DEVBIOL LE 3 - Developmental Biology PDF

DEVBIOL: Developmental Biology LE 2: Dr. Gliceria Ramos Term 2 AY 2022-2023 Transcribed: Berana, Capistrano, Dalapo, Delos Angeles, Juachon, Narbonita, Noval, Paclibar, Rabang, Sartorio, Silao, Soliman LECTURE 11A induction...

DEVBIOL: Developmental Biology LE 2: Dr. Gliceria Ramos Term 2 AY 2022-2023 Transcribed: Berana, Capistrano, Dalapo, Delos Angeles, Juachon, Narbonita, Noval, Paclibar, Rabang, Sartorio, Silao, Soliman LECTURE 11A induction increases the complexity of an EMBRYONIC INDUCTION embryonic cells “All that you touch…you change. All that Responders or Responding cells: you change, changes you. The only group of cells that undergoes changes lasting truth is change.” – Octavia Butler and eventually, whose fate will be (1998) determined One of the core aspects of embryology and of the broader course on animal EMBRYONIC CELL COMPETENCE developmental biology. A cell to cell communication or signaling between and among embryonic cells, and in the process, bringing changes INDUCTION AS A DEVELOPMENTAL MECHANISM Competence: ability of a cell or tissue to respond to a specific inductive signal Capacity: ability of the inducer to synthesize and send signals COMPETENCE The influence of one group of Can be established by: embryonic cells on adjacent group ○ Synthesis of new of embryonic cells, in the process, membrane receptors changes are being made ○ Synthesis of molecules to Interaction at close range allow membrane receptor ○ Interaction between cells or to function tissues of different histories Molecules and properties associated with Interactions can go on the same time membrane ○ Because of the close receptors proximity of interactions Inductive interactions occur during sensitive stages of embryonic development Inducers: group of cells that cause the changes ○ When acting on responders causing changes to Simple illustration establish their fate gives with membrane the bigger picture that receptors and the  associated formation of diacylglycerol (DAG) and molecules that inositol triphosphate (IP3). allows these Inositol triphosphate (IP3): acts as a receptors to function secondary messenger ○ Repression of an inhibitor ○ Activates the release of Simply removing calcium ions from storage molecules that block sites like Endoplasmic the binding sites of Reticulum (ER) the receptors ○ Calcium is released to the Repression of cytoplasm and also acts as transcriptional another group of secondary inhibitors ar a messengers activating nuclear level various of proteins ○ Activation depends on the SIGNAL TRANSDUCTION presence of calcium ○ Various proteins are activated, hence, cellular responses. INTRACELLULAR SIGNALS Signal Transduction: transducing the signal from outside into the cell ○ Extracellular signals are received at the membrane ○ Signal is then transduced in the cytoplasm We have a cell with a membrane receptor Triggers for the next step G-protein linked receptor: This Secondary messengers: act as receptor is associated with a G-protein intracellular signals that are molecule responsible for the next step in signal ○ G protein is phosphorylated transduction When an inductive signal binds with Can act at cytoplasmic level creating the receptor, the associated G protein cellular responses is activated – it gets phosphorylated. Yet still, others can be translocated ○ When it gets from the cytoplasm into the nucleus phosphorylated and and act at a nuclear level by activated, and activates activating or changing the rates of another yet membrane gene expression bound enzyme – Phospholipase C. Phospholipase C: cleaves phospholipid molecules resulting in the  SUMMARY Telencephalic and Ligand-Receptor (L-R) binding can diencephalic region cause a cascade of events that will Optic vesicles on lead to the regulation of the activities the sides of transcription factors and/or ○ At the level of the trunk -> cofactors at the nuclear level. dorsal trunk ectoderm These transcription factors can trigger gene expression programs 1. Normal induction of lens by optic New gene expression programs vesicle ultimately drives the cellular changes Optic vesicle: acting as within the embryonic cell inducer ○ If inducer is incised COMPONENTS OF THE I-R and transplanted at INTERACTION (SIGNAL-RESPONSE the level of the trunk SYSTEM) ectoderm, the Signal molecule dorsal ectoderm did ○ An inductive signal not form lens ○ A ligand placode Receptor (R in the cell membrane) ○ Even if it has the ○ Receptor on the membrane capacity to induce, of the responding cell ectoderm will not Mechanism to transport the signal have the ○ This is the signal competence to transduction mechanism respond to the Mechanism to translate the signal inducer ○ To cause or stimulate the 1. No formation cell behavior or cellular of lens response activation of gene placode activity ○ Head ectoderm will ○ Involves the intracellular have the signals competence to respond through an INDUCTION AND COMPETENCE inductive signal Dorsal ectoderm: acting as the responding group of cells Formation of a lens or lens placode At around the stage of neural tube formation ○ Maybe even after neural tube formation ○ Brain vesicles are already established Ectodermal competence Bottom line of induction is the exchange of signals between developing organs that coordinate their development in a given time in a specific location Primary induction Involves specific events associated with neural tube formation Specific to events assisted Chordamesoderm acting on the Pax6 protein: accounts for the overlying ectoderm to form the competence of the dorsal ectoderm primordium of the CNS ○ Gives rise to Pax6 protein Goal is to form adjacent precursor competence factor cells or rudiments of an organ Pax6 has been primed early on ○ Not yet functional ○ Even before formation of ○ No other complementary optic vesicle parts are there ○ Even before generation of Specific to events associated with late inductive signal gastrulation-neurulation (neural tube BMP4: inductive signal from inducer formation) in amphibians ○ Further activates another ○ Forming primordium of set of genes (Sox2 and CNS Sox3) Sox2 & Sox3: codes for transcription factors Combined activation (Pax6, Sox2, Sox3) are all needed for the formation of the lens placode All accumulations of competence factors are needed in the dorsal head ectoderm ○ Gives competence to the head ectoderm to respond to the BMP4 Secondary induction & Reciprocal Competence is not a passive state, it interactions is gradually acquired in a stepwise Proximate tissue interaction fashion one after the other Followed by a series of reciprocal ○ It is not a one shot deal interactions ○ Accumulation of ○ Basis for formation of competence factor different organs (organogenesis) EMBRYONIC INDUCTION ○ Ultimately laying down the Induction as an embryonic body plan phenomenon has different phases Form complementary parts of an ○ Primary induction organ ○ Secondary induction ○ Reciprocal interactions  Leading to the complexity of So when the right embryonic cell environment is near Proximity of inducer and responders the genes that are that gives the effective guarantee that allowed to be the organs and structures for which expressed the embryonic cells are fated for will ○ The genes are allowed to arise next to each other and in proper be expressed and these matching sizes embryonic cells will now commit to their SECONDARY TISSUE INDUCTION: developmental pathway PERMISSIVE VS. INSTRUCTIVE In the instructive signal, the signal is necessary; if the signal is not present, the cell cannot commit to its developmental pathway to follow ○ Instructive induction restricts the cell’s developmental options ○ Instructive induction is There are two modes: permissive and more on restriction instructive Whereas, in the permissive mode or In instructive induction: induction, the expression of the gene ○ Instructive induction depends on the optimal presence of operates among embryonic the external or necessary environment cells we're the genes to be ○ So it tends to regulate the expressed are not yet degree of expression specified (unspecified ○ Gene expression can be embryonic cells) up-regulated in an ○ For the genes to be optimal environmental specified, a signal is condition; if it is not necessary as signal from favorable, the degree of the inducing cell is gene expression is necessary to the specified downregulated subsets of the gene to be ○ Permissive induction is expressed more on regulation Permissive induction ○ Permissive induction INSTRUCTIVE INTERACTIONS operates among embryonic Instructive induction – the signal for cells where the genes to the inducing cell is necessary to be expressed are already initiate new gene expression in the specified – those genes responding cell are already tagged, marked General principles of instructive ○ These genes are just interactions: waiting for the go signal for the information signal Presence of tissue A, responding tissue B And the signal is the → develops in a certain way (let us call it proper the X way) environment Absence of tissue A, responding tissue B → does not develop in a certain way (in this case, the X way) In the absence of tissue A, but in the presence of tissue C, → tissue B does not develop in that way (Y way) Example: Optic vesicle placed under a new region of head ectoderm ○ Head ectoderm does not develop a lens placode When the optic vesicle was removed, without the inducing cell, the responding cell is not capable of differentiating (in that particular way) ○ The dorsal lens did not form the placode SUMMARY: Instructive interactions restrict the cell’s developmental options ○ Basement-membrane-direc PERMISSIVE INTERACTIONS ted gene expression In permissive induction, the ○ Cell shown in cell culture, responding tissue has already been mammary cells are capable specified and need only an optimal of cell division because the environment that allows the genes for cell division are expression of those traits ON: c-myc and cyclin D1 Permissive interactions tend to regulate the degree of expression of the remaining developmental potential of the cell ○ Waiting for the right conditions to express the chosen set of genes SUMMARY: It is more of regulating the degree of expression Example: ○ These mammary cells in Development of mammary gland cells culture are already in cell culture specified, so the genes waiting to be expressed are already specified: These are the genes that code for the gene products,  necessary for ○ To be turned on, p21 gene mammary glands: is turned on to inhibit cell lactoferrin, beta division casein, whey acidic This process is called gene activation protein (WAP) by extracellular matrix (ECM) ○ The aforementioned genes attachment are responsible for coding ○ All genes were expressed the protein products of the ○ The specified genes were mammary glands expressed in the presence In the image, (A) the genes that code of a conducive for the protein products are not yet environment: cell adhesion expressed (or turned OFF), only molecules, or substrate turned ON is the genes involved in cell adhesion molecules which division are components of ECM ○ These are mammary gland ○ An example of permissive cells in culture without induction basal lamina In (B), the cells are plated onto the WHERE CAN CASCADES OF culture dish with laminin-containing INDUCTION/RECIPROCAL BE basement membrane OBSERVED? ○ Laminin is a substrate adhesion molecule, a component of the extracellular matrix (ECM) ○ In the presence of laminin in the basement membrane, one of the specified gene was turned Mouse lens - reciprocal induction on or expressed, lactoferrin Figure A: Optic lens as the inducer Mammary gland cells wrap the acting on the dorsal ectoderm basement membrane and forms The dorsal ectoderm then forms the secretory epithelium lens placode Integrins eventually bind with the ○ Neural ectoderm is inducer laminin ○ Lens placode is the ○ Binding of the integrins with responder the laminin are necessary Comes next is respondent cell (RC) in for the transcription of the turn become inductive and change the beta casein gene fate of their neighbors by producing Eventually, gradually, in a stepwise new signals fashion, with the presence of the Thus, generating sequential inductive comment of the ECM events that increase cell-fate ○ All of the specified genes diversity in tissues are turned on (lactoferrin, Figure B: lens placode is now beta casein, whey acidic inductive and sends signals to the protein (WAP)) optic vesicle ○ Genes for cell division are Figure C: Optic vesicle turns to a turned off since their more complex structure forming the function is over  retinal layers: sensory layer and ○ Stomodaeum and pigmented layer proctodaeum only have Figure D: Optic lens acting on the ectoderm and endoderm ectoderm to form the prospective without intervening cornea mesenchyme Figure E: Cornea becomes present, optic lens become present, layers of EPITHELIA AND MESENCHYME the retina became well-developed as a REVIEW result of cascades of reciprocal and sequential inductions INDUCTIVE INTERACTIONS: RECIPROCAL TO SEQUENTIAL Epithelia: flat sheets or tubes of connected cells derived from any germ Competence is not a passive state, it layer is gradually acquired Inductive events increase cell-fate diversity in tissues EPITHELIAL-MESENCHYMAL INTERACTIONS Another type of embryonic induction Mesenchyme: loosely packed, Mesenchyme is the inducing tissue unconnected cells Epithelial cells are the responding ○ Derived from mesoderm or tissue, resulting to epithelial cells’ neural crest cells gene activity is initiated ○ Plenty of ECM All organs (regardless of germ layer origin) consist of an epithelium and an associated mesenchyme (except at stomodaeum and proctodaeum)  EPITHELIAL DERIVATIVES Branching structures made up of lobes, globules, ducts Epithelial-mesenchymal interactions form branched organs! EPITHELIAL-MESENCHYMAL INTERACTIONS: BIRD DERIVATIVE FORMATION Epithelial derivatives: ○ Feathers ○ Hair Same wing epithelium but different ○ Mammary glands mesenchyme source, resulting to ○ Scales different derivatives ○ Sweat glands Formation of the derivatives of the Derivative type depends on restrictions wing epidermal epithelium depends on by region and genetics the instruction coming from the Example: different sources of mesenchyme ○ Kung ang An example of instructive signal, epithelium-mesenchyme signal is necessary to activate the interaction ay nasa upper gene coding for the expression of the arm, the epithelium won’t formation of a wing feather, thigh respond to be mammary feather, etc. glands Tingnan mo na lang yung figure kasi… ○ In mammals or higher this highlights regional specificity vertebrates, we won’t have Regional specificity - source of scale derivatives because mesenchyme (inducing tissue) we do not have genes to do determines the structure of the so. epithelial derivative Just read the table above. These organs have a similarity, they are branching type of organs  GENETIC SPECIFICITY INDUCTION AS A DEVELOPMENTAL MECHANISM Increases complexity of organ structures An effective guarantee that structures for which cells they are fated will arise next to each other an in matching sizes ○ Due to the proximity of the I & the R Due to close range, many inductive interaction can go on at the same time ○ Inductive interactions occur during sensitive stages of embryonic development A property of epithelial-mesenchymal CLASSICAL EXPERIMENT OF HANS interaction epithelial response is SPEMANN & HILDE MANGOLD EMBRYONIC INDUCTIVE EXPERIMENT limited to genomic capability ○ The newt gastrula has the mesenchyme Frog ectoderm responded by making the kind of mouth it “knew” how to make, the suckers ○ The transplanted frog Interaction of the archenteron roof ectoderm responded by (ectoderm) with the making a kind of mouth it chordamesoderm: essential for knew how to make, the neural differentiation suckers CM with dorsalizing influence Newt ectoderm responded by making ○ Ability to determine the kind of mouth it “knew” how to antero-postero axis make, the balancers Dorsal lip/ DL (CM) called the “chief ○ Frog tadpole develop cell organizer” balancers ○ For the mesoderm, ○ New ectoderm transplanted homologous to the nodal into the frog gastrula cells of the henson’s node. responded by making the Hence the henson’s node, kind of mouth it knew how which has the nodal cells to make, the balancers are the chief cell organizer Mesenchyme induces epithelial of avian and mammalian structures but can only induce what embryos the epithelium is genetically able to So here, they got a recipient embryo, produce at the gastrula stage and a donor ○ So the ectodermal embryo also in the gastrula stage epithelium is able to The dorsal lip of blastopore was produce suckers and transplanted into the donor and they balancers kept track of the development. The  recipient embryo develop two neural Conclusion: only gastrula structures: stage-ectoderm is competent to ○ One coming from its own, respond to the inducer dorsal lip blastopore ○ Embryonic stage specificity ○ Transplanted one Q3: DOES DL TAKEN FROM ANY DISCOVERY OF THE ORGANIZER GASTRULA STAGE HAVE THE SAME INDUCING CAPACITY? Experiment: ○ DL from Early G → neural head structures ○ DL from Late G → posterior structures Conclusion: regional specificity of induction Remove dorsal lip blastopore lip cells LECTURE 11B from donor embryo CELL-CELL SIGNALING Transplant these cells to ventral side The components of this signal or of host embryo response system must include: A secondary embryo begins growing in ○ Signal ventral side of host embryo Inductive signal Two complete embryos result, from inducers or connected at the belly inducing cells Similar result in the chick embryo ○ Receptor experiment On the cell membrane of Q1: WILL ANY TISSUE RESPOND TO responding cells INDUCER? ○ Mechanism to transport the Experiment: Dorsal Lip signal (DL)→endoderm→ no differentiation Signal transduction Conclusion: only the ectoderm is mechanism competent to respond to the inducing ○ Mechanism to translate the effect of DL signal to cause or stimulate ○ Endoderm is not competent the cell behavior or cellular to respond to inducing response (activation of effect of the dorsal lip of gene activity) blastopore Intracellular signals, ○ Tissue specificity Extracellular signals, and Q2: CAN ECTODERM FROM ANY intermediates that EMBRYONIC STAGE COMPETENT TO trigger the activity of RESPOND TO THE INDUCER? Experiment: the genes ○ DL → neurula stage → no neural formation SIGNAL TRANSDUCTION Signal transduction cascades involve ○ DL → blastula stage → sets of pathways waited for gastrula stage 1. Intercellular and/or Intracellular ○ The phosphorylated protein signals: will become an active a. As activators or initiators of protein and will be ready to pathways carry out its function to 2. Intermediates or Activated products cause cellular response a. As triggers for succeeding steps in the pathways PROTEINS MEDIATING SECONDARY EXAMPLE: Receptor Tyrosine Kinase (2o) INDUCTION (INDUCTIVE SIGNALS) (RTK) Paracrine signaling factors or Growth ○ Very important member of and differentiation factors (GDFs) the cascade ○ Fibroblast Growth Factors ○ Enzyme that (FgFs) phosphorylates protein ○ Hedgehog family ○ Wingless family (Wnt) RECEPTOR TYROSINE KINASE (RTK) ○ Transformation Growth Factors (TgFs) Beta Superfamily Juxtacrine signaling Factors ○ Delta-notch (receptor) signal Delta: ligand or inductive signal Notch: receptor that can generate an intracellular signal Other ligands that can bind to notch How it works are Serrate and ○ A cell with the RTK with the Jagged extracellular and cytoplasmic domain – a PARACRINE FACTORS IN dimer. DEVELOPMENT ○ However, when the ligand Connection between paracrine is not yet there, it is factors and induction undimerized ○ Receptor → ligand binding → conformational change → gets phosphorylated (undergoes autophosphorylation) Ligand = hormone or paracrine factor (inductive signal) ○ Paracrine Factors: ○ It becomes an active diffusible molecules tyrosine kinase produced by inducing cells ○ Then, it can activate and affect cells on the side inactive responding protein Para = side  ○ Extracellular matrix glycoproteins (laminin, components: integrins, fibronectin, integrins) lamenins, fibronectins, Unique SAMs and CAMs characteristics: cell ○ Components of the ECM adhesion molecules are generated, produced, and substrate and secreted by an adhesion molecules inducing cell that cause changes to cell adjacent to JUXTACRINE SIGNALING them. How is this type of signaling different ○ Inducing cells: generally from paracrine signaling? produce and secrete ○ Cells are juxtaposed ligands, which in some against each other cases require complex ○ There is a signal molecule processing in the (does not leave the producing cells or in the signaling cell) that resides extracellular matrix in adjacent cell member (non diffuseable) INDUCING SIGNALS: PARACRINE AND Proteins from the inducing cell interact JUXTACRINE with receptor proteins of adjacent responding cells The signal does not diffuse from the inducer (B) Juxtacrine signaling ○ There is cell to cell contact ○ Cells are juxtaposed to each other (C) Classical Paracrine signaling (D) Paracrine signaling involving ECM components ○ No cell to cell contact ○ Contact is in the form of paracrine factors (diffusible molecules) ○ ECM which has Notch receptor: closely associated components synthesized with protease and secreted by an ○ Protease: enzyme that can inducing cell can also serve cleave protein as a paracrine signal on the Delta signal: binds with the notch neighboring cells receptor ○ ECM: source of critical ○ Notch receptor undergoes developmental signals conformational change, ○ ECM components: becomes accessible to a collagen, proteoglycans, protease  ○ Protease can cleave the ○ Activates transcription intracellular domain, factor serving as a intracellular ○ Transcribes target gene signal that gets Hes5 gene translocated into the ○ Hes5 gene is transcribed nucleus when it gets activated, ○ Notch activates a inhibiting activity of Mash 1 transcription factor of CSL -> blocked NeuroD family -> transcribe its Neural-determining target gene gene ○ Can also recruit other If NeuroD is participants in the blocked, there is no transcription process more neural ○ Transcription can proceed determination ○ Transcription of the gene ○ Hes5 also inhibits responsive to the delta neurogeneninD -> blocked signal NeuroD Notch receptors: important in the No more neural nervous system and in a wide diversity differentiation or of developmental contexts determination Notch proteins: important receptors Delta and Notch operates in in neuron development determining which embryonic cell is to Delta binds Notch receptor -> activates become a neural cell and which one proteolytic cleavage of Notch inner does not portion Classical example: vertebrate eye ○ Proteolytic fragment moves ○ D-N interaction regulates to nucleus and: which cells become optic Displaces the neurons and which become repressor glial cells Recruits p300 HAT ○ Juxtacrine signaling are P300: important in the histone development of the acetyl components of the nervous transferase system and the sense Activates organs transcription Responsive to delta signal INHIBITION OF NEUROGENESIS IN MOUSE The binding of delta to notch tells the receiving cell not to become neural ○ Proteolytic cleavage ○ Notch fragment gets translocated inside the nucleus  PARACRINE FACTORS OF NOTE: FGFs are more than a dozen DEVELOPMENT ○ FGF1, FGF2, FGF3, and so FGFs, hedgehog, Wnt, TGF-Beta on superfamily ○ Used repeatedly to regulate Functions of FGFs in mature systems development and (are involved in): differentiation of ○ Keratinocyte organization organ-systems ○ Wound healing FIBROBLAST GROWTH FACTORS HOW DO FGFs INDUCE CHANGES IN (FGFs) CELLS (FGF PATHWAY) They activate tyrosine kinase receptors known as FGFRs or fibroblast growth factor receptors ○ FGFRs are RTK (tyrosine kinase receptors) in nature Functions of FGFs in developmental processes: ○ Participate in mesoderm formation ○ Establishing antero-posterior patterning The image above is the FGF pathway of limbs The FGF pathway utilizes an RTK or ○ Limb development as a FGFRs whole ○ The dormant tyrosine ○ Neural development kinase domain is the (FGF8) undimerized RTK ○ The ligand comes along, Also an important factor in (these and bounds to the binding processes depends on the function/s site of the receptor of FGFs): ○ These receptors undergo ○ Angiogenesis (FGF2) – dimerization and endothelial cell proliferation auto-phosphorylation and organization of ○ It is now a very active endothelial cells into kinase, activates an tube-like structures inactive responding protein Started by forming ○ The protein gets the vasculators in phosphorylated, now the embryo, became an active establishing blood responding protein, ready vessels to carry out its function for ○ Vasculogenesis - follows the cellular response of the angiogenesis, which is the cell in response to FGF growth of new blood Examples of responding proteins vessels from preexisting (protein products of) : vasculature ○ Engrailed 1 and 2 genes ○ Wnt gene product  What are the functions of Engrailed 1 and 2 genes? The Wnt genes? This is a stained embryo in situ hybridization Dark areas indicate sites of Fgf8 expression ○ Somites, isthmus, pharyngeal regions, limb buds, etc. This is the isthmus, the depression between the mesencephalon and ESTABLISHMENT OF BODY AXES rhombencephalon FORMATION ○ This is an organizing center in the brain and secretes Fgf8 ○ Fgf8 is important for the development of midbrain and limbs ○ Fgf8 is highly expressed in the isthmus region, and activates the gene product of engrailed 1 and 2 and Fgf8 is important in establishing the Wnt genes left-sidedness of an embryo Engrailed 1 and 2 ○ Left side lateral (?) genes regulate the symmetry of an embryo development of (under body axis formation) dorsal midbrain and formation of cerebellum Wnt 1 gene assists in the development of cerebellum  WHAT IS THE HEDGEHOG FAMILY HEDGEHOG FAMILY Sites of activity Induce particular cell types and create boundaries between tissues ○ Sonic hedgehog (shh) Most widely used during embryonic development Extremely important in limb and neural differentiation ○ Desert hedgehog (dhh) ○ Indian hedgehog (ihh) Involves in ventral patterning of the neural tube Patterning of the ventral portion somites to cartilage of the spine Initiate antero-posterior axis in limbs ○ In the image (an embryo, subjected to in situ VENTRAL PATTERNING OF hybridization), in red is the THE NEURAL TUBE BY SHH nervous system ○ Blue participates in the gut development ○ Black participates in the limb buds development A family of genes Function: Induces or establishes Shh is secreted by the floor plate of particular tissues or tissue boundaries neural tube and notochord In the case of vertebrates, these are Shh activates a gradient. the commonly known hedgehogs: If high gradient expression of Shh, ○ Sonic hedgehog (SHH) motor neurons arise! ○ Desert hedgehog (DHH) If gradient expression of TGF-B, ○ Indian hedgehog (IHH) sensory neurons arise! Of these three, SHH is the most widely used in various developmental contexts DHH is expressed in sertoli cells of the testes ○ Participates in spermatogenesis IHH is expressed in gut and cartilage ○ Participates in the formation of the bones DHH and IHH, postnatally – regulate bone growth and sperm production  MOLECULAR SIGNALS FOR PATTERNING OF THE VENTRAL BRAIN DEVELOPMENT PORTION OF THE SOMITES GIVING RISE TO CARTILAGE OF THE SPINE The Side of the notochord is the paraxial mesoderm that gives rise to the… ○ dermatome (outer) Color-coded yellow is Shh, expressed ○ myotome(middle) by the prechordal plate and notochord ○ sclerotome (innermost ○ ventralizes the brain, where around the notochord) motor neurons arise on the ventral side Shh then activates the expression of NKX2.1 ○ Regulated development of the hypothalamus Adjacent non-neural ectoderm secretes BMP4 & 7 (blue code) ○ Control dorsal patterning of the brain ○ Sensory neurons on the dorsal side SHH and Noggin - induce the ventral somite to form sclerotome Fgf8 - secreted by the anterior neural ○ Sclerotome the express ridge which is the organizing center PAX1 gene during brain development. PAX1 gene - controls chondrogenesis -activates FOXG1 gene and vertebral formation Involved in patterning the ventral FOXG1 - expression in adjacent portion of the somites neuroectoderm ○ regulates development of telencephalon to cerebrum; diencephalon to hypothalamus ○ Involves in regional specification of prosencephalon  INITIATE ANTERIOR-POSTERIOR Panel B: Anterior-posterior AXIS IN LIMBS patterning ○ Anterior-posterior axis is from the thumb to the tiny finger ○ Controlled by cells in the ZPA ○ ZPA - zone of polarizing activity produces retinoic acid (RA) ○ RA - initiates expression of SHH ○ SHH - regulates A-P patterning HEDGEHOG PATHWAY HOW DOES THE SONIC HEDGEHOG PARACRINE FACTOR INFLUENCES LIMB AND NEURAL DEVELOPMENT IN VERTEBRATES? Panel A: Limb bud and apical ectodermal ridge (AER) formation ○ Fgf10 initiates limb bud formation ○ Rad fringe (Radical fringe) gene restricts location of AER ○ AER expressed Fgf4 & Fgf 8 which signals progressive zone of proliferation Patched receptor: inhibitor if smoothened proteins ○ Hedgehog signal is present ○ Smoothened is released from inhibition: inhibits activity of PKA and Slimb PKA and Slimb are cleavage proteins ○ Ci or intracellular signals is released from microtubules (uncleaved) translocated into the  nucleus → activates gene Ci is bound to microtubules (by Cos2 expression & Fused proteins) Ci gets translocated ○ Because it is bound to the from the cytoplasm microtubules, it is easily inside the nucleus accessible to: PKA and and it activates the Slimb and cleave Ci expression of genes ○ Ci becomes a that is responsive to transcriptional repressor; the hedgehog signal blocks gene transcription Hedgehog makes use of patched No activation of a proteins/receptor. And a patched gene. Thus, no receptor is associated or an inhibitor to gene product a smoothened protein. But in the Any misregulation in presence of a hedgehog signal, the the signalling inhibition is lifted off, the smoothened pathway can result become a very active proteins. Thus, it to embryonic can inhibit the activity of the cleavage abnormalities proteins. ○ Ci= Cubitus interuptus ○ Hindi sila makagalaw, so si homologous to GLI3 protein Ci buo siya, and it gets ○ When GLI3 is truncated, translocated into the gives rise to Greig nucleus and activates gene cephalopolydactyly (high expression. forehead and extra digits) Expression of a No signal= it inhibits smoothened gene is responsive proteins (hindi siya makagalaw). So to the hedgehog these are free to move or to excert signal their function. And their function is to cleave Ci WHAT HAPPENS IN THE ABSENCE OF SONIC HEDGEHOG? WNT FAMILY (FAMILY OF CYSTEINE-RICH GLYCOPROTEINS) Induces dorsal side of somites to become muscles ○ Wnt activate PAX3 (PAX3 demarcates dermamyotome)  INITIATE DORSO-VENTRAL AXIS IN LIMBS ○ Wnt acts on the dorsomedial portion of somite and induces the formation muscle precursor of cells that will soon express muscle-specific gene, MYF5 A. Limb bud and AER formation B. Anterior-posterior patterning C. Dorso-ventral patterning ○ Wnt in cooperation with ○ A function of the Wnt family BMP4 activates MyoD ○ Thumb (ventral side) and expression on the the back of the thumb Ventrolateral region of (dorso side) somite, then second group ○ It is directed by Wnt of muscles are formed (Wnt7a) Establishes polarity of vertebrate limbs ○ Wnt expressed in the Necessary for urogenital system dorsal ectoderm (colored development: purple in the image) ○ Kidney development ○ Wnt: induces expression of ○ Sex determination LMX1, transcription factor in the dorsal mesenchyme cells specifying these cells as dorsal  WNT PATHWAY When β-catenin is not freed, it will be degraded. Thus, no activation of the gene responsive to the Wnt This can happen to the older cells, not in the embryo There are cases where APC or How can the Wnt pathway mediate β-catenin is mutated, as shown on B, cell division? the conformation is different from the The Wnt pathway makes use of the normal conformation frizzled receptor associated with β-catenin causes activation of cell disheveled protein division genes, c-myc, that can lead to ○ Receptor-ligand binding tumor formation activates the associated It is important that these pathways are protein [disheveled] Wnt regulated When disheveled protein is active, it inhibits the activity of GSK-3 (glycogen APC - adenomatosis polyposis coli synthase kinase) (tumor suppressor); targets catenin for What are the ramifications of this degradation pathway? GSK-3 - Glycogen synthase kinase 3; When the function of GSK is inhibited, prevents β-catenin dissociation from β-catenin can be released from the APC complex, then an intracellular signal is Wnt binds to Frizzled receptor family generated, which gets translocated in and activates Disheveled the nucleus, binds with DNA-binding Disheveled blocks GSK-3 proteins, transcription of the gene ○ When GSK-3 is blocked, responsive to the Wnt signal proceeds β-catenin released from β-catenin causes activation of cell APC and enters the division genes, c-myc nucleus LEF/TCF = DNA binding proteins ○ β-catenin associates with In the absence of a Wnt signal, LEF/TCF disheveled is not active, GSK is active, Other possible participants: β-catenin cannot be freed from the ○ E.g.-at surface complex. The gene responsive to a -co-receptors, etc. Wnt signal is not activated -cytoplasmic -G-protein, other factors  TGF-𝛽 SUPERFAMILY Members: ○ TGF-𝛽 family ○ Activin family ○ BMPs BMP4, BMP7 ○ Glial-derived neurotrophic factor ○ Mullerian-inhibitory factor Regulate various developmental processes ○ BMPs are involved in inducing bone formation ○ Involved in embryonic skeletal and soft tissue development ○ Present where mesenchymal-epithelial inductive interactions may SMAD PATHWAY occur: Heart Tooth buds Skin Cranio-facial processes Central nervous system ○ Activins mediates mesoderm induction TGF-𝛽 ligands make use of the ○ Activins have been serine-kinase receptor proposed in a number of Two types of receptor: reproductive organs ○ Different dimers including the gonads, ○ Undergo dimerization in the uterus, and pituitary presence of the ligand ○ Regulate folliculogenesis, How the receptor works: spermatogenesis, ○ An activin or a TGF-𝛽 pregnancy ligand comes along TGFs-𝛽 are involved in the formation binding to the receptor of extracellular matrix; regulation of ○ The receptor kinase is cell division auto-phosphorylated and becomes active If this comes to activin family, activin member, or TGF-𝛽 ligand member, the Smad activated are Smad 2 and 3,  these are proteins RTK PATHWAY activated. Smad 2 and 3, can now form a complex with Smad 4 Smad 4 and Smad 2 or 3 has intracellular signals and get translocated from the cytoplasm to the nucleus and they activate gene expression Expression of a gene responsive to activin or TGF-𝛽 ligand ○ BMP ligand comes along Still a kinase Receptor-ligand Ligand-receptor binding binding RTK Auto-phosphorylated and later Auto-phosphorylatio recognized by adaptor protein that n activates another intermediate- GMRP In this case, if the The activated GMRP activates the ligand was a BMP, RAS protein that is associated to a the proteins GDP; the activated RAS with the GDP activated are Smad that is phosphorylated 1 and Smad 5 The activated RAS, in turn, activates a Smad 1 and 5 form series of kinases: MEK, ERK a complex with ○ These are kinases that may Smad 4 and gets phosphorylate transcription translocated into the factors in the nucleus → nucleus where it alter gene expression participates with Eventually/ ultimately leads to gene expression activation of transcription factor OTHER PATHWAYS OF CELL-TO-CELL SIGNALING DURING DEVELOPMENT JAK-STAT pathway Apoptosis pathway (programmed cell death) ROLE OF JAK-STAT PATHWAY Regulation fo fetal bone formation Extremely important in the differentiation of blood cells Activation of casein gene for milk production  EXAMPLE: ACTIVATION OF CASEIN GENE Ligand in the pathway may involve prolactin, cytokines, growth factors In the figure, prolactin comes along. JAK receptor dimerizes. It undergoes autophosphorylation and becomes active. It then activates STAT 5. This gets translocated. Shown in the image is the casein gene promoter. The casein gene is then activated. STAT PATHWAY Ligand and receptor binding wherein This plays for the regulation of fetal the receptor is JAK (Janus tyrosine bone growth kinase) Stat1 protein mediates signaling JAK is activated and gets protein - phosphorylated Stat pathway is also utilized by FGF JAK intern activates STAT (signal (fibroblast growth factor). FGF binds transducers & activators of with FGFReceptor3 interaction transcription) STAT is phosphorylated and Normal development undergoes dimerization ○ FGF-FGFR3 STAT gets translocated in the nucleus. (ligand-receptor binding) It undergoes nuclear translocation and ○ STAT1 protein is binds with DNA phosphorylated It then activates gene transcription ○ STAT1 is translocated in The gene that is activated is the the nucleus casein gene ○ It encodes for the transcription of cell cycle inhibitor, p21 ○ P21 gene is activated ○ Chondrocytes division stops ○ Chondrocytes starts to undergo chondrogenesis to establish the formation of cartilages  APOPTOSIS PATHWAY It is also called program cell death The time where mammalian cells make “life-or-death decisions” Who enters the suicidal program during development? ○ Cells who have embryonic cells that have reached the end of their functional lifespan It is necessary for the sculpting of organs It is necessary for the normal morphogenesis of hands and feets which results to proper spacing of digits It is essential for normal development of the nervous system thus there is proper spacing of neurons It is essential for normal operation of the immune system in adults Sculpting of the digits by apoptotic Nervous system and the immune machinery eliminates the interdigital system arise through overproduction webs and new shape is revealed of cells but gradually those who cannot form functional connections with target PCD PLAYS A CRUCIAL ROLE IN ORGANOGENESIS AND TISSUE cells they undergo program cell death REMODELING Also, cells in the immune system that The best known example is the failed to produce antigen specificity formation of digits in higher undergo program cell death vertebrates where PCD eliminates the interdigital webs primarily via the apoptotic machinery PCD is also involved in the conversion of solid structures to hollow tubes, thereby yielding a lumina such as in the creation of the proamniotic cavity (Coucouvanis and Martin 1995; Weil et al., 1997). It is observed when epithelial sheets invaginate, forming tubes or vesicles, for example in the Cell death adjusts number of nerve establishment of the neural tube or cells to size of target lens and when epithelial sheets Apoptosis adjusts the number of nerve fuse to construct the mammalian cells to match the number of target palate (Glucksmann, 1951). cells that require innervation PCD is involved in sculpting the future inner ear in chicks (Avallone et al., 2002)  Essential for generating the DEATH SIGNAL four-chamber architecture of the heart (Abdelwahid et al., 2002) APOPTOTIC PROTEINS: CED3, CED4, CED9 Apoptotic proteins Ced3, Ced4, and Ced9 were first identified in Caenorhabditis elegans which is an invertebrate (nematode) NO DEATH SIGNAL There is a signaling molecule binding Ced9 is inactivated ○ When it is inactive, it cannot inhibit the activity of Ced4 and Ced3 ○ Therefore, cascades of activation proceeds Proteases and nucleases are activated leading to the formation of blebs in the cell. EQUIVALENT OF APOPTOTIC The purple structure is the death PROTEINS IN MAMMALS receptor but there is no signaling molecule Ced9 protein is active ○ When it is active, it inhibits the activity of Ced4 and Ced3. If Ced4 and Ced3 are inactive, there is no induction of cell death CEDs are protease-activating factors CED9: Bcl-2 family ○ It helps regulate activation of procaspases CED4: Apaf-1 (apoptotic protease activating factor-1) CED3: caspase 9 and caspase 3  SCHEMATIC REPRESENTATION OF INTRINSIC PATHWAY APOPTOTIC EVENTS Coming from inside Non-receptor mediated stimuli Two main pathways There is a formation of intracellular ○ Extrinsic Pathway signals ○ Intrinsic Pathway Involves MPT (mitochondrial permeability transition) EXTRINSIC PATHWAY There is a generation of cytochromes and that will form the apoptosome Apoptosome activates caspase 9 Caspase 9 (activator of an execution pathway) activates caspase 3 After caspase 3 is activated, it then activates endonucleases and proteases ○ Endonuclease: enzymes that degrade chromosomal DNA in the nucleus Coming from outside ○ Protease: enzymes that Receptor-mediated stimuli; binding of breakdown proteins; a ligand to a receptor degradation of nuclear and Adaptor proteins are activated cytoskeletal proteins which in turn induces the formation which gradually results in of DISC (death-inducing signaling cytoskeletal complex) reorganization DISC formation activates caspase 8 After cytoskeletal reorganization, there Activation of caspase 8 activates an will be cytomorphological changes executioner which is caspase 3; which ○ Changes include: activates the execution pathway chromatin and cytoplasmic condensation, nuclear fragmentation, etc. Cytomorphological changes will then lead to the formation of apoptotic bodies  1. The cell shrinks; chromatin INTRINSIC MITOCHONDRIAL PATHWAY condenses 2. Gradually, the membrane starts blebbing; The cellular organelles disintegrate 3. Nucleus and organelles collapse; Membrane continues to bleb 4. Fragmentize; Apoptotic bodies form 5. Macrophages phagocytose apoptotic bodies 6. No inflammation The mitochondrial permeability INTEGRATING SIGNALING PATHWAYS transition (MPT) core will release two At the start of the embryonic main groups of normally sequestered induction, because of close pro-apoptotic proteins: proximity of induction, interactions ○ Cytochrome C (Orange can go on at the same time structure): Activates Apaf ○ Resulting to the integration 1 which will activate of the signaling pathways caspase 9 which will activate caspase 3 (execution pathway) gradually resulting in the apoptosis or formation of apoptotic bodies. ○ Smac (green structure): proteins that inhibit the Many of the mechanisms activity of IAPs. underlying cell-type specification IAPs: inhibitor of apoptosis and formation of distinct tissue rely ○ These are inhibited so they on interactions between signaling cannot inhibit apoptosis pathways thereby contributing to the (based on the illustration) fast-track onset of ○ Shh - yellow apoptosis. ○ Wnt7a - blue Pro-apoptotic proteins: they promote ○ Bmp4 - red apoptosis ○ Noggin - arrows Integration of signaling pathways FORMATION OF APOPTOTIC BODIES to exert their function in the formation of organs  MOLECULAR SIGNALS FOR BRAIN Adjacent non-neural ectoderm DEVELOPMENT secretes BMP4 & 7 ○ Control dorsal patterning of the brain ○ Sensory neurons on the dorsal side Molecular signals for brain development are initiated at neural plate stage As in the brain, BMPs initiate the BMPs = Establish sensory signaling cascade for sensory regions areas in the spinal cord, and SHH = establishes motor regions SHH regulates motor areas (based on illustration) ANR - anterior neural ridge, an organizing center secretes Fgf8 ○ FOXG1: expression in adjacent neuroectoderm Regulates development of telencephalon to How can the spatial temporal cerebrum interaction of signaling pathways Regional produce complex patterns during specification of development? prosencephalon ○ Example: somitogenesis Prechrodal plate (P) and notochord (process of somite (N) secrete SHH formation) ○ Ventralizes the brain Oscillating mechanism ○ Motor neurons on the (segmenation clock) ventral side ○ Drives pulses of NKX2.1 expression expression of a limited ○ Regulates development of number of genes the hypothalamus repeatedly in the  presomitic mesoderm Induces dorsal side of somites to (PSM) every time a new become muscles somite is formed Wnt activate PAX3 ○ Every time a new somite is ○ PAX3 demarcates formed in the presomitic demamyotome mesoderm, all the genes ○ Wnt → Dorsomedial portion involved undergo of somite → form muscle simultaneous expression precursor cells → express ○ Group of genes, oscillate, muscle-specific gene wherein they are all (MYF5) expressed at the same time Wnt + BMP4 → activates MyoD ○ Then another group of expression on the ventrolateral genes oscillate and get region of somite → second group expressed at the same time of muscles at the right location SHH & Noggin ○ Well-regulated ○ Ventral somite → As a result, spatial and highly sclerotome dynamic temporal regulations of Wnt, BMP4, SHH, PAX3, Noggin: these signaling activities all integrate for the signaling pathways NOTABLE POINTS FOR INTEGRATING SIGNALING PATHWAYS Each pathway does not specifically regulate a single biological process ○ No unique signal for induction of each cell type Each pathway can trigger diverse effects, depending on the Example: Wnt pathway state of the cell at the time the ○ Activated in the presomitic pathway is activated mesoderm (PSM) before The response of a cell to a segmentation; oscillation signal depends: with other genes like FGFs, ○ On amplitude Notch… ○ Duration Result: spatial and highly dynamic ○ Interactions between temporal regulations of these pathways signaling activities ○ Integration of transcription factor effectors at the promote region and enhance region of target genes  The same embryonic cell may respond differently to two different signals ○ Signal 1 = rate of cell division is fast ○ Signal 2 = rate of cell division is slow The same signal may trigger different response by different cells ○ Signal 1 = Fast rate of cell division ○ Signal 2 = slow rate of cell division Different cells of different embryonic stage may respond differently to the same signal ○ Signal 1 Embryonic stage 1= expression of gene is turned ON Embryonic stage 2= expression of gene is turned OFF  PART 12A Muscle Tissue Connective Tissue CELL DIFFERENTIATION Nervous Tissue (very limited) ○ Though, there are other subtypes Muscle tissue subtypes: skeletal, smooth, and cardiac Connective tissue subtypes: regular, irregular, and supporting connective tissues ○ Differentiated cell types can be defined by their histology and morphology Another core aspect of developmental and why the different types biology of proteins that they Explains how cells grow to look so synthesize is specific to different and perform such different themselves functions Differentiated state is generally stable ○ Considering that all cells of ○ When a cell has undergone an embryo are derived from the differentiated state, this a single fertilized egg that is a point of no return – can had undergone cleavage no longer revert its fate to divisions, mitotically become another type of creating genetically similar cell cells at the stage of Cells become different in a stepwise cleavage division. process which may begin during Cell differentiation: the process of embryonic period of histogenesis altering the pattern of gene expression ○ At first, primary induction, and thus becoming a cell of a secondary inducting giving particular type rise to the complementary Which genes are turned ON; and parts of an organ which genes are turned OFF ○ Reciprocal interactions The differences between cell types is make an organ more in the patterns of gene expression complex Some cells divide in the differentiated GENERAL CHARACTERISTICS OF state CELL DIFFERENTIATION ○ Not ALL Multicellular organisms develop ○ EXAMPLES limited catalog of differentiated cell Cells lining the types digestive tract ○ There are only four (4) (stomach and major types of tissues in intestine), cells that the body of a vertebrate are exposed to Epithelial Tissue erosion, as they  perform or function ○ Housekeeping genes in secretion and (common genes): genes absorption of that are expressed in all nutrients and cell types of the body; materials; they are expressed by all embryonic replenished when cells they are worn out Genes that encode Outer layer of the for proteins skin cells: replaced necessary for the by cells at the maintenance of the innermost layer of embryonic cell or epidermis – stratum any cell of the body basale Encode for proteins that maintain the STEPWISE PROCESS integrity and functioning of a cell ○ Effector genes: