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# 7. Klíčové molekulární komponenty vývoje II. Signály z vnějšího prostředí. TGF/BMP. Notch. Receptor tyrosin kinases (RTK). Wnt. Hedgehog. Signály z vnějšího prostředí. TGF/BMP. Notch. Receptor tyrosin kinases (RTK). Wnt. Hedgehog. Notch.

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#7

Klov molekulrn komponenty vvoje II


Sign ly z vn j ho prost ed

Signly z vnjho prosted

TGF/BMP

Notch

Receptor tyrosin kinases (RTK)

Wnt

Hedgehog


Sign ly z vn j ho prost ed1

Signly z vnjho prosted

TGF/BMP

Notch

Receptor tyrosin kinases (RTK)

Wnt

Hedgehog


Notch

Notch

  • Notch=zez podle prvnho fenotypu octomilky se zezy na kdlech (T.H. Morgan, 1919)


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receptory Notch1-4

Notch ligandy jsou vzny na bunn povrch


Notch dr ha overview

Notch drha - overview


Notch signalling step by step

Notch signalling step-by-step


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Notch drha, stejn jako ostatn morfogenetick systmy, reguluje (a u negativn nebo pozitivn) jednotliv vvojov procesy na nkolika stupnch. Pklad: vvoj jednotlivch bunnch typ v nervovm systmu


Notch and the segmentation clock

Notch and the segmentation clock


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Jak studovat somitogenezi u myi?


Sign ly z vn j ho prost ed2

Signly z vnjho prosted

TGF/BMP

Notch

Receptor tyrosin kinases (RTK)

Wnt

Hedgehog


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Wnts viz pednka . 10

  • rodina ligand

  • 19 len u lovka a myi

  • glykosylovan a palmitoylovan extracelularn proteiny

  • Nzev: z Wingless prvn mutace u Drosophily a Int onkogen penen MMTV-virem a zpsobujc prsn karcinomy


Wnt kateninov dr ha

Wnt/-kateninov drha

  • nejlpe poznan

  • Wnt=ligand

  • Frizzled=receptor

  • beta-katenin je hlavn soust tto drhy, po aktivaci aktivuje ve spoluprci s dalmi faktory transkripci


Sign ly z vn j ho prost ed3

Signly z vnjho prosted

TGF/BMP

Notch

Receptor tyrosin kinases (RTK)

Wnt

Hedgehog


Hedgehog dr ha

Hedgehog drha

  • hedgehog (Hh) u octomilky nzev jeek podle fenotypu larvy

  • u savc jsou ti homology sonic hedgehod (Shh), indian hedgehog (Ihh) a desert hedgehog (Dhh)

Sonic the Hedgehog


Sch ma shh dr hy

Schma Shh drhy

Gli1,2-aktivtor

Gli3 - represor u obratlovc

Ci (cubitus interruptus) u octomilky

Sonic hedgehog (SHH) is translated as a ~45kDa precursor and undergoes autocatalytic processing to produce an ~20kDa N-terminal signaling domain (referred to as SHH-N) and a ~25kDa C-terminal domain with no known signaling role (1 on figure 5). During the cleavage, a cholesterol molecule is added to the carboxyl end of the N-terminal domain, which is involved in trafficking, secretion and receptor interaction of the ligand. When SHH reaches its target cell, it binds to the Patched-1 (PTCH1) receptor(3). In the absence of ligand, PTCH1 inhibits Smoothened (SMO), a downstream protein in the pathway(4). It has been suggested that SMO is regulated by a small molecule, the cellular localisation of which is controlled by PTCH. PTCH1 has a sterol sensing domain (SSD), which has been shown to be essential for suppression of Smo activity. A current theory of how PTCH regulates SMO is by removing oxysterols from SMO. PTCH acts like a sterol pump and remove oxysterols that have been created by 7-dehydrocholesterol reductase. Upon binding of a Hh protein or a mutation in the SSD of PTCH the pump is turned off allowing oxysterols to accumulate around SMO.This accumulation of sterols allows SMO to become active or stay on the membrane for a longer period of time. The binding of SHH relieves SMO inhibition, leading to activation of the GLI transcription factors(5): the activatorsGli1 and Gli2 and the repressorGli3. The sequence of molecular events that connect SMO to GLIs is poorly understood. Activated GLI accumulates in the nucleus(6) and controls the transcription of hedgehog target genes(7).


Sch ma aktivace transkrip n ho faktoru gli sou asn pohled 2007

Schma aktivace transkripnho faktoru Gli souasn pohled (2007)

The 12-transmembrane domain protein patched (PTCH), the receptor for hedgehog proteins, negatively regulates the seven-transmembrane receptor smoothened (SMO) in the resting state. The binding of sonic hedgehog (SHH) to PTCH relieves this inhibition and SMO changes its localization to the membrane, thereby stimulating the Gli family of transcription factors, which are responsible for most of the effects of SMO. SMO activates Gli through G i proteins that inhibit adenylyl cyclases, and G activates phosphatidylinositol 3-kinase (PI3K) and Akt. Both of these mechanisms seem to prevent the protein kinase A (PKA)-regulated inhibitory phosphorylation of Gli. Gli is also constitutively suppressed by suppressor of fused (SUFU), and this inhibition is relieved by SMO activation through a still unclear mechanism that might involve G protein-coupled receptor kinase 2 (GRK2).


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Shh jeden z nejlpe popsanch klasickch morfogen (tzv. model francouzsk vlajky) v zvislosti na koncentraci morfogenu se spout odlin transkripn programy

Nap. specifikace jednotlivch prst konetiny


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Dorsalizujc faktory

BMP

Wnts

Inhibitory BMP (noggin, chordin)

Sonic hedgehog (Shh)

ventralizujc faktory produkovan notochordem

Shh jeden z nejlpe popsanch klasickch morfogen (tzv. model francouzsk vlajky) v zvislosti na koncentraci morfogenu se spout odlin transkripn programy

floor plate nejspodnj st nervov trubice

notochord zdroj Shh

Nap. specifikace jednotlivch neuronlnch typ ve vyvjejc se nervov trubici


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Elektroporace kuec nervov trubice umonila poznat jakm zpsobem buky bhem vvoje zskvj a udruj svou identitu

Cross section of the spinal cord of an embryonic day three chicken embryo stained with fluorescent antibodies. Shown here in red is the motor neuron progenitor domain (pMN), one of many precise domains established by earlier signaling events. The pMN domain is here labelled through the use of antibodies specific for Olig2, a critical regulator of motor neuron formation. Developing motor neurons emerging from the pMN are shown labelled in green.

Fig. A - A model for early spinal cord development. The neural tube which will form the spinal cord is patterned into specific domains by multiple external signals which include a ventralizing Sonic Hedgehog (Shh) signal from the notochord (N) and floor plate (F), a dorsalizing BMP signal from the roof plate (R), and retinoic acid (RA) signaling from the adjacent somites (S).


P irozen inhibitory shh dr hy

Pirozen inhibitory Shh drhy

cyclopamin teratogenn alkaloid z kchavice (Veratrum californicum), poprv identifikovn jako ltku zpsobujc cyklopii (= 1 oko) a holoprosencephalii u ovc


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Expression of Sonic hedgehog (Shh) protein and the determination of the midline structure in mouse embryo.An SEM micrograph of the frontal view of a mouse embryo (fetal age 7.75 days). Shh protein is green. The dotted line in the micrograph shows the region: Shh antibody reveals Shh. The part that will become the brain (head fold) is followed by the perchordal plate. Shh (in green) that is expressed in the prechordal plate induces midline structure formation.

Model mice with Holoprosencephaly due to a Sonic Hedgehog (Shh) deficiency.

An SEM micrograph of ten-day old mouse embryos (front view of face). The mouse deficient in Shh gene (right) has no midline structure and only one region (eye position shown in green). Note, too, the lack of nostril separation due to no midline structure. The normal embryo (left), by contrast, has both the eyes and nostrils separated to between the two hemispheres.


Hypoxie a hif

Hypoxie a HIF

st podklad dodali:

Eva Lincov

Jana Kamartov

Filip Trka


Hypoxie a hif1

Hypoxie a HIF

Hypoxie: snen parciln tlak O2 ve tkni x normoxie

HIF Hypoxia-Inducible Factor:

Heterodimerick TF aktivujc geny obsahujc v promotorov sekvenci HRE (Hypoxia response element), vlastn transkripce je iniciovna pomoc koaktivtor p300 a CBP (CREB-binding protein)

Prozatm je znmo kolem 60 (100) gen regulovanch HIF, ada z nich reguluje odpov na hypoxii (angiogeneze, proliferace, metabolismus glukzy, migrace, apoptza, erytropoeza, metabolismus Fe)

Heterodimer sestv ze t podjednotek (HIF1, 2, 3) a jedn podjednotky (HIF=ARNT)

podjednotky jsou pi normoxii siln labiln, podjednoteka je na koncentraci O2 nazvisl


Struktura hif podjednotek

Struktura HIF podjednotek

bHLH (basic Helix-Loop-Helix) motiv: vazba na DNA, dimerizace

PAS (Per/ARNT/Sim) domna: usnaduje heterodimerizaci

N-TAD (N-terminal transactivation domain)

C-TAD (T-terminal transactivation domain)

ODD(D) (Oxygen-dependent degradation domain)

P402, P564: msta specifick hydroxylace v ODD pi normoxii

N803: msto specifick hydroxylace asparaginu mimo ODD


Hif p i normoxii a hypoxii

HIF pi normoxii a hypoxii

http://www.chem.ox.ac.uk/oc/cjschofield/images/new-2.png

VHL(von Hippel-Lindau) - tumor supresorov gen


Modelov v vojov zm ny spojen s hypoxi hif syst mem

Modelov vvojov zmny spojen s hypoxi/HIF systmem

embryonln vvoj

angiogenese

rst chrupavek

krvetvorba aktivace EPO genu


Angiogenese

Angiogenese

Angiogenese

tvorba novch krevnch cv

HIF-2 se ve do oblasti promotoru a iniciuje transkripci receptoru VEGFR 2 i expresi VEGF(Vascular EndothelialGrowth Factor)

hlavn faktor angiogenese

v normlnm vvoji ale i bhem ndorovho rstu


R st chrupavek

Rst chrupavek

HIF ptomen pi rstu chrupavek

HIF-1 me hrt roli v adaptaci chondrocyt na nzk tlak kyslku

osteoartritis

vt procento HIF- mRNA


Hypoxie v karcinogenezi

Hypoxie v karcinogenezi

  • Hypoxie je ji ve vzdlenosti 100 mM od cvy

  • Aktivovan geny podporuj angiogenezi a metastzovn

www-ermm.cbcu.cam.ac.uk/05009129h.htm


P iny a d sledky aktivace hif

Piny a dsledky aktivace HIF

PINY

Hypoxie

Psoben rstovch faktor a onkogennch signlnch drah (EGF, Ras, Src, )

Mutace ndorovch supresor (p53, PTEN, pVHL)

Nedostaten aktivita hydroxylz

DSLEDKY

Podpora angiogeneze a rst ndoru

Metabolick adaptace na hypoxii (podpora glykolzy)


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