Matthew Bjerknes

Clonal analysis of mouse intestinal epithelial progenitors

BACKGROUND & AIMS Little is known about the cell lineages leading from stem cells to the various terminally differentiated cell types of the intestinal epithelium. In particular, the existence and characterization of intermediate progenitor types remain open issues, which are discussed in this study. METHODS Chemical mutagenesis was used to genetically mark random intestinal epithelial cells by somatic mutation of the Dlb-1 locus. Intact epithelium was isolated at various times thereafter, and the composition, size, and location of mutant clones were scored. RESULTS & CONCLUSIONS Analysis of clone dynamics showed short-lived (days) progenitors (C1, M1, and Mix) yielding one or two cell types and long-lived (months) mucous cell progenitors (M0), columnar cell progenitors (C0), and pluripotential stem cells (S) capable of giving rise to all epithelial cell types. Furthermore, study of clonal dispersion, during crypt branching morphogenesis or cell migration, shows that mutant progenitors usually partition into only one of the two daughter crypts and that cells are often widely dispersed in spite of the extensive intercellular junctions in the epithelium.

Modulation of specific intestinal epithelial progenitors by enteric neurons

The proglucagon-derived peptide glucagon-like peptide 2 (GLP-2), a product of a subset of gut epithelial cells, is pursued clinically for its ability to stimulate gut epithelial growth and repair. Here we show that although specific epithelial progenitors respond to GLP-2 administration, the epithelium does not express the GLP-2 receptor. Rather, enteric neurons express the receptor, respond to GLP-2, and transmit a signal (which can be blocked by the voltage-gated sodium channel inhibitor tetrodotoxin) back to the epithelium. Thus the nervous system is a key component of a feedback loop regulating epithelial growth and repair.

The crypt cycle. Crypt and villus production in the adult intestinal epithelium

We propose a model for the growth of individual crypts that is able to account for the observed changes in the number of cells in crypts under normal conditions, after irradiation, and after 30% resection. Parameter values for this model are estimated both for mouse and man, and detailed predictions of crypt growth rates are made. This model does not predict a steady-state crypt size; rather it suggests that crypts grow until they bifurcate. We therefore propose a crypt cycle (analogous to the cell cycle) and present evidence that most if not all crypts in the adult mouse are cycling asynchronously and independently. This evidence consists of four experiments that indicate that branching crypts are randomly distributed over the intestinal epithelium, that the plane of bifurcation of branching crypts is randomly oriented with respect to the villus base, and that the size distribution of crypts is consistent with an expanding crypt population. We also report for the first time evidence of villus production in the adult mouse intestinal epithelium. We conclude that the crypt and villus populations in the adult mouse are not in a steady state.

CRP‐ductin: A gene expressed in intestinal crypts and in pancreatic and hepatic ducts

A subtraction screen isolated CRP‐ductin (CRP), a gene expressed in intestinal crypts. METHODS: DNA sequencing, in situ hybridization, immunostaining, Western and Northern blotting were used to characterize murine CRP. RESULTS: CRP is restricted to the intestine and its associated glands. In the small intestine, CRP mRNA is expressed in crypt cells at all stages of differentiation from the stem cells to the terminally differentiating cells of the crypt top, but not in the mature cells of the villus. In the colon, CRP mRNA is most heavily expressed in the mid‐crypt. Expression is also seen in the pancreas and pancreatic ducts, and in the epithelium lining larger hepatic ducts, but not in the liver parenchyma or stomach. CRP protein is localized to the lumenal aspect of crypt cells in the small intestine. In the colon, the protein is seen in the lumenal aspect of surface epithelial cells. CRP protein is similarly found in the lumenal aspect of epithelial cells lining the pancreatic duct system and the larger hepatic ducts. Two cDNA variants, CRP‐α and CRP‐β, were cloned from mouse jejunal epithelium. Their 3′‐sequence differs in an 82‐bp domain unique to CRP‐β. CONCLUSIONS: The CRP‐α sequence predicts a protein with a short cytoplasmic region, a transmembrane domain, and a large extracellular region composed of many repeats (8 scavenger receptor domains, 5 CUB‐domains, 1 ZP‐domain, and 6 copies of a previously unreported domain which we call the CRP‐domain). The structure of the CRP protein suggests a role in ligand interaction; possible functions are discussed.

Origin of the brush cell lineage in the mouse intestinal epithelium

Mix progenitors are short-lived multipotential cells formed as intestinal epithelial stem cells initiate a differentiation program. Clone dynamics indicates that various epithelial cell lineages arise from Mix via a sequence of progressively restricted progenitor states. Lateral inhibitory Notch signaling between the daughters of Mix (DOM) is thought to break their initial symmetry, thereby determining whether a DOM invokes a columnar (absorptive) or granulocytic (secretory) cell lineage program. This is supported by the absence of granulocytes following enforced Notch signaling or Atoh1 deletion. Conversely, granulocytes increase in frequency following inhibition of Notch signaling or Hes1 deletion. Thus reciprocal repression between Hes1 and Atoh1 is thought to implement a Notch signaling-driven cell-fate-determining binary switch in DOM. The brush (tuft) cells, a poorly understood chemosensory cell type, are not incorporated into this model. We report that brush cell numbers increase dramatically following conditional Atoh1-deletion, demonstrating that brush cell production, determination, differentiation and survival are Atoh1-independent. We also report that brush cells are derived from Gfi1b-expressing progenitors. These and related results suggest a model in which initially equivalent DOM progenitors have three metastable states defined by the transcription factors Hes1, Atoh1, and Gfi1b. Lateral inhibitory Notch signaling normally ensures that Hes1 dominates in one of the two DOMs, invoking a columnar lineage program, while either Atoh1 or Gfi1b dominates in the other DOM, invoking a granulocytic or brush cell lineage program, respectively, and thus implementing a cell fate-determining ternary switch.

Asymmetric distribution of actin mRNA and cytoskeletal pattern generation in polarized epithelial cells

We analysed the distribution of actin mRNA in intestinal epithelial cells using in situ hybridization of 35S-labelled cytoplasmic beta-actin RNA. We found that the distribution of actin mRNA generally parallels that of polymerized actin, i.e. there is an accumulation of actin mRNA in the apical end of villous epithelial cells. Furthermore, the development of this asymmetric localization of actin mRNA appears to parallel the elaboration of the cytoskeleton during cellular differentiation. We discuss the possibility that the interaction between actin and its mRNA may be important for the establishment and maintenance of cytoskeletal pattern in polarized epithelial cells.

Cell Lineage metastability in Gfi1-deficient mouse intestinal epithelium

Elucidating the mechanisms determining multipotent progenitor cell fate remains a fundamental project of contemporary biology. Various tissues of mice and men with defects in the zinc-finger transcriptional repressor Gfi1 have dramatic perturbations in the proportions of their differentiated cell types. In Gfi1-deficient intestinal epithelium there is a shift from mucous and Paneth towards enteroendocrine cells, leading to the proposal that Gfi1 functions in the allocation of the progeny derived from a hypothetical common granulocytic progenitor. However, studies of clones have yielded no evidence of such a common progenitor prompting us to investigate alternate mechanisms explaining the Gfi1-deficient phenotype. We report that mucous and Paneth but not enteroendocrine lineage cells normally express Gfi1. Sporadic mucous and Paneth lineage cells in the crypts of Gfi1-deficient mice aberrantly express the pro-enteroendocrine transcription factor Neurog3, indicating that stable repression of Neurog3 in these lineages requires Gfi1. Importantly, we also find mucous and Paneth lineage cells in various stages of cellular reprogramming into the enteroendocrine lineage in Gfi1-deficient mice. We propose that mucous and Paneth cell lineage metastability, rather than reallocation at the level of a hypothetical common granulocytic progenitor, is responsible for the shifts in cell type proportions observed in Gfi1-deficient intestinal epithelium.

Colossal Crypts Bordering Colon Adenomas in ApcMin Mice Express Full-Length Apc

Enlarged but nondysplastic crypts are frequently observed at the margins of colon tumors, forming what has been called a transitional epithelium. It is now thought that this is a reactive state and not a preneoplastic condition as previously suggested. We have used the mouse familial adenomatous polyposis model, ApcMin, to study these abnormal adenoma-associated crypts. We report that these nondysplastic crypts are enormous (as much as 10 times normal length) and branch more frequently than normal crypts. They express wild-type Apc protein and display the wild-type Apc allele. We conclude that the colossal crypts at adenoma margins have normal Apc gene function, consistent with the suggestion that their phenotype is a reactive state. The cause remains an open question, but the dramatic epithelial response hints at the presence of potent epithelial trophic factors in the vicinity of colon tumors.

Three-dimensional reconstruction of biological objects using a graphics engine

A common problem in the study of biological material is the determination of three-dimensional structure from serial sections. The large number of sections required to obtain sufficient internal detail of a structure results in enormous processing requirements. These requirements can now be satisfied by current graphics engine technology in combination with image-digitizing hardware. The previously onerous tasks of manipulating and displaying 3D objects become routine with this combination of technologies. We report a computer-assisted reconstruction system on a graphics engine-based workstation. The system accepts images from any video source and includes a utility for aligning adjacent video images. Also available is an editor for geometric object entry and editing. More novel in our approach is the use of video interiors in 3D displays in addition to contours and tiled surfaces. Video interiors is a form of display in which digitized pixels interior to objects are revealed by cutaway blocks.

The crypt cycle and the asymptotic dynamics of the proportion of differently sized mutant crypt clones in the mouse intestine

The dynamics of clones of intestinal epithelial crypts populated by mutant stem cells offers the hope of new and independent evidence of continued crypt replication throughout adult life, an important prediction of recent models of intestinal stem cell biology. It is shown here that the experimentally most tractable measurement - scoring of the fraction of groups of mutant crypts found as isolated singletons, pairs, or clusters of ≥3 mutant crypts — should tend to an asymptotic distribution as animals age. In particular, if the rate of mutation is low relative to the rate of crypt production then 1/2, 1/6 and 1/3 of groups of mutant crypts should be found as singletons, pairs, and larger clusters, respectively, as animals reach old age. Such a result is not immediately obvious, and if obtained from an experiment could easily be misinterpreted as implying that the crypt cycle must slow radically as animals age.