6. Endoderm Flashcards
What are the roles of the endoderm?
Adult = gut responsible for food uptake and respiratory tube responsible for respiration
Mammal embryo = food and oxygen provided by placenta
Lower vert embryo = yolk provides nutrients and gas exchange performed through skin.
Endodermal cell layer of embryo::
- provides signal to nearby mesoderm e.g. cardiogenic mesoderm
- forms lining of gut and respiratory tube
- forms epithelium of several glands e.g. tonsils, thyroids, thymus, parathyroid glands
What are the steps in endodermal organ development?
- Endoderm specification occurs prior to gastrulation.
- Endoderm is patterned along AP and DV axis
- The gut tube undergoes morphogenesis.
- Endodermal organ buds are induced.
- Organ buds grow out and undergo morphogenesis.
- Endodermal progenitors differentiate into mature cell types
Describe endoderm specification occurring prior to gastrulation.
E develops from vegetal cells in xenopus, blastoderm marginal cells in zebrafish and the first cells that go through the anterior primitive streak in the mouse/human.
E specification in all species involved activation of Nodal in some way (in general, high nodal, activation of endodermal gene expression programme).
E cells specified along blastoderm margin by high levels of Nodal signalling –> Initially, sheet of cells involutes at the blastoderm margin –> cells dissociate from the sheet and individual cells ingress, forming a dispersed cell sheet –> cells converge at the midline as the axis extends –> at midline, cells form a rod that turns into a tube.
Definitive endodermal cells leave primitive streak, switching off E-cadherin.
GFP-+ visceral endoderm cells are replaced and most become extraembryonic.
Definitive endodermal cells and remaining visceral endodermal cells form a solid cell sheet
Describe how the endoderm is patterned along the AP and DV axis.
Solid sheet gives rise to a tube (definitive and some visceral contribution) which is progressively patterned along AP and DV axes. Organ buds develop in defined positions.
Zebrafish: endodermal progenitors are located at the blastoderm margin prior to
gastrulation; clear correlation between dorsoventral location of endodermal progenitors at the start of gastrulation and the final AP position of the mature endodermal cell (dorsal progenitors generate the anterior pharynx and ventral progenitors give rise to the posterior intestine).
Refined AP and DV patterning in broad domains - progenitors in foregut becomes thyroid, lung, liver, biliary tree, pancreas and stomach
Anterior part of the endoderm experiences high levels of Nodal
Posterior: Wnt, FGF and BMP signalling induce expression of caudal gene (encodes TF that activates hindgut genes):
- Hindgut – Cdx expression
- Midgut – Pdx1 expression
- Foregut – Hhex/Sox1 expression
- Little bit of overlap between these broad domains
Describe how cells that express enzyme retinaldehyde deydrogenase are a source of RA.
RA is made from vitamin A (retinol) which is transported into cell and oxidised, turning the alcohol into an aldehyde –> retinaldehyde.
Retinaldehyde oxidised futher into RA.
Enzyme controls this rate-limiitng step.
RA then diffuses out and can be used by other cells if they express RAR and RXR (form complex with RA and promote expression of particular target genes downstream)
Cells in vicinity also express CYP26 which can destroy RA into inactive RA metabolites
Anterior somites express RALDH2 and are source of RA
RA patterns heart field into atrial and ventricular parts
Describe how the gut tube undergoes morphogenesis.
Ventral folding starts anteriorly and posteriorly, giving rise to 2 pits (anterior and caudal intestinal portal - AIP and CIP) –> AIP and CIP elongate and approach each other –> form midgut –> connection of YS is eventually lost.
Stomach forms where Wnt signalling is blocked and RA is present.
Low Wnt/FGF/BMP4 and high RA = Posterior endoderm = Cdx2 expression = hindgut
Med Wnt/FGF/BMP4 and med RA = Pdx1 expression = midgut
High Wnt/FGF/BMP4 and low RA = Anterior endoderm = Hhex/Sox2 expression = foregut
Mesoderm close to ectoderm = somatic/parietal mesoderm
Mesoderm close to endoderm = splanchnic/visceral mesoderm
Wnt signalling defines endoderm that generates the intestine –> Posteriorly, Wnt signal blocks Hhex expression in the gut epithelium –> Hhex can’t inhibit Cdx expression –> Cells express Cdx and differentiate as intestinal epithelium.
Mesodermal cells that express Wnt inhibitors define the stomach domain of the gut:
Anteriorly mesenchymal cells of mesodermal origin express TF Barx1 –> these cells release Wnt inhibitors sFRP1 and 2 –> they act as a sink and scavenge Wnt ligands –> nearby anterior endodermal cells can’t receive a Wnt signal –> these endodermal cells express Hhex and Sox2 but not Cdx.
Describe how endodermal organ burds are induced.
By mouse E9.5, surrounding mesenchyme has induced formation of organ buds - blood circulation has already started (E8.25).
Mesenchymal signals induce local refinements in gene expression profiles that lead to organ bud formation - patterning along AP axis reflected in expression in different buds.
Pharynx develops from most anterior part of the gut tube and forms 4 pairs of pharyngeal
pouches that are separated by pharyngeal arches.
NCC seed pharyngeal arches and pouches and contribute a number of different cell types
Sonic Hedgehog – secreted by endoderm promotes NCC survival
FGF – signals from mesoderm and ectoderm support survival and guide migration; FGF
signals also important for pouch formation itself
What do the pharyngeal pouches develop into in fish?
Develop into slits that turn into gills
What do the pharyngeal pouches develop into in mammals?
1st pair give rise to auditory cavities of middle ear and eustachain tubes.
2nd pair forms walls of tonsils
3rd pair forms thymus and 1st pair of parathyroids.
4th pair forms 2nd pair of parathyroids.
Central diverticulum between 2nd pair of pouches forms thyroid gland.
A sprout between the 4th pair forms the lungs
Describe how the lung bud forms
Lung bud forms groove in pharynx ventral floor between 4th pair of pouches –> bud forms trachea that bifurcates to form the bronchi –> lead to larger right lung and smaller left lung.
Folding of the embryo puts heart near this endoderm –> lung bud is induced by nearby heart mesoderm – high FGF2 signal induces pharyngeal endoderm to express TF Nkx2.1 –> this activates expression of genes involved in lung development
Mesenchyme next to oesophagus expresses Barx1 –> induces sFRPs that block Wnt signalling –> oesophagus epithelium maintains Sox2 expression and develops as a squamous epithelium.
Developing tracheal epithelium comes into contact with mesenchyme that doesn’t express Barx1 - instead Wnt signalling is present –> lung epithelium expressed Nkx2.1 and becomes ciliated respiratory epithelium of the trachea.
In Barx1 mutant mice, tubes don’t separate properly.
Once specified by a heart FGF signal, lung bud outgrowth is triggered by RA (same signal that specifies atrial part of heart tube) –> RA induces expression of Tbx4 and 5 in ventral mesoderm –> activate expression of FGF10 –> triggers proliferation and chemotaxis of the lung tissue
In the neck region, the respiratory tube grows straight and forms the trachea
In the thorax, it branches and forms the two bronchi and the lungs - In explant experiments it was shown that In the absence of the thoracic mesenchyme, there is no branching –> signals from the trunk mesenchyme induce the branching
Describe how the liver forms
Hepatic diverticulum is induced just posterior to the stomach and gives rise to the liver, gallbladder and ventral pancreatic bud.
Dorsal pancreatic bud induced on dorsal side. The two pancreatic buds eventually fuse.
In humans, only ventral duct is retained and drains digestive enzymes into midgut.
FGF2 from heart mesoderm and high levels of BMP from septum transversum induce liver cells
Ventral mesoderm supports liver induction, dorsal mesoderm (notochord) blocks liver induction.
Gut cells need to be competent to respond to FGF and BMP signals – expression of FoxA1 and FoxA2 TFs establish this competence – Mouse foxA1/A2 double mutants generate no liver
Describe how the pancreas forms from ventral foregut progenitors.
Liver cells maintain Hhex expression. Pancreato-biliary cells maintain expression of Sox17 and Pdx1. Notch signalling and expression of Hes1 allow expansion of pancreatic progenitors then specify biliary cells maintaining Sox17 expression. Cells that see a reduced notch signal maintain Pdx1 and become pancreatic cells. Loss of Pdx1 expression causes loss of pancreas formation and defects in stomach/duodenum development. Forced expression of Pdx1 can divert prospective liver cells to a pancreatic fate.
Initially, pancreatic development requires RA signal from notochord and the most anterior somites.
As development proceeds, dorsal posterior foregut endoderm receives signals from notochord at E8 –> sends FGF2 and Activin signals –> these block Shh expression in future pancreatic endoderm (in presence of Shh, endoderm cannot express Pdx1) –> by E8.5 the two dorsal aorta have fused and send signals to dorsal bud to form and maintain Pdx1 expression. Ventrally, vitelline vein sends same signals to ventral pancreatic bud.
What does the exocrine pancreas do?
Generates digestive enzymes that it secretes into duodenum, transported by the duct.
Acinar cells produce proteases, lipases, amylases, etc.
What does the endocrine pancreas do?
Produces hormones that maintain glucose homeostasis.
Alpha cells produce glucagon.
Beta cells produce insulin.
Delta cells produce somatostatin.
Epsilon cells produce ghrelin.
PP cells produce pancreatic polypeptide
Describe molecular programming of pancreas development.
Cells that receive a Wnt signal express TF Ptf1a and become exocrine pancreatic cells
Cells that receive TGF-beta signal switch on TF Ngn3 (Neurogenin 3) and become endocrine pancreatic cells that differentiate into the different cell types
Describe how Cdx2 is required for the formation of the intestine.
Wnt signalling establishes expression of Cdx2 in posterior gut - required to establish and maintain posterior endodermal identity:
- high levels = large intestine
- low levels = small intestine
- loss of cdx transforms intestine into oesophagus
Simple epithelium –> at E10.5, epithelium starts to look pseudostratified (nuclei no longer neatly arranged at middle) –> becomes stratified –> secondary lumina form within epithelium –> lumina fuse with main lumen of intestinal tube –> formation of crypts –> endoderm between crypts is pushed up by mesenchyme underneath to form a villus –> now have crypts and villi alternating in the gut by E18.5.
Stem cells and Paneth cells are found at bottom of the crypt. New cells form there and move up the villi. About 250 cells per crypt and 10 crypts contribute cells to a single villus.
Intestinal stem cells sit at the bottom of the crypt which give rise to proliferative progenitors (transit-amplifying cells) – these divide 2-5 times and differentiate as they leave the crypt. Cells get lost at the top of the villus and die
What types of cells do proliferative progenitors differentiate into?
Absorptive:
- enterocytes (absorb nutrients and water)
- microfold cells (pick up luminal antigens and pass onto immune cells)
Secretory:
- goblet cells (produce mucin - released and form mucus)
- paneth cells (secret antimicrobials, provide stem cell niche)
- enteroendocrine cells (secrete hormones)
- tuft/brush cells (chemosensory, induce type 2 immune responses)
Describe how notch is involved in endodermal progenitors differentiating into mature cell types.
Cells that receive notch signal become absorptive cells and cells that express notch ligand delta become secretory cells.
Loss of notch signalling leads to a complete loss of all absorptive cells and an increase in secretory cells.
Cre-mediated conditional loss of RBP-J in all intestinal cells prevents Notch target genes expression and leads to:
–> A loss of Ki67-positive cells in the crypt loss of proliferative cells in the crypt
–> Increased expression of Atonal (Atoh) in all trans-amplifying cells
–> A massive increase in mucus-secreting PAS-positive goblet cells
Notch signalling maintains undifferentiated crypt base columnar cells -TF Atonal counteracts Notch signalling to promote differentiation of progenitors into secretory cell lineages
Describe how lgr5 is involved in endodermal progenitors differentiating into mature cell types.
Encodes leucine-rich repeat G-protein coupled receptor 5.
Expressed in crypt base columnar cells (CBCC)
Binds ligand R-spondin which is an enhancer of Wnt signalling, turns on Wnt target genes
Lgr5:GFP and Lgr5:lacZ knock in genes are expressed in crypt base columnar cells and
no expression outside the crypt base.
Use CreERT2:LoxP system to lineage trace progeny of lgr5+ cells. Cre expressed under control of promoter in CBCC and fusion protein translocates to nucleus and recombines loxP sites under tamoxifen, deleting stop cassette.
Progeny of lgr5-expressing crypt base columnar cells give rise to cells all the way up the villus –> crypt base columnar cells are stem cells
What do paneth cells secrete and why is it important?
EGF, Wnt and Delta-like 4 signals
Form niche for CBCC - R-spondin, Wnt and Delta actively promote cellular proliferation while BMP signalling supports cellular differentiation
