Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA.

BACKGROUND: HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. METHODS: We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. RESULTS: Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. CONCLUSION: Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Tril targets Smad7 for degradation to allow hematopoietic specification in Xenopus embryos.

In Xenopus laevis, bone morphogenetic proteins (Bmps) induce expression of the transcription factor Gata2 during gastrulation, and Gata2 is required in both ectodermal and mesodermal cells to enable mesoderm to commit to a hematopoietic fate. Here, we identify tril as a Gata2 target gene that is required in both ectoderm and mesoderm for primitive hematopoiesis to occur. Tril is a transmembrane protein that functions as a co-receptor for Toll-like receptors to mediate innate immune responses in the adult brain, but developmental roles for this molecule have not been identified. We show that Tril function is required both upstream and downstream of Bmp receptor-mediated Smad1 phosphorylation for induction of Bmp target genes. Mechanistically, Tril triggers degradation of the Bmp inhibitor Smad7. Tril-dependent downregulation of Smad7 relieves repression of endogenous Bmp signaling during gastrulation and this enables mesodermal progenitors to commit to a blood fate. Thus, Tril is a novel component of a Bmp-Gata2 positive-feedback loop that plays an essential role in hematopoietic specification.

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo.

Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.

GATA2 regulates Wnt signaling to promote primitive red blood cell fate.

Primitive erythropoiesis is regulated in a non cell-autonomous fashion across evolution from frogs to mammals. In Xenopus laevis, signals from the overlying ectoderm are required to induce the mesoderm to adopt an erythroid fate. Previous studies in our lab identified the transcription factor GATA2 as a key regulator of this ectodermal signal. To identify GATA2 target genes in the ectoderm required for red blood cell formation in the mesoderm, we used microarray analysis to compare gene expression in ectoderm from GATA2 depleted and wild type embryos. Our analysis identified components of the non-canonical and canonical Wnt pathways as being reciprocally up- and down-regulated downstream of GATA2 in both mesoderm and ectoderm. We show that up-regulation of canonical Wnt signaling during gastrulation blocks commitment to a hematopoietic fate while down-regulation of non-canonical Wnt signaling impairs erythroid differentiation. Our results are consistent with a model in which GATA2 contributes to inhibition of canonical Wnt signaling, thereby permitting progenitors to exit the cell cycle and commit to a hematopoietic fate. Subsequently, activation of non-canonical Wnt signaling plays a later role in enabling these progenitors to differentiate as mature red blood cells.

EGT022, an RGD-containing recombinant disintegrin, inhibits the VEGF-induced angiogenic process by targeting integrin ß3 in endothelial cells.

OBJECTIVES: EGT022, an RGD-containing recombinant disintegrin from human ADAM metallopeptidase domain 15 (ADAM15), has been reported to stimulate vascular maturation of retinal blood vessels with promotion of pericyte coverage through binding to integrin αIIbß3. Previous studies have reported that angiogenesis can be inhibited by several RGD motif-containing disintegrins; however, the effect of EGT022 on Vascular endothelial growth factor (VEGF)-induced angiogenesis has not yet been determined. This study was conducted in order to evaluate the anti-angiogenic function of EGT022 in VEGF-induced endothelial cells. METHODS: A proliferation and migration assay was performed using human umbilical vein endothelial cells (HUVEC) cells stimulated with VEGF to determine whether the angiogenic process was suppressed by EGT022. An in vitro trans-well assay and Mile's permeability assay were performed to determine the effect of EGT022 on permeability. Western blot was performed in order to further determine whether EGT022 can inhibit phosphorylation of VEGF receptor-2 (VEGFR2) and Phospholipase C gamma1 (PLC-γ1). An integrin binding assay and luciferase assay were performed for identification of the integrin target of EGT022. RESULTS: Angiogenesis including proliferation, migration, tube formation, and permeability was significantly inhibited by EGT022 in HUVEC cells. Our findings also demonstrated that EGT022 binds directly to integrin αvß3, induces dephosphorylation of integrin ß3, and inhibits phosphorylation of VEGFR2. In addition, phosphorylation of PLC-γ1 and activation of Nuclear Factor of Activated T-cell (NFAT), a downstream pathway of VEGF, are inhibited by EGT022 in HUVEC cells. CONCLUSION: These results clearly demonstrate the anti-angiogenic role played by EGT022 as a potent antagonist of integrin ß3 in endothelial cells.

MYC Deregulation and PTEN Loss Model Tumor and Stromal Heterogeneity of Aggressive Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) patients have a poor prognosis and few treatment options. Mouse models of TNBC are important for development of new therapies, however, few mouse models represent the complexity of TNBC. Here, we develop a female TNBC murine model by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. This Myc;Ptenfl model develops heterogeneous triple-negative mammary tumors that display histological and molecular features commonly found in human TNBC. Our research involves deep molecular and spatial analyses on Myc;Ptenfl tumors including bulk and single-cell RNA-sequencing, and multiplex tissue-imaging. Through comparison with human TNBC, we demonstrate that this genetic mouse model develops mammary tumors with differential survival and therapeutic responses that closely resemble the inter- and intra-tumoral and microenvironmental heterogeneity of human TNBC, providing a pre-clinical tool for assessing the spectrum of patient TNBC biology and drug response.

Sortilin associates with transforming growth factor-beta family proteins to enhance lysosome-mediated degradation.

Transforming growth factor (TGF)-ß family proteins are synthesized as precursors that are cleaved to generate an active ligand. Previous studies suggest that TGF-ß activity can be controlled by lysosomal degradation of both precursor proteins and ligands, but how these soluble proteins are trafficked to the lysosome is incompletely understood. The current studies show that sortilin selectively co-immunoprecipitates with the cleaved prodomain and/or precursor form of TGF-ß family members. Furthermore, sortilin co-localizes with, and enhances accumulation of a nodal family member in the Golgi. Co-expression of sortilin with TGF-ß family members leads to decreased accumulation of precursor proteins and cleavage products and this is attenuated by lysosomal, but not proteosomal inhibitors. In Xenopus embryos, overexpression of sortilin leads to a decrease in phospho-Smad2 levels and phenocopies loss of nodal signaling. Conversely, down-regulation of sortilin expression in HeLa cells leads to an up-regulation of endogenous bone morphogenic protein pathway activation, as indicated by an increase in phospho-Smad1/5/8 levels. Our results suggest that sortilin negatively regulates TGF-ß signaling by diverting trafficking of precursor proteins to the lysosome during transit through the biosynthetic pathway.

Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain.

BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 sites of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range.

A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP.

Zip code-binding protein 1 (ZBP-1) and its Xenopus laevis homologue, Vg1 RNA and endoplasmic reticulum-associated protein (VERA)/Vg1 RNA-binding protein (RBP), bind repeated motifs in the 3' untranslated regions (UTRs) of localized mRNAs. Although these motifs are required for RNA localization, the necessity of ZBP-1/VERA remains unresolved. We address the role of ZBP-1/VERA through analysis of the Drosophila melanogaster homologue insulin growth factor II mRNA-binding protein (IMP). Using systematic evolution of ligands by exponential enrichment, we identified the IMP-binding element (IBE) UUUAY, a motif that occurs 13 times in the oskar 3'UTR. IMP colocalizes with oskar mRNA at the oocyte posterior, and this depends on the IBEs. Furthermore, mutation of all, or subsets of, the IBEs prevents oskar mRNA translation and anchoring at the posterior. However, oocytes lacking IMP localize and translate oskar mRNA normally, illustrating that one cannot necessarily infer the function of an RBP from mutations in its binding sites. Thus, the translational activation of oskar mRNA must depend on the binding of another factor to the IBEs, and IMP may serve a different purpose, such as masking IBEs in RNAs where they occur by chance. Our findings establish a parallel requirement for IBEs in the regulation of localized maternal mRNAs in D. melanogaster and X. laevis.

Modeling and analysis of an energy-efficient mobility management scheme in IP-based wireless networks.

An energy-efficient mobility management scheme in IP-based wireless networks is proposed to reduce the battery power consumption of mobile hosts (MHs). The proposed scheme manages seven MH states, including transmitting, receiving, attention/cell-connected, attention/paging area(PA)-connected, idle, off/attached, and detached states, to efficiently manage battery power, radio resources, and network load. We derive the stationary probabilities and steady state probabilities of the seven MH states for the proposed scheme in IP-based wireless networks in compact form. The effects of various input parameters on MH steady state probabilities and power consumption are investigated in the proposed scheme compared to the conventional scheme. Network costs such as cell updates, PA updates, binding-lifetime-based registrations, and paging messages are analyzed in the proposed and conventional schemes. The optimal values of PA size and registration interval are derived to minimize the network cost of the proposed scheme. The combined network and power costs are investigated for the proposed and conventional schemes. The results provide guidelines to select the proper system parameters in IP-based wireless networks.

Oligonucleotide conjugated antibody strategies for cyclic immunostaining.

A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.

Simultaneous Detection of RNAs and Proteins with Subcellular Resolution.

We describe the detailed methods of "immunoFISH" to analyze the expression level and the spatial localization of RNA transcripts and proteins on cultured cells and formal-fixed, paraffin-embedded (FFPE) tissue sections. On cultured cells, we detect specific transcripts using the Stellaris fluorescence in situ hybridization (FISH) probes labeled with fluorophores that target multiple regions along the desired transcripts and proteins combining the immunofluorescent staining. On FFPE tissue sections, we use the RNAscope FISH probes, modified branched DNA (bDNA) probes to amplify the RNA signals, followed by immunofluorescent staining for protein detection. The abundance, composition, and spatial distribution are determined by signals from fluorescently labeled proteins and individual transcripts of images acquired using high-resolution fluorescence microscopy.

Oligonucleotide conjugated antibodies permit highly multiplexed immunofluorescence for future use in clinical histopathology.

SIGNIFICANCE: Advanced genetic characterization has informed cancer heterogeneity and the challenge it poses to effective therapy; however, current methods lack spatial context, which is vital to successful cancer therapy. Conventional immunolabeling, commonplace in the clinic, can provide spatial context to protein expression. However, these techniques are spectrally limited, resulting in inadequate capacity to resolve the heterogenous cell subpopulations within a tumor. AIM: We developed and optimized oligonucleotide conjugated antibodies (Ab-oligo) to facilitate cyclic immunofluorescence (cyCIF), resulting in high-dimensional immunostaining. APPROACH: We employed a site-specific conjugation strategy to label antibodies with unique oligonucleotide sequences, which were hybridized in situ with their complementary oligonucleotide sequence tagged with a conventional fluorophore. Antibody concentration, imaging strand concentration, and configuration as well as signal removal strategies were optimized to generate maximal staining intensity using our Ab-oligo cyCIF strategy. RESULTS: We successfully generated 14 Ab-oligo conjugates and validated their antigen specificity, which was maintained in single color staining studies. With the validated antibodies, we generated up to 14-color imaging data sets of human breast cancer tissues. CONCLUSIONS: Herein, we demonstrated the utility of Ab-oligo cyCIF as a platform for highly multiplexed imaging, its utility to measure tumor heterogeneity, and its potential for future use in clinical histopathology.

Fluorescent Imaging for In Situ Measurement of Drug Target Engagement and Cell Signaling Pathways.

Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and identify druggable therapeutic targets. Tyrosine kinase inhibitors (TKI) are a class of drugs seeking to inhibit signaling pathways critical to sustaining proliferative signaling, resisting cell death, and the other hallmarks of cancer. While tumors may initially respond to TKI therapy, disease progression is near universal due to mechanisms of acquired resistance largely involving cellular signaling pathway reprogramming. With the ultimate goal of improved TKI therapeutic efficacy our group has developed intracellular paired agent imaging (iPAI) to quantify drug target interactions and oligonucleotide conjugated antibody (Ab-oligo) cyclic immunofluorescence (cycIF) imaging to characterize perturbed signaling pathways in response to therapy. iPAI uses spectrally distinct, fluorescently labeled targeted and untargeted drug derivatives, correcting for non-specific drug distribution and facilitating quantitative assessment of the drug binding before and after therapy. Ab-oligo cycIF exploits in situ hybridization of complementary oligonucleotides for biomarker labeling while oligonucleotide modifications facilitate signal removal for sequential rounds of fluorescent tagging and imaging. Ab-oligo CycIF is capable of generating extreme multi-parametric images for quantifying total and phosphorylated protein expression to quantify protein activation, expression, and spatial distribution. Together iPAI and Ab-oligo cycIF can be applied to interrogate drug uptake and target binding as well as changes to heterogenous cell populations within tumors that drive variable therapeutic responses in patients.

Signal removal methods for highly multiplexed immunofluorescent staining using antibody conjugated oligonucleotides.

Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and have vastly affected how cancer is diagnosed and treated. However, the translation of complex genomic analyses to pathological diagnosis remains challenging using conventional immunofluorescence (IF) staining, which is typically limited to 2-5 antigens. Numerous strategies to increase distinct antigen detection on a single sample have been investigated, but all have deleterious effects on the tissue limiting the maximum number of biomarkers that can be imaged on a single sample and none can be seamlessly integrated into routine clinical workflows. To facilitate ready integration into clinical histopathology, we have developed a novel cyclic IF (cycIF) technology based on antibody conjugated oligonucleotides (Ab-oligos). In situ hybridization of complementary oligonucleotides (oligos) facilitates biomarker labeling for imaging on any conventional fluorescent microscope. We have validated a variety of oligo configurations and their respective signal removal strategies capable of diminishing fluorescent signal to levels of autofluorescence before subsequent staining cycles. Robust signal removal is performed without the employment of harsh conditions or reagents, maintaining tissue integrity and antigenicity for higher dimensionality immunostaining of a single sample. Our platform Ab-oligo cycIF technology uses conventional fluorophores and microscopes, allowing for dissemination to a broad audience and congruent integration into clinical histopathology workflows.

Transcriptional Programming of Normal and Inflamed Human Epidermis at Single-Cell Resolution.

Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ∼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+ myeloid dendritic cell population in psoriatic epidermis, and IL1ßhiCCL3hiCD14+ monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation.

UUCAC- and vera-dependent localization of VegT RNA in Xenopus oocytes.

Localized mRNAs are directed to their destinations by "localization elements" (LEs) in their 3'UTRs. LEs harbor multiple, functionally redundant localization "signals." These signals are poorly defined, hence it is unclear whether the signals-and their cognate factors-are unique to each RNA or employed generally. Five "E2s" (UUCACs) in the 366 nt Vg1 LE (VgLE) direct this transcript to the vegetal pole of Xenopus oocytes via the binding of a protein-Vera/Vg1RBP/ZBP. Here we show that a different vegetal RNA, VegT, employs the same signal and factor. Five E2s within a 440 nt subregion (VegT440) of the VegT 3'UTR predict its LE and are both necessary and sufficient (in the context of antisense VegT440) for directing localization. The E2s in VegT440 and VgLE function similarly to recruit Vera protein: (1) in both contexts, E2 nt substitutions partially (UU to AC) or completely (CA to UG) inhibit localization in accordance with the sequence selectivity of Vera protein for E2s; (2) VegT440 and VgLE crosscompete, in an E2-dependent manner, for localization and Vera binding; (3) injection of anti-Vera antibody into oocytes inhibits localization of both injected transcripts. These findings imply that general localization signals traffic diverse RNAs.

Quantitative, in situ analysis of mRNAs and proteins with subcellular resolution.

We describe here a method, termed immunoFISH, for simultaneous in situ analysis of the composition and distribution of proteins and individual RNA transcripts in single cells. Individual RNA molecules are labeled by hybridization and target proteins are concurrently stained using immunofluorescence. Multicolor fluorescence images are acquired and analyzed to determine the abundance, composition, and distribution of hybridized probes and immunofluorescence. We assessed the ability of immunoFISH to simultaneous quantify protein and transcript levels and distribution in cultured HER2 positive breast cancer cells and human breast tumor samples. We demonstrated the utility of this assay in several applications including demonstration of the existence of a layer of normal myoepithelial KRT14 expressing cells that separate HER2+ cancer cells from the stromal and immune microenvironment in HER2+ invasive breast cancer. Our studies show that immunoFISH provides quantitative information about the spatial heterogeneity in transcriptional and proteomic features that exist between and within cells.

A fully integrated, three-dimensional fluorescence to electron microscopy correlative workflow.

While fluorescence microscopy provides tools for highly specific labeling and sensitive detection, its resolution limit and lack of general contrast has hindered studies of cellular structure and protein localization. Recent advances in correlative light and electron microscopy (CLEM), including the fully integrated CLEM workflow instrument, the FEI CorrSight with MAPS, have allowed for a more reliable, reproducible, and quicker approach to correlate three-dimensional time-lapse confocal fluorescence data, with three-dimensional focused ion beam-scanning electron microscopy data. Here we demonstrate the entire integrated CLEM workflow using fluorescently tagged MCF7 breast cancer cells.

Expression pattern of bcar3, a downstream target of Gata2, and its binding partner, bcar1, during Xenopus development.

Primitive hematopoiesis generates red blood cells that deliver oxygen to the developing embryo. Mesodermal cells commit to a primitive blood cell fate during gastrulation and, in order to do so the mesoderm must receive non-cell autonomous signals transmitted from other germ layers. In Xenopus, the transcription factor Gata2 functions in ectodermal cells to generate or transmit the non-cell autonomous signals. Here we have identified Breast Cancer Antiestrogen Resistance 3 (bcar3) as a gene that is induced in ectodermal cells downstream of Gata2. Bcar3 and its binding partner Bcar1 function to transduce integrin signaling, leading to changes in cellular morphology, motility and adhesion. We show that gata2, bcar3 and bcar1 are co-expressed in ventral ectoderm from early gastrula to early tailbud stages. At later stages of development, bcar3 and bcar1 are co-expressed in the spinal cord, notochord, fin mesenchyme and pronephros but each shows additional unique sites of expression. These co-expression and unique expression patterns suggest that Bcar3 and Bcar1 may function together but also independently during Xenopus development.