BI410 final

Bone regeneration

- (1) Hematoma (bruise/clotted blood cells) formation - (2) Fibrocartilaginous callus formation --> new chondrocytes are drawn to that region and proliferate --> replace with cartilage, which undergoes ossification - (3) Bony callus formation --> cartilage ossifies into spongy bone - (4) Bone remodeling

Lung branching morphogenesis

- (1) Mesenchyme produces FGF10 - (2) Epithelium grows - (3) After growth, epithelium --> Shh - (4) Shh inhibits FGF10, bifurcating the FGF signal - (5) Mesenchyme produces FGF10 - (6) Epithelium grows - (7) After growth, epithelium --> Shh - (8) Shh inhibits FGF10, bifurcating the FGF signal

Overview of somite clock model

- (1) WAVEFRONT: Fgf8 leads to activation of system (always on, functions to start each cycle) - (2) BRIDGE: Fgf8 --> Wnt3a --> axin --I Wnt3a --> axin degrades and Wnt3a can be active again (this makes a cycle) - (3) CLOCK: Wnt3a --> Notch --> Lfng --I Notch activation (Notch activity will cycle as well) - (4) EFFECTOR: Notch --> Hairy1 --> --> EphrinA4 --> segmentation Somite clock model is built into paraxial mesoderm as it is formed --> if transplanted elsewhere, will segment on time; if transplanted in an inverted manner, it will segment according to its original location

Male gonadal development

- 4 weeks: genital ridge is between mesonephric ridge and aorta/dorsal mesentary - 6 weeks: when gonad is getting ready to develop, primordial germ cells migrate through the hindgut and dorsal mesentary to populate the genital ridge - 8 weeks: testis development --> Wolffian duct transforms into epididymis and vas deferens - 16 weeks: Mullerian duct degenerating; Wolffian duct tubules reach into testis - At birth: testes descend, seminal vesicles are formed, external genitalia has differentiated into a penis

Female gonadal development

- 8 weeks: PGCs arrive at genital ridge - 10 weeks: Mullerian ducts fuse to make beginning of uterus and top half of vagina - 20 weeks: follicles present; mesonephros and Wolffian duct completely degenerate (mesonephros does not form ducts into ovary like with testes); Mullerian ducts form fallopian tubes

Adaptive immune response vs. innate response

- Adaptive response (days): antigen presenting cell --> T lymphocyte --> bosses around B lymphocyte --> B cells and T cells give rise to memory cells (involves antibodies) - Innate response (hours): macrophages can phagocytose viruses, cytokine-mediated reactions, epithelial barriers, NK cells, etc. (doesn't involve antibodies) - Adaptive immunity is a relatively recent innovation in evolution --> if looking at metazoans, only see adaptive immunity starting with jawless fish --> only vertebrates have adaptive immune systems

Teratogen

- An agent which perturbs normal development - Thalidomide --> severe limb growth suppression - Alcohol --> fetal alcohol syndrome --> discriminating features (short palpebral fissures, flat midface, short nose, indistinct philtrum, thin upper lip) and associated features (epicanthal folds, low nasal bridge, minor ear anomalies, micrognathia) - Alcohol --> ROS (reactive O2 species) --> apoptosis in neural tube, which is rescued by SOD (superoxide dismutase, removes ROS) - Agent Orange is held responsible by many for the local elevation in birth defects in Vietnam - Aquatic pollutants include bioactive substances whose teratogenic effects are becoming increasingly appreciated --> less neurite outgrowth after exposure to PCB

Endothelial cells

- Aorta on both sides of notochord fuse to form primary dorsal aorta - Some sclerotome cells will migrate just below notochord and contribute to the aorta as well as making vessels that go between the somites (intersomitic vessels, which eventually become intervertebral vessels) - Sclerotome cells from the posterior somite contribute to the dorsal aorta and intervertebral arteries

Pancreas

- Arises from similar region of gut as liver - Big inducer is proximity to blood vessels - Pdx1 is pancreas TF - Blood supply induces islet cell differentiation - Pancreas initiates as two separate organs that later fuse together - Pancreas buds (dorsal and ventral) develop --> ventral is connected to common bile duct - Gut undergoes morphogenesis and is a bit twisted --> ventral side comes into contact with dorsal side and they fuse - Mature pancreas has common bile duct underneath it or penetrating it - Pancreatic lineages: ductal, acinar (exocrine), islet (endocrine) --> all arise from one multipotent progenitor lineage - Pancreas makes a system of ducts --> ducts terminate in exocrine organs (acini) --> secrete digestive enzymes - Islets are endocrine part of pancreas --> secretes hormones - Pancreas development: start building bud --> bud becomes more layered and has central duct --> small lumens arise between cells --> later, the lumens connect to form the system of ducts - Pdx1-positive cells give rise to all pancreatic lineages - Liver: FGF (heart) and BMP (ventral) --> pancreas: FGF (heart), not BMP (notochord) and endothelial induction (unknown signal) - Early endoderm --> inputs (including blood supply) --> induce Pdx1 --> pancreatic progenitor cell --> first makes ducts, acinar, or endocrine progenitor cells --> progenitor cell differentiates into alpha cell (glucagon), B cell (insulin), epsilon cell (ghrelin), delta cell, PP cell

Mesoderm development

- As the embryo develops, signaling changes from global to local - At the time when neural plate is developing, the mesoderm is sort of amorphous plane of cells reaching from dorsal midline to ventral midline - By the time the neural tube has closed, we have somites that came from the pre-somitic/paraxial mesoderm - Intermediate mesoderm more ventral to somites --> will eventually make kidneys and gonads - Ventral to intermediate mesoderm is lateral plate mesoderm, which has split into two layers --> upper layer closer to skin is called somatic mesoderm and lower layer is called splanchnic mesoderm - Somatic mesoderm is mostly going to make body wall and limbs - Splanchnic mesoderm is going to give rise to heart and circulatory system - Space between somatic and splanchnic mesoderm is called coelom - As development continues, the dorsal edges where somatic meets splanchnic come together underneath --> splanchnic ends up in middle with somatic/lateral plate wrapping around - Chorion and amnion begin to wrap around outside of somatic mesoderm --> chorioamniotic fold - Dorsal aorta tissue under notochord will eventually fuse to become aorta --> comes from splanchnic mesoderm on both sides - Tissue below dorsal aorta will become heart

Thymic selection

- Auto-reactive T cells are eliminated in thymus gland during late development - Positive selection in thymus cortex --> MHC is recognized by TCR (if not, apoptosis) - Negative selection in thymus medulla --> self antigens are not recognized (if so, apoptosis) - Aire and Fezf2 are thymus-specific proteins that produce self-antigens (TRA peptides) - If this process doesn't work well, you end up with autoimmune disease

BMP and bone development

- BMP is a critical bone-inducing signal - Need BMP inputs to make osteochondro progenitor cells that give rise to chondrocytes and pre-osteoblasts - Need BMP to promote the conversion of osteoblasts to embedded osteocytes - BMP triggers proliferation of chondrocytes to hypertrophy and initiate the bone-making process

Osteoporosis

- Bone loss - Can be caused by some diseases or drugs (steroids), menopause (no longer producing estrogens) - Bones become very fragile

Splanchnic mesoderm

- Bottom layer of lateral plate mesoderm - Gives rise to lymphatic system and circulatory system --> heart, blood vessels, blood - During development, lateral plate mesoderm curls down and splanchnic forms heart tube (somatic mesoderm becomes ventral body wall)

Achondroplasia

- Brakes applied too soon - FGFR3 activity is normally growth inhibitory - A GOF mutation in FGFR3 leads to constitutively active receptor, which leads to severe growth inhibition - Axial skeleton is pretty normal

Marfan syndrome

- Brakes are too weak - Fibrillin is mutated in Marfan syndrome --> this ECM component may normally reduce the amount of available growth factor by soaking it up

Stomach

- Breaks food down - Extremely acidic to promote digestion of food --> need a lot of mucus to protect tissue from acid - Don't have villi --> instead have deep branching crypts - Deeper tissue layers are similar to small intestine

Antibodies

- Built out of two heavy chains and two light chains - Antigens bind to variable regions (light and heavy) - Each antibody has two variable regions --> other regions are constant regions - To make variable regions, antibodies undergo genomic recombination (VDJ recombination) to generate diversity - Both heavy and light chains undergo recombination --> light chains don't have D segments - Variable regions are built out of V (variable, largest), D (diversity), and J (joining) segments - During embryonic diversity, one V, one D, and one J segment are selected and are recombined with each other at the level of the genome --> this means every B lymphocyte has a slightly unique genome in the region of its antibody genes - Further mutations can enhance specificity and diversity

Somite development

- Called paraxial mesoderm before it segments into somites - Somites make cartilage of vertebra and ribs, most muscles (body, limbs, tongue), tendons, dorsal dermis, and vascular cells (aorta, intervertebral vessels) - Somites are specified by the notochord (BMP inhibition) --> adding noggin-secreting cells results in extra somites - AP patterning of somites is controlled by Hox gene expression - Once presomitic mesoderm is specified, its AP fate is determined --> this is before segmentation has occurred - Transplant unsegmented mesoderm to a younger donor's anterior position --> end up with vertebrae in neck making ribs - Always make somites anterior to posterior - S0 is the somite we're currently making, SI is the somite we just made, and SII is the one we made before SI --> somite we're about to make is S-I and the one we make after that is S-II - In chickens, somites form every 90 minutes - Every somite can be divided into an anterior and posterior compartment - As time progresses, we gain more posterior cells, so what started out as posterior ends up in the middle of the body --> due to body extension - Delta gene KO --> perturb vertebrae and rib development - LOF for Delta-like ligand --> neural tube and somites are messed up - LOF for Notch --> same kind of defects (abnormal segmentation, problems with NT) - Notch and Delta are very important for appropriate specification and development of somites - Transplant mid-somite (to be) from quail to chicken --> nothing happens - Transplant of somite border (to be) from quail and transplant to middle of a different somite in a chicken --> tissue makes an ectopic border - Boundary information is already there - Transplant mid-somite (to be) from quail to chicken and electroporate activated Notch --> also get ectopic boundary - NOTCH ACTIVITY IS SUFFICIENT TO SPECIFY THE SOMITE BOUNDARY - Clock-and-wavefront model for somite formation: at the beginning, Notch activity (clock) turns on everywhere, then turns off and leaves a stripe, then turns on everywhere, then turns off and leaves a stripe, etc. --> each time it leaves a progressively more posterior stripe --> wavefront seems to be defining what the anterior-most reach of the Notch wave --> wavefront is thought to be FGF --> clock oscillates and wavefront moves progressively more posterior - Somite clock: reflected by the expression of hairy1 (a TF) downstream of active Notch (via NICD in nucleus binding CSL) - Notch activation --> hairy1 expression --> --> ephrin - Posterior half of each somite has Notch --> this is why posterior half of somites have ephrin - Ephrin ligands are present in posterior halves of somites, while ephrin receptors are turned on in anterior half --> Eph4A receptor expressed in anterior halves of somites-to-be - Ephrin induced immediately before separation in posterior of "new" somite, downstream of Notch --> Hairy - The combination of ephrin in posterior of new somite plus Eph (receptor) in anterior of trailing somite promotes their separation - Ephrins promote separation into somites by defining the posterior boundary via repulsive reactions to Eph receptor in trailing somite - Lfng (lunatic fringe) GOF or LOF --> perturb somite boundaries - Lfng is a glycosylase that operates in the Golgi --> adds sugars to Notch - Glycosylated Notch does not bind to Delta - Unglycosylated Notch binds Delta --> NICD binds CSL --> CSL drives hairy1 and Lfng --> Lfng glycosylates Notch --> Notch is inactivated - Lfng expression oscillates in presomitic mesoderm in same pattern as Notch --> this is why Notch oscillates

Tubulogenesis by cavitation

- Cavitation involves apoptosis --> cord and cell hollowing do not - (1) Polarizing signal --> basolateral - (2) Vesicles produced with apical markers - (3) Fuse internally to generate cavity - Podocalyxin (gp135) is associated with apical membranes, and is required for lumen formation --> KO either doesn't make lumen or makes several unconnected lumens - Vessel anastomosis employs VE-cadherin --> tip cells on the apical ends of vesicles send out filopodia that find each other and coalesce --> make internalized singular vesicle --> new apical vesicle fuses with lumens on either side --> complete open conduit - VE-cad KO --> still make filopodia but they never coalesce --> little lumens form but can't fuse with each other or larger approaching lumens --> VE cadherin is required for normal anastomosis

C. elegans vulva development

- Classic developmental model - Two sexes: hermaphrodites and males - Hermaphrodites have a pair of ovaries that produce eggs --> eggs enter uterus - Hermaphrodites also produce sperm, which are delivered to uterus - Eggs are fertilized and undergo divisions in uterus --> during development, eggs are laid through the vulva - Males only make sperm and do not have ovaries or oocytes - Most of the time, C. elegans are clones of their hermaphroditic parent, but they can have sex with males and get more genetic diversity that way - There is a naturally occurring phenotype called vulvaless --> vulva never forms and developing embryos cannot be laid, so the parent explodes to release them - Gonadal (ovarian) tissue has a cell in it called an anchor cell - Anchor cell sends out an inductive signal that is received by vulval precursor cells (VPCs) next to gonadal tissue - Cell nearest to anchor cell gets the most inductive signal and becomes primary vulva fate - Two adjacent cells get next highest dose of inductive signal --> make secondary vulva fate - Three distant cells are competent to respond to the signal but too far away to receive it --> develop as skin (hypodermis) - Cells undergoing primary and secondary vulva fates have more cleavages from tertiary cells --> primary has the most cleavages, secondary makes one fewer daughter cell than primary - Anchor cell specification depends on Notch signaling - Start with two cells, either one of which could become the anchor cell --> undergo lateral inhibition to define one cell as anchor cell (cell sending signal) and ventral uterine precursor (cell receiving signal) - Inductive signal sent by anchor cell is EGF --> VPC fates are controlled by EGF and Notch - EGF from anchor cell strongly inhibits Notch in primary cell (P6.p)--> low Notch, high Delta --> sends Delta signal - Weaker diffusion of EGF allows Notch signaling in secondary cells (P5.p and P7.p) --> high Notch, low Delta --> receive Delta signal

Limb patterning (Hox)

- Classical disaggregation/re-aggregation experiment in chick wing: if you take PZ mesenchyme out and mix it up and see what happens when you put it back, you see that limb develops fairly normally (distal structures at distal end, etc.) --> this is a patterning problem - PD patterning --> Hox genes expressed along limb --> note that Hox11 marks zeugopod and Hox13 marks autopod (same in forelimb and hindlimb) - Deletion of limb bone elements by deletion of paralogous Hox genes --> take hox11 out and end up skipping zeugopod - Deletion of paralogous Hox13 genes reduces the bones in the autopod (digits are small and fused, single knuckle) --> syndactyly - RA treatment proximalizes limb grafts --> take limb bud and graft onto head region of embryo --> more proximal development in RA-treated grafts than untreated - FGF and Wnt treatment distalizes limb buds --> expression of meis1 (stylopod), hoxa11 (zeugopod), hoxa13 (autopod) moves backwards after treatment with FGF/Wnt --> distal expansion - Early Hox gene expression in limb bud is curved --> later in development, it shifts and you get clean stripes with hox13 very distally expressed - HoxD cluster 3' end has cis-regulatory module ELCR (early limb control regulatory element) --> 3' ELCR drives nearest Hox genes first (9, 10, etc.) --> 5' POST restricts nearest Hox genes (12 and 13) to smallest spatial pattern --> together ELCR and POST produce pattern with small Hox13 domain at bottom, then 12 above (also tight domain), 11 above that (more spread), and 10 at top - Phase 1 of Hox expression sets up stylopod and zeugopod - Next, the terminal Hox genes collaborate to turn on ZRS enhancer (ZPA regulatory region) --> ZRS is long range response element in CRR for Shh --> Hox genes collectively turn on Shh expression in posterior of limb --> this defines ZPA - Next, Shh signaling (via Gli) activates Hox expression using the GCR (global control regulatory region), which drives the nearest genes first --> coming from 5' end so activates 13 first, then 12, etc. --> this shifts gene expression to "move" Hoxd13 distally and anteriorly --> Hoxd10-12 are nested, progressively more posteriorly --> future thumb expresses only Hoxd13 - This shifting of expression and setting up patterning on distal end is phase 2 --> phase 2 patterns autopod - Changes to 5' Hox enhancers are likely to underlie the evolution of limb elaboration and digit evolution --> AER region and autopod are shaped differently --> more autopod with more 5' enhancers

Digestive system

- Comes from endoderm - Digestive tract differs between different vertebrates --> different animals have different versions of cecum - Appendix or cecum in mammals and marsupials --> in most tetrapod mammalian animals, see cecum at junction between large and small intestine - Don't know function of cecum --> a reserve site for microbiome? - Amniotic embryos start of with endoderm looking mostly like yolk sac (2 weeks) --> start narrowing the yolk sac opening and tucking gut into body on ends (3 weeks) until opening to yolk sac is very small and most of the gut has been internalized (4 weeks) - Gut development: gut becomes progressively more internalized and distinct from yolk sac --> as this happens, endoderm -associated organs (thyroid, lung, liver, pancreas, gallbladder) bud from gut tube --> this occurs well after neural and heart development has progressed - The anterior digestive tract and associated tissues are derived from NC/prechordal plate and pharyngeal arches --> anterior end of formal endoderm is pharynx - Regional specification along digestive tract is marked by specific gene expression - Endoderm cellular morphology changes progressively as development proceeds --> squamous epithelium to cuboidal epithelium (while being internalized) to pseudo-stratified epithelium (starting to undergo differentiation and development) - Digestive system is very long and needs to be folded up --> small intestines are coiled - Looping is executed largely by the mesentery (from splanchnic) --> surface layer around inside of cavity and around all digestive organs - Mesentery is now considered to be an organ - Mesentery supports GI tract and its vascular and lymphatic supply - Separation of gut tube from mesentery --> intestine unfolds - Length of mesentery is shorter than length of intestinal tissue --> stretch mesentery and fuse two together --> get folding --> mesentery acts to contract and fold intestine at primary level of folding

Gene therapy

- Correcting genetic problems at level of gene - Nonviral vector --> transfection --> lipid vesicle fuses with cell and delivers plasmid --> mRNA made --> not very good approach because not long term - Adenoviral vector --> cell has to have right receptor --> once inside, make therapeutic protein --> adenoviruses aren't permanent so it's never incorporated into genomic DNA --> also, cells tend to recognize adenoviral infection and launch immune response - Adenoviral vector good for respiratory or liver tissues if you wanted a temporary change - Retroviral vector --> RNA genome with reverse transcriptase/integrase enzyme, protein shell, everything inside of lipid membrane --> bind to receptor --> endocytosis --> unpacking and RNA genome released --> reverse transcriptase causes RNA to be copied into DNA --> DNA enters nucleus and is inserted into host genome (integration, done by integrase) --> mRNA --> therapeutic protein - Advantages to retroviral vector: permanent change to genome - Caveats of retroviral: only infects cells that are proliferating (not differentiated cells); where therapeutic gene integrates is random, so there is the possibility it could integrate and disrupt important gene - Some researchers want to make adenoviruses that deliver genes to make anti-HIV antibodies --> anti-HIV antibodies expressed by tissue of person --> works in animals - Gene therapy for lymphocytic leukemia and acute lymphoblastic leukemia: patient's T cells are collected, genetically engineered to attack B-cells, then reintroduced into patient's circulation (CAR-T) --> 4 of 10 recipients in complete remission and 4 others have strongly reduced disease --> as of 2016, attempts to improve results via combination with chemotherapy are not successful since patients are dying (cytokine storm in response to re-engineered cells), so clinical trial halted in 2016 but back on now (give IL10 with re-engineered cells to prevent cytokine storm) - Gene therapy for hemophilia: adeno-associated virus expressing clotting factor IX used to infect patient's liver, which increases factor IX protein levels in the blood to normal levels - People who lose large pieces of bone to injury or cancer are candidates for gene therapy (adenoviral), and would otherwise require amputation or bone-shortening surgery --> autologous stem cells (fatty or bone marrow) are engineered to express BMP2 --> BMP2 cells imbedded into fibrin gel --> gel implanted into bone gap --> bones heal in response to BMP2 extremely quickly - Virus can rebuild heart's own pacemaker in animal tests via viral introduction of Tbx18 gene into heart - Dogs have been cured of type 1 diabetes via viral introduction of insulin and glucokinase --> both required for cure - Gene therapy for junctional epidermolysis bullosa via viral introduction of laminin --> stable for over 6 years - Engineering normal skin for junctional EB: skin biopsy --> viral correction of laminin expression --> expansion and transplantation --> nearly 100% recovery of normal skin - CRISPR/Cas9-mediated restoration of dystrophin in MD dog model

Development of aortic system

- Day 29: right dorsal aorta and left dorsal aorta connect to truncus arteriosus via aortic arches - Day 49: remodeling of carotid arteries, subclavian arteries, etc. --> remodeling not done in fish - Day 56: anterior circulatory system --> normal aorta with ascending and descending loops, subclavians go to arms, carotids go to head, pulmonary to lungs, artery and vein pairs for most major organs

Dermomyotome

- Dermomyotome is what's left over after sclerotome differentiates - Specification of dermomyotome is mostly Wnt-mediated - Muscle stem cells (myoblasts) proliferate between the dermomyotome and sclerotome and will migrate out to form muscles - The part of dermomyotome closest to notochord is dorsomedial lip (DML)/epaxial region - Epaxial region gives rise to dorsal body wall muscles --> less migration for cells due to proximity - The other end of the dermomyotome is called the ventrolateral lip (VLL)/hypaxial region - Hypaxial region makes ventral body wall muscles and limb muscles --> significant migration - In between DML/epaxial region and VLL/hypaxial region is central dermomyotome (cDM) - Central region gives rise to muscle stem cells and dermis

Lungs

- Develop from foregut epithelium - Starts out by creating a secondary tube (laryngotracheal tube) --> respiratory diverticulum - Tube branches off and separates from pharynx --> ends of tube branch into tracheal buds --> undergo branching morphogenesis to form ducts - Surrounded by mesenchyme - Takes about 8 weeks in mice - Progressive specialization and reduction of size/diameter --> develop over time - One of the last things to develop in embryo - Mesendoderm --> endoderm --> foregut --> lung/trachea --> distal --> proximal and distal - Nkx2.1 defines lung specification - Lung cell fates bifurcate into proximal (trachea, bronchi) and distal (branches and alveoli) - "Proximal-ness" is progressive --> "distal-ness" is at the actively branching region, so is constantly moving more distally - Wnts required for trachea and lung development --> Wnt2/2b KO doesn't make trachea or lungs - Wnt2/2b and BMP activate Nkx2.1 expression on the ventral side of the foregut - Dorsally, Noggin inhibits BMP (so Sox2 is expressed), and Sfrp inhibits Wnt - Sox2 inhibits Nkx2.1 and is ventrally inhibited by BMP - Branching is regulated by local mesenchyme --> if you replace lung mesenchyme with tracheal mesenchyme, inhibit branching - Have boundary between proximal and distal in tissue - Proximal epithelium re-expresses Sox2, while distal tips express Sox9 and Id2 --> YAP is involved in forming this boundary - FGF10 in distal mesenchyme promotes growth - FGF10 induces Sox9 in endoderm, which maintains proliferative stem-like state of distal cells, and blocks them from expressing Sox2, suppressing proximal fate - In proximal mesenchyme, RA and TGFB restrain FGF (and growth) to distal region - Once branched, add cartilaginous rings at larger diameters and cartilage plates at smaller diameters --> keeps airway open - Alveoli are lined with surfactant, a lipoprotein mixture that coats the cell surface and reduces tension at the water-air interface - Without surfactant, the sacs would completely collapse upon exhaling and be very hard to reinflate - Surfactant is produced at end of development, and pre-term babies sometimes don't have enough, leading to respiratory distress - Surfactant production in embryonic lungs triggers childbirth --> surfactant secreted into amniotic fluid --> macrophages there are stimulated and migrate into uterine muscle and execute signaling cascade that leads to uterine muscle contraction and labor

Brittle bone disease

- Due to defective collagen I --> recall that osteoid is a collagen I-rich ECM secreted by osteoblasts and osteocytes that is subsequently mineralized to produce bone - Bones are misshapen and fragile

Heart specification in mice

- E0 (E1 for humans): embryonic stem cells (ESC) of mouse blastocyst (expressing Oct4, Sox2, Nanog) or human epiblast (expressing Oct4, Sox2, Nanog, Brachyury) --> Nodal and Wnt (canonical) signaling to get to epiblast - Nodal, BMP2, Wnt (canonical) signaling to get to mesendoderm - E2: mesendoderm --> Oct4, Sox27, Gsc, FoxH1, Brachyury - BMP2 signaling to get to mesoderm - E4: mesoderm --> Mesp1/2, Tbx6, Brachyury, SRF - FGF and BMP2 signaling (E4-8) to get to cardiac mesoderm - E6: early streak --> primitive streak is just forming - E6.5: mid-streak --> embryo is undergoing gastrulation and is pushing mesoderm in - E7: late streak --> cranial cardiac mesoderm is already specified and is in anterior lateral position - E8: cardiac mesoderm --> Gata4/6, Nkx2.5, Mef2c, Hand1/2, Tbx5 - E8: 1-4 somites --> cardiac crescent present (will give rise to heart) - FGF, Shh, BMP, Wnt (non-canonical) signaling to get to cardiac myocyte - E12-16 (mouse-human): cardiac myocyte --> myosins, actinin, a-actin

Structure of a long bone

- Ends of the bone are called epiphysis - Middle part is called diaphysis - Epiphysial line is where growth plates are --> growth plates are eventually replaced by bone as you mature - Outer part of bone is called compact bone and is very dense - Inner part of bone is called spongy bone - Layer of cartilage around the outside of the compact bone - In the middle of the bone is bone marrow --> site of hematopoiesis (generation of blood cells) - Compact bone is organized into osteons - An osteon is a concentric circle of bone with a hollow opening n the middle called the central/Haversian canal --> this is where blood vessels go - The diameter of an osteon is limited by the diffusion of oxygen from its blood vessel to the cells around it - Each osteon is made of layers of osteocytes and bone called lamellas - Osteocytes extend a lot of fine processes that extend to other cells

Progress zone (PZ)

- FGF10 is expressed in the limb bud mesenchyme and drives proliferation to lengthen limb - FGF10 marks progress zone - FGF10 is the gas --> other factors act as brakes to control limb length - FGF10 also induces FGF8 in AER - PZ defines limb identity --> take block of mesodermal tissue that would have formed thigh structure (back of bud) and transplant to tip of wing bud --> resulting wing has upper wing and forearm with toes with terminal claws --> transplanted tissue recognizes that it's distal and makes distal stuff, but makes distal stuff from the limb it's originally from --> positional specificity along limb is plastic, while limb identity is not - Note that transplanted mesenchyme is not from distal region but produces distal structures - A variation of this experiment is to transplant heterochronically (times don't match) --> transplant young PZ to old limb bud --> young PZ makes a complete limb on top of what the old limb bud had already made - Transplant old PZ to young limb bud --> old PZ only makes digits --> thalidomide? - This shows that PZ contains proximal-distal information

Sexual dimorphism in Drosophila

- Females are slightly larger - Females have slightly larger wings - Males have extra barbs on front legs called sex combs --> important for the way they mate

Kidney

- Functional unit of a kidney is called a glomerulus - Glomerulus has blood vessels coming in, transiting around, then coming out --> blood comes in here to be cleaned - Glomerulus is surrounded by podocytes --> podocytes control filtration through capillaries by interlocking to form filtration slits - Capillaries of glomerulus are fenestrated and let fluids and solutes through - Net filtration pressure favors movement out of capillaries and into glomerular lumen - Fluid then enters tubule - Blood supply continues around and good stuff is reabsorbed from blood later in tubule system - Tube gets bigger and bigger and eventually goes to bladder

Treatments for birth defects

- Gene therapy - Stem cells - Regenerative medicine and tissue engineering

Intermediate mesoderm

- Gives rise to kidneys (urinary system) and gonads (reproductive system) - Intermediate mesoderm (IM) is specified between the somites and the lateral plate mesoderm in the primitive streak - Signaling environment: FGFs and Wnts around origin of IM help drive migration of cells away from primitive streak - When somites are epithelialized, IM is to the side of somites - When somites have differentiated, IM has moved downwards below the sclerotome --> gonadal/nephric ridge above the coelom (peritoneal cavity at this point) - RA signaling but doesn't get all the way posterior because we turn on Cyp26 --> gradients of FGFs and RAs help specify mesoderm tissues - FGF9 is important for specifying IM - Somites specified by combination of FGF9 and Cerberus - Lateral plate specified by BMP and Nodal - Notochord is source of Noggin - If you use a needle to separate presomitic mesoderm from intermediate mesoderm --> uninduced intermediate mesoderm - Intermediate mesoderm is specified by somites --> Lim1 and Pax2 TFs define kidney-producing IM - IM goes all the way down the body

Genes involved in sex determination/gonadal development in mammals

- Gonads produce gametes and are a critical part of the endocrine system - Gonad co-occurs with mesonephros (not identical tissues but adjacent) - Female is the default state, whereas male development requires input - Start with genital ridge --> differentiate gonad - PGCs migrate in - End up with three different cell types (whether male or female) --> germ line, supporting cells, and sex-hormone-secreting cells - In males, sex-hormone-secreting cells secrete testosterone, which pushes development in male direction - In males, Sertoli cells are the supporting cells --> express Sry - Sry from Y chromosome is expressed specifically in males - In response to Sry, males turn on sox9 --> inhibits B-catenin and drives AMF and Fgf9 (reinforces sox9) - Males express sry, sox9, fgf9, and AMH - Sertoli cells also secrete anti-Mullerian hormone --> promotes degeneration of Mullerian duct - In females, supporting cells are follicle cells - In females, hormone-secreting cells are called theca cells and produce estrogen --> pushes in female direction - Default female gonad activates B-catenin, which inhibits sox9 --> B-cat promotes female development - Females express Wnt4, B-catenin, and Rspo1 --> Rspo1 is marker of ovary and is involved in onset of meiosis in ovum - Females expressing Sry develop as males - Sox9 is sufficient to induce male-appearing testis development in females --> not sufficient to make fertile male gonads in females (unlike Sry) - Male but not female gonad attracts mesonephros migration --> take some mesonephros tissue and put it with gonad to see if tubules go into gonad --> if you have Sry, allows migration of mesonephric cells; no Sry, no mesonephric cell migration - Fgf9 treatment is sufficient to induce migration into female gonads --> take female gonad and make it express Fgf9 --> allows migration of mesonephric cells OVERVIEW: - Start with genital ridge - Get input signals --> mainly Wnt4 (female) or Sox9 (male) - Bipotential gonad --> XX has Wnt4 (B-cat) and Rspo1; XY has Sry and Sox9 - Ovary --> granulosa and thecal cells --> follicles --> estrogen - Estrogen promotes survival of Mullerian duct and causes it to differentiate into female internal genitalia (uterus, oviducts, cervix, upper vagina) - Estrogens also promote genital tubercle and urogenital sinus to become female external genitalia (labia, clitoris, lower vagina) - Wolffian duct degenerates by default in females - Females don't make androgens, so later in development, produce Wnt inhibitors (Dickkopf, etc.) --> Wnt/B-catenin activity is inhibited --> estrogen and estrogen receptor promote differentiation of female external genitalia - Testis --> Sertoli and Leydig cells --> AMF and testosterone, respectively - T preserves Wolffian duct and induces it to make male internal genitalia (epididymis, vas deferens, seminal vesicle) - AMF promotes Mullerian duct regression - Later, T is turned into DHT --> DHT promotes genital tubercle and urogenital sinus to become penis, prostate, and scrotum - Later during genital development, androgens binding androgen receptor inhibit Wnt inhibitor (Dickkopf, etc.) --> leads to activation of Wnt/B-catenin activity, which contributes to development of male external genitalia

Heart development

- Heart arises from heart fields --> two separate, anterior and lateral regions - What is the signaling environment for these cells? --> anterior, so Wnt is low; lateral/ventral, so BMP is high - Making cardiogenic mesoderm from lateral plate mesoderm - Heart is induced where Wnt is inhibited (more anterior) due to DKK/crescent from N. center, but where BMP is present (more lateral) - Hemangiogenic mesoderm also comes from lateral plate mesoderm (but from posterior) --> blood is induced where Wnt and BMP are both present (lateral and posterior) - Have early progenitor cells in splanchnic that could become cardiac precursor cell or hemangioblast - Important genes here are Bry (brachyury) and Flk1 (VEGFR) --> VEGF receptor --> vascular endothelial cell growth factor - To induce cardiac fate, first turn on Bry but not Flk1 --> later on, turn on Flk1 --> diversify and turn on various TFs --> endothelial differentiation (line heart), cardiomyocyte differentiation (contract heart), smooth muscle differentiation (around vessels) - To induce blood/blood vessel fate, turn on both Bry and Flk1 --> hematopoietic cells (blood) or angioblasts (vessels) - Cardiac fate is all downstream of BMP - Blood fate is downstream of BMP and Wnt - Cardiogenic mesoderm gives rise to all the heart lineages --> endothelial cells (line heart), cushion cells (part of septum), atrial myocyte, ventricular myocyte, purkinje fiber (conductive fibers) - Purkinje fibers are specialized conductive myocardial fibers --> coordinate heart muscle contractions - Heart development: splanchnic lateral plate mesoderm --> cardiogenic mesoderm --> form a tube (top part is called truncus arteriosis and will become ventricle; middle will become atrium; bottom is called sinus venosus and is inflow tract) --> bottom of tube bends --> atrium positioned above ventricles --> twisting and further morphogenesis --> make septum and divide chambers --> heart - When NT is in process of closing, notochord is beneath it and endoderm beneath that, with mesoderm between the ectoderm and endoderm on the sides --> amnion and chorion on top, yolk sac and allantois on bottom - Call the ectoderm and mesoderm on top the somatopleure (gives rise to amnion and chorion), and endoderm and mesoderm on bottom the splanchnopleure (gives rise to yolk sac and allantois) - Splanchnic mesoderm starts proliferating and differentiating into cardiogenic mesoderm cells - Endocardial cells between splanchnic and endoderm form one tube on each side - Tubes migrate toward each other/midline --> two tubes end up next to each other - At the same time, the mesoderm around the tube area differentiates into muscle (muscle and endocardial tube fates come from same progenitor cells) --> muscle forms a tube around the two tubes - Endocardial tubes fuse into one tube (endocardium) surrounded by myocardium --> myocardium pinches off from splanchnic mesoderm at bottom to close bottom of muscle tube, leaving yolk sac below - Miles apart gene (sphingosine-phosphate receptor) is required for heart fusion --> expressed in endoderm on either side of the midline and is required for correct response to fibronectin cues along migration pathway - Miles apart mutant results in cardia bifida - In cardia bifida, two functioning hearts develop, but they don't connect to vasculature properly --> lethal - Foxp4 (TF) is expressed in foregut endoderm along the migration path --> in mutants, each side develops a separate heart - ECM components are required for cardiac migration --> fibronectin KO develops hearts on separate sides --> fibronectin is required - ECM remodeling is required for cardiac migration --> migrating cells express mmps in front of them --> mmp (matrix metalloproteinase) remodels ECM --> mmp inhibitor at stage 5, no migration --> stage 7, block end of migration --> 3 somite stage, no blocking --> mmps are important during stage 5 and 7 but finished by 3 somite stage - Dorsal aorta also fuses - We have now formed heart tube --> next we have to loop and twist it up - Day 21: heart tube formed --> ventricles and truncus arteriosus (becomes aorta and pulmonary arteries) are up and atria and sinus venosus are down --> tube splits at the bottom into vitelline veins (become vena cava/atria?) - Day 28: looping --> atria ends up on top and ventricles on bottom - Looping depends on asymmetric cell divisions (left faster than right --> causes it to bend), cytoskeletal changes, and ECM remodeling by MMPs - Twist truncus arteriosus in front of atria, build septa to separate chambers and truncus arteriosus - 33 days: heart is one big tube --> start making cushions that are beginning of septa --> most of septa comes from NC cells that migrate in - Looping is conserved in vertebrates --> separates inflow and outflow into two different planes --> this has significant effects on the way blood flows (less turbulence, less valve damage due to contact with flow) --> protostomes have linear, deuterostomes have S shape (more dramatic as you progress to amniotes) - Third month: heart has four chambers - As an embryo, we keep an opening between the two atria (foramen ovale) --> also keep an opening between aorta and pulmonary artery (ductus arteriosus) --> this is because we're not using our lungs for gas exchange yet (doing gas exchange through placenta) - Foramen ovale and ductus arteriosus close by the time you're born --> separates oxygenated and deoxygenated blood once we start using our lungs - Babies born with holes in their hearts have foramen ovale that hasn't closed - Aorta and left side of heart deliver oxygenated blood from lungs to body - Pulmonary artery and right side of heart deliver deoxygenated blood from body to lungs

Heart development in chick vs. mouse

- Heart development occurs early and follows defined stages - Early cardiogenesis: 12-22 h in chick (split into 12-13, 18-19, and 19-22); day 6-7 in mice - Specification during early cardiogenesis - Determination overlaps with specification during final third of early cardiogenesis --> determination ends 60% into heart field formation - Heart field formation: 23-29 h in chick (split into 23-25, 26-27, and 27-29); day 7-8 in mice - Patterning overlaps with determination during middle third of heart field formation --> patterning ends 30% into heart tube formation - Heart tube formation: 29-45 h in chick (split into 29-33, 33-38, and 40-45); day 8-9 in mice - Differentiation starts with heart tube formation and overlaps with patterning during the first third of heart tube formation - Each of these stages of development is marked by the expression of different sets of genes (signaling molecules --> myocardial transcription factors --> differentiation products --> morphogenetic regulators --> morphogenetic effectors)

Migration of PGCs in discoidal embryos

- In chicks, PGCs from germinal crescent (marked by vasa) - The germinal crescent is just anterior of the embryo itself --> here, PGCs are specified through early signaling - At cup-shaped stage (before gastrulation), PGCs are just outside the posterior end of embryo - After gastrulation, migrate into embryo --> migrate to allantoic region, and later migrate further into embryo - Like with flies, PGCs are often associated with the hindgut --> often migrate in hindgut, up the gut tube, then go across tissue bridge (dorsal mesentery) that connects to genital ridge --> go into genital ridge (developing gonad) - In mouse, the migration depends on fibronectin

Regenerative medicine and tissue engineering

- Initially defined by Kaiser 1992: "A new branch of medicine will develop that attempts to change the course of chronic disease and in many instances will regenerate tired and failing organ systems" - Often involves SCs - Direct transplantation of cells to replace damaged/diseased organs - Injection of cells that secrete regeneration-inducing signals - Directly providing sources of regeneration-inducing signals (e.g., ECM) - Transplantation of in vitro-grown organs (tissue engineering) - Basic principles of tissue engineering: cells from a biopsy --> monolayer cell culture --> expanded cells --> culture on a 3D polymeric scaffold --> generation of a graft --> transplantation - Artificial bladder for spina bifida patients (Anthony Atala) --> surgeon takes small, full-thickness biopsy from bladder --> in lab, isolate urothelial cells (bladder lining) and smooth muscle cells (outer surface) --> isolated cells cultured separately --> cells attach and grow properly throughout scaffold --> neo-bladder implanted --> biodegradable scaffold dissolves and is eliminated from body - Artificial trachea --> scaffold mimics sponginess of the endogenous stroma and also has hard rings, mimicking the usual cartilaginous rings - Currently controversial --> issues with patient consents and high rates of patient deaths - Engineering a kidney: take a kidney and flush proteases in to decellularize --> scaffold of ECM is left --> scaffold seeding --> put endothelial cells into renal artery and they become artery (HUVECs), blood vessels repopulate --> put epithelial cells in ureter and they become ureter (neonatal rat kidney) --> maintain negative pressure to make cells want to go into organ --> recellularized - In vitro analysis of kidney engineering: with added pressure, regenerated kidneys produce nearly the urine output as cadaveric kidneys --> in vivo analysis: engineered kidneys produce 37.5% of the urine as the control (endogenous kidney) - Advances in skeletal muscle engineering: mesangioblasts (Mab cells) are combined with polyethylene glycol and fibrinogen in hydrogel, then injected directly into sites of muscle injury in mouse muscular dystrophy model ---> the use of gel increases Mab cell survival and integration, and the organization of resulting muscle fibers - Artificial skin has lots of different types of cells, but not going to get hair follicles or sweat glands - Skin gun: spray skin SCs - Cartilage is very poorly regenerative, and damaged cartilage is very difficult to repair (because it's mostly ECM and pretty much acellular) --> 3D printed engineered cartilage implants will have profound medical benefits, offering an alternative to joint replacements - 3D printer can be used to create new bones: combine artificial ECM (like bone) and take bone marrow and cells from patient --> put it all together and 3D print bone --> implant in different place inside patient and let it vascularize --> scaffold dissolves --> then transplant to where it belongs and connect vessels - Organ printing: cluster of human cells behaves like a liquid --> when placed next to one another, clumps fuse to form layers or other shapes --> printer assembles cells into shape --> printer creates living bio paper and prints cell clusters onto it, drop by drop --> cell clusters fuse and form more complex tissue structures --> after each layer is completed, another layer of cell clusters is added --> eventually, bio paper separating layers dissolves and individual layers fuse - Capillaries are hard to print because they're so small --> one of the major limitations of organ printing is getting them fully vascularized at microscopic scale

Two-step model for amphibian limb regeneration

- Injury - Wound closure - Proliferation and cell migration - If no input signal, scar forms --> scar remodeling --> skin regeneration (this is what happens in us) - (1) Signals from nerves --> proliferation and de-differentiation - Formation of blastema - If no signal, synthesize ECM and cartilage --> blastema regresses - (2) Signals from fibroblasts and AEC --> promote growth and pattern formation --> limb regeneration

Body systems derived from mesoderm

- Integumentary system (dermis) --> from somites - Muscular system --> from somites - Skeletal system --> from somites - Circulatory system --> lateral plate mesoderm - Lymphatic system --> lateral plate mesoderm - Urinary system --> intermediate mesoderm - Reproductive system --> intermediate mesoderm

Vertebrate kidney development

- Kidneys are derived from intermediate mesoderm, which goes all the way down the body - Pronephros (most anterior): initial embryonic kidney in anamniotes (vestigial in amniotes) --> pronephric duct is required for induction of the rest of the system in amniotes - Mesonephros: secondary embryonic kidney, becomes opisthonephros (adult kidney) in anamniotes --> embryonic kidney, degenerates/contributes to gonads in amniotes - Metanephros (most posterior) --> adult kidney in amniotes - Need to have a kidney at all times during development to filter waste from blood - Like the somites, kidney segments from anterior to posterior, starting with pronephros and then mesonephros --> metanephros does not segment - Along with segmentation, we also make a duct - For all IM, first divide into one side that makes a duct all the way down and one part that segments all the way down --> segments then connect to duct - Paramesonephric duct is pre-gonad (also part of IM) - In the beginning, the IM is just a block of tissue, which is divided into anterior and posterior parts (marked by specific TFs) - Next, segregate out the duct part (nephric duct) from the mesenchyme part (nephrogenic mesenchyme) --> maintain AP specification in NM - Then start having segmentation, differentiation, connection to ducts (elongating as this is happening) - When we first make the pronephros, the embryo is still fairly primitive --> aorta is right below notochord and sends out vessel to make primitive glomerulus against coelom --> secrete stuff from blood into coelom - When we make pronephros, we make one nephron on each side - Make pronephros to reabsorb from coelom - Pronephros eventually degenerates - Make mesonephros, which has its own glomerulus --> glomerulus feeds into duct, which goes into bladder - With mesonephros, get 10-50 nephrons - Eventually make metanephros, with all the kidney stuff and one million nephrons - Compare gene expression in mammalian metanephros, zebrafish pronephros, and xenopus pronephros --> see differences in TFs that are expressed along the whole system --> those TFs are conserved across evolution --> nephron organization conserved/metanephric and pronephric nephrons share a similar segmentation pattern - To form mesonephros, mesonephric duct and mesonephric vesicle fuse --> aorta sends out capillary bed --> fused vesicle/duct wraps around capillary bed to form receiving end of nephron --> tube behind folds (recollecting system) and leads to duct - Formation of metanephros is similar - Glial-derived neurotrophic factor (GDNF) from metanephrogenic mesenchyme induces ureteric bud --> Ret is GDNF receptor on metanephrogenic duct - GDNF KO ---> make nephric duct, but never make ureteric bud --> never enter MM and make kidney - One GDNF allele knocked out --> weak formation - Ret-expressing cells give rise to ureteric buds --> made chimera with some cells with Ret and some without --> only Ret-expressing cells are competent to become bud - Duct enters mesenchyme and undergoes branching morphogenesis - Wnt9 is required to drive the condensation of the metanephrogenic mesenchyme --> if that doesn't happen, no development of kidneys (still get bud) - Wnt11 is required for branching morphogenesis (mechanism unknown) - TGFß1 delays branching (stabilizes existing branches?) - BMP4 is inhibitory (branch site selection?) - When condensation and branching are done, still need to make glomerulus and tubules - Mesenchyme undergoes mesenchymal to epithelial transition (MET) --> MM condenses onto tips of ducts after branching and forms tubules and glomeruli - Tubule system (glomerulus, bowman's capsule, etc.) all comes from MM and connects to the collecting duct (which comes from the ureteric duct), which goes to the bladder - Switch from mesonephric to metanephric systems, and the formation of the bladder --> at first, the ureter is connected to the nephric duct, which connects to the bladder --> over time, these resolve and the ureter connects directly to the bladder - In males, some of the nephric duct is preserved and transforms into the gonads - Nephric duct also called Wolffian duct - Initially have one common excretory opening called the cloaca --> hindgut, bladder, and allantois connect to it - Gradually separate cloaca into urogenital sinus and rectum

Limb DV patterning

- Limb DV patterning depends on Wnt - Dorsal ectoderm expresses Wnt7a - Wnt drives Lmx1 (TF), which is expressed in dorsal mesoderm - Ventral side expresses engrailed - This patterning is highly conserved - Wnt7a mutant mice show limb DV transformations --> ventral structures (tendons, footpads) are dorsally duplicated in mutant - Lmx1b mutant mice show limb DV transformations

Limb development

- Limb develops primarily from somatic mesoderm - Early limb buds --> flattened paddle --> digits - Mouse vs. chicken timing: chicken goes a little faster and a little earlier, but comparable - Mesoderm in PZ undergoes proliferation - Hypaxial myoblasts migrate down and into limb - Motor neuron axons, neural crest derivatives, and myoblasts all migrate into developing limb - Initial limb musculature: dorsal and ventral blocks - Mature limb musculature: refinement and redistribution - Specialized motor neurons at regions for limbs - Dermatomes: regions of skin from which sensory neurons enter spinal cord (not to be confused with somite regions also called dermatomes) --> along trunk, stacked disks; along limbs, longitudinal - In humans, forelimb develops first --> as arms and legs come out, they turn --> dermatomes shift dramatically when turning happens

Limb bud positioning

- Limb positioning is dictated by Hox gene expression --> forelimbs always at anterior thoracic (where Hox6 starts), hindlimbs always at sacral - Transplantation of somites changes limb bud sizes --> thus, somites define limb field (position and size) --> transplant somites from limb to flank level and get enlarged limb bud --> transplant somites from flank to limb and get smaller limb bud - FGF induces limb buds and drives Shh expression --> implant FGF bead and induce ectopic limb bud - Fgf10 is expressed in limb bud, and ectopic Fgf10 induces extra limbs - Specification of limb type in chick by Tbx4 and Tbx5 (TFs) --> Tbx5 expressed in forelimb bud and Tbx4 in hindlimb bud --> specifies forelimb vs. hindlimb - In ectopic FGF10 experiments, if you put it in the middle between the forelimb and hindlimb, you can get it so that the top half is expressing Tbx5 and lower half is expressing Tbx4 --> get a chimera limb - Pitx1 is as important as Tbx4 for hindlimb positioning - RA is required to suppress FGF8 expression in torso region and to drive Tbx5 --> FGF8 strongly expressed in heart and caudal plate --> RA and FGF8 are expressed in mutually exclusive spatial domains - When RA synthesis is blocked, FGF8 expression expands --> thus, RA is required to repress FGF8 - When RA synthesis is blocked, Tbx5 in forelimb bud is lost --> thus, RA is required to induce Tbx5 expression - Model for how embryo knows where to put limbs: (1) Hox --> specify somites as trunk; (2) somites --> RA; (3) RA --I Fgf8, permitting forelimb bud development; (4) RA --> Tbx5 - Prospective forelimb fields of salamander and mouse: RA in trunk region gives field of cells that can give rise to the limb --> shoulder girdle anterior to free limb and flank tissue posterior to it; ring of tissue around it that is also competent to become limb - If you laser ablate everything making limb, ring of tissue steps in and makes limb --> if you laser ablate everything (including ring of tissue), no limb formed - FGF10-expressing mesoderm is called the progress zone (PZ) - FGF8-expressing ectoderm is called the apical ectodermal ridge (AER) - As the PZ proliferates, it undergoes EMT and becomes migratory --> start with somatopleure (somatic mesoderm plus ectoderm), then undergo EMT --> somatopleure ends up bent over on side (still maintain epithelial layer at bottom near coelom) - Three crucial regions, AER, PZ, and ZPA (zone of polarizing activity --> expresses Shh) --> this system is what grows out and makes bones towards back of limbs, then bones for digits --> progression from 1 bone to 2 bones to many bones - Both PZ mesenchyme and AER collaborate to produce the limb - AER controls the extent of growth and some patterning --> add extra AER, wing is duplicated - Mesenchyme controls identity --> add leg mesenchyme into wing bud, make leg stuff - Mesenchyme required to maintain AER --> put non-limb mesenchyme in, cease development (because no FGF10, so AER regresses) - FGF sufficient to replace AER --> replace AER with FGF8 bead is sufficient to rescue development

Lineages from mesenchymal stem cells

- MSCs are multipotent --> could become adipocyte, muscle cell (MyoD), chondrocyte (Sox9), or osteoblast (Runx2) - ENDOCHONDRAL OSSIFICATION (axial skeleton, extremeties): if it makes an osteo-chondro progenitor cell, it can differentiate into a chondroblast --> chondroblast will make a chondrocyte --> chondrocyte can become a hypertrophic chondrocyte --> hypertrophic chondrocyte can differentiate into a preosteoblast --> preosteoblast can turn into osteoblast --> osteoblast can turn into osteocyte - ENDOCHONDRAL OSSIFICATION (axial skeleton, extremities): osteo-chondro progenitor cell can also differentiate into a perichondral cell --> perichondral cell can differentiate into preosteoblast --> preosteoblast can turn into osteoblast --> osteoblast can turn into osteocyte - INTRAMEMBRANOUS OSSIFICATION (skull bones, clavicles): MSC can turn into preosteoblast (Runx2) --> preosteoblast can turn into osteoblast --> osteoblast can turn into osteocyte

Liver

- Made of hepatocytes and biliary epithelium - Starts out budding from foregut --> liver diverticulum --> liver bud growth --> hepatocyte/biliary differentiation --> hepatic maturation - Develops anterior, right near heart --> cardiogenic mesoderm sends inductive signals for liver to develop (FGF) --> local mesenchyme in area secretes BMP - Induced by heart (FGF) and local mesoderm (BMP) - Suppression from notochord (Wnt/FGF4) --> develop away from posterior part of body - Liver will involve collaboration of mesoderm, endothelial cells, hepatocytes, and cholangiocytes - Start with mesendoderm cell --> mesoderm (Bry) and definitive endoderm (FoxA and Gata) --> endothelial cells (blood vessel cells) - Definitive endoderm can then specify progenitors that give rise to liver or pancrease (depends on Hhex) --> Hhex expression defines hepato-pancreatic lineage - This lineage diverges into pancreatic (Pdx1) and hepatoblasts (HNFs) --> liver fate specified by HNFS - Hepatoblasts give rise to hepatocytes (cells that make up liver, HNF4 and Prox1) or cholangiocytes (make biliary system, HNF6, Sox9) - Inductive signals from heart and STM (septum transverse mesenchyme) induce differentiation as progenitor/hepatoblasts --> some endothelial cells develop too - Hhex turns on - Prox1 comes on --> now genuinely hepatocytes - At this stage, also see MMPs that help break down ECM into patches --> hepatocytes invade mesoderm - Bile duct formation from cholangiocytes: in beginning, have hepatoblasts with endothelial cells lining lumen in middle with mesenchyme - Differentiate some biliary epithelium (cholangiocytes) - Continue and make lots of biliary epithelium --> make some little lumens - Some lumens persist and are surrounded by cholangiocytes and specialized mesenchyme --> make continuous tubes that run along middle lumen - The rest of the cells become hepatocytes - Endothelial cells lining lumen are fenestrated --> blood bathes hepatocytes - Between hepatocytes, see bile duct tubes - Tubules gather into bile duct, which feeds into small intestine - Blood enters from portal vein and hepatic artery and leaves through central vein - Portal vein comes from gut --> first pass effect --> gives liver chance to remove anything pathogenic or toxic that you might have consumed that made it into your blood - Kupffer cells (macrophages) surveil blood as it comes in - Blood also comes in from hepatic artery (arteriole), which comes from aorta (oxygenated blood) --> supports liver cells - Between endothelial cells and hepatocytes is space of Disse --> where blood goes to bathe hepatocytes - Stellate cells (fibrogenic) store vitamin A and respond to liver damage --> cirrhosis through overactive stellate cells

Endoderm fates

- Mesoderm --> hepatic mesenchyme - Endoderm --> foregut, midgut, hindgut - Foregut --> lung, liver (hepatoblast), gall bladder, pancreas - Liver --> hepatocyte, biliary epithelium

Mesoderm in head

- Mesoderm in head makes most head muscles - Prechordal mesoderm makes muscles that move our eyes - 3rd and 4th pharyngeal pouches make muscles towards the back of our head - Cheek muscles come from pouches 1 and 2

Migration of Drosophila pole cells

- Migration path: repulsive and attractive cues - Pole cells migrate through midgut as germ band wraps around --> hindgut invaginates and pole cells go with it - Migrate out of hindgut and into mesoderm - Alignment with gonadal mesoderm - Somatic cells of gonadal mesoderm bind to PGCs, and surround them --> gonad is being formed

Model for limb patterning in PD axis

- Model: FGFs and Wnts from AER/ectoderm antagonize RA from flank - These opposed gradients define the PD pattern along the limb - RAs --> Meis --> stylopod - FGFs/Wnts --> Hox13 --> autopod - Low RAs and low FGFs/Wnts --> Hox11 --> zeugopod - Zeugopod defined by two distinct thresholds: one for RA ( for proximal end) and one for FGF8/Wnt (for distal end)

How do bones develop?

- Most bones begin as cartilage models (chondrocytes, not osteoblasts) --> chondrocytes from sclerotome make Hyaline cartilage model that's shaped like the bone - Cartilage model has a primary ossification center in the middle - (1) Formation of bone collar around hyaline cartilage model --> compact bone around cartilage model - (2) Cavitation of the hyaline cartilage within the cartilage model --> spongy bone forming within cartilage model - (3) Invasion of internal cavities by the periosteal bud and spongy bone formation, vessels enter - (4) Formation of the medullary cavity as ossification continues; appearance of secondary ossification centers in the epiphyses in preparation for stage 5 - (5) Ossification of the epiphyses --> when completed, hyaline cartilage remains only in the epiphyseal plate and articular cartilages

Amyotrophic lateral sclerosis (ALS)

- Motor neuron disease in which motor neurons die --> muscles are not triggered to contract - ALS mechanism remains unknown - Neural degeneration: inclusion bodies in motor neurons - Genes associated involved in protein degradation, RNA processing, cytoskeleton

Collaboration between endoderm and mesoderm for liver development

- Multiple inductive signals are exchanged between endoderm and mesoderm in development of liver --> each germ layer sequentially instructs the other - Shh in endoderm --> reception of Shh in mesoderm --> Hox gene expression in mesoderm --> specification of mesoderm --> BMPs, FGFs made by mesoderm --> reception of paracrine signals by endoderm --> specification and differentiation of endoderm --> paracrine factors in endoderm --> differentiation of mesoderm

Muscular dystrophy

- Muscles degenerate - Proteins with mutations implicated in MD - Problems with proteins in connections between cells --> not holding cells together properly - Comparing healthy skeletal muscle to Duchenne skeletal muscle: muscle cells not ensheathed normally --> fat deposits fill spaces between cells --> collagen-matrix (scar tissue) begins to accumulate between cells --> increased inflammation reduces the weakened cells further --> cycle repeats

Androgen insensitivity

- Mutation in androgen receptor - Phenotypic female, genetic male - Undescended and sterile testes - Only have bottom half of vagina --> still have Sry and Sox9 so undergo male internal development early on

Bat vs. mouse

- Note the larger and differently shaped distal limb bud in bats - Bat forelimbs show increased Gremlin (inhibits apoptosis) and FGF8 (promotes growth) expression in interdigital regions

Notochord development

- Notochord is segregated from paraxial mesoderm --> boundary formation through replacement of C-cadherin clusters at border with actin-myosin contractile cables - The embryo body elongates via 3 mechanisms - (1) Enlargement (inflation) of internal vacuoles within the notochord - (2) Posterior migration of the dorsal medial zone (DMZ) cells --> some of these will become neural, some somites --> sets up progenitor zone on posterior end - (3) Cell division (triggered by cdc25) of presomitic mesoderm (PSM) in lateral/posterior regions - Integrins expressed by notochord tie it to the adjacent tissue --> if integrins are knocked out, then the notochord buckles (because not able to pull other tissue with it via ECM) - Notochord requires collagen 8a to develop straight --> when col8a is knocked out, notochord and neural tube are kinked and some vertebrae fuse - The notochord becomes the nuclei pulposi (within intervertebral disks) as development proceeds --> this process requires Shh

Primordial germ cells (PGCs)

- PGCs are set aside very early in development - PGCs usually develop outside the embryo proper --> this probably protects them from signaling influences - PGCs always express vasa, an RNA helicase that promotes translation of certain mRNAs - PGCs are generally repressed for transcription and translation, and this requires vasa function --> this probably keeps them stem-cell like (keeps germ line genome protected) - PGCs migrate into future gonads later in development

Myotome

- Part of somite that develops into muscle - Muscles are made of syncytial cells with nuclei pushed off to the side --> cells are filled with myofibrils containing actin and myosin - Muscle cell is surrounded by ECM/laminin layer and has Dystrophin inside plasma membrane --> together, the laminin, membrane, and dystrophin make overall structure of fibril unit - Satellite-like cells are marked by Pax7 - Mitochondria are all aligned in between the fibers --> need a lot of mitochondria to make ATP - NMJ is muscle side of synapse

Dermatome

- Part of somite that makes dermis of skin - Central region of dermomyotome, right below epidermis - Common fibroblast progenitor cells from dermatome - These cells differentiate into a variety of different fibroblasts (most of the cells that make up the dermis are fibroblasts) --> papillary fibroblasts (papillary layer), reticular fibroblasts (reticular layer), pre-adipocytes (towards bottom), and adipocytes (bottom of skin) - Dermal papilla is a specialization of papillary fibroblast cells - Dermal sheath cells are associated with the papilla - Dermamyotome makes only the dorsal dermis, while the ventral and limb dermis is made by lateral plate mesoderm --> limb muscles from somites, limb skeleton and dermis from lateral plate

Syndetome

- Part of somite that makes tendons - Syndetome is specified between sclerotome (bones) and myotome (muscles) --> in the correct anatomical location for tendons - Gene that marks the syndetome is called scleraxis --> in situ shows it in every somite and tendons - Myotome (muscle) secretes FGF --> which induces sclerotome --> sclerotome also gets positive inputs from Shh - Sclerotome gives rise to tendon --> more Shh gives sclerotome, less Shh gives tendons - This fits with syndetome position in regards to notochord --> sclerotome is closer and syndetome is farther away - Sclerotome that doesn't become tendon has mutual exclusion

Sclerotome

- Part of somites - Sclerotome region is specified primarily by notochord (Shh) - If you transplant notochord, convert the entire somite into a sclerotome - Sclerotome produces the axial skeleton (ribs, vertebrae, back of skull, not the face) --> lateral somatic mesoderm produces the limbs, including the appendicular skeleton - Sclerotome cells migrate around the neural tube to form vertebrae - The notochord is obliterated in the bones, but retained as the nucleus pulposus of the intervertebral discs - Nerves, muscles and sclerotome are all lined up --> nerves need to reach muscles but sclerotome is in the way - Split sclerotome into rostral and caudal segments so nerves can reach muscles --> merge caudal segment of one sclerotome with rostral segment of sclerotome below it to form vertebrae - Muscles and nerves remain segmented, while bone offsets to get out of the way (not unlike segments and parasegments in flies, except flies are specified offset and we just rearrange to offset) - Final vertebrae are offset from original segmentation pattern to allow the passage of nerves

Pax3

- Pax3 drives expression of myoD family of TFs (bHLH type) --> myf5 (early, specification), mrf4 (early/late, specification), myoD (mid, specification), myogenin (late, differentiation) - Pax3 and myf5 are required for muscle specification in the body and limbs, but not in the head - Myf5 heterozygote --> normal musculature - Heterozygous mutant for myf5 and pax3 --> have face and body muscles but lose diaphragm and muscles in limbs --> no development of posterior muscles - Homozygous loss of myf5 and pax3 --> retain muscles in head but no muscles in limbs or body -

PGCs in Drosophila

- Pole cells - Granules from pole cells: cytoplasmic determinants that are differentially inherited (flies and worms) --> granules define pole cells as PGCs - Normally, have oskar localized to posterior end of embryo and bicoid localized to anterior end - Take 3' UTR from bicoid gene (contains bicoid localization signal) and stick it onto oskar gene --> make oskar go to anterior end of embryo --> pole cells induced at both ends of embryo --> oskar is sufficient to drive specification of PGCs in flies

Hutchinson-Gilford progeria

- Premature aging - Defective lamins (proteins that make nuclear envelope) - (1) Arrested fibroblast proliferation and death - (2) Defective Wnt signaling (canonical) - (3) Defective ECM production - Rescue fibroblast proliferation with exogenous ECM proteins - CRISPR/Cas9 targeting defective lamin restores health and life span in mouse progeria model - Subjects who receive Cas9 and guide mRNAs have largest improvements

Myogenesis timeline

- Primary myogenesis in embryonic development --> primary myotome/dermomyotome with stem cells proliferating right underneath it --> cell fusion - Pax3 and Pax7 specify muscle in somitic stem cells --> if only expressing Pax3, become muscle; if expressing Pax3 and Pax7, become a muscle stem cell - Secondary myogenesis during fetal development --> progenitor migration to limb buds and body wall --> basal lamina assembly; innervation/NMJ formation; MTJ formation - Pax7, Myf5, and Pax3 turn on in fetal stem cell - MyoD turns on in muscle cells during migration - During neonatal development --> no somite left --> adult fiber type specification; satellite cell niche establishment - Adult --> homeostasis; atrophy/hypertrophy; regeneration - Head muscles come from prechordal mesoderm

Sites of hematopoiesis

- Progress through 1-4 over time - (1) Yolk sac - (2a) AGM (aorta/gonad/mesonephros region) - (2b) Placenta - (3) Fetal liver - (4) Bone marrow

Python vs. anole

- Pythons exhibit deletions in ZPA-specific Shh enhancer (ZRS) that reduce Shh expression, leading to apoptosis of limb bud and loss of most of limb - Pythons produce a rudimentary limb, while anoles develop a normal limb - Comparing Shh CRR ZRS module across species shows that pythons have deletions in three major regions: A, B, and C - Testing python vs. anole ZRS for transcriptional activity shows that python ZRS is significantly less functional - Python limb buds undergo normal early development, then apoptosis

Large intestine

- Reabsorbs water - Mostly crypts (like stomach), but not as mucosal because not as acidic

Limb regeneration

- Regeneration of salamander forelimb: if you amputate through zeugopod or stylopod, limb regrows to same proportions - Regenerating limbs --> a blastema (cells undergoing proliferation) --> cells at tip of limb de-differentiate - (1) Blastema (comparable to PZ) induces apical ectodermal cap (AEC), which is analogous to AER both structurally and molecularly - (2) Blastema proliferates and grows - (3) Blastema re-differentiates - A region of de-differentiated cells is just proximal to the proliferating blastema - Regeneration is always toward distal, regardless of upstream anatomy --> amputate hand, take stump end and reattach to body and allow to heal --> reamputate through stylopod --> now have two regeneration stumps (distal end in body wall, proximal end extending out) --> both regenerate to make distal stuff - Donor and host limbs amputated and blastemas form --> proximal blastema and distal blastema --> proximal blastema removed, distal blastema grafted to proximal stump --> all proximal structures in regenerated limb mostly come from proximal stump tissue - Take distal blastema and insert along with proximal blastema --> get extra autopod (rotated) - Blastema cells retain their specification state despite de-differentiation --> (1) GFP expressed specifically in limb cartilage --> (2) labeled tissue transplanted to different animal --> (3) recipient animal amputated through labeled tissue --> (4) determine fate of labeled tissue --> upon re-differentiation, the labeled cells all become cartilage - The same result occurs if dermis or muscle are GFP-labeled prior to amputation --> each tissue strictly retains its prior specification state - This implies that you're shutting of expression of differentiation genes but not turning off lockdown loops (can't go backwards) - Intact neural fibers are required for regeneration --> if you cut nerve before amputation, limb won't regenerate - Regeneration of newt limbs depends on nAG (newt anterior gradient protein) --> nAG normally supplied by limb nerves - nAG is supplied by glia associated with limb nerves and is required for blastema cell proliferation --> de-innervated amputation without nAG does not regenerate but treatment with nAG rescues --> its expression is specifically induced by amputation - Depolarization of blastema is required for regeneration and likely promotes proliferation - Interestingly, the contralateral limb is also depolarized, at the same PD position as the amputated limb - This effect does not involve the nervous system, and results from specific ion channel activity at amputation site (and on contralateral side) --> a mechanism for defining the extent of PD regeneration?

Apical ectodermal ridge (AER)

- Ridge of tissue on top of limb bud - FGF8 in AER --> as limb bud grows, FGF8 expression along edge of tip - Cells are packed more tightly in AER --> outer edge is periderm and below it is AER - AER controls limb development --> if you remove it, you block limb development --> if you split it, you cause two limbs to develop (can also do this by transplanting another AER) - Multi-limbed Pacific tree frog resulting from infestation of tadpole-stage developing limb buds by trematode cysts --> splits AER - This can happen with people too - AER is required for wing development --> AER loss leads to immediate cessation of limb development - Ectrodactyly, or split hand, results from various defects in maintaining medial AER --> where the middle digit would arise, instead the hand/foot splits and neighboring digits usually fuse - Dlx (distal-less) mutant mice have an ectrodactyly-like phenotype (middle part of AER is lost)

Muscle regeneration following injury

- Satellite cells on outside of muscle fibers express Pax3/7 (marked by Pax7 specifically) --> normally quiescent (contain myf5 mRNA but no protein) - When you get an injury, you wake satellite cells up --> express myoD, myf5, pax3/7 - Satellite cells migrate into the wounded area and proliferate --> make new myotubes to regenerate muscle - This only works if the injured region is small enough

Digits

- Shh-expressing (and thus secreting) cells form digits 4 and 5 (pinky and digit next to it) - Adjacent digits 2 and 3 are specified mostly by Shh reception - Digit 1 is Shh-independent (needs Hox13) - Natural polydactyly mutant displays ectopic Shh expression --> the mutation is in enhancer for Shh, ~1000 kb upstream from coding region (in ZRS) - There are two effects in this mutant: posterior signal enhanced and anterior signal is induced --> suggests the site of mutation is repressive (would normally block Shh expression) and disabled by mutation --> note larger distal buds in mutant - Gli is a repressor of digits - Normal Shh signaling: Gli protein is in gradient of repressor form to activator form - No Shh protein --> Gli in repressor form only --> only make thumb (Shh-independent) - No Gli protein --> loss of repression (polydactyly, amorphous digits) - As limb continues to grow, FGF8 from AER and Shh from ZPA drive BMP behind AER --> BMP might auto-activate to maintain itself --> Shh also indirectly induces expression of Gremlin (BMP inhibitor) --> BMP inhibits FGF8 - FGF drives growth, Shh drives patterning - Interdigital mesenchyme regulates digit identity --> removal of posterior mesenchyme leads to conversion to anterior digit (e.g., 2 --> 1 or 3 --> 2) - Noggin-soaked beads (BMP inhibitor) have a similar effect, indicating that BMP signaling from the space posterior to the digit provides its identity - Noggin bead in mesenchyme also leads to webbing in chicken foot - Chicken vs. duck: Gremlin inhibits BMP and apoptosis in duck but not chicken feet --> Gremlin co-expressed with BMP in duck but not chicken - Digits and joints sculpted by apoptotic cell death - GDF5 (growth and differentiation factor) is a BMP involved in joint development --> GDF5 blocks bone formation (makes it stay cartilage) and is expressed in presumptive joints --> noggin mouse does not express GDF5 or form joints - Model of hyoid joint formation: GDF5 signaling turns on Irx5/7 --> in future joint regions, cartilage is maintained by Irx5/7 TFs, which block Sox9-mediated expression of collagen (Col2a1), and therefore inhibit bone formation in future joint regions

Intramembranous ossification

- Skull, jaw, clavicles - (1) An ossification center appears in the fibrous connective tissue membrane --> selected centrally located mesenchymal cells cluster and differentiate into osteoblasts, forming an ossification center - (2) Bone matrix (osteoid) is secreted within the fibrous membrane --> osteoblasts begin to secrete osteoid, which is mineralized within a few days; trapped osteoblasts become osteocytes - (3) Woven bone and periosteum form --> accumulating osteoid is laid down between embryonic blood vessels, which form a random network and results in a network (instead of lamellae) of trabeculae --> vascularized mesenchyme condenses on the external face of the woven bone and becomes a periosteum - (4) Bone collar of compact bone forms and red marrow appears --> trabeculae just deep to the periosteum thicken, forming a woven bone collar that is later replaced with mature lamellar bone --> spongy bone (diploe), consisting of distinct trabeculae, persists internally and its vascular tissue becomes red marrow

Fibrodysplasia ossificans progressiva (FOP)

- Soft tissue ossification in response to injury (ligaments, tendons, muscles) - As soft tissue converts to bone, joints become frozen - Surgeries to remove excess bone accelerate the pathology by introducing further soft tissue injuries - FOP is characterized pediatrically by deformities of the big toe - FOP caused by GOF mutation in BMP receptor

Somite segmentation

- Somites undergo epithelialization (mesenchymal to epithelial transition) - Start making two parallel edges of epithelium, then fill in to connect around the edges - This process is downstream from Notch --> Notch makes fibronectin (ECM) and N-cadherins (adhesion proteins), which drive epithelialization - Posterior part epithelializes before anterior part --> epithelialization is posterior to anterior - Staining for F-actin reveals epithelialization - After epithelializing, most of the somite goes back to being mesenchymal (upper part stays epithelial) - This really segments the somites - Somite is divided into three regions, sclerotome, dermatome, and myotome - The cells that undergo the EMT after epithelializing become sclerotome, which makes bone - Dermomyotome is left over

Sox9

- Sox9 is a critical chondrocyte TF --> need it to be a chondrocyte - Neural crest cells that give rise to chondrocytes for facial bones turn on Sox9 - Sox9 is triggered again at onset of chondrocyte hypertrophy - Heterozygous Sox9 mutation --> captomelic dysplasia --> don't make cartilage models correctly and thus make deformed/bent bones

Hematopoietic stem cell lineages

- Start with pluripotent HSC - Lineages branch - One branch makes blood --> erythrocytes (RBCs), platelets, and white blood cells/leukocytes (basophils, eosinophils, neutrophils, monocytes) - Other branch makes immune system (lymphocytes) --> NK cells, dendritic cells, Tc lymphocytes, T helper lymphocytes, B cells - Blood lineage is innate response, lymphocyte lineage is adaptive response --> makes sense to split into ancient branch (blood) and new branch (lymphocyte) - Multipotency and self-renewal of SCs decreases as differentiation increases

Muscle development

- Start with progenitor cell that could become dermis, smooth muscle, endothelial cell, or brown fat - Specification of muscle initiates via Pax3 (Foxc2 for other lineages) --> mutual exclusion between Pax3 and Foxc2 - Myoblast proliferation --> express Pax3/7; sprouty is important for proliferation - Myoblasts halt proliferation upon aligning for fusion - ERK (MAPK) activity is required for muscle fusion --> no fusion if given Erk inhibitor - Myoblast differentiation after receiving Fgfr4 signals --> start to turn on myoD family genes (Myf5, MyoD) - Myotube --> express myogenin - Innervation

Intestinal differentiation

- Start with pseudostratified epithelium - The endoderm secretes Hh and PDGF, which stimulate mesenchymal clustering --> this clustering coincides with villus growth - The clusters stay with the elongating tips of the villi, and secrete BMP --> invagination/proliferation --> BMP prevents proliferation of local cells --> this limits proliferation of the cells between villi (crypts) because they are too far away to receive BMP - The crypt develops last, containing ISCs and Paneth cells (make SC niche) --> the SCs higher in the crypt (towards villi) are close enough to start proliferating --> transit amplifying zone - Migrate up the villus --> arrive at top and get pushed off --> apoptosis - The mature villi has goblet cells (secrete mucus) and enteroendocrine cells (secrete hormones) - At bottom, have strong Wnt signaling --> promotes proliferation --> helping maintain SC character at bottom - At top, have strong BMP signaling --> promotes differentiation --> promotes differentiation after exit from transit amplifying zone - Notch activity in base blocks proliferative response --> ISCs aren't undergoing mitotic divisions, undergoing SC renewal --> daughters move into transit zone (low Notch) --> rapid proliferation - All lineages present in villus and crypt come from ISCs - SCs have Notch activity and divide to make first progenitor cell - Progenitor cell divides to make absorptive and secretory progenitor - Secretory progenitor expresses Atoh1 --> gives rise to all types except enterocytes - Absorptive progenitor maintains some Notch until it starts proliferating --> enterocyte - Notch important to maintain SC character and intestinal epithelial fate of enterocytes - Beneath monolayer of epithelial cells, have layer of fibroblasts (connective tissue) --> ECM (with vascular supply) - Underneath that are two layers of smooth muscle, oriented perpendicularly to each other - Below that is another (thinner) layer of connective tissue - At the bottom is another layer of epithelial tissue - In small intestine, there are big folds (plica) - Small intestine absorbs nutrients from food

Stem cells

- Stem cell therapy: take stem-like cells from a biopsy of your tissue, put them in a dish, and induce them to differentiate into the tissue you want, then transplant - Initially, the only place we could get totipotent SCs was from embryos (ICM) or from fetus (PGCs) - People discovered that if you express four key TFs (OSKM --> Oct4, Sox2, Klf4, c-Myc) in basic cells, you can convert them into pluripotent SCs --> OSKM sufficient to induce pluripotency to get iPSCs (induced pluripotent SCs) - Can express OSKM through a variety of methods --> viruses, plasmids, injecting proteins, RNA transfection - Culturing SCs: (1) isolate and culture host cells (e.g., mouse embryonic fibroblasts and adult human dermal fibroblasts); (2) introduce ES specific genes (iPS factors) into cells using retrovirus vector or other method; (3) harvest and culture cells according to method for ES cell culture using feeder cells (provide survival signals); (4) a subset of the cells generate ES-like colonies --> iPS cells - Mouse iPSCs demonstrate important characteristics of pluripotent SCs, including expressing SC markers, forming tumors containing cells from all three germ layers (very pluripotent), and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development - Separated iPS cells undergo apoptosis - 2007: MIT and Kyoto-based scientists cure a mouse with sickle cell anemia --> collect skin cells --> reprogram into ES like-iPS cells --> genetically identical iPS cells --> correct mutation --> genetically corrected iPS cells --> differentiate into blood SCs (HSCs) --> transplant --> recovered mouse - Mini-kidneys from iPS cells and metanephric mesenchyme --> took iPS cells and made duct, then added MM - iPS-generated blood vessels - iPS cells can be induced to become cardiomyocytes --> contractile in culture dish - Origins of CPCs during heart development: epiblast --> mesoderm --> pre-cardiac mesoderm --> cardiac progenitors --> first heart field and second heart field - Generation of CPCs in plate (stage-specific method): ES/iPS cels --> EBs (embryoid bodies)--> cardiac mesoderm --> CPCs --> cardiomyocytes, smooth muscle cells, endothelial cells - Generation of CPCs in plate (one-step method): ES/iPS cells --> CVPCs --> cardiomyocytes, smooth muscle cells, endothelial cells - Dopaminergic neurons from iPS cells - Lab-grown model brains - Light-sensing retina in a dish - Optic cup formation from ES cell aggregates - Can make a variety of organoids in a dish --> each has a different clinical application - "Human" on a chip has future possibilities as a means to better test systemic drug effects

Limbs

- Thalidomide babies --> limb defects (arms, legs, or both) - Bionic prosthetics --> perform functions simply by thinking about them --> detect movement of muscles, which have been rewired

Axes of limbs

- The one bone (humerus or femur) is generically called the stylopod - Middle pair of bones (ulna/radius, tibia/fibula) are called zeugopod - Everything else (carpals of wrist, digits of hand, etc.) is called autopod - AP axis: thumb is anterior, pinky is posterior - Proximal end of limb is closest to body (shoulder) - Distal end of limb is farthest from body (fingers/digits) - Dorsal surface is back of hand/top of foot - Ventral surface is palm of hand/bottom of foot - Each axis is patterned independently and uses different signaling molecules - PD axis is primarily patterned by FGFs - AP axis involves RA, mostly Shh, later BMPs - Both PD and AP also use Hox genes - DV axis uses Wnts

Major histocompatibility complex (MHC)

- This is how we get B cells to make antibodies - (1) Antigen presenting cell (APC) engulfs pathogen and breaks it up - (2) APC presents antigen peptides on its surface using MHC - (3) T cell receptor (TCR) bind MHC and may recognize antigen --> activation - TCR V regions also undergo VDJ recombination --> T cells need to be specific enough to recognize MHC and not recognize your own cells - If T cell activated is TC (killer cell), it kills infected cells - (4) Meanwhile, B cell receptor (Ab) also binds antigen --> processes and presents it on MHC - (5) TH (T helper, aka CD4+) recognizes antigen on B cell --> activation - B cell differentiates into plasma cell and starts secreting antibody

GRNs for muscle

- Three different skeletal muscle GRNs have been described for muscles that form in different regions of the organism (vertebrates) --> body, limb, head - Body and limb both include Pax3 myf5 - GRN for head muscle does not include Pax3 --> it does include Myf5 but it isn't really needed because Mrf4 is present and is a duplication of it

Evolution of hearts across deuterostomes

- Top to bottom: echinoderm, hemichordates, urochordates, cephalochordates, vertebrates - Sea urchins (echinoderm) don't have anything resembling a singular centralized heart - In vertebrates, pumping blood is a two pump process (lub dub) --> echinoderms, hemichordates, urochordates, and cephalochordates are simpler than that (squeeze and release/peristaltic pump) - Echinoderms have diffuse, primitive circulatory system - In hemichordates, blood is "pumped" via a contractile vesicle - Urochordates have something more like a pump --> contractile V-shaped sinus venosus - Amphioxus (cephalochordate) also has a sinus venosus --> S-shaped morphology is thought to represent the heart --> SV here isn't contractile --> the vessels around the SV are contractile - Vertebrate heart has inflow into ventricles --> move blood into atrium --> push blood out of SV (split in humans) - Heart field specification is conserved across chordates --> looking at ciona (urochordate), chicken, and mouse (amniotes) --> labeling for Tbx5 (TF that marks anterior heart field, which corresponds with outflow/ventricle) and RADH2 (RA synthesizing enzyme marking posterior heart/inflow/atria) --> specification between inflow and outflow tracts is very conserved

Blood

- Undifferentiated mesenchymal cells under yolk sac endoderm differentiate into blood islands --> form vasculature around them - Blood is mostly red blood cells but also contains platelets (cell fragments) and lymphocytes (immune system) - Lots of types of cells in our blood --> all come from hematopoietic stem cells, which branch into lineages - HSCs are maintained in marrow by multiple interactions with osteoblasts (Wnt, angiopoietin, Notch/Jagged, cadherin)

Sex determination in Drosophila

- Use the same genes but splice them differently --> alternative splicing - First gene in cascade is Sxl (sex-lethal) --> Sxl is a TF and a splicing factor - Sxl gene has two different promoters --> SxlPe (establishment) and SxlPm (maintenance) - In Drosophila, sex is determined by the ratio of X and autosomal chromosomes - SxlPe can be bound by two families of early promoter TFs (2X and 2A) FEMALES: - 2X:2A - (1) In females, SxlPe promoter is activated - 2X homodimers initiate Sxl expression from Pe, leading to alternative splicing and Sxl protein --> splice out male exon with stop codon in it - Later use SxlPm to make late Sxl protein --> late Sxl protein needs to be spliced by early Sxl protein - Sxl drives its own expression from Pl, and Sxl induces alternative splicing to maintain its own late expression --> splices out male exon - Presence of both early and late Sxl --> female - (2) Make tra (transformer) --> also a splicing factor - (3) Tra splices dsx (doublesex) to dsxf - Female dsx promotes female differentiation genes and suppresses male differentiation genes --> female phenotype - Female Dsx and Ix (intersex, a TF) turns on Wg (wingless) and turns off Dpp and FGF --> promotes female genitalia - Female Dsx drives yolk proteins to be made, spermathecal ducts, and female pigmentation patterns - (4) Tra is present, so females retain exon with stop codon in fruitless --> no fruitless made - (5) Sxl suppresses msl protein from being expressed - Loss of msl slows transcription from x chromosome, balancing x-linked genes between males and females --> mammals do this via X inactivation; C. elegans does this by slowing down transcription rates in hermaphrodites MALES: - Default state - 1X:2A --> only one X chromosome, so can only make heterodimer TFs --> unable to bind early promoter - In males, SxlPe promoter cannot be activated --> no early Sxl protein in males - Only turn on SxlPm --> nonproductive splicing - Male exon with stop codon is not spliced out, so transcription terminates and no protein is made - No late Sxl protein --> male - Do not make tra (transformer) - No tra, so dsxm is spliced per male-specific splicing --> male - Remove exon with stop codon (because default is to do this) --> make fruitless - Male-specific dsx and fruitless proteins drive male differentiation genes and suppress female differentiation genes --> male phenotype - Male Dsx turns off Wg and turns on Dpp and FGF --> male-specific growth genital disc, paragonia - Male Dsx suppresses yolk genes and drives production of clasper (sex combs) and male pigmentation patterns - No sxl, so msl is present - Msl protein speeds up transcription from X chromosome, balancing x-linked genes between males and females

Lymphatic system development

- VEGF-C required for lymphatic vessel formation --> VEGF-C deficient embryo has edema - Lymphatic system buds from the veinous system --> sprouts form on cardinal vein and coalesce to form system - Expression pattern in lymphatic endothelial cell precursor is different from that of migrating LEC --> adding a few more things as you go arterial to migrating LEC --> being arterial endothelial cell is simplest, then venous, then LEC precursor, then migrating LEC

Limb evolution

- Vertebrates with limbs are called tetrapods - All vertebrates came from fish - In order for limbs to evolve, lungs had to evolve first so fish could walk on land - Swim bladder is air sack that fish use to stay buoyant --> this is what was converted to lung - Lungfish have lungs --> pair air sacs and send blood supply - Fish system: two chambered heart --> send blood to gills to get oxygenated --> send to capillaries of body --> back to heart - Lungfish system: swim bladder converted to lung is vascularized so we get a capillary bed there and can do gas exchange --> separate circuit established for lungs --> two chambered heart separates circulatory system for body and for lungs - In lungfish, walking and swimming movements are different - Lungfish show developmental plasticity to pectoral girdle (bone system that supports front fins) when reared on land --> bones are larger and more angled - In primitive fish, see beginnings of major bones of forearm --> evolved to make early amphibians with full limb - Several evolutionary intermediates between lobe fin fish (lungfish) and vertebrates - Tiktaalik is first with a true limb (but not really digits) - Tiktaalik a fish with wrists and fingers, lived in shallow waters about 375 million years ago - Tiktaalik's "arms" were jointed, and the joints were integral to force generation by the "arms" - Fossilized tetrapod tracks controversy --> when did this all happen? --> prior view: 375-386 mya --> revised view with new fossils: 397.5 mya? --> seems limb tetrapods have been on earth for 390ish-400 million years

Development of vasculature

- Want to push blood around and use it for gas exchange --> want to be able to have diffusion between blood and tissues, but also want to move it around affectively - Hydraulics tells us that larger diameter is better, since resistance to flow increases by radius^4 as diameter decreases - Physical chemistry tells us that slower flow is better for diffusion of stuff in and out - Biology utilizes both - In large vessels, flow is 100x faster than in capillaries --> this works as a closed system because total volume of capillaries >> total volume of large vessels - Process of making blood vessels starts with vasculogenesis --> endothelial cells arrange themselves in vascular formation and then merge to form internal lumen --> gives first form of vasculature, which can then be pruned and remodeled - Angiogenesis --> make new blood vessels --> triggered by perception of low oxygen --> sprouting from pre-existing vessels - Hemangiogenic mesoderm comes from lateral plate mesoderm (but from posterior) --> blood is induced where Wnt and BMP are both present (lateral and posterior) - Have early progenitor cells in splanchnic that could become cardiac precursor cell or hemangioblast - Important genes here are Bry (brachyury) and Flk1 (VEGFR) --> VEGF receptor --> vascular endothelial cell growth factor - To induce blood/blood vessel fate, turn on both Bry and Flk1 --> hematopoietic cells (blood) or angioblasts (vessels) - TF Etsrp/Etv2/ER71 is required for angioblast differentiation --> a "master regulator" of vasculogenesis --> etsrp MO has a big pile of blood in posterior because nowhere for blood to go - Still vascularize yolk sac in mice even though it's empty and vestigial --> we make umbilical vein and umbilical artery that go to placenta - Blood vessel development requires VEGF --> VEGF mutant has no vessels and embryo is tiny because starving to death (fatal) - In the yolk sac, exposure to a gradient of VEGF induces arterial, then venous outgrowths --> outgrowths run parallel - Multiple signals regulate vasculogenesis --> VEGF for vessel formation, Ang1/Tie2 for mature vessels in a primary capillary plexus - First form vessels, then start joining together the vessels in a capillary plexus --> then prune and remodel to produce a juvenile vascular system, and then continue to mature and refine/remodel to produce a mature vascular system throughout life (vessel formation --> capillary plexus --> refinement --> remodeling - Take endothelial cells from umbilical vein --> takes about 6 hours to start lining up, joining together, forming lumen --> capillary plexus --> if done under hypoxia, everything goes faster (hypoxia induces expression of VEGF) - FAK is focal adhesion kinase --> knock-in of kinase-dead FAKR454 but not FAKWT inhibits capillary formation in yolk sac (messes up focal adhesions) --> need focal adhesions to organize at first level - Lateral angioblasts migrate to the midline to produce DA first, then PCV second - 16 somite stage: artery vs. vein --> artery experiences more Hh and VEGF signaling than vein (VEGF from sclerotome and Hh from notochord) - 22 somite stage: dorsal aorta above and posterior cardinal vein below it - Endothelial cells are induced by VEGF and Hh --> differentially specified as arteries vs. veins by stimulation of Notch by Hh --> arteries get Hh and stimulate Notch; veins do not stimulate Notch --> differential expression of ephrin-B2 and EphB4 receptor --> arteries express ephrin-B2 and veins express EphB4 receptor - Yolk sac vasculature fails to mature in ephrin B2 KO mouse - Arteries and veins both have endothelial lining surrounded by smooth muscle, which is surrounded by connective tissue - Veins have valves inside --> important to stop blood from moving backwards - Ephrins in arteries and ephrin receptors in veins prevents arteries and veins from fusing together (forbidden fusion) --> somehow, at capillary level, the repulsion doesn't take effect and there is permitted fusion - Roles for ephrins in vascular development: organizing capillary beds, patterning vessels (especially between somite), and inducing sprouting - Semaphorin (repulsive cue for neural patterning and vascular system) expression in lens inhibits vascularization of the cornea --> blood vessels in cornea would block vision - Angiogenesis: in response to angiogenic stimulus (VEGF), endothelial cell becomes motile and extends filopodia that guide the development of a capillary sprout --> the leading/tip cell continues to move away from the capillary as cells behind it migrate in and divide, forming a stalk --> sprout begins to hollow out, forming a tube --> pinocytic vesicles fuse with one another to create internal vacuoles --> internal vacuoles then fuse to create lumen that runs through capillary sprout - In culture, endothelial cells can spontaneously develop internal vacuoles that join up from cell to cell, creating a single lumen shared by many cells --> even though cells share a lumen, they do not share a cytoplasm --> remain separate cells after fusion

Bone cells

- We make bones out of osteoblasts and osteocytes, which are derived from mesenchymal stem cells - Osteoclasts are also important, and are derived from macrophages, which come from hematopoietic stem cells - If cells are on the surface of bone (outer or inner), they are osteoblasts - If cells are embedded in the bone, they are osteocytes - Osteoclasts digest bone during bone remodeling - Osteoid is a collagen 1-rich ECM secreted by osteoblasts and osteocytes that is subsequently mineralized to produce bone - Cement line is the edge between calcified and uncalcified bone - Types of bones in humans: long bones (humerus), short bones (triquetral), flat bones (sternum), and irregular bones (vertebra)

Zone of polarizing activity (ZPA)

- ZPA helps maintain AER (especially posterior AER, which starts to express more FGFs (especially FGF4)) --> FGFs from posterior AER feed back to maintain ZPA - Shh protein is expressed in ZPA - Transplant of ZPA to anterior limb bud --> mirrored duplicated digits - Shh is sufficient to induce digit duplication --> took fibroblast cells and transfected so they expressed Shh, then implanted in anterior portion of limb bud and get mirror duplication of autopod (Shh GOF) - Add normal pellet of Shh and get normal duplication --> add small pellet, only duplicate digit 2 --> leads to idea that there's a threshold of Shh these digits have to experience to be specified

Five layers of epiphyseal plate

- Zone 1: zone of resting cartilage --> normal resting hyaline cartilage - Zone 2: zone of proliferating cartilage --> chondrocytes undergo rapid mitosis - Zone 3: zone of hypertrophic cartilage --> chondrocytes stop mitosis and begin to hypertrophy - Zone 4: zone of calcified cartilage --> chondrocytes start undergoing mineralization - Zone 5: zone of ossification --> chondrocytes transdifferentiate into osteocytes

Model for limb bud initiation

CHICK/MOUSE/FISH FORELIMB BUD INITIATION: - In forelimb mesenchyme, RA --> Tbx5 - Tbx5 induces EMT - Tbx5 induces FGF10 (via Wnt2b) in mesoderm --> mesoderm proliferation --> mesoderm becomes mesenchymal (individualized cells that are adhering to each other) --> proliferate and migrate --> drives expansion of limb bud - FGF10 --> FGF8 in ectoderm (via Wnt3a) - FGF8 in ectoderm activates FGF10 in mesoderm, which activates FGF8 in ectoderm --> positive feedback loop that self-maintains CHICK AND MOUSE HINDLIMB BUD INITIATION: - In hindlimb, a signal (unknown) --> Tbx4, pitx1 (in chicken), Islet (only in mice) - This induces EMT (probably) - This also induces FGF10 (via Wnts) in mesoderm --> mesoderm proliferation - FGF10 in mesoderm --> FGF8 in ectoderm (via Wnt3a) --> feedback loop

Major vessels of embryo

EMBRYONIC CIRCULATION: - Aorta (dorsal aorta) - Ventral aorta (temporary) - Aortic arch arteries --> connect ventral and dorsal aorta - Anterior cardinal vein - Common cardinal vein (turns into vena cava) - Posterior cardinal vein VITELLINE CIRCULATION (yolk sac): - Vitelline vein - Vitelline artery PLACENTAL CIRCULATION: - Left umbilical vein - Umbilical artery

Female vs. male gonads

FEMALE: - Gonadal type: ovary - Germ cell location: inside follicles of ovarian cortex - Remaining duct: Mullerian - Duct differentiation: oviduct, uterus, cervix, upper portion of vagina MALE: - Gonadal type: testis - Germ cell location: inside testis cords (in medulla of testis) - Remaining duct: Wolffian - Duct differentiation: vas deferens, epididymis, seminal vesicle

Bone growth vs. remodeling

GROWTH --> bone grows in length because: - (1) Cartilage grows at the outer edge of articular cartilage on the ends of bones - (2) Cartilage is replaced by bone on the inner edge of articular cartilage - (3) Cartilage grows at the surface of the epiphyseal growth plate closest to the end of the bone - (4) Cartilage is replaced by bone at the inner surface of the epiphyseal plate REMODELING --> growing shaft is remodeled by: - (1) Bone resorbed on the outer surface below epiphyseal plate (osteoclasts do this) - (2) Bone added by appositional growth below (1) - (3) Bone resorbed on inner surface --> straighter because no longer where the epiphysis is

Limb skeleton patterning

REACTION-DIFFUSION MODEL FOR PD LIMB SPECIFICATION: - In Turing RD, elements are added as size increases (1 bone --> 2 bones --> many bones) - Interaction between TGFB and BMPs - Developing limb divided into three zones - (1) Progress zone ("apical"): FGF inhibits fibronectin expression (fibronectin normally used to start cartilage model), inhibiting patterning (development of bone) --> now called inhibitory zone - (2) Active zone: here, a TGFB-based Turing TD system produces peaks of fibronectin, which "seed" cartilaginous nodules that presage the condensations and bones - (3) Frozen zone: the final cartilaginous nodule is produced, and no further patterning occurs --> stylopod being formed here - Form of the bone depends on shape of the bud --> broader bud means more bones (more condensation of foci) --> change in distal bud shape means change in number of digits EARLY LIMB BUD: - Apical zone = PZ - Rest of bud is active zone, undergoing TRD to establish stylopod (e.g., humerus) cartilage model - In TRD, activators are BMPs and TGFBs, inhibitors are Noggin, FGFs, Notch --> promoting making of cartilage OLDER LIMB BUD: - Apical zone = PZ - Intermediate region = active zone (TRD for zeugopod) - Proximal region = frozen zone where bone is forming MATURING LIMB BUD: - No apical zone - No active zone - Frozen zone is where bones are --> everything is frozen zone PATTERNING: - Turing model does a good job of recapitulating what we saw experimentally (AER intact vs. AER removed early vs. removed late vs. add extra ZPA) - Talpid encodes a ciliary protein that negatively regulates Shh, although the mechanism is still under investigation - Talpid mutant animals exhibit craniofacial and limb patterning defects - Hoxd11-13 expression dose sets the Turing wavelength for digits --> gradually removing terminal Hox genes --> dialing down Hox dosage dials up number of digits produced in Gli-/- animal - Stronger Hox dosage --> longer wavelength --> fewer digits - This led to hypothesis that there is also TRD mechanism for digits/patterning autopod TRD MODEL FOR DIGIT PATTERNING: - Sox9 is pro-cartilage - Wnt pathway genes and BMP2 are expressed in stripes that alternate with Sox9 - This was recently discovered by RNA-seq comparisons of Sox9-positive and Sox9-negative cells - TRD model was proposed, in which Sox9 is activated by BMP and inhibited by Wnt, while distal Hoxd13 and FGF8 (AER) define TRD wavelength - Comparisons of TRD simulations based on this model and biological outcomes are very good - Regions that express Sox9 make digit - Regions between Sox9 express BMP and Wnt --> inhibits Sox9 expression and controls how many digits we make - Wnt is expressed only on dorsal side

Types of tubulogenesis

REQUIRES AN EPITHELIUM: - Wrapping (ex: neural tube) - Budding (ex: branching morphogenesis) DE NOVO: - Cavitation (ex: salivary gland and mammary gland, mouse embryo) --> kill off internal cells; basal surface on outside, apical on inside; cells joined by cell junctions - Cord hollowing (ex: vasculature) --> create lumen on inside of smaller cord of cells; basal on outside, apical inside; joined by autocellular junction - Cell hollowing (ex: vasculature) --> create lumen on inside of single cells; basal outside, apical inside; no junction because one cell