Madeline Hayes

Ptk7 promotes non-canonical Wnt/PCP-mediated morphogenesis and inhibits Wnt/β-catenin-dependent cell fate decisions during vertebrate development

Using zebrafish, we have characterised the function of Protein tyrosine kinase 7 (Ptk7), a transmembrane pseudokinase implicated in Wnt signal transduction during embryonic development and in cancer. Ptk7 is a known regulator of mammalian neural tube closure and Xenopus convergent extension movement. However, conflicting reports have indicated both positive and negative roles for Ptk7 in canonical Wnt/β-catenin signalling. To clarify the function of Ptk7 in vertebrate embryonic patterning and morphogenesis, we generated maternal-zygotic (MZ) ptk7 mutant zebrafish using a zinc-finger nuclease (ZFN) gene targeting approach. Early loss of zebrafish Ptk7 leads to defects in axial convergence and extension, neural tube morphogenesis and loss of planar cell polarity (PCP). Furthermore, during late gastrula and segmentation stages, we observe significant upregulation of β-catenin target gene expression and demonstrate a clear role for Ptk7 in attenuating canonical Wnt/β-catenin activity in vivo. MZptk7 mutants display expanded differentiation of paraxial mesoderm within the tailbud, suggesting an important role for Ptk7 in regulating canonical Wnt-dependent fate specification within posterior stem cell pools post-gastrulation. Furthermore, we demonstrate that a plasma membrane-tethered Ptk7 extracellular fragment is sufficient to rescue both PCP morphogenesis and Wnt/β-catenin patterning defects in MZptk7 mutant embryos. Our results indicate that the extracellular domain of Ptk7 acts as an important regulator of both non-canonical Wnt/PCP and canonical Wnt/β-catenin signalling in multiple vertebrate developmental contexts, with important implications for the upregulated PTK7 expression observed in human cancers.

Nedd4-1 binds and ubiquitylates activated FGFR1 to control its endocytosis and function

Fibroblast growth factor receptor 1 (FGFR1) has critical roles in cellular proliferation and differentiation during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4‐1), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR1. Nedd4‐1 binds directly to and ubiquitylates activated FGFR1, by interacting primarily via its WW3 domain with a novel non‐canonical sequence (non‐PY motif) on FGFR1. Deletion of this recognition motif (FGFR1‐Δ6) abolishes Nedd4‐1 binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4‐1 knockdown. Accordingly, FGFR1‐Δ6, or Nedd4‐1 knockdown, exhibits sustained FGF‐dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk1/2, and PLCγ). Expression of FGFR1‐Δ6 in human embryonic neural stem cells strongly promotes FGF2‐dependent neuronal differentiation. Furthermore, expression of this FGFR1‐Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR1 signalling. These results identify Nedd4‐1 as a key regulator of FGFR1 endocytosis and signalling during neuronal differentiation and embryonic development.

Imaging tumour cell heterogeneity following cell transplantation into optically clear immune-deficient zebrafish

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2E450fs (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal dominance in T-cell acute lymphoblastic leukaemia and tumour evolution resulting in elevated growth and metastasis in BRAFV600E-driven melanoma. Cell transplantation approaches using optically clear immune-compromised zebrafish provide unique opportunities to uncover biology underlying cancer and to dynamically visualize cancer processes at single-cell resolution in vivo.

ptk7 mutant zebrafish models of congenital and idiopathic scoliosis implicate dysregulated Wnt signalling in disease.

Scoliosis is a complex genetic disorder of the musculoskeletal system, characterized by three-dimensional rotation of the spine. Curvatures caused by malformed vertebrae (congenital scoliosis (CS)) are apparent at birth. Spinal curvatures with no underlying vertebral abnormality (idiopathic scoliosis (IS)) most commonly manifest during adolescence. The genetic and biological mechanisms responsible for IS remain poorly understood due largely to limited experimental models. Here we describe zygotic ptk7 (Zptk7) mutant zebrafish, deficient in a critical regulator of Wnt signalling, as the first genetically defined developmental model of IS. We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis. Furthermore, we demonstrate that embryonic loss-of-gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with CS. Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.

Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth. DOI: http://dx.doi.org/10.7554/eLife.19214.001

The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growth and Self-Renewal in Embryonal Rhabdomyosarcoma

Summary Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)—a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 up-regulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.

In Vivo Imaging of Cancer in Zebrafish.

Zebrafish cancer models have greatly advanced our understanding of malignancy in humans. This is made possible due to the unique advantages of the zebrafish model including ex vivo development and large clutch sizes, which enable large-scale genetic and chemical screens. Transparency of the embryo and the creation of adult zebrafish devoid of pigmentation (casper) have permitted unprecedented ability to dynamically visualize cancer progression in live animals. When coupled with fluorescent reporters and transgenic approaches that drive oncogenesis, it is now possible to label entire or subpopulations of cancer cells and follow cancer growth in near real-time. Here, we will highlight aspects of in vivo imaging using the zebrafish and how it has enhanced our understanding of the fundamental aspects of tumor initiation, self-renewal, neovascularization, tumor cell heterogeneity, invasion and metastasis. Importantly, we will highlight the contribution of cancer imaging in zebrafish for drug discovery.

tp53 deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish

The TP53 tumor-suppressor gene is mutated in >50% of human tumors and Li-Fraumeni patients with germ line inactivation are predisposed to developing cancer. Here, we generated tp53 deleted zebrafish that spontaneously develop malignant peripheral nerve-sheath tumors, angiosarcomas, germ cell tumors, and an aggressive Natural Killer cell-like leukemia for which no animal model has been developed. Because the tp53 deletion was generated in syngeneic zebrafish, engraftment of fluorescent-labeled tumors could be dynamically visualized over time. Importantly, engrafted tumors shared gene expression signatures with predicted cells of origin in human tissue. Finally, we showed that tp53del/del enhanced invasion and metastasis in kRASG12D-induced embryonal rhabdomyosarcoma (ERMS), but did not alter the overall frequency of cancer stem cells, suggesting novel pro-metastatic roles for TP53 loss-of-function in human muscle tumors. In summary, we have developed a Li-Fraumeni zebrafish model that is amenable to large-scale transplantation and direct visualization of tumor growth in live animals.

Abstract 5153: Myogenic regulatory factors and their role in embryonal rhabdomyosarcoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rhabdomyosarcoma (RMS) is a pediatric sarcoma of muscle. RAS pathway activation is the dominant oncogenic driver event in fusion negative RMS, which includes the Embryonal RMS (ERMS). We have previously shown in a transgenic zebrafish model of kRASG12D induced ERMS that tumor-propagating potential is confined to molecularly defined cells that express Myf5, m-Cadherin, c-Met and additional satellite cell markers. MYF5 and MYOD1 are bHLH myogenic regulatory factors (MRFs) that orchestrate skeletal muscle differentiation during development and regeneration. Both are sufficient to reprogram human mesenchymal cells into a myogenic fate. Importantly, these same factors are highly expressed a subset of mouse and human ERMS. Given that Myf5 is highly and specifically expressed in the TPC compartment in our ERMS model, we hypothesized that Myf5 and its transcriptional targets may regulate self-renewal and growth of TPCs. To address this question, we utilized transgenic zebrafish and expressed Myf5 in differentiated ERMS cells that lack proliferative capacity and can not make tumors when transplanted into recipient fish. Induced expression of Myf5 was sufficient to confer tumor-propagating ability to differentiated populations of ERMS cells. These “Induced TPCs” proliferate and generate aggressive tumors that re-express satellite cell markers and yet retain expression of mature muscle markers. Next, we assessed if Myf5 is required for ERMS initiation in the zebrafish model. Remarkably, kRASSG12D-induced ERMS could be generated in Myf5 loss-of-function zebrafish. Moreover, Myf5-deficient ERMS could regrow following transplantation into rag2E450fs immune-compromised zebrafish; however, these tumors are histologically and molecularly distinct when compared with ERMS arising in wild-type fish. Given the redundancy of function between Myf5 and MyoD in muscle and their differential expression in satellite cells and muscle progenitors, our current hypothesis is that Myf5-deficient zebrafish ERMS likely recapitulate a molecularly-distinct class of human ERMS. This idea is consistent with the fact that MYF5 is expressed in only 50% of primary human ERMS and a small fraction of human cell lines. Importantly, knock down experiments in human ERMS cell lines confirm independent roles of either MYF5 or MYOD1 in maintenance of ERMS cell viability in vitro. Collectively, our results support a previously unappreciated role for bHLH MRFs in ERMS cell survival and intra-tumor functional heterogeneity and suggest that myogenic factor expression may define unique subtypes of ERMS. Citation Format: Ines M. Tenente, Myron Ignatius, Eleanor Chen, Madeline Hayes, David M. Langenau. Myogenic regulatory factors and their role in embryonal rhabdomyosarcoma. [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 5153. doi:10.1158/1538-7445.AM2015-5153

Abstract 4733: Notch signaling increases the number of relapse-driving tumor propagating cells in embryonal rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is a devastating pediatric muscle cancer with extremely poor prognosis at relapse. Work from our group has identified the tumor-propagating cell (TPC) in a transgenic zebrafish model of kRASG12D-induced ERMS that is responsible for driving continued tumor growth and relapse. The TPC is molecularly similar to an activated muscle satellite-cell and expresses myf5, c-met, and m-cadherin. Building on these observations, we have identified the Notch pathway as a potent enhancer of ERMS self-renewal and TPC number. Specifically, bulk tumor limiting dilution cell transplantation experiments revealed that TPCs are increased 10-fold in kRASG12D expressing ERMS that co-express activated intracellular Notch1 (ICN1). This increase in TPC number is partly the result of ICN1 expressing ERMS exhibiting a 3-fold expansion of relapse-driving myf5-GFP+/mylz2-mCherry-negative ERMS-cell population. Unexpectedly, cell transplantation experiments revealed that Notch pathway activation also conferred tumor-propagating ability to the myf5-GFP+/mylz2-mCherry+ mid-differentiated ERMS cells - a population of cells previously shown to lack self-renewal capacity. Single cell engraftment studies uncovered that NOTCH activation caused cells to oscillate between the TPC and mid-differentiated ERMS molecular states that was not observed in kRASG12D-expressing ERMS, suggesting that Notch has important roles in both self-renewal and cell state transitions. Next, we validated our findings in human ERMS where NOTCH1 is highly expressed both in tumors and ERMS-cell lines. We show important roles for NOTCH in regulating self-renewal and differentiation in human ERMS. Specifically, human ERMS cells that expressed activated NOTCH1 had elevated sphere-colony formation, a surrogate for self-renewal in vitro. By contrast, shRNA knockdown of NOTCH1 resulted in decreased sphere-colony formation and robust terminal differentiation of ERMS cells into late-stage, myosin-expressing myoblasts. Moreover, we identified that NOTCH1 directly activated SNAI1 expression and was required for both efficient sphere formation and differentiation in ERMS cell lines. SNAI1 is commonly over-expressed in human ERMS and its expression is positively correlated with NOTCH1. Taken together, our data indicate that Notch signaling is an important modifier of human ERMS acting to regulate both TPC self-renewal and differentiation. Notch and/or SNAl1 pathway inhibition may have potential benefit for a subset of patients with relapsed ERMS. Citation Format: Myron Ignatius, Riadh Lobbardi, Madeline Hayes, Eleanor Chen, Karin McCarthy, G. Petur Nielsen, Brian Beleyea, Corinne Linardic, Javed Khan, Charles Keller, David M. Langenau. Notch signaling increases the number of relapse-driving tumor propagating cells in embryonal rhabdomyosarcoma. [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 4733. doi:10.1158/1538-7445.AM2015-4733