a stereotypical pattern The metapterygial arch - basic plan for forming the limb  It Doesn't matter what organism you're looking at, the order and pattern in which cartilage condensation is formed is the same. Didn't matter what the outcome was Always occurred in the same order. Condensation that gives rise to humorous, branch to form 2 condensation that would separate, coming from ulna get a sequence of budding off then branching that would give rise to the digits - form in an arch like progression Conserved features Homologies of bone structure Can directly compare a human 4 limb to fin of whale and find the same development but via the metapterygial arch that gives rise to analogous structures Homology is arising from a fundamental pattern of building limb buds Pattern of morphogenesis is highly conserved Hox genes are also highly conserved Fin to limb transition Progression from linear to arched development of bony elements How did this evolve? Develops in a linear way Ancestrally out in one direction with side branches, or boney elements forming one after another in serial progression - no development of metapterygial arch Shubin, coates et al, fossil record on ellesmere island Were not a lot of specimens in record to go looking for this Ellesmere island - deposits of rock that would be the same time creatures were climbing out of the oceans and finding their ways on lands Vacant spot on fossil record and had to find how these limbs developed Found in 5th ont to the right - Beginning of metapterygial arch forming Found progressively more Pattern over evolution, some fossils have 11 digits - AER and ZPA had to of been there, metapterygial arch forming but had to of been big Why make more digits than we need? So down to 4 digits - standard state of the plan for building limbs Hox mutant mice demonstrated limb defects For example, Knocked out a member of Hox 9 orthologues, would have defect/absence of elements in the shoulder Hox 10, humerus defected Hox 11, skeletal Hox 12, carpels Hox 13, digits Just as the overlapping and nested hierarchy of expression domains is patterning their address, the hox genes are a tool kit to be called out to perform a similar structure to specify the address What areas within the cluster play a role in regulating the activity of hox genes in the later stages of development? Found that most regulatory activity of these hox genes was directed by sequences outside of the cluster. Hox gene regulation Three phases of hox gene regulation 1. Early, anterior Hox expression largely regulated within cluster (unwinding of cluster chromatin) 2. Control of posterior axis genes by 3’ remote enhancer (centromeric C-DOM) 3. Control of limb, genital tract expression by 5’ remote enhancer (telomeric T-DOM) Early control phase (anterior axis) Topologically associated domains - genomic regions where DNA sequences interact more with each other than with sequences outside the region Diagram B: Early hox cluster, all genes locally controlled and being subdivided in discrete domains, sequences that act as insulators Late control Two different domains. Early regulation by 3’ enhancer acting upon the earlier genes along the axis The late regulatory domain, acting later, and turning on distal most of hox genes Enhancers working by folding in close proximity to the hox genes Proteins that will bind enhancers and activate hox genes Topologically associated domains (TADs) TADs - co-localise genes and their regulatory elements as well as forming the unit of genome switching between active and inactive compartments Produce them with heat maps Relates frequencies in which one gene domain acts with another TADS- boundaries define chromatin interaction domains/possibilities Remote 3’ enhancer separated by hox gene by what's called a gene desert When the 3’ enhancer bind their sequence, there's a topological change that permits that enhancer region and its attached proteins to come to close contact with hox 9,10,11,12 This then activates those genes A way of bringing in remote activity to turn on remote genes in a time and space manner When this happened, genes turn on in the posterior end ZRS domain ahs hox responsive elements When hox genes are being activated by the 3’ activation enhancer, will be turning on hox 9,10,11 which will turn on shh and ZPA will be activated Domains of hox genes being turned on one after the other in a posterior gene These domains are overlapping and nesting hierarchies Later in development, secondary activation of hox genes by the 5’ enhancer region, also spaced from the hox domain by relatively gene sparse stretch of chromating that will turn on the later genes for a second time Hox deployment explains metapterygial arch Early and late phase of hox gene expression If you were to look at early stage hox expression in fish or in mouse or chick, it would look pretty much the same early in development, nested hierarchy. Hox10 turned on  earlier and posteriorly. Later in development, hox 11, 12 and 13 turn on providing pre combinatorial domains to identify different parts of the limn What differentiates fish from other tetrapods like human, chick, frog - lack a second period of hox gene activation In chick, get extra extended arch like domain of expression, this doesn't happen in fish The phylotype is being regulated by the zoolotype Metapgrgial arch being regulated by the genes and their activity Late stage of gene promoter enhancer activity Redeployment of hox genes that is enabling them to pattern what becomes metaphrgyail arch Metaphrgyail arch - extra time and space deployment of hox genes, and consequence effect it has on Expression of hox genes, then arch like progression of condensation patterns Hox deployment variation explains limb morphologies Hoxd13 mutations - last of hox gene to be expressed, most posterior and distal domain. When it is expressed ectopically, you get pure digits or fusion of bone elements that normally give rise to more than 5 digits Gli1 and gli3 play a role in restricting posteriorization by providing inhibitory cues in the anterior region Suppress gli3 - that anteriorizing cue is lost, get extra digits that form as a consequence Gain of function – synpolydactyly or brachydactyly (fewer, sometimes broader digits) Loss of function – polydactyly (extra digital bony elements) Gonad development - female Hox genes are being expressed in a segment specific way also during formation of your gut and also genetal tract Females, precursor to fallopian tubes is mullerian ducts Hox a9 is expressed and important in development of upper most regions of that duct Hox 13 to posterior part - vagina Hoxa10 and hoxa11 middle regions Hoxa9 - important for flower like ending of fallopian Hoxa11 - cervix Gonad development - male Different hox genes evolved more in an overlapping expression domain Hoxa9 playing a role in differentiation of epididymis and vas deferens and meis2 plays a role in that specification Mutations that will have effects on the ability to reproduce Hands and gonads: sexual dimorphism IVF clinics – noticed people with fertility problems were slightly more likely to have asymmetric hand size (left versus right) Index versus Ring finger length ration (2D:4D) was slightly more likely to be off Index finger versus ring finger length Tend to be roughly equal in females Index finger shorter than ring finger in males Hypothesis is that Testosterone: estrogen levels in utero affects Hox gene expression More Androgen (testosterone) and Estrogen Receptors (AR and ER) in fourth digit than in 2nd Some reported links to sexual behavior/preferences, psychology, autism, left handedness etc. L23: Gender Determination and development The indifferent stage Aristotle was the first to comment upon it - early stage (indifferent stage), involved formation of a cloaca Many organisms have a single orifice for excretory and reproductive functions eg; cloaca excretes feces, urine, but also gametes in 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 Amusing Fact: Aristotle, later Lucretius (Roman), thought males the more evolved and more fully developed side of humanity because females retain the cloaca-like opening, but this sutures up and grows into a phallus in males. (reflected societal roles for women – no vote, chattel etc.) Indifferent stage: external appearance  Start with cloaca, in male this soutures up Scar that runs from base of testis up the phallus - scar thats left of cloaca Labium elaborating from edges of cloaca slit Indifferent stage: internal plumbing Start off with two parallel sets Epigenetic landscape - cloaca either develops vagina or phallus One or the other parallel sets disappear Millerian glands will serve as the primordia for the female reproductive tract - give rise to ampulla (flower like ending of fallopian tube), fallopian tubes, cervix In male, rudiments are wolffian ducts - precursors to epididymis, vas deferens Both rudiments are present in indifferent embryo stage, only one or the other is supported and the other deteriorates First steps to gonad development Primordial germ cells migrate to genital/gonadal ridge from allantois (external sac) via hindgut Mesonephric mesenchyme migrates to ridge - cells proliferate and assemble into sex cords Look at genital ridges, they are on either side of a bump caused by neural tube/aorta enclosed. Cells migrate to genital ridges, and genital ridges and mesenchyme will also contribute cells - will enclose germ cells and divide into testes or oocytes in consequence First steps (both genders in common) Kidney system develops in 3 iterations Nephric system undergoes change – starts as primitive system (pronephros), which dismantles as the mesonephros builds, and this in turn dismantles as the metanephros (kidney) system assembles. Pronephros - glomerular forming, then begins to disintegrate then mesonephros forms, it disintegrates then metanephros forms A good example of “ontogeny recapitulating phylogeny” (Haeckel) = embryogenesis goes through the same steps as evolution Could be zootype, genetic mechanisms put in place are based on something that is old and shared amongst us. As we evolve more modules/partners, cause the same genes to be redeployed Could be that while primitive states represent stuff in far distant past, they haven't been removed, since same genetic pathway is used Mesonephric system begins to dismantle, and mesenchyme migrates to the genital/gonadal ridge Mesonephric mesenchyme cells assemble into rows (columns) called the sex cords. Indifferent stage in 3D Some tissues on genetal ridge contribute to ovary and testes, so does mesenchyme Decision time Females Males PGCs migrate to genital ridge PGCs migrate to genital ridge Mesonephric mesenchyme migrates to Mesonephric mesenchyme migrates to genital genital Ridge ridge Mesenchyme abortively forms sex cords, Mesenchyme proliferates and forms doesn’t proliferate much substantial sex cords, Cells of genital ridge assemble into cortex, Cells of genital ridge contribute only slightly proliferate become supporting granulosa cells to cortex, become thick interstitial tissue, probably also Leydig cells, future source of testosterone Sex cords eventually form into Sertoli cells of seminiferous tubules – future source of Müllerian inhibiting substance/hormone (MIH/MIS) In the absence of testosterone, Wolffian ducts MIS/MIH inhibits Müllerian development, degenerate ducts degenerate Müllerian ducts differentiate into Fallopian Wolffian ducts differentiate into epididymis, tubes, uterus, cervix, upper vagina which is segmented, the seminal vesicle and the vas deferens What directs gender determination? Hormones? Frog embryo experiment In frog embryos, take eggs and fertilize them in petrie dish and flood them with media containing estradiol or testosterone Roughly 50% of the eggs that develop will be male If you treated form early stages with estradiol, 100% would be phenotypically female even if they were genetically male Those that have been administered testosterone, would be automatically male Parabiosis Let embryo grow to certain stage Scratch ectoderm off developing embryo Press them together while healing Presto! Conjoined twins Frog embryo (Rana pipiens - XY, XX frog) Where you have XX joined to an XY, they were always anatomically male Despite there being a 50/50 genetic distribution, they develop 75% anatomically male Similar chimeras in mice Put 2 embryos together under a bit of pressure, they will merge Chimeras will form In some cases, chimeras will be XY and XY cells, or XX and XX Something about male cells that drives determination and masculinizes irrespective of genotype Masculinized genetic females are not fertile Similar to freemartin cattle (mixed gender twins with shared placenta, shared blood supply). If one is male and one is female, the female will be subjected to abnormally high levels of testosterone from her sibling Human chromosomal anomalies Chromosomal Status Anatomical Phenotype XY missing short arm of Y chromosome Female XY missing long arm of Y chromosome Male XXshort arm Y translocation Male XXlong arm Y translocation Female XO (no Y chromosome) Female What is the factor produced by the small arm on the Y chromosome? SRY – a high mobility group class of transcription factor (includes Sox genes, of which Sox9 is a major player in gonad determination  Expresses on the ridge in males Loss of function mutants (of SRY) fail to support male gonadal development Ectopic expression in females causes male gonadal development The genetic hierarchy of action SRY turns on SOX9 turns on SF drives the differentiation of Leydig cells that secrete testosterone SOX9 also turns on FGF which supports continued proliferation of sex cords and their differentiation into sertoli cells that start to secrete MIS/MIH Comparison Females Males No SRY (so no SOX9, noSF) High SRY, high SOX9, high SF, high FGF Low mesonephric recruitment, sex cords High mesonephric recruitment and dissolve proliferation. Sex cords grow Higher Cortical recruitment, Granulosa cells Low cortical recruitment, Leydig cells develop develop No Testosterone, Wolffians degenerate High SF = Leydig cells produce testosterone Wollfians persist No MIH/MIS, Müllerians persist (Wolfians High FGF = high Sertoli and MIH/MIS die). Müllerians degenerate  Ovaries, fallopian tubes, uterus, cervix, upper Testes, epididymis, seminal vesicle and the vagina form vas deferens form Anal and “cloacal” openings separate Anal and “cloacal” openings separate External features elaborate – outer labial Labia-scrotal folds suture, scrotum develops, folds (labia majora),inner fold(s labia glans tops a growing phallus minora) form, clitoris forms Is there a female SRYY? Yes! Dax1, XIST DAX1 Steroid nuclear receptor family Gene resides on X Chromosome – XY have one copy, XX have 2 In females one of the X chromosomes gets compacted into a barr body, the compression is somewhat leaky and DAX1 is one of the few genes that expresses from the second X chromosome XY males with autosomal translocation of Dax1 can be dose-dependent sex reversed - gender differentiation of the genetically male, get instances of intersex Lacks DNA binding domain - has to bind to a partner Binds to and competitively removes SF from action – inhibits testosterone Female expressed gene that is acting to absorb and remove SF from play and removing a fundamental element of male deriving genetic activity XIST Y chromosome is very small and carries few genes If both X chromosomes are active, there would be many more copies of genes in females than males The way we have evolved to keep the number of genes on the X chromosome regulated so we both develop normally with exception of anatomical differences One X chromosome gets compacted - XIST is the factor that does this XIST is an RNA transcribing gene, the RNA is not translated - no protein that arises XX females must inactivate one X chromosome (randomly at blastula stage) to attain a sort of gene dosage compensation – if both active – embryonic lethal XIST expressed from both X at first, but then more from one than the other XIST RNA gloms onto X and serves to nucleate epigenetic repression – Barr body One X chromosome normal, and one condensed that essentially most of the genes are rendered inactive In female twins, X chromosome inactivation occurs late in blastula development. Each blastomere has 2 X chromosomes, the one that gets inactivated happens randomly (some  maternal, some paternal). When you grow up, represented by clonally different patches of cells - some from maternal X some from paternal X Calico cats - pigmented difference. Random inactivation in blastula stage = different patching XIST expressed in female nucleus Cell nucleus that has been stained with PI Wherever you have DNA, you have blue The yellow is gene for XIST If you have 2 X chromosomes, 2 yellow spots where the gene is (one where Xa is, one where Xi is) Example 1 Androgen insensitivity syndrome (lack functional testosterone receptor) - may be making testosterone, the signal is not received and transmitted so its as if they dont have testosterone High SRY, high SOX9, high SF, high FGF High mesonephric recruitment and proliferation. Sex cords grow Low cortical recruitment, Leydig cells develop High SF = Leydig cells produce testosterone Testosterone not sensed, Wolffians degenerate, so testes, epididymis, seminal vesicle and the vas deferens fail to form High FGF = high Sertoli and MIH/MIS - Müllerians degenerate Anal and “cloacal” openings separate External – feminine, but blind end vagina Sterile Other examples Sex Chromosome Number/Identity Associated Syndrome XO Turner Syndrome (X)XXY Klinefelter Syndrome XY (No SRY) Swyer Syndrome Klinefelter - extra X chromosomes. Another version that can be XYY Swyer sometimes a point mutation, but many linked to expansion of trinucleotide repeats Turner Syndrome Klinefelter Syndrome Swyer Syndrome SRY No Yes Dysfunctional Mesonephric Little Yes Little Recruitment Sex Cords No Yes No Sertoli Cells No Yes No Mullerian Inhibiting No Yes No Substance (MIS) Mullerian Tubules Yes No Yes Leydig Cells No Yes No Testosterone No Yes No  Wolffian Tubules No Yes No Internal Plumbing Fallopian Tubes, Normal, smaller testes Female Uterus, Cervix, Upper Vagina External Phenotype Female Male Female Fertility Not fertile Not fertile (Abnormal Not fertile (Streak number of gonad, delayed onset chromosomes - puberty, need Meiosis does not occur hormones) properly) Alternative modes of sex determination Fly - don't have a lot of chromosome. Gender determined by ratio of X chromosomes to autosomes If they have two X chromosomes = female, one X chromosome=male The Y chromosome isn't necessary to gender determination. Necessary for fertility but not development of gonads Take away and X chromosome, the ratio of X to autosome will be low = male If you have extra X, more or less females that are intersex dependent on number of chromosomes Can do this by either changing number of sex chromosomes or autosomes Autosome to sex chromosome ratio (although XY, Y only contains fertility factors - not involved in gender development per se) Sex Chromosomes Autosome sets X;A ratio Sex XX 2 1.0 Female XY 2 0.5 male XO 2 0.5 male XXY 2 1.0 female XXX 2 1.5 metafemale XX 3 0.67 intersex Alternative modes of sex determinants Nematode worm C. elegans Two sexes 1. Hermaphrodite (most) 5 pairs of autosomes, 2 X chromosomes Can self fertilize 2. Male 5 pairs of autosome, 1 X chromosome - sperm half X, half are null 0 Must mate - how you get possibility of genetic recombination to occur Fish Some, like salmonids, are XY (males heterogametic) or ZW (females heterogametic) determined Others, by social status, maturity, and size – reef fishes are sequential hermaphrodites 1. Protoandrous – start male, but transform to females later in life 2. Protogynous – start female, but transform to male later Chickens ZW Only one of the pair of gonads active On rare occasions, females can activate the silent gonad and become male Z is more like a mammalian autosome than like X Monotremes Platypus 5 pairs of X and Y - tiny Reptiles Genotypic: Snakes – by ZW sex chromosomes (females ZW, males ZZ) Some determined by heterogametic but some determined by temperature Temperature dependent Crocodile and Alligators Below threshold, 31 oC in Crocs,– females, above 31oC male – laying site and position in clutch matters Gender distribution varies according to environmental conditions Snapping turtle Below 22 or above 28 – female. In between… male L24: Induction of eye Sensory structures and placode formation Senses of hearing, smell, and sight result from very similar progression of early differentiative events, namely, formation of ectodermal placodes. Placodes are columnar patches of ectoderm that thicken in response to neural cues Placodes: disk of thicker cells, usually forming in response to neural cues. Typically in head region where placodes are forming is where you have direct contact of ectoderm and neurectoderm Where theres an absence of mesoderm, tend to be where you have a placode forming Placodes are precursors of olfacatory - give rise to hearing Presage: Otic vesicle (hearing) Lens (Eye) Lateral line organs (mechanosensory system along flank of aquatic animals) Olfactory epithelium (smell) Pituitary - forms where the roof/mouth ectoderm coming into contact with neural Sensory ganglia of face, tongue, oesophagus and visceral tissues. Mutual induction/communication of neural and ectodermal tissues starts where they make direct contact (no interposing mesoderm) Where the eye is developing, ectoderm forming from placode giving rise to lens, sends signals to underlying neural tissues -> cell shape changes, remodelling for what will become optic For eye, ectodermal cells are induced by underlying neural tube to thicken - form into region of columnar cells. Cells invaginate to form specialized structures which associate with sensory neuron aggregations. Eye development - classic example of induction In head, rare mesoderm-free zone permits neurectoderm to directly contact ectoderm. Optic vesicle grows, lens placode thickens and then invaginates At the side of the head, have ectoderm overlying, a bulge coming out of developing brain and ectoderm begins to thicken and form the lens placode The lens placode sends signal to underlying neural ectoderm to cause it to thicken and induce it to form a pit/depression Both ectoderm and neural ectoderm invaginate to form a big bowl Lens pit then lens vesicle forming (rounded ball of cells), inducing underlying neural structure to form a cup Neural structure has 2 layers: outer pigment epithelium of the eye, the inner is photosensory retina  By the end, have vesicle filling up with lens fibers, ectoderm left behind becomes cornea, surrounding hole is mesoderm forming connective tissue to move the eye around in its orbit Neural crest cells are migrating in and form a scaffold around the lens This scaffold forms temporary plates on which muscle migrates and forms In chickens, have plates permanently, but in us they dissolve Lens induction in X-section OV Optic Vesicle PLE Presumptive Lens Ectoderm LP Lens Placode Lpi Lens Pit - rolls up to form vesicle LV Lens Vesicle LF Lens Fiber LE Lens Epithelium Cup like structure of the eye is forming, and pinches off towards the rear Step wise process Optic vesicle induces placode Placode induces vesicle to invaginate to form optic cup Placode invaginates to form lens Outer optic cup forms pigmented epithelia retina Inner optic cup layer forms neural retina  As optic cup develops, pinched off proximal region becomes optic stalk which makes connections with optic tectum (brain) Other contributors: Neural crest – cartilage scaffold to form iris musculature, sclera Proof defect experiments Black = Ectoderm Red = Mesoderm Blue = Neural epithelium (neurectoderm) If you remove pre lens ectoderm, in the absence of lens placode (precursor not there) wont induce neural tissue to form that cup, no eye Can take optic vesicle and transplant it further on back, does not create an extra eye Only head ectoderm is confident to receive these signals and form these eye structures Matters where the mesoderm is and where ectoderm and neurectoderm come into contact Eye induction is a two way street Experiments confirm optic vesicle induces lens placode Confirms lens placode induces invagination and formation of retinas Moreover, flank ectoderm is not competent to form placode, lens. Ectopic eyes not possible  And all head ectoderm is competent to form eye – removal of interposing mesoderm leads to huge eye induction and cyclopia Processivity of competent cells Jacobsen- noted that lens placode derived from tissue which has come into sequential contact with: Anterior endoderm Heart mesoderm Optic vesicle Examine competence of various stages of ectoderm to undergo induction after association with vesicle: there is early competence Taking ectoderm at different stages and bringing into contact with neural vesicle to see if it can form a placode then a lens Even tho ectoderm was from an early stage (ex: early gastrula), if into contact 4% of the time can induce a lens, late gastrula is about a quarter, early neurula 83% inducible Ectoderm that will contribute to head acquire competence to react to these signals really early Donor stage % lens formation Early Gastrula 4% Late Gastrula 24% Early Neurula 83% Late Neurula 100% What regulate eye development? Is it an evolutionary conserved element? Unlikely for several reasons: eye structures very different - flies - ommatidia, humans - retina induction path very different flies - eye forms as imaginal disk furrows, cephalopod - lens and retina form from a single placode, mammals from vesicle and placode Surprise - common genes! Pax6 among them Flies derive from ectoderm, us from ectoderm and neurectoderm Pax6 Master regulatory gene - turns on early in development and its absence in any organism severely degrades the ability of the eye to form Presumptive lens ectoderm and neural components fall within adjacent field, are subjected to anterior endoderm, then separate, before coming to lie one atop the other. Purple indicates that cell has been expressing mRNA for PAX6 Early in the left panel is an embryo, broad at anterior region, sides narrowing, PAX6 expressed at periphery of anterior neural ridge and a bit ore along the sides Later in development, PAX6 can see it where neural tube is souturing up but also breaking into discrete fields in ectoderm and presumptive neural tissue Arising in similar area, coming apart then coming together again PAX6 is in the lens ectoderm and expressed in neural component Pitc3 - Pituitary and lens Pitx3 only expressed in ectoderm that gives rise to lens Expressing at anterior most margin of neural plate Pitx3 playing a role early to respond to cues and respond to placode Pax6 homology and conserved function Pax6 in mouse/human, and homolog in flies maps to eyeless locus. - Human - PAX6 Expresses in presumptive retina, stalk Mutants have abnormal eye development (some heterozygotes lack irises (aniridia) homozygotes lack eyes  - Mouse Pax6 Expresses in presumptive retina, stalk mutants = small eyes mutation. Homozygotes lack eyes, nose - Fly Pax6 (Eyeless) Lack eyes Fly PAX6 (eyeless) experiments Gain of function-experiment Turn fly pax6 on in wing or antenna imaginal disk. What happens? Eye structures grow in ectopic sites even on wings or legs Promoters to drive expression - get eyes forming in the antenna Fly meets mouse! Are genes conserved enough to play a role in reciprocal swap experiments? Gene encodes a protein that is so conserved in function that it can rescue the mutant phenotype In a fly that will not be able to form PAX6, will rescue eye development The protein it encodes and the partners it interacts and targets it interacts must be highly conserved Turn mouse or ascidian (a basal chordate at larval stages - has notochord and light sensitive ocellus - cells that express something like rods and cones and sense light) Pax6 on in fly imaginal discs - same result as with fly pax6 – phenotypic rescue - ectopic eyes. Express ascidian in a mutant -> rescues mutant phenotype Modularity and Conservation 1. Pax6 and eye gene regulatory networks are very ancient and conserved 2. Play a role in development of visual organs of all eyed creatures Pitx3 more recent - Human mutants  Aniridia (no iris), congenital cataracts, anterior segment dysgenesis (things messed up at front end of the eye - the iris) Small eyes - Mouse mutants aphakia (ak) – regulatory deficit – coding sequences normal small eyes that lack a lens Parkinson’s-like symptoms (expresses in substantia nigra - dopaminergic neurons). Activates enzyme necessary to synthesis of dopamine, namely tyrosine kinase. - Frog Impair Pitx3 using either repressor chimeras or morpholinos - will prevent cells from translating Lack lens, fails to induce retina, no eye Why the difference? Processivity? Morpholinos Morpholino will bind to the mRNA for Pitx3 and target it for destruction Used a lot in fish and frog studies Might be more potent than gene knockout – impairs translation, not transcription so RNA binding protein effects not seen (ie; miRNA-mRNA, RNA Binding proteins. doesn’t leave partial mRNA or polypeptide remnant) Is a modified antisense RNA analog that is resistant to RNase degradation Can be labelled with fluorescent markers Can be injected into individual blastomeres Permits contralateral controls  Take ectoderm form an embryo that is expressing huge amount of morpholino and switch it with ectoderm from a normal embryo? Take normal embryo and put morpholino lens ectoderm and ask if it responds to cues? Answer is no Take morpholino injected embryo and put normal ectoderm over top? If the ectoderm alone is normal, is it capable of responding to the cues coming from the neural? Yes normal eye forms Putx3 acting specifically and critical in eye Ectoderm and neurectoderm The way different fields of cells come into contact, separate and come back into contact Example of experimental serendipity Pitx3 perturbation disturbs laterality or situs: patterning of left -right body asymmetry Heart and gut coil the wrong way Leads us to the next lecture – Heart “It is only with the heart that one can see rightly; what is essential is invisible to the eye” Saint-Exupery L25: Cardiac Development and Asymmetry Circulatory system Centrally, in mammals have a central tube that forms now the ventral mid lane, the ventral tube will loop asymmetrically At the ends, it ramifies - Have vessels emerging from either end - all derives from mesoderm and ectoderm. Will give rise to cardiac muscle More ventrally, have appearance of blood iron (many beginning to express fetal hemoglobin genes. Sprout lots of minor off shoots, ramify contribute to major vessels) - look gross. Where will become gut see blotches of blood. They will give rise to ? ramify, ends meet up with ends and have one integrative system that develops A merger of two components: 1. From the developing heart - major vessels 2. From de novo aggregation of mesoderm - capillary beds 1. Heart derives from: Splanchnic mesoderm - muscle Somatic mesoderm - pericardium (sac that encloses the heart) Cardiac neural crest – ascending aorta and mitral valve Endoderm – endocardial cushion Frog cardiac development Going around periphery, two layers of mesoderm that differentiate that go all the way around  As they are developing, reach down, fuse at ventral mid line As they are growing out, consolidating two layers of somatic mesoderm and meets at midline to suture The inner part is going to give rise to the muscle Outer layer is going to give rise to the pericardium sac Between gut and mesoderm, cluster of endodermal cells gives rise to epithelial cushion Endocardium forming on inside, myocardium surrounding it and pericardium cavity enclosing that Above have floor gut and developing cardiac tube Start as a single long tube, loops asymmetrically Chick cardiac development Looking at embryo that develop from top, have arms that fold Don't form a single tube - Two parallel tubes on either side that swing down around and fuse ventrally Beginnings of two vessels on either side of the midline and fuse to form single vessel If you treat chick embryo with vitamin A early in development, arrest fusion of two vessels - will form two autonomous parallel tubes never a normal heart, embryo dies Fusion of central vessel At top and bottom have big major vessels Us and chicks - Vessels at bottom will end up at top of heart and form the atria and the ones at the top are going to move down Asymmetrical looping and displacement top to bottom  Aorta and aortic arches where neural crest cells going to migrate and form ascending aorta and mitral valve Like in frog, as mesoderm forms to become precursor to heart muscle, enclose endodermal cells that will populate heart to form endocardial cushion Field of cells competent to contribute to heart is larger than what actually comprises the heart (like limb development) Mammals (mouse and human) 2 overlapping cardiac fields of cells: cardiac and periphery Cardiac field is smaller on top that will contribute to cardiac muscle. Peripherally, cells will contribute to nature of vessels Like chick start with central tube, ventricles on top, atria will develop from ramifying major vessels - larger below Bottom loop displaces up top Capillary beds Capillary bed formation Have two populations, open and merge to create a website Genetics of cardiogenesis Drosophila Tinman – homeobox gene. Possess a homeodomain, repressor region Acts to turn off specific targets Is first expressed in most mesoderm (almost universally during fly development)  Progressively restricted to cardiac mesoderm (a long ventral, pulsatile and open-ended tube) Homozygote null - neither copy of the gene active = Deletion results in absence of heart and gut muscle Can be induced by dpp, but not persistent enough to form heart ectopically Tinmann seems to be necessary for heart development Mouse Nkx2.5 (mouse and human) = tinman Expressed in mesoderm in a more restrictive way Expressed in pre-cardiomyocytes Mutation results in arrested development at cardiac tube stage- ventricles are underdeveloped and looping doesn’t occur. Ventricles remain on top. Molecular markers of heart, for the most part, are till expressed Interacts with Tbx genes, some of which play a role in septation Knockout mice can be rescued by ubiquitous over expression, will get rescue of cardiac development but curiously, although heart is r