Physiological DNA damage promotes functional endoreplication of mammary gland alveolar cells during lactation.

Lactation insufficiency affects many women worldwide. During lactation, a large portion of mammary gland alveolar cells become polyploid, but how these cells balance the hyperproliferation occurring during normal alveologenesis with terminal differentiation required for lactation is unknown. Here, we show that DNA damage accumulates due to replication stress during pregnancy, activating the DNA damage response. Modulation of DNA damage levels in vivo by intraductal injections of nucleosides or DNA damaging agents reveals that the degree of DNA damage accumulated during pregnancy governs endoreplication and milk production. We identify a mechanism involving early mitotic arrest through CDK1 inactivation, resulting in a heterogeneous alveolar population with regards to ploidy and nuclei number. The inactivation of CDK1 is mediated by the DNA damage response kinase WEE1 with homozygous loss of Wee1 resulting in decreased endoreplication, alveologenesis and milk production. Thus, we propose that the DNA damage response to replication stress couples proliferation and endoreplication during mammary gland alveologenesis. Our study sheds light on mechanisms governing lactogenesis and identifies non-hormonal means for increasing milk production.

Nuclear VANGL2 Inhibits Lactogenic Differentiation.

Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, transmembrane PCP proteins are trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in the HCC1569 breast cancer cell line, and in undifferentiated, but not differentiated, HC11 cells that serve as a model for mammary lactogenic differentiation. The loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate recruitment of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a, Ccnd1 and Csn2, larger acini and organoids, and precocious differentiation; phenotypes are rescued by overexpression of Vangl2, but not Vangl2ΔNLS. Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.

Nuclear VANGL2 Inhibits Lactogenic Differentiation.

Planar cell polarity (PCP) proteins coordinate tissue morphogenesis by governing cell patterning and polarity. Asymmetrically localized on the plasma membrane of cells, PCP proteins are also trafficked by endocytosis, suggesting they may have intracellular functions that are dependent or independent of their extracellular role, but whether these functions extend to transcriptional control remains unknown. Here, we show the nuclear localization of transmembrane, PCP protein, VANGL2, in undifferentiated, but not differentiated, HC11 cells, which serve as a model for mammary lactogenic differentiation. Loss of Vangl2 function results in upregulation of pathways related to STAT5 signaling. We identify DNA binding sites and a nuclear localization signal in VANGL2, and use CUT&RUN to demonstrate direct binding of VANGL2 to specific DNA binding motifs, including one in the Stat5a promoter. Knockdown (KD) of Vangl2 in HC11 cells and primary mammary organoids results in upregulation of Stat5a , Ccnd1 and Csn2 , larger acini and organoids, and precocious differentiation; phenotypes rescued by overexpression of Vangl2 , but not Vangl2 ΔNLS . Together, these results advance a paradigm whereby PCP proteins coordinate tissue morphogenesis by keeping transcriptional programs governing differentiation in check.

DeepSea is an efficient deep-learning model for single-cell segmentation and tracking in time-lapse microscopy.

Time-lapse microscopy is the only method that can directly capture the dynamics and heterogeneity of fundamental cellular processes at the single-cell level with high temporal resolution. Successful application of single-cell time-lapse microscopy requires automated segmentation and tracking of hundreds of individual cells over several time points. However, segmentation and tracking of single cells remain challenging for the analysis of time-lapse microscopy images, in particular for widely available and non-toxic imaging modalities such as phase-contrast imaging. This work presents a versatile and trainable deep-learning model, termed DeepSea, that allows for both segmentation and tracking of single cells in sequences of phase-contrast live microscopy images with higher precision than existing models. We showcase the application of DeepSea by analyzing cell size regulation in embryonic stem cells.

Subfunctionalized expression drives evolutionary retention of ribosomal protein paralogs Rps27 and Rps27l in vertebrates.

The formation of paralogs through gene duplication is a core evolutionary process. For paralogs that encode components of protein complexes such as the ribosome, a central question is whether they encode functionally distinct proteins or whether they exist to maintain appropriate total expression of equivalent proteins. Here, we systematically tested evolutionary models of paralog function using the ribosomal protein paralogs Rps27 (eS27) and Rps27l (eS27L) as a case study. Evolutionary analysis suggests that Rps27 and Rps27l likely arose during whole-genome duplication(s) in a common vertebrate ancestor. We show that Rps27 and Rps27l have inversely correlated mRNA abundance across mouse cell types, with the highest Rps27 in lymphocytes and the highest Rps27l in mammary alveolar cells and hepatocytes. By endogenously tagging the Rps27 and Rps27l proteins, we demonstrate that Rps27- and Rps27l-ribosomes associate preferentially with different transcripts. Furthermore, murine Rps27 and Rps27l loss-of-function alleles are homozygous lethal at different developmental stages. However, strikingly, expressing Rps27 protein from the endogenous Rps27l locus or vice versa completely rescues loss-of-function lethality and yields mice with no detectable deficits. Together, these findings suggest that Rps27 and Rps27l are evolutionarily retained because their subfunctionalized expression patterns render both genes necessary to achieve the requisite total expression of two equivalent proteins across cell types. Our work represents the most in-depth characterization of a mammalian ribosomal protein paralog to date and highlights the importance of considering both protein function and expression when investigating paralogs.

Alveolar progenitor differentiation and lactation depends on paracrine inhibition of notch via ROBO1/CTNNB1/JAG1.

In the mammary gland, how alveolar progenitor cells are recruited to fuel tissue growth with each estrus cycle and pregnancy remains poorly understood. Here, we identify a regulatory pathway that controls alveolar progenitor differentiation and lactation by governing Notch activation in mouse. Loss of Robo1 in the mammary gland epithelium activates Notch signaling, which expands the alveolar progenitor cell population at the expense of alveolar differentiation, resulting in compromised lactation. ROBO1 is expressed in both luminal and basal cells, but loss of Robo1 in basal cells results in the luminal differentiation defect. In the basal compartment, ROBO1 inhibits the expression of Notch ligand Jag1 by regulating ß-catenin (CTNNB1), which binds the Jag1 promoter. Together, our studies reveal how ROBO1/CTTNB1/JAG1 signaling in the basal compartment exerts paracrine control of Notch signaling in the luminal compartment to regulate alveolar differentiation during pregnancy.

Acute and endothelial-specific Robo4 deletion affect hematopoietic stem cell trafficking independent of VCAM1.

Hematopoietic stem cell (HSC) trafficking is regulated by a number of complex mechanisms. Among them are the transmembrane protein Robo4 and the vascular cell adhesion molecule, VCAM1. Endothelial VCAM1 is a well-known regulator of hematopoietic cell trafficking, and our previous studies revealed that germline deletion of Robo4 led to impaired HSC trafficking, with an increase in vascular endothelial cell (VEC) numbers and downregulation of VCAM1 protein on sinusoidal VECs. Here, we utilized two Robo4 conditional deletion models in parallel with Robo4 germline knockout mice (R4KO) to evaluate the effects of acute and endothelial cell-specific Robo4 deletion on HSC trafficking. Strikingly similar to the R4KO, the acute deletion of Robo4 resulted in altered HSC distribution between the bone marrow and blood compartments, despite normal numbers of VECs and wild-type levels of VCAM1 cell surface protein on sinusoidal VECs. Additionally, consistent with the R4KO mice, acute loss of Robo4 in the host perturbed long-term engraftment of donor wild-type HSCs and improved HSC mobilization to the peripheral blood. These data demonstrate the significant role that endothelial Robo4 plays in directional HSC trafficking, independent of alterations in VEC numbers and VCAM1 expression.

Generation of Mosaic Mammary Organoids by Differential Trypsinization.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

VANGL2 regulates luminal epithelial organization and cell turnover in the mammary gland.

The VANGL family of planar cell polarity proteins is implicated in breast cancer however its function in mammary gland biology is unknown. Here, we utilized a panel of Vang1 and Vangl2 mouse alleles to examine the requirement of VANGL family members in the murine mammary gland. We show that Vang1CKOΔ/Δ glands display normal branching while Vangl2flox/flox and Vangl2Lp/Lp tissue exhibit several phenotypes. In MMTV-Cre;Vangl2flox/flox glands, cell turnover is reduced and lumens are narrowed. A Vangl2 missense mutation in the Vangl2Lp/Lp tissue leads to mammary anlage sprouting defects and deficient outgrowth with transplantation of anlage or secondary tissue fragments. In successful Vangl2Lp/Lp outgrowths, three morphological phenotypes are observed: distended ducts, supernumerary end buds, and ectopic acini. Layer specific defects are observed with loss of Vangl2 selectively in either basal or luminal layers of mammary cysts. Loss in the basal compartment inhibits cyst formation, but has the opposite effect in the luminal compartment. Candidate gene analysis on MMTV-Cre;Vangl2flox/flox and Vangl2Lp/Lp tissue reveals a significant reduction in Bmi1 expression, with overexpression of Bmi1 rescuing defects in Vangl2 knockdown cysts. Our results demonstrate that VANGL2 is necessary for normal mammary gland development and indicate differential functional requirements in basal versus luminal mammary compartments.

Making Connections: Guidance Cues and Receptors at Nonneural Cell-Cell Junctions.

The field of axon guidance was revolutionized over the past three decades by the identification of highly conserved families of guidance cues and receptors. These proteins are essential for normal neural development and function, directing cell and axon migration, neuron-glial interactions, and synapse formation and plasticity. Many of these genes are also expressed outside the nervous system in which they influence cell migration, adhesion and proliferation. Because the nervous system develops from neural epithelium, it is perhaps not surprising that these guidance cues have significant nonneural roles in governing the specialized junctional connections between cells in polarized epithelia. The following review addresses roles for ephrins, semaphorins, netrins, slits and their receptors in regulating adherens, tight, and gap junctions in nonneural epithelia and endothelia.

Extracellular Regulation of the Mitotic Spindle and Fate Determinants Driving Asymmetric Cell Division.

Stem cells use mode of cell division, symmetric (SCD) versus asymmetric (ACD), to balance expansion with self-renewal and the generation of daughter cells with different cell fates. Studies in model organisms have identified intrinsic mechanisms that govern this process, which involves partitioning molecular components between daughter cells, frequently through the regulation of the mitotic spindle. Research performed in vertebrate tissues is revealing both conservation of these intrinsic mechanisms and crucial roles for extrinsic cues in regulating the frequency of these divisions. Morphogens and positional cues, including planar cell polarity proteins and guidance molecules, regulate key signaling pathways required to organize cell/ECM contacts and spindle pole dynamics. Noncanonical WNT7A/VANGL2 signaling governs asymmetric cell division and the acquisition of cell fates through spindle pole orientation in satellite stem cells of regenerating muscle fibers. During cortical neurogenesis, the same pathway regulates glial cell fate determination by regulating spindle size, independent of its orientation. Sonic hedgehog (SHH) stimulates the symmetric expansion of cortical stem and cerebellar progenitor cells and contributes to cell fate acquisition in collaboration with Notch and Wnt signaling pathways. SLIT2 also contributes to stem cell homeostasis by restricting ACD frequency through the regulation of spindle orientation. The capacity to influence stem cells makes these secreted factors excellent targets for therapeutic strategies designed to enhance cell populations in degenerative disease or restrict cell proliferation in different types of cancers.

Diverse regulation of mammary epithelial growth and branching morphogenesis through noncanonical Wnt signaling.

The mammary gland consists of an adipose tissue that, in a process called branching morphogenesis, is invaded by a ductal epithelial network comprising basal and luminal epithelial cells. Stem and progenitor cells drive mammary growth, and their proliferation is regulated by multiple extracellular cues. One of the key regulatory pathways for these cells is the ß-catenin-dependent, canonical wingless-type MMTV integration site family (WNT) signaling pathway; however, the role of noncanonical WNT signaling within the mammary stem/progenitor system remains elusive. Here, we focused on the noncanonical WNT receptors receptor tyrosine kinase-like orphan receptor 2 (ROR2) and receptor-like tyrosine kinase (RYK) and their activation by WNT5A, one of the hallmark noncanonical WNT ligands, during mammary epithelial growth and branching morphogenesis. We found that WNT5A inhibits mammary branching morphogenesis in vitro and in vivo through the receptor tyrosine kinase ROR2. Unexpectedly, WNT5A was able to enhance mammary epithelial growth, which is in contrast to its next closest relative WNT5B, which potently inhibits mammary stem/progenitor proliferation. We found that RYK, but not ROR2, is necessary for WNT5A-mediated promotion of mammary growth. These findings provide important insight into the biology of noncanonical WNT signaling in adult stem/progenitor cell regulation and development. Future research will determine how these interactions go awry in diseases such as breast cancer.

IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC.

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) expression correlates with malignancy, but its role(s) in pathogenesis remains enigmatic. We interrogated the IGF2BP3-RNA interaction network in pancreatic ductal adenocarcinoma (PDAC) cells. Using a combination of genome-wide approaches, we have identified 164 direct mRNA targets of IGF2BP3. These transcripts encode proteins enriched for functions such as cell migration, proliferation, and adhesion. Loss of IGF2BP3 reduced PDAC cell invasiveness and remodeled focal adhesion junctions. Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) revealed significant overlap of IGF2BP3 and microRNA (miRNA) binding sites. IGF2BP3 promotes association of the RNA-induced silencing complex (RISC) with specific transcripts. Our results show that IGF2BP3 influences a malignancy-associated RNA regulon by modulating miRNA-mRNA interactions.

Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness.

Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence.

Mammary Stem Cell Self-Renewal Is Regulated by Slit2/Robo1 Signaling through SNAI1 and mINSC.

Tissue homeostasis requires somatic stem cell maintenance; however, mechanisms regulating this process during organogenesis are not well understood. Here, we identify asymmetrically renewing basal and luminal stem cells in the mammary end bud. We demonstrate that SLIT2/ROBO1 signaling regulates the choice between self-renewing asymmetric cell divisions (ACDs) and expansive symmetric cell divisions (SCDs) by governing Inscuteable (mInsc), a key member of the spindle orientation machinery, through the transcription factor Snail (SNAI1). Loss of SLIT2/ROBO1 signaling increases SNAI1 in the nucleus. Overexpression of SNAI1 increases mInsc expression, an effect that is inhibited by SLIT2 treatment. Increased mInsc does not change cell proliferation in the mammary gland (MG) but instead causes more basal cap cells to divide via SCD, at the expense of ACD, leading to more stem cells and larger outgrowths. Together, our studies provide insight into how the number of mammary stem cells is regulated by the extracellular cue SLIT2.

SLIT/ROBO2 signaling promotes mammary stem cell senescence by inhibiting Wnt signaling.

WNT signaling stimulates the self-renewal of many types of adult stem cells, including mammary stem cells (MaSCs), but mechanisms that limit this activity are poorly understood. Here, we demonstrate that SLIT2 restricts stem cell renewal by signaling through ROBO2 in a subset of basal cells to negatively regulate WNT signaling. The absence of SLIT/ROBO2 signaling leads to increased levels of nuclear ß-catenin. Robo2 loss does not increase the number of stem cells; instead, stem cell renewal is enhanced in the absence of SLIT/ROBO2 signaling. This is due to repressed expression of p16(INK4a), which, in turn, delays MaSC senescence. Together, our studies support a model in which SLITs restrict the expansion of MaSCs by countering the activity of WNTs and limiting self-renewal.

Changes in cell and tissue organization in cancer of the breast and colon.

Most cancers arise in epithelia, the tissue type that lines all body cavities. The organization of epithelia enables them to act as a barrier and perform vectorial transport of molecules between body compartments. Crucial for their organization and function is a highly specialized network of cell adhesion and polarity proteins aligned along the apical-basal axis. Comparing breast and intestinal tissue as examples of common cancer sites, reveals an important contribution of polarity proteins to the initiation and progression of cancer. Defects in polarity are induced directly by mutations in polarity proteins, but also indirectly by changes in the expression of specific microRNAs and altered transcriptional programs that drive cellular differentiation from epithelial to more mesenchymal characteristics. The latter is particularly important in the metastatic process.

Mammary gland development.

The mammary gland develops through several distinct stages. The first transpires in the embryo as the ectoderm forms a mammary line that resolves into placodes. Regulated by epithelial­mesenchymal interactions, the placodes descend into the underlying mesenchyme and produce the rudimentary ductal structure of the gland present at birth. Subsequent stages of development­pubertal growth, pregnancy, lactation, and involution­occur postnatally under the regulation of hormones. Puberty initiates branching morphogenesis, which requires growth hormone (GH) and estrogen, as well as insulin-like growth factor 1 (IGF1), to create a ductal tree that fills the fat pad. Upon pregnancy, the combined actions of progesterone and prolactin generate alveoli, which secrete milk during lactation. Lack of demand for milk at weaning initiates the process of involution whereby the gland is remodeled back to its prepregnancy state. These processes require numerous signaling pathways that have distinct regulatory functions at different stages of gland development. Signaling pathways also regulate a specialized subpopulation of mammary stem cells that fuel the dramatic changes in the gland occurring with each pregnancy. Our knowledge of mammary gland development and mammary stem cell biology has significantly contributed to our understanding of breast cancer and has advanced the discovery of therapies to treat this disease.

A roundabout way to cancer.

The Slit family of secreted proteins and their transmembrane receptor, Robo, were originally identified in the nervous system where they function as axon guidance cues and branching factors during development. Since their discovery, a great number of additional roles have been attributed to Slit/Robo signaling, including regulating the critical processes of cell proliferation and cell motility in a variety of cell and tissue types. These processes are often deregulated during cancer progression, allowing tumor cells to bypass safeguarding mechanisms in the cell and the environment in order to grow and escape to new tissues. In the past decade, it has been shown that the expression of Slit and Robo is altered in a wide variety of cancer types, identifying them as potential therapeutic targets. Further, studies have demonstrated dual roles for Slits and Robos in cancer, acting as both oncogenes and tumor suppressors. This bifunctionality is also observed in their roles as axon guidance cues in the developing nervous system, where they both attract and repel neuronal migration. The fact that this signaling axis can have opposite functions depending on the cellular circumstance make its actions challenging to define. Here, we summarize our current understanding of the dual roles that Slit/Robo signaling play in development, epithelial tumor progression, and tumor angiogenesis.