Natasza A. Kurpios

ERM is required for transcriptional control of the spermatogonial stem cell niche

Division of spermatogonial stem cells produces daughter cells that either maintain their stem cell identity or undergo differentiation to form mature sperm. The Sertoli cell, the only somatic cell within seminiferous tubules, provides the stem cell niche through physical support and expression of surface proteins and soluble factors. Here we show that the Ets related molecule (ERM) is expressed exclusively within Sertoli cells in the testis and is required for spermatogonial stem cell self-renewal. Mice with targeted disruption of ERM have a loss of maintenance of spermatogonial stem cell self-renewal without a block in normal spermatogenic differentiation and thus have progressive germ-cell depletion and a Sertoli-cell-only syndrome. Microarray analysis of primary Sertoli cells from ERM-deficient mice showed alterations in secreted factors known to regulate the haematopoietic stem cell niche. These results identify a new function for the Ets family transcription factors in spermatogenesis and provide an example of transcriptional control of a vertebrate stem cell niche.

On the growth and form of the gut

The developing vertebrate gut tube forms a reproducible looped pattern as it grows into the body cavity. Here we use developmental experiments to eliminate alternative models and show that gut looping morphogenesis is driven by the homogeneous and isotropic forces that arise from the relative growth between the gut tube and the anchoring dorsal mesenteric sheet, tissues that grow at different rates. A simple physical mimic, using a differentially strained composite of a pliable rubber tube and a soft latex sheet is consistent with this mechanism and produces similar patterns. We devise a mathematical theory and a computational model for the number, size and shape of intestinal loops based solely on the measurable geometry, elasticity and relative growth of the tissues. The predictions of our theory are quantitatively consistent with observations of intestinal loops at different stages of development in the chick embryo. Our model also accounts for the qualitative and quantitative variation in the distinct gut looping patterns seen in a variety of species including quail, finch and mouse, illuminating how the simple macroscopic mechanics of differential growth drives the morphology of the developing gut.

The PEA3 subfamily of Ets transcription factors synergizes with β-catenin-LEF-1 to activate matrilysin transcription in intestinal tumors

ABSTRACT The matrix metalloproteinase matrilysin (MMP-7) is expressed in the tumor cells of a majority of mouse intestinal and human colonic adenomas. We showed previously that matrilysin is a target gene of β-catenin–Tcf, the transcription factor complex whose activity is thought to play a crucial role in the initiation of intestinal tumorigenesis. Here we report that overexpression of a stable mutant form of β-catenin alone was not sufficient to effect expression of luciferase from a matrilysin promoter-luciferase reporter plasmid. However, cotransfection of the reporter with an expression vector encoding the PEA3 Ets transcription factor, or its close relatives ER81 and ERM, increased luciferase expression and rendered the promoter responsive to β-catenin–LEF-1 as well as to the AP-1 protein c-Jun. Other Ets proteins could not substitute for the PEA3 subfamily. Luciferase activity was induced up to 250-fold when PEA3, c-Jun, β-catenin, and LEF-1 were coexpressed. This combination of transcription factors was also sufficient to induce expression of the endogenous matrilysin gene. Furthermore, all matrilysin-expressing benign intestinal tumors of the Min mouse expressed a member of the PEA3 subfamily, as did all human colon tumor cell lines examined. These data suggest that the expression of members of the PEA3 subfamily, in conjunction with the accumulation of β-catenin in these tumors, leads to coordinate upregulation of matrilysin gene transcription, contributing to gastrointestinal tumorigenesis.

The Chirality of Gut Rotation Derives from Left-Right Asymmetric Changes in the Architecture of the Dorsal Mesentery

We have investigated the structural basis by which the counterclockwise direction of the amniote gut is established. The chirality of midgut looping is determined by left-right asymmetries in the cellular architecture of the dorsal mesentery, the structure that connects the primitive gut tube to the body wall. The mesenchymal cells of the dorsal mesentery are more condensed on the left side than on the right and, additionally, the overlying epithelium on the left side exhibits a columnar morphology, in contrast to a cuboidal morphology on the right. These properties are instructed by a set of transcription factors: Pitx2 and Isl1 specifically expressed on the left side, and Tbx18 expressed on the right, regulated downstream of the secreted protein Nodal which is present exclusively on the left side. The resultant differences in cellular organization cause the mesentery to assume a trapezoidal shape, tilting the primitive gut tube leftward.

The direction of gut looping is established by changes in the extracellular matrix and in cell:cell adhesion

The counterclockwise coiling of the intestines is initiated by a leftward tilt of the primitive gut tube, imparted by left–right asymmetries in the architecture of the dorsal mesentery. In silico analysis suggests that this is achieved by synergistic changes in its epithelium and mesenchyme. Within the mesenchymal compartment, cells are more densely packed on the left than on the right. In silico results indicate that this property can result from asymmetries in both extracellular matrix (ECM) and cell:cell adhesion. We find that the dorsal mesentery ECM is indeed left–right asymmetric and moreover that the adhesion molecule N-cadherin is expressed exclusively on the left side. These asymmetries are regulated by the asymmetrically expressed transcription factors Pitx2 and Isl1. Functional studies demonstrate that N-cadherin acts upstream of the changes in the ECM and is both necessary and sufficient to explain the asymmetric packing of the mesenchymal cells.

Function of PEA3 Ets Transcription Factors in Mammary Gland Development and Oncogenesis

The Ets gene families of mice and man currently comprise 27 genes that encode sequence-specific transcription factors. Ets proteins share an ∼85 amino acid structurally conserved ETS DNA binding domain. Genetic analyses in model organisms suggest roles for Ets proteins in embryonic development and various adult physiological processes. Chromosomal translocations involving several ETS genes are associated with Ewings sarcomas and leukemias, whereas the overexpression of some ETS genes is linked with numerous malignancies, including breast cancer. Indeed PEA3, ETS-1, PDEF, and ELF-3 transcripts have all been reported to be elevated in human breast tumors. Some of the ETS genes that are overexpressed in human breast tumors are also overexpressed in mouse models of this disease. Notably, pea3, as well as its close paralogs er81 and erm, which comprise the pea3 subfamily of ets genes, are coordinately overexpressed in mouse mammary tumors. Genetic analyses in mice reveal required roles for one or more of the PEA3 subfamily Ets proteins in the initiation and progression of mouse mammary tumors. The pea3 subfamily genes are normally expressed in the primitive epithelium of mouse mammary buds during embryogenesis, and these three genes are expressed in epithelial progenitor cells during postnatal mammary gland development. Loss-of-function mutations in the mouse pea3 gene results in increased numbers of terminal end buds and an increased fraction of proliferating cells in these structures, suggesting a role for PEA3 in progenitor cell renewal or terminal differentiation. Taken together these observations suggest that the PEA3 subfamily proteins play key regulatory roles in both mammary gland development and oncogenesis.

The Left-Right Pitx2 Pathway Drives Organ-Specific Arterial and Lymphatic Development in the Intestine

The dorsal mesentery (DM) is the major conduit for blood and lymphatic vessels in the gut. The mechanisms underlying their morphogenesis are challenging to study and remain unknown. Here we show that arteriogenesis in the DM begins during gut rotation and proceeds strictly on the left side, dependent on the Pitx2 target gene Cxcl12. Although competent Cxcr4-positive angioblasts are present on the right, they fail to form vessels and progressively emigrate. Surprisingly, gut lymphatics also initiate in the left DM and arise only after-and dependent on-arteriogenesis, implicating arteries as drivers of gut lymphangiogenesis. Our data begin to unravel the origin of two distinct vascular systems and demonstrate how early left-right molecular asymmetries are translated into organ-specific vascular patterns. We propose a dual origin of gut lymphangiogenesis in which prior arterial growth is required to initiate local lymphatics that only subsequently connect to the vascular system.

Large-Scale Expansion of Mammary Epithelial Stem Cell Aggregates in Suspension Bioreactors†

Mutations in the pathways regulating mammary epithelial stem cell (MESC) self‐renewal and differentiation are currently hypothesized to result in uncontrolled cell division and, in turn, breast tumor formation. Although research is aggressively being pursued to understand how such pathways result in breast cancer formation, current studies have been greatly limited by MESC scarcity. To address this issue, this study has successfully developed large‐scale expansion protocols for MESC through the subculture of murine mammary epithelial tissue aggregates, called mammospheres, in suspension bioreactors. Growth kinetics of mammospheres cultured in 125 mL suspension bioreactors and T‐flasks were found to be comparable, achieving cell densities of 3.10 × 105 and 2.75 × 105 cells/mL, respectively. This corresponded to a 4‐fold expansion over 8 days. Yields were also found to be strongly affected by liquid shear forces, where high agitation rates reduced overall cell numbers. Bioreactor cultures were scaled up to 1000 mL operating volumes, resulting in the production of 4.21 × 108 total cells (5.6‐fold expansion) from a single passage. Furthermore, intermittent replacement of culture medium with fresh medium dramatically improved maximum cell densities, resulting in an 11‐fold expansion, thereby enabling the generation of stem cells in quantities sufficient for standard biochemical and genetic analyses. After being cultured in suspension bioreactors for several passages, analysis by flow cytometry of Ki‐67 revealed that 85% of the population was composed of proliferating cells. The successful development of expansion protocols for MESC aggregates in suspension bioreactors makes available experimental avenues that were not previously accessible for breast cancer research, thereby facilitating future investigations into elucidating the role of MESCs in breast cancer tumorigenesis.

Integration of left-right Pitx2 transcription and Wnt signaling drives asymmetric gut morphogenesis via Daam2.

A critical aspect of gut morphogenesis is initiation of a leftward tilt, and failure to do so leads to gut malrotation and volvulus. The direction of tilt is specified by asymmetric cell behaviors within the dorsal mesentery (DM), which suspends the gut tube, and is downstream of Pitx2, the key transcription factor responsible for the transfer of left-right (L-R) information from early gastrulation to morphogenesis. Although Pitx2 is a master regulator of L-R organ development, its cellular targets that drive asymmetric morphogenesis are not known. Using laser microdissection and targeted gene misexpression in the chicken DM, we show that Pitx2-specific effectors mediate Wnt signaling to activate the formin Daam2, a key Wnt effector and itself a Pitx2 target, linking actin dynamics to cadherin-based junctions to ultimately generate asymmetric cell behaviors. Our work highlights how integration of two conserved cascades may be the ultimate force through which Pitx2 sculpts L-R organs.

The Pea3 Ets transcription factor regulates differentiation of multipotent progenitor cells during mammary gland development

The Pea3 Ets transcription factor is overexpressed in breast tumors suggesting that it plays a role in mammary oncogenesis. However, the normal biological function of Pea3 in the mammary gland is not known. Here we report that Pea3 was expressed in the epithelium of the mouse mammary anlagen commensurate with their genesis, and at later times in the nipple and mammary ducts of female embryos. In adult mice Pea3 transcripts peaked at the onset of puberty and early pregnancy, times of active epithelial cell proliferation and differentiation. Pea3 was expressed in all progenitor cap cells and rare body cells of terminal end buds, and in the myoepithelial cells of ducts and alveoli. Analyses of the mammary glands of Pea3-null mice during puberty revealed an increased number of terminal end buds and an increased fraction of proliferating progenitor cells within these structures compared to their wild type littermates. Tissue transplant experiments demonstrated that these phenotypes were intrinsic to the Pea3-null mammary epithelium. During pregnancy, mammary glands isolated from Pea3-null females had impaired alveolar development as revealed by a decreased fraction of alveolar structures. We performed in vitro colony forming assays of mammary epithelial cells and discovered that loss of Pea3 altered the distribution of specific multipotent progenitor cells. Double-immunofluorescence confirmed that multipotential progenitors co-expressing markers of the myoepithelial and luminal epithelial lineage were amplified in the mammary glands of Pea3-null mice by comparison to their wild type counterparts. We propose that Pea3 functions in multipotential progenitors to regulate their lineage-specific differentiation potential.