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Parallels among embryonic development, stem cells, and cancer have long been recognized. We identified, isolated, and characterized stem cells that first become committed to a mammary fate during embryogenesis; we refer to these cells as fetal mammary stem cells (fMaSCs). Lineage tracing, in vitro sphere formation, and in vivo transplantation studies by our group and many others all confirm that cells in the embryo are the bipotent progenitors of the mammary gland. There is debate, however, on whether such bipotent cells persist into the adult, or whether the luminal and basal lineages are maintained by unipotent progenitors. To gain insight into the relationships between fMaSCs and breast cancer, and to investigate their potential persistence in the adult, we have applied bulk and single cell RNA-sequencing (sc-RNA-seq) and single nucleus ATAC-sequencing (snATAC-seq) throughout mammary development. The results to be discussed demonstrate that fMaSC transcriptomes are heterogeneous, but all share co-expression of genes associated with luminal and basal cell fates. This fits a model in which the bipotent state is created by a balance of lineage specifiers. We also find that the fMaSC transcriptome is highly enriched in basal-like human breast cancers and identify potential embryonic pathways that correlate with poor prognosis. We used a variety of computational tools to infer the gene expression programs that ensue when fMaSCs commit to luminal and basal states. The data from scRNA-seq and snATAC-seq demonstrate that the transitions are gradual, not precipitous, and that luminal and basal cells exhibit significant transcriptomic and epigenetic heterogeneity. This challenges the notion that the mammary gland consists of discrete cell types defined by rigid transcriptomic parameters, and reveals a potential for intrinsic phenotypic plasticity of normal mammary cells. Using the combined databases, we identified Sox10 as a significantly differentially expressed cell state regulator. We show that tumors are heterogeneous with regard to Sox10 expression, and that locally invasive cells tend to express high Sox10 levels. Elevated Sox10 correlates with acquisition of a neural-crest like, EMT-related state. Implications for interception of metastasis by targeting neural crest-like cells will be discussed. Finally, we have generated a web resource that is available to the scientific community to enable the transcription and epigenetic characteristics of any gene of interest to be tracked through mammary development (https://wahl-labsalk.shinyapps.io/Mammary_snATAC/). Citation Format: GM Wahl, Z Ma, C Chung, C Dravis, BT Spike, RR Giraddi, O Balcioglu, C Fan, B Hagos, R Heinz, Herrera-Valdez J, X Hou, J Hwang, R Lasken, G Luna, NE Lytle, EM Mehrabad, M Novotny, CM Perou, O Poirion, S Preissl, B Ren, T Reya, CL Trejo, KT Varley. Understanding breast cancer using a developmental perspective [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES10-2.

SummaryIt has only recently become possible to obtain single-cell level resolution of the epigenetic changes that occur during organ development. We reasoned that precision single-cell chromatin accessibility mapping of mammary gland development could provide needed insight into the epigenetic reprogramming and transcriptional regulators involved in normal mammary gland development. Here, we provide the first 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, and we present a web application as a scientific resource for facilitating future analyses. Strikingly, these single-cell chromatin profiling data reveal that fMaSCs can be separated into basal-like and luminal-like lineages, providing evidence of early lineage segregation prior to birth. Such distinctions were not evident in analyses of single-cell transcriptomic data.

SUMMARY 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., In Brief Basal-like breast cancer (BLBC) preferentially originates from ER-negative luminal stem/progenitor cells (LSPCs). Christin et al. show that the transcription factor SOX9 acts as a key regulator for these LSPCs. Inactivation of BLBC tumor suppressors co-opt SOX9 upregulation to promote luminal-basal reprogramming and tumor progression., Graphical Abstract

Intra-tumoral heterogeneity complicates accurate diagnosis, reduces the effectiveness of precision therapeutics, and fuels metastatic dissemination. Discovering the genes and epigenetic mechanisms that produce developmentally plastic cells that engender heterogeneity presents an important challenge and need. As cancer is a caricature of development, we used chromatin accessibility assays and transcriptional profiling to infer genes and pathways that underlie the lineage potential of normal fetal and adult mammary cell populations. We show that fetal and adult basal cells share epigenetic features consistent with multi-lineage differentiation potential. We show that Sox family transcription factor DNA-binding motifs are enriched in the open chromatin of stem/progenitor cells, and provide multiple levels of evidence that Sox10 expression level correlates with multiple indicators of cell state plasticity. We used multiple animal models to show that Sox10 directly contributes to tumor development and correlates with their intratumoral heterogeneity. Strikingly, we provide the first evidence that Sox10-positive mammary tumor cells exhibit neural crest cell features, and that Sox10 binds to and regulates genes that elicit a neural crest cell-like phenotype. Collectively, these data rationalize the ability of adult basal but not luminal mammary cells to exhibit stem cell activity in transplantation assays, reveal how Sox10 is involved in the cell state plasticity that generates intratumoral heterogeneity and metastatic clones, and demonstrates the remarkable scope of cell state reprogramming that can occur during tumorigenesis.

SummaryTo 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.

SUMMARY 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., Graphical Abstract, In Brief Single-cell RNA sequencing of developing mouse mammary epithelia reveals the timing of lineage specification. Giraddi et al. find that fetal mammary stem cells co-express factors that define distinct lineages in their progeny and bear functionally relevant metabolic program signatures that change with differentiation and are resurrected in human breast cancers and metastases.

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., Stem cells: protein not needed for stem cell activity in mammary gland A protein considered necessary for mammary stem cell activity turns out to be dispensable. What’s more, it’s not required for tumor development either. Geoffrey Wahl, Christy Trejo, and colleagues from the Salk Institute for Biological Studies in La Jolla, CA, USA, investigated whether the expression of a protein called Lgr5 denotes mammary stem cells and whether it’s truly needed for the cells to be capable of giving rise to two cell lineages—a subject of active debate among researchers. Wahl’s team showed that mouse fetal mammary stem cells expressing Lgr5 had this dual-lineage capacity; their counterparts in the adult mouse did not. Yet, even though Lgr5 expression marks a population of fetal mammary stem cells, the protein is not required for stem cell activity or for tumors to form in a mouse model of breast cancer.

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.

SUMMARY 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., Graphical Abstract, In Brief The ability to deconstruct complex tissues into their constituent cell states and identify molecular mechanisms involved in cell differentiation is enabling deeper understanding of normal development and disease. Chung et al. use snATAC-seq to agnostically determine the chromatin states correlated with cell-state changes during embryonic and postnatal mammary development.

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

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.

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-B26YFΔV and ephrin-B2ΔV 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.

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.

Many foundational features of tumor malignancy such as tumor initiation, tumor heterogeneity, therapeutic resistance, and metastasis are driven through dysregulated cell plasticity. Thus, the development of strategies to counter mechanisms of reprogramming in cancer cells is a critical area of need in the field. To address this challenge, we have focused on discovering how cell plasticity is specified in normal mammary development and tissue, and how these processes may be co-opted to drive tumor progression in breast cancer. To facilitate the study of these complex phenomena, we have developed a CRISPR- and tissue engineering-based platform system to rapidly generate de novo, in vivo, mammary glands and mammary tumors that carry any conceivable genomic modification. Here, we have used this system to study the function of Sox10 in breast cancer, as we have previously shown that the transcription factor Sox10 is a critical mammary cell plasticity factor that labels and is functionally required for stem/progenitor activity in normal mammary cells. We now demonstrate that Sox10 is also a major plasticity factor in breast cancer. We find that Sox10 is expressed in both human breast cancer and multiple murine mammary tumor models, and demonstrate critical roles for Sox10 in driving both tumor initiation and progression. Sox10 expression in these tumors correlates with multiple indicators of elevated cell plasticity, which include features of de-differentiation and EMT, specific enrichment of mammary stem/progenitor identity, and expanded lineage potential compared to normal mammary cells. In addition to its role in specifying stem/progenitor activity in mammary cells, Sox10 is also a dominant specifier of neural crest cells, a primitive and highly plastic cell state found only in the early embryo. Strikingly, we find that Sox10+ mammary tumor cells also exhibit transcriptional and epigenetic features of neural crest cells that are not found in normal mammary cells. Collectively, these data reveal that Sox10 is a major plasticity factor that contributes to mammary tumor progression, and demonstrate the remarkable scope of cell state reprogramming that can occur during tumorigenesis when potent plasticity factors are dysregulated. Note: This abstract was not presented at the conference. Citation Format: Christopher Dravis, Geoffrey Wahl. Sox10 drives tumor progression in breast cancer by reprogramming mammary tumor cells into a state of plasticity resembling primitive neural crest cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B64.

Cell state reprogramming during tumor progression significantly contributes to the genesis of intra-tumoral heterogeneity. Discovering how cell state plasticity is specified during normal mammary development, and how this relates to breast cancer, could provide new treatment approaches. We used chromatin accessibility assays and transcriptional profiling to infer lineage potential within distinct normal mammary cell populations. We show that fetal and adult basal cells exhibit features consistent with multi-lineage differentiation potential. SOX family transcription factor DNA-binding motifs were enriched in the open chromatin of stem/progenitor cells, implicating SOX factors in mammary cell plasticity. We provide evidence that SOX10 expression correlates with multiple indicators of cell state plasticity, and that it directly contributes to tumor development in multiple animal models. Strikingly, we show that SOX10-positive tumor cells uniquely exhibit neural crest cell features, and that SOX10 binds to genes that regulate neural crest cell identity. Collectively, these data help to resolve conflicts concerning cell fate and cell potential in the mammary cell hierarchy, reveal that SOX10 is a plasticity factor driving mammary tumor progression, and demonstrate the remarkable scope of cell state reprogramming that can occur during tumorigenesis. Citation Format: Christopher Dravis, Chi-Yeh Chung, Geoff Wahl. Epigenetic and transcriptomic profiling of mammary gland development and tumor models disclose regulators of cell state plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1992.

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.

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.

Cytokinesis in Saccharomyces cerevisiae involves coordination between actomyosin ring contraction and septum formation and/or targeted membrane deposition. We show that Mlc1p, a light chain for Myo2p (type V myosin) and Iqg1p (IQGAP), is the essential light chain for Myo1p, the only type II myosin in S. cerevisiae. However, disruption or reduction of Mlc1p–Myo1p interaction by deleting the Mlc1p binding site on Myo1p or by a point mutation in MLC1, mlc1-93, did not cause any obvious defect in cytokinesis. In contrast, a different point mutation, mlc1-11, displayed defects in cytokinesis and in interactions with Myo2p and Iqg1p. These data suggest that the major function of the Mlc1p–Myo1p interaction is not to regulate Myo1p activity but that Mlc1p may interact with Myo1p, Iqg1p, and Myo2p to coordinate actin ring formation and targeted membrane deposition during cytokinesis. We also identify Mlc2p as the regulatory light chain for Myo1p and demonstrate its role in Myo1p ring disassembly, a function likely conserved among eukaryotes.

Determine if differences in right and left semicircular size account for phenotypic behavior, indicating vestibulopathy in EphB deficient mice.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.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.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 (P0.0001). 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.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.N/A.

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-β-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.

To discover mechanisms that mediate the initiation and progression of aggressive and stem-like breast cancers, 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 report that Sox10 is specifically expressed in mammary cells that exhibit 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, while its overexpression increases stem/progenitor activity. Intriguingly, we also discover that Sox10 overexpression elicits epithelial-to-mesenchymal transition (EMT) in mammary organoids in an FGF signaling-dependent manner. Further, modulation of Sox10 levels can induce sequential EMT, migratory, and clonogenic behaviors that strikingly resemble proposed mechanisms of metastasis. Consistent with these findings, we report that Sox10 is preferentially expressed in the most stem- and EMT-like triple negative breast cancer subtypes, which suggest that Sox10 may reprise these same functions during tumorigenesis. Indeed, we find that in an autochthonous mouse model of basal-like breast cancer, Sox10 is expressed at high levels and that these Sox10+ tumor cells exhibit characteristics that are similar to mammary cells in which Sox10 is ectopically expressed. Collectively, these results demonstrate a signaling mechanism through which stem and mesenchymal-like states are acquired in mammary cells, and indicate possible therapeutic targets to counter these functions in breast cancers for which targeted therapies are currently unavailable. Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 regulates stem- and mesenchymal-like features in mammary cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 914.

To discover mechanisms that mediate the initiation and progression of aggressive and stem-like breast cancers, 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 report that Sox10 is specifically expressed in mammary cells that exhibit 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, while its overexpression increases stem/progenitor activity. Intriguingly, we also discover that Sox10 overexpression elicits epithelial-to-mesenchymal transition (EMT) in mammary organoids in an FGF signaling-dependent manner. Further, modulation of Sox10 levels can induce sequential EMT, migratory, and clonogenic behaviors that strikingly resemble proposed mechanisms of metastasis. Consistent with these findings, we find that Sox10 is preferentially expressed in the most stem- and EMT-like triple negative breast cancer subtypes, and that Sox10+ tumor cells utilize FGF signaling for growth and invasive behaviors, which suggest that Sox10 may reprise these same functions during tumorigenesis. Collectively, these results demonstrate a signaling mechanism through which stem and mesenchymal-like states are acquired in mammary cells, and indicate possible therapeutic targets to counter these functions in breast cancers for which targeted therapies are currently unavailable. Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 regulates stem/progenitor and mesenchymal cell states in mammary epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr IA10.

The tail of yeast myosin II is localized to the division site by two distinct molecular pathways and sufficient for promoting actomyosin ring assembly, furrow ingression, and guidance in ECM remodeling., 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.

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 EphB3rtTA-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. Developmental Dynamics 241:1008–1019, 2012. © 2012 Wiley Periodicals, Inc.

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.

Molecular pathways that guide cell proliferation and programming events during embryonic development may be aberrantly re-activated in the cancer state to drive tumor progression. To identify mechanisms that drive progression of aggressive breast cancers, we identified molecules that are present in the fetal mammary stem cells (fMaSCs) that drive early development of the mammary gland. These analyses revealed that Sox10 is one of the most highly expressed transcription factors within fMaSCs, and prompted us to investigate the function of Sox10 in the mammary gland and in subtypes of cancer originating from it. Our studies described here indicate that Sox10 is specifically expressed in mammary cells that exhibit higher levels of stem or progenitor cell functions using in vitro or in vivo assays. This includes fetal and adult mammary cells in vivo, as well as mammary cells cultured in vitro as organoids, and for the first time, male fetal mammary stem cells. Sox10 appears to contribute to these stem/progenitor behaviors, as the genetic deletion of Sox10 limits stem/progenitor activities, while the overexpression of Sox10 significantly expands clonogenic behavior. Intriguingly, ectopic Sox10 expression is also able to elicit an EMT-like response in mammary organoids. Sox10 thus represents a single molecule capable of directly mediating both stem-like and EMT-like properties in mammary cells. Furthermore, by modulating Sox10 levels in 3D mammary organoids, we are able to induce cells into sequential motile and stem/progenitor states. This provides a striking in vitro surrogate for metastatic behavior. Consistent with these findings, we find that Sox10 is highly expressed in basal-like and claudin-low subtypes of breast cancer, which are the most stem-like and EMT-like manifestations of the disease, respectively. Finally, we show that Sox10 expression is regulated through a feedback loop involving FGF signaling, and that inhibition of FGF signaling can block EMT-like cell behaviors mediated by Sox10. These findings have important implications in how stem-like and metastatic properties may be specified in mammary cells. Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 promotes both stem-like and EMT properties in mammary cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2015-2326

BACKGROUND: The 13q-deletion syndrome causes human congenital birth defects due to the loss of regions of one long arm of human chromosome 13. A distal critical region for severe genitourinary and anorectal birth defects in the region of 13q32.2-34 has been suggested; we sought to narrow this critical region. METHODS: From patients with karyotypes revealing haploinsufficiency for distal chromosome 13q and their parents, peripheral blood was obtained and lymphocytes were immortalized for DNA isolation. Genetic and molecular cytogenetic methods were used to map deletions. Patient and parental samples were genotyped with a panel of 20 microsatellite markers spanning 13q31.3 qter and deletions identified by loss of heterozygosity. Deletions were also mapped using a panel of 35 BAC clones from the same region as probes for fluorescence in-situ hybridization on patient lymphoblastoid metaphase preparations. The data were synthesized and a deletion map defining the critical region was generated. RESULTS: Eight patients with known deletions around 13q32qter and their parents were analyzed, and categorized into three groups: three patients with anorectal and genitourinary anomalies (hypospadias, penoscrotal transposition), four male patients without anorectal and genitourinary anomalies, and one XY patient with ambiguous genitalia without anorectal anomalies. We mapped the critical region for anorectal and genitourinary anomalies to a approximately 9.5-Mb interval of 13q33.3-q34 delineated by markers D13S280-D13S285; this spans approximately 8% of the chromosome and contains 20 annotated genes CONCLUSION: The critical region of chromosome 13q mediating genitourinary/anorectal anomalies has been mapped, and will be narrowed by additional patients and further mapping. Identification of the gene(s) mediating these syndromic genitourinary defects should further our knowledge of molecular mediators of non-syndromic hypospadias, penoscrotal transposition and anorectal malformations.

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.

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.

Molecular pathways that guide cell proliferation and programming events during embryonic development may be aberrantly re-activated in the cancer state to drive tumor progression. We have demonstrated the applicability of this tenet to studies of breast cancer. We have isolated fetal mammary stem cells (fMaSCs) from embryonic mammary rudiments, and verified that particular subtypes of breast cancers, including Basal-like triple-negative breast cancers, show strong correlation with expression signatures found in fMaSCs. This suggests that elucidation of the biology behind fMaSC function will yield insight into better treatment of these aggressive breast cancers. Through transcriptome analysis of fMaSCs, the transcription factor Sox10 was identified as a potential mediator of MaSC-activity. By analyzing a Sox10-Venus reporter line, we have discovered that Sox10 labels fMaSCs in the embryo, and improves recovery of fMaSC-enriched populations threefold over previous markers. Intriguingly, Sox10 also permits the first identification and recovery of male fMaSCs, which also demonstrate stem cell behaviors in vitro and in vivo. In the adult mammary gland, Sox10 only labels a subset of mammary cells, yet these cells possess all in vitro colony-forming potential compared to Sox10-negative cells. Of interest, both Sox10+ and Sox10-negative adult mammary cells demonstrate in vivo stem cell behaviors, suggesting the presence of unique multipotent MaSC populations. We find that Sox10 is also highly enriched in Basal-like breast cancers. Per previous studies with Sox9 and Sox10 in pancreatic and skin cancer models, Sox10 may play critical roles in the progression of Basal-like breast cancer. Molecular pathways that regulate Sox10 expression may therefore be useful therapeutic targets for treating Basal-like breast cancers. We find that perturbation of FGF signaling significantly attenuates Sox10 expression in organoids grown in vitro that are derived from either fMaSCs or luminal progenitors. As Sox10+ cells in mammary organoids specifically possess and label retained MaSC properties that are not seen in Sox10-negative cells, this may indicate a mechanism to attenuate MaSC specification or function in mammary tissue. Our discoveries with Sox10 thus represent a molecular focal point around which to build signaling and transcriptional networks underlying fMaSC state and function. To capitalize on this, we have performed RNA-sequencing on fetal and adult mammary populations using Sox10 as a marker. These analyses combine significantly purer cell fractions with a sequencing tool capable of most accurately profiling transcription events. This is expected to provide highest quality transcriptome data describing signaling events in these mammary tissues, which will identify potential therapeutic targets for different subtypes of breast cancer. Citation Format: Christopher Dravis, Geoff Wahl. Sox10 expression labels mammary stem cell activity in fetal, adult, cultured, and male mammary tissues and is attenuated by FGF signaling inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1960. doi:10.1158/1538-7445.AM2014-1960

Purpose Persistent cloaca is a rare, severe congenital anomaly. The Eph family receptor tyrosine kinases and their ligands, the ephrin family, mediate diverse embryonic cell-cell recognition events. We previously reported ephrin-B2lacZ/lacZ mice manifest male high imperforate anus and female persistent cloaca (Dravis et al. Dev Biol 2004; 271(2): 272-290). We studied and demonstrated via 3D-reconstruction whether altered ephrin-B2 signaling in female ephrin-B2 lacZ/lacZ mutant mice affect apoptosis, thereby possibly contributing to persistent cloaca malformation. Material and methods Female embryonic days 11.5, 13.5 and 16.5 (E11.5, E13.5 and E16.5) wild type (WT), ephrin-B2lacZ/+ (Z/+) and ephrin-B2 lacZ/lacZ (Z/Z) mice sections were dual labeled for ephrin-B2 and TUNEL immunohistochemistry. Anti-galactosidase antibody detected the ephrin-B2-gal fusion protein in Z/+ and Z/Z mice. WinSurf software was used for 3D reconstructions of the cloacal region. Results In WT E11.5 females, the cloacal membrane (CM) is intact. The urorectal septum (URS) is partially separating the cloaca into urogenital sinus and hindgut; no vagina has developed. In contrast, Z/Z females had poor cloacal development. Z/+ embryos showed widened URS. Apoptosis in WT females occurred prominently in CM, dorsal cloaca, and hindgut. In contrast, apoptosis in Z/Z females was limited to the endoderm of the poorly formed cloaca. These changes progressed throughout E13.5 and finally at E16.5, Z/Z females exhibited persistent cloaca. Conclusions The 3D reconstruction of the cloacal region provides a better understanding of the mechanism by which altered ephrin-B2 signaling yields persistent cloaca via diminished apoptosis in Z/Z mice.

Molecular pathways that guide cell proliferation and programming events during embryonic development to generate specific organs may be aberrantly re-activated in the cancer state of those organs to drive tumor progression. Recently, our laboratory has successfully demonstrated the applicability of this tenet to studies of breast cancer. We have developed an enrichment protocol for fetal mammary stem cells (fMaSCs) from embryonic mammary rudiments, and verified that particular subtypes of breast cancers, including Basal-like triple-negative breast cancers for which targeted therapies do not exist, show strong correlation with expression signatures found in fMaSCs. This suggests that elucidation of the biology behind fMaSC function will yield important insight into better treatment of these aggressive breast cancers. To identify mechanisms that govern fMaSC function, we have performed transcriptome analyses on cell populations enriched for fMaSCs and their surrounding stroma. From this data, we have identified candidate signaling and transcription networks that we are currently validating. Here we describe ongoing work with one candidate to emerge from our analyses, Sox10. Sox10 is specifically expressed in the enriched fMaSC population, and is of particular interest given the well-documented roles for other Sox family transcription factors in: 1) regulating cell fate decisions in embryonic and adult tissue, and 2) mediating the progression of some types of cancer. We show that Sox10 expression specifically correlates with MaSC function in fetal and adult mammary. We further show that Sox10 expression can be utilized to identify the cells maintaining MaSC stem cell properties in culture. Importantly, incorporation of Sox10 into our existing fMaSC purification protocol results in a twofold improvement in fMaSC enrichment. Our discoveries with Sox10 thus represent a molecular focal point around which to build signaling and transcriptional networks underlying fMaSC state and function. They further demonstrate the promise of our approach in identifying novel targets for the development of new therapies for poorly treated cancers. Citation Format: Christopher Dravis, Claire Johns, Benjamin T. Spike, Michelle Southard Smith, Geoffrey M. Wahl. Sox10 expression labels in vitro mammary stem cell activity. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B136.

Somatic stem cells govern the development, maintenance and regeneration of tissues, and their dysregulation is associated with diverse pathologies including cancer. Given the significance of these cells both biologically and therapeutically, it is critical to elucidate the signaling mechanisms that dictate their behavior. Our recent work on the fetal mammary gland has dissected complementary roles for the stem cell compartment and stromal cues in fetal mammary stem cell function. We also identified significant similarities between the gene expression profiles of some of the most aggressive and untreatable forms of human breast cancer and those of the mouse fetal stem cell enriched mammary epithelial and stromal fractions. These similarities suggest that stem cell regulatory mechanisms operative during organogenesis, including those specified by the stromal niche, may serve as novel therapeutic targets in these cancers. We also discovered significant gene expression heterogeneity within the fetal stem cell compartment when analyzed at single cell resolution. While such heterogeneity likely reflects the natural diversity of stem and non-stem cell states comprising the system, it confounds the interpretation of expression profiles from mixed populations and the delineation of stem cell specific and niche specific molecular components. Our current objective is to utilize genome wide single cell RNA-Sequencing to hasten the identification of functionally relevant, stem cell specific regulators and markers in the context of this multi-cellular/multi-signal system. Among other candidates, we have identified the Cripto/GRP78 signaling axis as a critical regulator of mammary stem cell activity, and have developed novel molecular genetic tools to temporally control the activity of candidate stem cell regulators in vivo in normal and neoplastic contexts. Integration of these tools promises to deliver a new molecular understanding of stem cell biology in the mammary gland and other systems and of stem cell-like cancer cells. Citation Format: Benjamin T. Spike, Danielle D. Engle, Jennifer C. Lin, Jonathan A. Kelber, Justin La, Samantha K. Cheung, Evan Booker, Rose Rodewald, Christopher Dravis, Peter C. Gray, Geoffrey M. Wahl. Deconstructing stemness from the fetal mammary perspective. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A136.

Mammary stem cells (MaSCs) self-renew and generate the different epithelial cell types of the breast. Impressively, a single MaSC can regenerate an entire functional mouse mammary gland when transplanted into a cleared fat pad. Intriguingly, stem-like cells have been identified in some types of breast cancer using bioinformatic strategies. These observations raise a number of important questions: Do some breast cancers arise by mutations occurring in mammary stem cells, as such cells have a high capacity for self-renewal, proliferation and differentiation? Or, do breast cancers arise by de-differentiation of cells to a stem-like state, as such cells may be particularly able to generate the heterogeneity that fuels tumor progression? If stem cells contribute to cancer, what is the nature of such cells, and can we use this information to devise better treatment strategies or prognostic molecular signatures? Finally, cancer cells that seem particularly competent at generating tumors upon xenografting into nude mice have also been suggested to exhibit stem cell characteristics such as multi-lineage differentiation and self-renewal. Are these “cancer stem cells” mutant stem cells, or a different type of cell that is particularly tumorigenic in the xenograft assay? Here, we integrate approaches involving developmental, stem cell, and cancer biology to gain insight into these challenging questions. We took a new direction to isolate and characterize MaSCs during fetal mammogenesis when undifferentiated cells are expected to be enriched. We microdissected and dissociated fetal mammary cells across different stages of development and conducted limiting dilution, orthotopic mammary transplantation analyses to determine fetal MaSC (fMaSC) frequency. We found that the fMaSCs are not measurable until mid-gestation, when an elongating duct that invades through the adjacent mesenchyme is formed. We used flow sorting to significantly enrich fMaSCs, and then used microarray and single cell RNA-seq to begin to interrogate the molecular pathways that drive their proliferation. We find that the pathways they express, and that are expressed by the fetal stroma that surround them, are enriched in several very aggressive types of human breast cancer, as well as in several genetically engineered mouse models. These data and powerful experimental strategies are opening new ways of understanding cancer from a developmental perspective, and for using this knowledge to develop new diagnostic and treatment strategies. Citation Format: Geoffrey Myles Wahl, Christopher Dravis, Claire Johns, Rose Rodewald, Benjamin T. Spike, Christy Trejo. Dissecting cancer through the lens of development. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA11.

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 β-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.