Cellular architecture and well-orchestrated upwards columnar movement of

Cellular organization of the intestinal crypt:


The inner lumen of the human
colon is comprised of a single cell columnar epithelial layer, arranged into finger
like invaginations, known as the crypts of Lieberkühn. The self-renewing intestinal
stem cells (ISC’s) at the crypt base give rise to a proliferating progenitor population
of transit-amplifying cells (TA). As they migrate towards the crypt/surface
epithelium border, these TA cells terminally differentiate to acquire either a
secretory cell or absorptive enterocyte fate. They finally undergo senescence
or apoptosis on reaching the surface
epithelium, hence balancing the continuous proliferation of the ISC’s. This
hierarchical crypt architecture and well-orchestrated upwards columnar movement
of cells enables rapid epithelial self-renewal.

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Adult intestinal stem cells: Two
putative populations


The stem cell compartment of the small intestine has been
widely studied and is a model for the adult intestinal crypt, while the colonic
crypt stem cells remain poorly defined. Two populations of stem cells
have been postulated to exist within the intestinal crypt: the crypt base columnar cells
(CBC’s) and the +4 cells.


Cheng and Leblond identified CBC’s,
which were actively cycling cells that populated the crypt base. Given their
multipotent and self-renewal capabilities, they were postulated to be intestinal
stem cells. (Cheng and Leblond, 1974). Barker et al, 2007 showed that these
cells, which were interspersed between Paneth cells (intestine) or goblet-like
cells (colon), uniquely expressed a Wnt target gene, LGR5 (Leucine-rich-repeat-containing
G-protein-coupled receptor 5). This was visualised by creating a knock-in
allele, wherein an EGFP-IRES-Cre-ERT2 cassette was inserted downstream of the
start codon of the LGR5 gene and EGFP expression was detected in LGR5+ cells.

Furthermore, lineage tracing using an Lgr5-EGFP-ires-CreERT2/Rosa26RlacZ
mouse model revealed that the crypt base stem cells could generate multiple epithelial
cell lineages as well as maintain long-term self-renewing capacity. This was
evidenced by the presence of Lac-Z labelled cells in the crypt-villi axes even
after 6-10 months (Barker, 2007). In comparison with the small intestine, LGR5+
cells of the colon showed slower cycling kinetics, taking longer to generate Lac-Z
stained crypts. This, perhaps, reflects the difference in epithelial turnover
rate, which is slower in the colon. (Barker, 2007). Following the identification
of LGR5, gene signature studies have revealed various markers that label CBC
cells. Ascl2, a Wnt target gene, that encodes a transcription factor involved
in stem cell fate determination was expressed at higher levels in crypt base
stem cells of the human small intestine and colon, when compared to TA and
Paneth cells. (Van der Flier, 2009). Similarly, Olfm4, was also established as
a marker for CBC’s in the mouse small intestine and was later found to be
expressed in the human colon as well (Van der flier, 2009)



The + 4 stem cells, characterized by the expression of
BMI1, TERT, HOPX and LRIG1, were originally identified by Potten et al as
actively dividing, DNA label retaining cells. However, they are now considered
to represent a relatively quiescent stem cell pool.


Lineage Tracing of BMI1 – regeneration, slow-cycling


Intestinal stem cell niche:
Homeostasis is maintained by major signalling pathways




Wnt signalling: Pathway


Secreted Wnt glycoproteins act as
long and short distance morphogens, establishing Wnt gradients which play an
integral role in directing cell proliferation and fate determination; both
during development and in regulating adult tissue homeostasis.


The canonical Wnt pathway
(described in Figure 2), which promotes the nuclear localization of B-catenin
and its subsequent interaction with the Tcf family of transcription factors
(Tcf1,Tcf3, Tcf4 and Lef1) to influence gene expression, is a key player in
driving homeostasis of the intestinal crypt. In the absence of Wnt, free B-catenin
in the cytoplasm is sequestered by a destruction complex consisting of core
proteins including Axin, APC, CK1a and GSK3B. CK1a and GSK3B phosphorylate B-catenin,
leading to its degradation. The binding of a Wnt ligand to Frizzled, a seven-pass
transmembrane receptor and its co-receptor lipoprotein receptor related protein
(LRP) activates the canonical Wnt pathway.  Dishevelled proteins are recruited to the
cytoplasm, which inhibit the destruction complex. Cytosolic B-catenin,
therefore accumulates and is translocated into the nucleus, where it interacts
with the TCF/Lef complex to displace the transcriptional repressor, transducin-like
Enhancer of split (TLE), which is the mammalian homolog of Groucho, thereby
activating Wnt target genes. In the absence of nuclear B-catenin, TCF3 and TCF4
are bound to TLE, which recruits histone deacetylases (HDAC’s) (Brantjes et al,2001).

This represses the chromatin state of the downstream target genes, preventing
their transcription. (Chen
et al,2001). When B-catenin binds to the TCF/Lef complex, it interacts
with chromatin activators such as CBP/p300 and Brg1, which in turn induce
target gene transcription. (Barker
et al,2001)




Wnt signalling drives
intestinal crypt homeostasis


The involvement of Wnt signalling
in preserving intestinal architecture was proposed in early studies by Korinek
et al,1998, who showed that disruption of Tcf7l2,
the gene encoding Tcf4, resulted in the loss of crypt formation and stem cell maintenance
in murine embryonic small intestine. This was further substantiated in experiments
by Van et al, 2012, where floxed Tcf4 mouse models were generated to inactivate
Tcf4 function. These Tcf4LoxP mice were crossed with a tamoxifen
inducible CreErt2 VillinCreert2  line
 and ki67 expression was used to detect
cell proliferation within the crypt. A loss of ki67+ cells was seen 7 days
after cre-induction had occurred, within the colonic crypts. Similar results were
observed in the small intestine, where the number of proliferating cells
reduced within 3 days of cre-activation. To determine if the proliferating
cells that were lost were in fact stem cells, Olfm4 (intestinal stem cell
specific marker) expression was studied. It was seen that within 7 days of
cre-induction, all Olfm4 expressing cells had been eliminated. Furthermore,
Lgr5 expression was lost after 7 days, whereas Bmi1, which is not a Wnt target
gene, was not affected. This, therefore, highlights the importance of downstream
factors of the canonical Wnt pathway in maintaining intestinal stem cells. Crypt
based stem cells express high levels of nuclear B-catenin (van de Wetering et
al. 2002) and deletion of B-catenin led to loss of intestinal crypts and goblet
cells (Ireland et al, 2004). Furthermore, inhibition of the Wnt pathway by overexpression
of Dickkopf (Dkk), a Wnt antagonist, caused the intestinal architecture to




Transcription factors involved
in Wnt signalling



Notch signalling is essential
for cell fate determination


Notch signalling plays a key role
in mediating cell proliferation and cell differentiation as blocking of the
notch pathway leads to a loss of proliferating cells and differentiation
towards a secretory lineage. This mechanism is determined by Notch ligands and
receptors that are present on adjacent cells, through which neighbouring cells
influence the fate of each other.  In
mammals, four Notch genes have been identified, which encode a single
transmembrane receptor each, known as Notch 1-4. These receptors in turn,
interact with one of five Notch ligands – Delta-like (Dll) 1,3 and 5 and
Jagged-1 and 2. In the intestine, Notch 1 and 2, are expressed, albeit in a redundant
fashion, as only inactivation of both receptors together has a phenotypic
impact. High levels of Notch 1 are localized in the proliferating crypt-base
stem cells, in both the colon and the small intestine. Dll1 and Dll4, along
with Jagged-1 are found at the bottom of the intestinal crypts, while Jagged-2
is found in cells underneath absorptive colonocytes (Sander et al, 2004).


Upon ligand binding, y-secretase
(and other proteases) cleaves the Notch receptor, thus freeing the Notch
intracellular domain (NICD), which is then translocated into the nucleus. Once,
in the nucleus, NICD binds to a transcriptional regulator, CSL (CBP/RBP-Jk).

This relieves transcriptional repression by CSL on Notch target genes, hence activating
the canonical notch pathway. This allows Notch target genes, Hairy/Enhancer of
Split (Hes) 1,5 and 7, HEY1,5 and HEYL to be transcribed. The intestinal
epithelium of Hes1 null mice showed an increase in goblet cells and Paneth cells,
but a decrease in absorptive cells, indicating that Notch signalling is
required for absorptive cells specification and represses the formation of
secretory cells (Jensen et al, 2000). The Hes family act as transcriptional
factors which repress transcription of basic helix-loop-helix (bHLH) transcription
factors including ATOH1, Math1 and HATH1, that are involved in inducing cell differentiation
and lineage specification. Math1 is highly expressed in secretory cells, and
the intestinal epithelium of mutant Math1 mice consist of only absorptive
cells, demonstrating its role in secretory cell specification. Another
transcription factor, kruppel-like factor 4 (Klf4) that is repressed by Hes1
activity, is essential for terminal differentiation of goblet cells, as
deletion of Klf4 in mice, caused a 90% reduction in goblet cells in the colon
(Katz et al,2002).   Zheng et al, 2009, showed that blocking Notch
signalling via a ?-secretase inhibitor significantly increased Klf4 mRNA levels
as well as the number of Klf4-positive cells (goblet-cells), in both the small
intestine and colon. However, Pellegrinet et al, 2012 argued that ,while loss
of function of Dll1 caused an increase in goblet cell numbers and decreased
proliferative progenitors, inactivation of Klf4 did not decrease goblet cell
numbers. Furthermore, pharmacological notch inhibition in both, a control and
Klf4 mutant mouse model, resulted in excessive goblet cells and a breakdown of
the proliferative crypt, thus questioning the role of Klf4 in notch-mediated
goblet cell differentiation.



Notch signalling maintains
intestinal stem cells


In addition to repressing the
secretory cell phenotype, notch signalling has been implicated in maintaining
the proliferative state of intestinal progenitors and stem cells.

Co-localisation of Hes1, with the intestinal stem cell marker Musashi-1 (Msi1)
in cells at the bottom of the crypt, indicated the presence of notch activity
in CBC’s. A similar observation was made at the +4 stem cells in the small
intestine. Lineage tracing studies using a tamoxifen induced Notch1 Cre-mouse
model (NIP1::CreERT2) revealed that, after eight months, labelled
cells occupied the entire crypt (and villus) and expressed markers of all the
differentiated cell types in the adult intestine (Pellegrinet et al, 2012).  This showed that cells expressing Notch1
activity were capable of self-renewal and differentiation, therefore,
expressing stem-cell properties. A lower number of Notch1 labelled cells were
found in the colon as compared to the small intestine. This phenomenon is
similar to the aforementioned observation in Lgr5 lineage tracing experiments,
where slower cycling rates were attributed to colonic CBC’s (Barker et
al,2007). The discovery of Lgr5 as a unique marker of CBC’s paved way for the identification
of Notch activity in intestinal stem cells as well. Dll1 and Dll4 double knockout
and RBP-J inactivated mice, along with increased goblet cell numbers, lost the
expression of stem cell markers including Lgr5, Olfm4 and Ascl2. Furthermore,
proliferating Ki67-positive cells in the crypts were ablated (Pellegrinet et
al, 2012).