The TGFß superfamily in Lisbon: navigating through development and disease.

The 10th FASEB meeting 'The TGFß Superfamily: Signaling in Development and Disease' took place in Lisbon, Portugal, in July 2017. As we review here, the findings presented at the meeting highlighted the important contributions of TGFß family signaling to normal development, adult homeostasis and disease, and also revealed novel mechanisms by which TGFß signals are transduced.

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.

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.

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.

The cell giveth and the cell taketh away: an overview of Notch pathway activation by endocytic trafficking of ligands and receptors.

Notch signaling is firmly established as a form of cell-to-cell communication that is critical throughout development. Dysregulation of Notch has been linked to cancer and developmental disorders, making it an important target for therapeutic intervention. One aspect of this pathway that sets it apart from others is its apparent reliance on endocytosis by signal-sending and signal-receiving cells. The subtle details of endocytosis-mediated molecular processing within both ligand- and receptor-presenting cells that are required for the Notch signal to maintain fidelity remain unclear. The endosomal system has long been known to play an important role in terminating signal transduction by directing lysosomal trafficking and degradation of cell surface receptors. More recently, endocytic trafficking has also been shown to be critical for activation of signaling. This review highlights four models of endocytic processing in the context of the Notch pathway. In ligand-presenting cells, endocytosis may be required for pre-processing of ligands to make them competent for interaction with Notch receptors and/or for exerting a pulling force on the ligand/Notch complex. In receptor-presenting cells, endocytosis may be a prerequisite for Notch cleavage and thus activation and/or it could be a means of limiting ligand-independent Notch activation. Recent advances in our understanding of how and why endocytosis of Notch receptors and ligands is required for activation and termination of signaling during normal development and in disease states are discussed.

BMP4 regulates the hematopoietic stem cell niche.

Bone morphogenetic protein 4 (BMP4) is required for mesoderm commitment to the hematopoietic lineage during early embryogenesis. However, deletion of BMP4 is early embryonically lethal and its functional role in definitive hematopoiesis is unknown. Consequently, we used a BMP4 hypomorph to investigate the role of BMP4 in regulating hematopoietic stem cell (HSC) function and maintaining steady-state hematopoiesis in the adult. Reporter gene expression shows that Bmp4 is expressed in cells associated with the hematopoietic microenvironment including osteoblasts, endothelial cells, and megakaryocytes. Although resting hematopoiesis is normal in a BMP4-deficient background, the number of c-Kit+, Sca-1+, Lineage- cells is significantly reduced. Serial transplantation studies reveal that BMP4-deficient recipients have a microenvironmental defect that reduces the repopulating activity of wild-type HSCs. This defect is even more pronounced in a parabiosis model that demonstrates a profound reduction in wild-type hematopoietic cells within the bone marrow of BMP4-deficient recipients. Furthermore, wild-type HSCs that successfully engraft into the BMP4-deficient bone marrow show a marked decrease in functional stem cell activity when tested in a competitive repopulation assay. Taken together, these findings indicate BMP4 is a critical component of the hematopoietic microenvironment that regulates both HSC number and function.

Fanconi anemia proteins are required to prevent accumulation of replication-associated DNA double-strand breaks.

Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

Ectodermally derived steel/stem cell factor functions non-cell autonomously during primitive erythropoiesis in Xenopus.

Signals derived from nonhematopoietic tissues are essential for normal primitive erythropoiesis in vertebrates, but little is known about the nature of these signals. In Xenopus, unidentified factors secreted by ectodermal cells during gastrulation are required to enable the underlying ventral mesoderm to form blood. Steel is expressed in the ectoderm of early Xenopus embryos and is known to regulate definitive erythroid progenitor survival and differentiation in other organisms, making it an excellent candidate regulator of primitive erythropoiesis. In this study, we tested whether steel signaling is required for primitive red blood cell differentiation in mice and frogs. We show that Xsl is expressed in the ectoderm in Xenopus gastrulae and that c-kit homologs are expressed in the underlying mesoderm at the same stages of development. We present loss of function data in whole Xenopus embryos and explants that demonstrate a requirement for ectodermally derived steel to signal through c-kit in the mesoderm to support early steps in the differentiation of primitive erythroid but not myeloid cells. Finally, we show that primitive erythropoiesis is not disrupted in mouse embryos that lack c-kit function. Our data suggest a previously unrecognized and unique function of steel/c-kit during primitive erythropoiesis in Xenopus.

Calmodulin-dependent protein kinase IV mediated antagonism of BMP signaling regulates lineage and survival of hematopoietic progenitors.

In the current study, we show that bone morphogenetic proteins (BMPs) play a role in hematopoiesis that is independent of their function in specifying ventral mesodermal fate. When BMP activity is upregulated or inhibited in Xenopus embryos hematopoietic precursors are specified properly but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of BMP activity induces erythroid precursors to undergo apoptotic cell death, whereas constitutive activation of BMPs causes an increase in commitment of hematopoietic progenitors to myeloid differentiation and a concomitant decrease in erythrocytes that is not due to enhanced apoptosis. These blood defects are observed even when BMP activity is misregulated solely in non-hematopoietic (ectodermal) cells, demonstrating that BMPs generate extrinsic signals that regulate hematopoiesis independent of mesodermal patterning. Further analysis revealed that endogenous calmodulin-dependent protein kinase IV (CaM KIV) is required to negatively modulate hematopoietic functions of BMPs downstream of receptor activation. Our data are consistent with a model in which CaM KIV inhibits BMP signals by activating a substrate, possibly cAMP-response element-binding protein (CREB), that recruits limiting amounts of CREB binding protein (CBP) away from transcriptional complexes functioning downstream of BMPs.