Signal Transduction
A similar role has been assigned to Wnt in Drosophila embryos, both at the level of segment polarity and of wing development. Here, unlike the more conventional inductive morphogens (such as Spatzle, BMP, Activin, Dpp, or Sonic Hedgehog), it acts to modulate the effects of inductive molecules.26
Wnt organizes the villous epithelium of the small intestine
Maintenance of the stem cell compartment requires Wnt signalling
The surface of the absorptive epithelium of the small intestine is lined with villi and with crypts of Lieberkühn (see Figure 14.14). Differentiated cells that
FIG 14.14 Migration of epithelial cells in intestinal crypts.
(a) The crypts of the small intestine contain stem cells that continuously produce committed progenitors. These move either downwards to transform into Paneth cells or up the villi to form the absorptive epithelium. (b) Wnt contributes to the maintenance of the stem cell compartment by ensuring the proliferation of the stem cells and their progenitors and also their localization within the crypt. It does this by induction of the ephrin receptor B2 and by
inhibiting expression of the ephrin ligands -B1 and -B2. This creates opposing gradients of both receptor and ligand. Stem cells and progenitors expressing high levels of the receptor are prevented from migrating out of the crypt through the repulsive action of the ephrins. Only when committed progenitor cells have switched off the canonical Wnt pathway do they escape repulsion and move up to the villus.
Panel (a) adapted from Sancho et al.81 Adapted and reprinted, with permission, from the Annual Review of Cell and Developmental Biology Volume 20 © 2004 by Annual Reviews (www.annualreviews.org).
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reside on the villi are enterocytes, whose role is to transport metabolites towards the interior. Here, enteroendocrine cells secrete the hormones gastrin, secretin, and cholecystokinin in response to the intestinal content and its acidity, and goblet cells secrete the mucin that lubricates the gut lining. Paneth cells, functionally similar to neutrophils, contribute to the extrinsic gastrointestinal barrier by releasing -defensins (cryptdins), lysosyme, and phospholipase A2. The crypts also harbour the stem and progenitor cell compartment, situated halfway between the bottom of the crypt and the start of the villi. The colonic epithelium has a flat surface pitted with crypts. The upper segment of the crypt contains goblet cells and mature enterocytes, whereas the lower segment is dominated by the stem cell/progenitor cell compartment.
The lifespan of intestinal epithelial cells is less than a week, during which time they migrate from the base of the crypts to the tips of the villi. Here they die and are shed into the faecal stream. Stem cells and Paneth cells escape migration. The stem cells cycle slowly, continuously producing progenitor cells which proliferate rapidly. As they attain the crypt–villus axes in the small intestine (or the upper crypt segments in the colon), they undergo cell cycle arrest and commence expressing differentiation markers.82 Cells of the crypt epithelium possess nuclear -catenin, the hallmark of Wnt signalling.
Wnt aligns committed progenitor cells along the crypt–villus axis
A screen for genes induced or repressed by -catenin/TCF-4 in intestinal epithelial cells revealed both Ephrin-B receptors (EphB) and their ligands (B-type ephrins) amongst the targets.83 Here, -catenin/TCF exerts inverse control, enhancing expression of the receptors and suppressing the ligands. This creates opposed gradients, with high receptor expression at the base of the crypts and high ligand expression at the points of the villi (see Figure 14.14).
Expression of EphB receptors is essential for the correct positioning of epithelial cells along the crypt–villus axis. Although they maintain their intestinal villi, mice carrying EphB2/EphB3 double mutants lose the characteristic localization of the Paneth cells in the crypts and the
characteristic compartmentalization of proliferating and differentiated cells along the axis (put simply, everything is all higgledy-piggledy).85 EphB
receptors, which are highly expressed at the bottom of the crypt, may thus act to restrict the upward migration of stem and Paneth cells.
Canonical Wnt signalling is vital for the maintenance of the epithelial stem cell compartment. Neonatal mice, deficient in TCF-4, lack the crypt progenitor compartment86 and transgenic mice expressing Dickkopf, an inhibitor of Wnt signalling, also show a loss of crypts.87,88
Other examples of Wnt signalling include the self-renewing capacities of mammalian hair follicles 89 and of haematopoietic stem cells.20,90 Of note,
Ephrin receptors represent the largest subfamily of receptor tyrosine kinases
(14 members in human). Based on their ligand binding specificities, these receptors are grouped into two subclasses. EphA receptors bind A-type ephrins, EphB bind B-type ephrins. Ephrin ligands are GPI-anchored proteins and the interaction between Eph receptors and their ligands therefore involves direct cell–cell interactions.
Eph receptors and their ligands are said to be involved in directed migration through repulsion. They constitute repulsive cues in processes such as axon path-finding, migration of neural crest cells and boundary formation between segmented structures such as rhombomeres.84
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Paneth cells are insensitive to the Wnt signal and remain non-proliferative differentiated cells.
Wnt and the asymmetric division of stem cells
In order to maintain their capacity for self-renewal, stem cells must undergo asymmetric division. The segregating proteins and mRNA are distributed in a way that generates one daughter stem cell and one daughter progenitor cell. The progenitor may still divide a number of times but is committed to
differentiate into a non-proliferating cell with a specialized function. The stem cell does not differentiate and retains its capacity to divide.
Molecular insight into the asymmetric distribution of cellular products during division has been provided by studies on brain neuroblasts. These cells achieve an unequal distribution of two proteins, brain tumour (Brat) and prospero (Pros), assisted by miranda (Mira, a scaffold protein). The daughter cell containing Brat and Pros becomes the precursor of ganglion mother cells, whereas its sister that lacks them, retains its identity as a neuroblast. Pros is a homeobox transcription factor that represses cyclins E and A as well as Cdc25, a dual-specificity phosphatase required for activation of cyclin-dependent kinases. Brat prevents translation of c-myc mRNA, a transcription factor involved in RNA-polymerase 1-dependent transcription of rRNA, thus a driver of ribosome production.91,92
To achieve the asymmetric distribution of cellular products, correct orientation of the microtubule spindle, prior to division, is essential (see Figure 14.15). Wnt and its 7TM Fz receptor are clearly implicated, but not the coreceptor LRP5/6.93–95 The signal is transmitted to Dsh, but is without effect on axin binding, instead switching towards Daam1 which is associated with RhoA.96 The RhoGEF involved remains to be identified.
Rho: regulator of the actin cytoskeleton
Rho is a key regulator in the organization of the actin cytoskeleton.97 Through its effector mDia, RhoA facilitates nucleation and polymerization of actin into long filaments. Through another effector, RhoA coiled-coil kinase (ROCK), it converts these filaments into stress fibres, cross-linking them with actinin and activating the motor protein myosin II. Both ROCK and mDia also operate in centrosome positioning during prometaphase.98 They control the connection of the astral microtubules with the cell cortex and hence they determine the orientation
of the mitotic spindle.99 Interestingly, APC, a component of the -catenin destruction complex (see page 424), binds the microtubules and is involved in organizing their linkage to the cortical network (see also page 589).100 It is not known what determines the site of fixation of the astral microtubules in epithelial stem cells. How this pathway signals to the ubiquitously expressed and evolutionarily conserved Par proteins that determine cell polarity is also unresolved.101 The non-canonical pathway involved in the induction of cell polarity is referred to as the‘planar cell polarity’(PCP) pathway.
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FIG 14.15 Wnt and asymmetric distribution of cellular products.
By activating the non-canonical pathway, through Dsh, Daam1, RhoGEF, and RhoA, Wnt is involved in asymmetric production of cortical actin. Downstream of RhoA, mDia assists the local polymerization of actin. One consequence is the asymmetric distribution of the scaffold protein Miranda which binds to cortical actin bringing with it, Brat and Pros. Brat suppresses translation of c-myc thereby limiting the protein synthetic capacity of the future committed progenitor cell. Pros suppresses the transcription of cyclins A and E and the dual specificity phosphatase Cdc25. This prevents repetitive cell division. The asymmetric distribution of cortical actin also determines the orientation of the mitotic spindle, through orientation of the astral microtubules. Attachment of these to the cell cortex is facilitated by the RhoA-controlled kinase ROCK, another component of the non-canonical pathway. Finally, this pathway also operates in the formation of the contractile actin ring,
positioned perpendicular to the mitotic spindle, which ultimately divides the cell in two. This occurs through the action of ROCK1 which phosphorylates and activates myosin II. The events depicted represent a compilation of data from different species and cell types and in different contexts.
Mutations of -catenin, Axin, and APC in human cancers
Several mutations in components of the Wnt signal transduction pathway give rise to cancer.50 Most of these result in the expression of non-functional proteins at both alleles. A prime example is the APC gene. Here, most of the mutations accumulate in the so-called mutation cluster region in the central part of the peptide chain. The truncated proteins that arise are unable to bind-catenin and axin.102 Cancer generating mutations also occur in axin and in-catenin. -Catenin is protected against degradation because the mutations either replace the phosphorylation sites for CK1 or GSK3 in the destruction
Different mutations in APC. The penetrance of germline mutations that increase the risk of colorectal cancer varies. The APC polymorphism I1307K has a penetrance
of 20%, meaning that 1 in 5 persons who carry it will develop adenomatous polyposis coli. This mutation does not directly affect the activity of APC, but enhances the chance of further ‘somatic’ mutations. The reason for this is that in the mutated protein, the codon ATA (isoleucine) has been changed to AAA (leucine). This gives rise to a DNA sequence consisting
of eight adenines in a row, which increases the risk of slippage during replication. It therefore increases the chance
of further mutations of which some will eventually give rise to a frame shift and loss of APC function. The
penetrance of truncation mutations approaches 100%.
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