The molecular chronology of mammary epithelial cell fate switching.

The adult mammary gland is maintained by lineage-restricted progenitor cells through pregnancy, lactation, involution, and menopause. Injury resolution and transplantation-associated mammary gland reconstitution are unique exceptions, wherein mammary basal cells gain the ability to reprogram to a luminal state. Here, we leverage newly developed cell-identity reporter mouse strains, and time-resolved single-cell epigenetic and transcriptomic analyses to decipher the molecular programs underlying basal-to-luminal fate switching in vivo. We demonstrate that basal cells rapidly reprogram toward plastic cycling intermediates that appear to hijack molecular programs we find in bipotent fetal mammary stem cells and puberty-associatiated cap cells. Loss of basal-cell specifiers early in dedifferentiation coincides with activation of Notch and BMP, among others. Pharmacologic blockade of each pathway disrupts basal-to-luminal transdifferentiation. Our studies provide a comprehensive map and resource for understanding the coordinated molecular changes enabling terminally differentiated epithelial cells to transition between cell lineages and highlights the stunning rapidity by which epigenetic reprogramming can occur in response to disruption of tissue structure.

Stem Cell Determinant SOX9 Promotes Lineage Plasticity and Progression in Basal-like Breast Cancer.

Lineage plasticity is important for the development of basal-like breast cancer (BLBC), an aggressive cancer subtype. While BLBC is likely to originate from luminal progenitor cells, it acquires substantial basal cell features and contains a heterogenous collection of cells exhibiting basal, luminal, and hybrid phenotypes. Why luminal progenitors are prone to BLBC transformation and what drives luminal-to-basal reprogramming remain unclear. Here, we show that the transcription factor SOX9 acts as a determinant for estrogen-receptor-negative (ER-) luminal stem/progenitor cells (LSPCs). SOX9 controls LSPC activity in part by activating both canonical and non-canonical nuclear factor κB (NF-κB) signaling. Inactivation of TP53 and RB via expression of SV40 TAg in a BLBC mouse tumor model leads to upregulation of SOX9, which drives luminal-to-basal reprogramming in vivo. Furthermore, SOX9 deletion inhibits the progression of ductal carcinoma in situ (DCIS)-like lesions to invasive carcinoma. These data show that ER- LSPC determinant SOX9 acts as a lineage plasticity driver for BLBC progression.

Single-Cell Chromatin Analysis of Mammary Gland Development Reveals Cell-State Transcriptional Regulators and Lineage Relationships.

Technological improvements enable single-cell epigenetic analyses of organ development. We reasoned that high-resolution single-cell chromatin accessibility mapping would provide needed insight into the epigenetic reprogramming and transcriptional regulators involved in normal mammary gland development. Here, we provide a single-cell resource of chromatin accessibility for murine mammary development from the peak of fetal mammary stem cell (fMaSC) functional activity in late embryogenesis to the differentiation of adult basal and luminal cells. We find that the chromatin landscape within individual cells predicts both gene accessibility and transcription factor activity. The ability of single-cell chromatin profiling to separate E18 fetal mammary cells into clusters exhibiting basal-like and luminal-like chromatin features is noteworthy. Such distinctions were not evident in analyses of droplet-based single-cell transcriptomic data. We present a web application as a scientific resource for facilitating future analyses of the gene regulatory networks involved in mammary development.

Epigenetic and Transcriptomic Profiling of Mammary Gland Development and Tumor Models Disclose Regulators of Cell State Plasticity.

Cell state reprogramming during tumor progression complicates accurate diagnosis, compromises therapeutic effectiveness, and fuels metastatic dissemination. We used chromatin accessibility assays and transcriptional profiling during mammary development as an agnostic approach to identify factors that mediate cancer cell state interconversions. We show that fetal and adult basal cells share epigenetic features consistent with multi-lineage differentiation potential. We find that DNA-binding motifs for SOX transcription factors are enriched in chromatin that is accessible in stem/progenitor cells and inaccessible in differentiated cells. In both mouse and human tumors, SOX10 expression correlates with stem/progenitor identity, dedifferentiation, and invasive characteristics. Strikingly, we demonstrate that SOX10 binds to genes that regulate neural crest cell identity, and that SOX10-positive tumor cells exhibit neural crest cell features.

Single-Cell Transcriptomes Distinguish Stem Cell State Changes and Lineage Specification Programs in Early Mammary Gland Development.

The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.

Lgr5 is a marker for fetal mammary stem cells, but is not essential for stem cell activity or tumorigenesis.

The search for the bipotent mammary stem cells that drive mammary development requires markers to enable their prospective isolation. There is general agreement that bipotent mouse mammary stem cells are abundant in late fetal development, but their existence in the adult is vigorously debated. Among markers useful for mammary stem cell identification, the Wnt co-receptor Lgr5 has been suggested by some to be both "necessary and sufficient" for bipotency and transplantation of adult mammary stem cell activity, though other studies disagree. Importantly, the relevance of Lgr5 to the bipotency of fetal mammary stem cells has not been studied. We show here that expression of a fluorescent protein driven by the endogenous Lgr5 promoter enables significant fetal mammary stem cell enrichment. We used lineage tracing to demonstrate embryonic cells expressing Lgr5 are bipotent, while their adult counterparts are myoepithelial restricted. Importantly, our data conclusively demonstrate that Lgr5 is dispensable for both fetal and adult mammary stem cell activity and for the development of mammary tumors.

Sox10 Regulates Stem/Progenitor and Mesenchymal Cell States in Mammary Epithelial Cells.

To discover mechanisms that mediate plasticity in mammary cells, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor Sox10. Here, we show that Sox10 is specifically expressed in mammary cells exhibiting the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence whereas its overexpression increases stem/progenitor activity. Intriguingly, we also show that Sox10 overexpression causes mammary cells to undergo a mesenchymal transition. Consistent with these findings, Sox10 is preferentially expressed in stem- and mesenchymal-like breast cancers. These results demonstrate a signaling mechanism through which stem and mesenchymal states are acquired in mammary cells and suggest therapeutic avenues in breast cancers for which targeted therapies are currently unavailable.

EphB receptor forward signaling regulates area-specific reciprocal thalamic and cortical axon pathfinding.

In early brain development, ascending thalamocortical axons (TCAs) navigate through the ventral telencephalon (VTel) to reach their target regions in the young cerebral cortex. Descending, deep-layer cortical axons subsequently target appropriate thalamic and subcortical target regions. However, precisely how and when corticothalamic axons (CTAs) identify their appropriate, reciprocal thalamic targets remains unclear. We show here that EphB1 and EphB2 receptors control proper navigation of a subset of TCA and CTA projections through the VTel. We show in vivo that EphB receptor forward signaling and the ephrinB1 ligand are required during the early navigation of L1-CAM(+) thalamic fibers in the VTel, and that the misguided thalamic fibers in EphB1/2 KO mice appear to interact with cortical subregion-specific axon populations during reciprocal cortical axon guidance. As such, our findings suggest that descending cortical axons identify specific TCA subpopulations in the dorsal VTel to coordinate reciprocal cortical-thalamic connectivity in the early developing brain.

Asymmetry in semicircular canal diameters may account for circling behavior in EphB-deficient mice.

OBJECTIVES/HYPOTHESIS: Determine if differences in right and left semicircular size account for phenotypic behavior, indicating vestibulopathy in EphB deficient mice. STUDY DESIGN: The diameters of the superior semicircular canals (SCC) were measured. The differences in the right and left superior SCC diameters were analyzed in homozygous EphB2 and EphB3 double knockout mice known to have head bobbing and circling behavior. Results were compared to similar analysis in wild type controls that displayed no signs of vestibulopathy. METHODS: Axial frozen sections through the superior (SCC) were analyzed by light microscopy; and the diameters of the left and right canals were measured in µm for both EphB2 and EphB3 double knockout mice, as well as in wild type control mice. The differences in diameter between the left and right superior SCC was determined for each animal. RESULTS: Overall, the EphB2 and EphB3 double knockout mice had smaller superior SCC diameters compared to wild type (109.0±21.4 µm vs. 185.0±5.2 µm (P<0.0001). The mean difference in left and right diameter of the superior SCC of EphB2/EphB3 double knockout mice was 29.0±8.7 µm; in wild-type controls this difference was 6.0±5.1 µm (P=0.002). In addition, the direction of circling appeared to be independent of the laterality of the smaller (or larger) superior SCC. CONCLUSION: Mice deficient in EphB2/EphB3 signaling have smaller superior SCC and asymmetry in lumen sizes between the left and right sides. The laterality of the larger versus smaller is not correlated with the direction of circling behavior. LEVEL OF EVIDENCE: N/A.

Ephrin-B2 reverse signaling increases α5ß1 integrin-mediated fibronectin deposition and reduces distal lung compliance.

Alveolar growth abnormalities and severe respiratory dysfunction are often fatal. Identifying mechanisms that control epithelial proliferation and enlarged, poorly septated airspaces is essential in developing new therapies for lung disease. The membrane-bound ligand ephrin-B2 is strongly expressed in lung epithelium, and yet in contrast to its known requirement for arteriogenesis, considerably less is known regarding the function of this protein in the epithelium. We hypothesize that the vascular mediator ephrin-B2 governs alveolar growth and mechanics beyond the confines of the endothelium. We used the in vivo manipulation of ephrin-B2 reverse signaling to determine the role of this vascular mediator in the pulmonary epithelium and distal lung mechanics. We determined that the ephrin-B2 gene (EfnB2) is strongly expressed in alveolar Type 2 cells throughout development and into adulthood. The role of ephrin-B2 reverse signaling in the lung was assessed in Efnb2(LacZ/6YFΔV) mutants that coexpress the intracellular truncated ephrin-B2-ß-galactosidase fusion and an intracellular point mutant ephrin-B2 protein that is unable to become tyrosine-phosphorylated or to interact with either the SH2 or PDZ domain-containing downstream signaling proteins. In these viable mice, we observed pulmonary hypoplasia and altered pulmonary mechanics, as evidenced by a marked reduction in lung compliance. Associated with the reduction in lung compliance was a significant increase in insoluble fibronectin (FN) basement membrane matrix assembly with FN deposition, and a corresponding increase in the α5 integrin receptor required for FN fibrillogenesis. These experiments indicate that ephrin-B2 reverse signaling mediates distal alveolar formation, fibrillogenesis, and pulmonary compliance.

Stem cells and the developing mammary gland.

The mammary gland undergoes dynamic changes throughout life. In the mouse, these begin with initial morphogenesis of the gland in the mid-gestation embryo followed by hormonally regulated changes during puberty and later in adulthood. The adult mammary gland contains a hierarchy of cell types with varying potentials for self-maintenance and differentiation. These include cells able to produce complete, functional mammary glands in vivo and that contain daughter cells with the same remarkable regenerative potential, as well as cells with more limited clonogenic activity in vitro. Here we review how applying in vitro and in vivo methods for quantifying these cells in adult mammary tissue to fetal mammary cells has enabled the first cells fulfilling the functional criteria of transplantable, isolated mammary stem cells to be identified a few days before birth. Thereafter, the number of these cells increases rapidly. Populations containing these fetal stem cells display growth and gene expression programs that differ from their adult counterparts but share signatures characteristic of certain types of breast cancer. Such observations reinforce growing evidence of important differences between tissue-specific fetal and adult cells with stem cell properties and emphasize the merits of investigating their molecular basis.

Characterization of the larynx in ephrin-B2 knockout mice: a novel animal model for laryngeal clefts.

OBJECTIVE: To identify and classify laryngeal clefts in a novel mouse model. DESIGN: In vivo animal study. SETTING: Academic research laboratory. SUBJECTS: 129/CD1 mice with the ephrin-B2 gene disrupted by the ß-galactosidase (lacZ) gene were humanely killed at embryonic day 18 (E18) and evaluated for the presence and characterization of a laryngeal cleft. Homozygous and heterozygous lacZ knockout mice as well as wild-type littermates were evaluated. MAIN OUTCOME MEASURES: Microsurgical dissection of the oral cavity and pharynx allowed for a pseudoendoscopic view of the larynx to determine the presence or absence of a cleft. The specimens were also histologically sectioned and examined for characterization and classification of the cleft. RESULTS: A laryngeal cleft was identified in 12 of 27 ephrin-B2 homozygous lacZ knockout mice (44%). Laryngeal clefts were not identified in heterozygous ephrin-B2 knockout mice or in wild-type littermates. CONCLUSIONS: Disruption of ephrin-B2 reverse signaling results in laryngeal clefts in lacZ knockout mice. This presents a novel mouse model in which future investigations into etiology of laryngeal clefts may be examined.

EphB3 marks delaminating endocrine progenitor cells in the developing pancreas.

BACKGROUND: Understanding the process by which pancreatic beta-cells acquire their "fate" is critical to the development of in vitro directed differentiation protocols for cell replacement therapies for diabetics. To date, these efforts are hampered by a paucity of markers that distinguish pancreatic endocrine cells at different stages of differentiation. RESULTS: Here, we identify EphB3 as a novel pro-endocrine marker and use its expression to track delaminating islet lineages. First, we provide a detailed developmental expression profile for EphB3 and other EphB family members in the embryonic pancreas. We demonstrate that EphB3 transiently marks endocrine cells as they delaminate from the pancreatic epithelium, prior to their differentiation. Using a Tet-inducible EphB3(rtTA-lacZ) reporter line, we show that short-term pulse-labeled EphB3(+) cells co-express Pdx1, Nkx6.1, Ngn3, and Synaptophysin, but not insulin, glucagon, or other endocrine hormones. Prolonged labeling tracks EphB3(+) cells from their exit from the epithelium to their differentiation. CONCLUSIONS: These studies demonstrate that pro-endocrine cell differentiation during late gestation, from delamination to maturation, takes approximately 2 days. Together, these data introduce EphB3 as a new biomarker to identify beta-cells at a critical step during their step-wise differentiation and define the timeframe of endocrine differentiation.

Ephrin-B reverse signaling controls septation events at the embryonic midline through separate tyrosine phosphorylation-independent signaling avenues.

We report that the disruption of bidirectional signaling between ephrin-B2 and EphB receptors impairs morphogenetic cell-cell septation and closure events during development of the embryonic midline. A novel role for reverse signaling is identified in tracheoesophageal foregut septation, as animals lacking the cytoplasmic domain of ephrin-B2 present with laryngotracheoesophageal cleft (LTEC), while both EphB2/EphB3 forward signaling and ephrin-B2 reverse signaling are shown to be required for midline fusion of the palate. In a third midline event, EphB2/EphB3 are shown to mediate ventral abdominal wall closure by acting principally as ligands to stimulate ephrin-B reverse signaling. Analysis of new ephrin-B2(6YFΔV) and ephrin-B2(ΔV) mutants that specifically ablate ephrin-B2 tyrosine phosphorylation- and/or PDZ domain-mediated signaling indicates there are at least two distinct phosphorylation-independent components of reverse signaling. These involve both PDZ domain interactions and a non-canonical SH2/PDZ-independent form of reverse signaling that may utilize associations with claudin family tetraspan molecules, as EphB2 and activated ephrin-B2 molecules are specifically co-localized with claudins in epithelia at the point of septation. Finally, the developmental phenotypes described here mirror common human midline birth defects found with the VACTERL association, suggesting a molecular link to bidirectional signaling through B-subclass Ephs and ephrins.

Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II.

Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR). However, how myosin II is targeted to the division site and promotes AMR assembly, and how the AMR coordinates with membrane trafficking during cytokinesis, remains poorly understood. Here we show that Myo1 is a two-headed myosin II in Saccharomyces cerevisiae, and that Myo1 localizes to the division site via two distinct targeting signals in its tail that act sequentially during the cell cycle. Before cytokinesis, Myo1 localization depends on the septin-binding protein Bni5. During cytokinesis, Myo1 localization depends on the IQGAP Iqg1. We also show that the Myo1 tail is sufficient for promoting the assembly of a "headless" AMR, which guides membrane deposition and extracellular matrix remodeling at the division site. Our study establishes a biphasic targeting mechanism for myosin II and highlights an underappreciated role of the AMR in cytokinesis beyond force generation.

Hypospadias and anorectal malformations mediated by Eph/ephrin signaling.

PURPOSE: Despite extensive research, the molecular basis of hypospadias and anorectal malformations is poorly understood, likely due to a multifactorial basis. The incidence of hypospadias is increasing, thus making research in this area warranted and timely. This review presents recent molecular work broadening our understanding of these disorders. MATERIALS AND METHODS: A brief review of our recent work and the literature on the role of Eph/ephrin signaling in hypospadias and anorectal malformations is presented. RESULTS: Genetically engineered mice mutant for ephrin-B2 or EphB2;EphB3 manifest a variety of genitourinary and anorectal malformations. Approximately 40% of adult male heterozygous mice demonstrate perineal hypospadias. Although homozygous mice die soon after birth, 100% of homozygous males demonstrate high imperforate anus with urethral anomalies and 100% of homozygous females demonstrate persistent cloaca. Male mice compound homozygous for EphB2(ki/ki);EphB3(Delta/Delta)/ also demonstrate hypospadias. CONCLUSIONS: These mouse models provide compelling evidence of the role of B-class Eph/ephrin signaling in genitourinary/anorectal development and add to our mechanistic and molecular understanding of normal and abnormal embryonic development. As research on the B-class Ephs and ephrins continues, they will likely be shown to be molecular contributors to the multifactorial basis of hypospadias and anorectal malformations in humans as well.

EphB2 and ephrin-B2 regulate the ionic homeostasis of vestibular endolymph.

The ability to transport cations and anions across epithelia is critical for the regulation of pH, ionic homeostasis, and volume of extracellular fluids. Although the transporters and channels that facilitate ion and water movement across cell membranes are well known, the molecular mechanisms and signal transduction events that regulate these activities remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are well known to transduce bidirectional signals that control axon guidance and other cell migration/adhesion events during development. However, these molecules are also expressed in non-motile epithelial cells, including EphB2 in K(+)-secreting vestibular dark cells and ephrin-B2 in the adjacent transitional cells of the inner ear. Consistent with these expression patterns, mice with cytoplasmic domain mutations that interfere with EphB2 forward signaling or ephrin-B2 reverse signaling exhibit a hyperactive circling (waltzing) locomotion associated with a decreased amount of endolymph fluid that normally fills the vestibular labyrinth. Endolymph is unusual as an extracellular fluid in that it is normally high in K(+) and low in Na(+). Direct measurement of this fluid in live animals revealed significant decreases in K(+) concentration and endolymphatic potential in both EphB2 and ephrin-B2 mutant mice. Our findings provide evidence that bidirectional signaling mediated by B-subclass Ephs and ephrins controls the production and ionic homeostasis of endolymph fluid and thereby provide the first evidence that these molecules can control the activities of mature epithelial cells.

EphB receptor tyrosine kinases control morphological development of the ventral midbrain.

EphB receptor tyrosine kinases and ephrin-B ligands regulate several types of cell-cell interactions during brain development, generally by modulating the cytoskeleton. EphB/ephrinB genes are expressed in the developing neural tube of early mouse embryos with distinct overlapping expression in the ventral midbrain. To test EphB function in midbrain development, mouse embryos compound homozygous for mutations in the EphB2 and EphB3 receptor genes were examined for early brain phenotypes. These mutants displayed a morphological defect in the ventral midbrain, specifically an expanded ventral midline evident by embryonic day E9.5-10.5, which formed an abnormal protrusion into the cephalic flexure. The affected area was comprised of cells that normally express EphB2 and ephrin-B3. A truncated EphB2 receptor caused a more severe phenotype than a null mutation, implying a dominant negative effect through interference with EphB forward (intracellular) signaling. In mutant embryos, the overall number, size, and identity of the ventral midbrain cells were unaltered. Therefore, the defect in ventral midline morphology in the EphB2;EphB3 compound mutant embryos appears to be caused by cellular changes that thin the tissue, forcing a protrusion of the ventral midline into the cephalic space. Our data suggests a role for EphB signaling in morphological organization of specific regions of the developing neural tube.

Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development.

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.