DiGeorge syndrome gene tbx1 functions through wnt11r to regulate heart morphology and function

DiGeorge syndrome (DGS) is the most common microdeletion syndrome, and is characterized by congenital cardiac, craniofacial and immune system abnormalities. The cardiac defects in DGS patients include conotruncal and ventricular septal defects. Although the etiology of DGS is critically regulated by TBX1 gene, the molecular pathways underpinning TBX1's role in heart development are not fully understood. In this study, we characterized heart defects and downstream signaling in the zebrafish tbx1^(−/−) mutant, which has craniofacial and immune defects similar to DGS patients. We show that tbx1^(−/−) mutants have defective heart looping, morphology and function. Defective heart looping is accompanied by failure of cardiomyocytes to differentiate normally and failure to change shape from isotropic to anisotropic morphology in the outer curvatures of the heart. This is the first demonstration of tbx1's role in regulating heart looping, cardiomyocyte shape and differentiation, and may explain how Tbx1 regulates conotruncal development in humans. Next we elucidated tbx1's molecular signaling pathway guided by the cardiac phenotype of tbx1^(−/−) mutants. We show for the first time that wnt11r (wnt11 related), a member of the non-canonical Wnt pathway, and its downstream effector gene alcama (activated leukocyte cell adhesion molecule a) regulate heart looping and differentiation similarly to tbx1. Expression of both wnt11r and alcama are downregulated in tbx1^(−/−) mutants. In addition, both wnt11r^(−/−) mutants and alcama morphants have heart looping and differentiation defects similar to tbx1^(−/−) mutants. Strikingly, heart looping and differentiation in tbx1^(−/−) mutants can be partially rescued by ectopic expression of wnt11r or alcama, supporting a model whereby heart looping and differentiation are regulated by tbx1 in a linear pathway through wnt11r and alcama. This is the first study linking tbx1 and non-canonical Wnt signaling and extends our understanding of DGS and heart development

Notch signaling expands a pre-malignant pool of T-cell acute lymphoblastic leukemia clones without affecting leukemia-propagating cell frequency

NOTCH1 pathway activation contributes to the pathogenesis of over 60% of T-cell acute lymphoblastic leukemia (T-ALL). While Notch is thought to exert the majority of its effects through transcriptional activation of Myc, it also likely has independent roles in T-ALL malignancy. Here, we utilized a zebrafish transgenic model of T-ALL, where Notch does not induce Myc transcription, to identify a novel Notch gene expression signature that is also found in human T-ALL and is regulated independently of Myc. Cross-species microarray comparisons between zebrafish and mammalian disease identified a common T-ALL gene signature, suggesting that conserved genetic pathways underlie T-ALL development. Functionally, Notch expression induced a significant expansion of pre-leukemic clones; however, a majority of these clones were not fully transformed and could not induce leukemia when transplanted into recipient animals. Limiting-dilution cell transplantation revealed that Notch signaling does not increase the overall frequency of leukemia-propagating cells (LPCs), either alone or in collaboration with Myc. Taken together, these data indicate that a primary role of Notch signaling in T-ALL is to expand a population of pre-malignant thymocytes, of which a subset acquire the necessary mutations to become fully transformed LPCs

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. In the absence of CCT-controlled protein folding, Th2 polarization diverges from normal differentiation with paradoxical continued IFN-γ expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity

Zebrafish screen identifies novel compound with selective toxicity against leukemia

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells

A model 450 million years in the making: zebrafish and vertebrate immunity

Since its first splash 30 years ago, the use of the zebrafish model has been extended from a tool for genetic dissection of early vertebrate development to the functional interrogation of organogenesis and disease processes such as infection and cancer. In particular, there is recent and growing attention in the scientific community directed at the immune systems of zebrafish. This development is based on the ability to image cell movements and organogenesis in an entire vertebrate organism, complemented by increasing recognition that zebrafish and vertebrate immunity have many aspects in common. Here, we review zebrafish immunity with a particular focus on recent studies that exploit the unique genetic and in vivo imaging advantages available for this organism. These unique advantages are driving forward our study of vertebrate immunity in general, with important consequences for the understanding of mammalian immune function and its role in disease pathogenesis

The zebrafish as a model for cancer

For the last three decades significant parts of national science budgets, and international and private funding worldwide, have been dedicated to cancer research. This has resulted in a number of important scientific findings. Studies in tissue culture have multiplied our knowledge of cancer cell pathophysiology, mechanisms of transformation and strategies of survival of cancer cells, revealing therapeutically exploitable differences to normal cells. Rodent animal models have provided important insights on the developmental biology of cancer cells and on host responses to the transformed cells. However, the rate of death from some malignancies is still high, and the incidence of cancer is increasing in the western hemisphere. Alternative animal models are needed, where cancer cell biology, developmental biology and treatment can be studied in an integrated way. The zebrafish offers a number of features, such as its rapid development, tractable genetics, suitability for in vivo imaging and chemical screening, that make it an attractive model to cancer researchers. This Primer will provide a synopsis of the different cancer models generated by the zebrafish community to date. It will discuss the use of these models to further our understanding of the mechanisms of cancer development, and to promote drug discovery. The article was inspired by a workshop on the topic held in July 2009 in Spoleto, Italy, where a number of new zebrafish cancer models were presented. The overarching goal of the article is aimed at raising the awareness of basic researchers, as well as clinicians, to the versatility of this emerging alternative animal model of cancer

Regional differentiation defects in <i>tbx1^−/−</i> mutants.

<p>48 hpf embryos stained for region-specific markers (A–H) and their schematic representation (I–L). Red lines indicate myocardium, grey lines indicate endocardium and blue lines indicate gene expression. <i>bmp4</i> (OFT, AVC and IFT), <i>notch1b</i> (AVC endocardium) and <i>versican</i> (AVC myocardium) expression is down-regulated in <i>tbx1^−/−</i> mutants (E–G) as compared to WT siblings (A–C). The insets in C and G show <i>versican</i> expression in the ear as a control for ISH. <i>anf</i> expression is localized to the outer parts of the chamber myocardium in WT (D), but is broadly expressed in the ventricle and atrium in <i>tbx1^−/−</i> mutants (H). Arrows point to the AVC in all panels; v, ventricle; a, atrium. The plots in M–P show the penetrance of the phenotype as percentage embryos with absent/mis-localized expression of the respective gene. N depicts the total number of embryos represented in the plot. Scale bars: 50 µm.</p

Heart looping and regional differentiation defects in <i>wnt11r^−/−</i> mutants.

<p>(A) <i>cmlc2</i> ISH showing the looping defect in <i>wnt11r^−/−</i> mutants. (B) Atrio-ventricular axis angle in wild-type (left, green) and <i>wnt11r^−/−</i> mutants (right, blue). N indicates the total number of embryos from 3 experiments. (C) Defective regional differentiation of heart in <i>wnt11r^−/−</i> mutants, similar to those seen in <i>tbx1^−/−</i> mutants. Scale bars: 50 µm.</p

Bulbous arteriosus defects in <i>tbx1^−/−</i> embryos.

<p>Dissected hearts from 72 hpf embryos showing the BA region (A, D) outlined in white, with the bidirectional arrowheads in black and red showing the length and width of BA, respectively. (B, E) The corresponding images showing staining for Alcama antibody (red) and DAF-2DA (green). (C, F) <i>tbx1^−/−</i> mutants at the same stage have absent <i>eln2</i> expression. Black arrows indicate the BA. (G) Quantification of BA width in WT and <i>tbx1−/−</i> mutants, while (H) demonstrates that BA length is reduced in <i>tbx1^−/−</i> mutants; * p-value = 0.0045. N = 13 for all measurements. Scale bars: 25 µm.</p

<i>wnt11r</i> and <i>tbx1</i> cooperate to regulate heart looping and differentiation.

<p>(A–C) ISH showing <i>wnt11r</i> expression in the fusing heart fields (21 somites, A), linear heart tube (24 hpf, B) and fully looped heart (48 hpf, C) in WT embryos. (D, E) Dissected hearts from WT (D) versus <i>tbx1^−/−</i> mutant (E) embryos stained for <i>wnt11r</i> expression at 48 hpf. The heart boundaries are indicated by dotted lines. Arrows point to the AVC. Scale bars: 50 µm. (F, G) Quantification of penetrance of looping and regional differentiation defects in double heterozygotes as compared to single heterozygotes of <i>tbx1</i> and <i>wnt11r</i>. WT is shown as negative control, and <i>tbx1^−/−</i> and <i>wnt11r^−/−</i> mutants as positive control for looping and regional differentiation defects. N depicts the total number of embryos from 3 experiments.</p