Vesa Kaartinen

Increased neutrophil respiratory burst in bcr-null mutants

Philadelphia (Ph)-positive leukemias invariably contain a chromosomal translocation fusing BCR to ABL. The BCR-ABL protein is responsible for leukemogenesis. Here we show that exposure of bcr-null mutant mice to gram-negative endotoxin led to severe septic shock and increased tissue injury by neutrophils. Neutrophils of bcr (-/-) mice showed a pronounced increase in reactive oxygen metabolite production upon activation and were more sensitive to priming stimuli. Activated (-/-) neutrophils displayed a 3-fold increased p21rac2 membrane translocation compared with (+/+) neutrophils. These results connect Bcr in vivo with the regulation of Rac-mediated superoxide production by the NADPH-oxidase system of leukocytes and suggest a link between Bcr function and the cell type affected in Ph-positive leukemia.

Cardiac outflow tract defects in mice lacking ALK2 in neural crest cells

Cardiac neural crest cells are multipotent migratory cells that contribute to the formation of the cardiac outflow tract and pharyngeal arch arteries. Neural crest-related developmental defects account for a large proportion of congenital heart disorders. Recently, the genetic bases for some of these disorders have been elucidated, and signaling pathways required for induction, migration and differentiation of cardiac neural crest have emerged. Bone morphogenetic proteins comprise a family of secreted ligands implicated in numerous aspects of organogenesis, including heart and neural crest development. However, it has remained generally unclear whether BMP ligands act directly on neural crest or cardiac myocytes during cardiac morphogenesis, or function indirectly by activating other cell types. Studies on BMP receptor signaling during organogenesis have been hampered by the fact that receptor knockouts often lead to early embryonic lethality. We have used a Cre/loxP system for neural crest-specific deletion of the type I receptor, ALK2, in mouse embryos. Mutant mice display cardiovascular defects, including persistent truncus arteriosus, and abnormal maturation of the aortic arch reminiscent of common forms of human congenital heart disease. Migration of mutant neural crest cells to the outflow tract is impaired, and differentiation to smooth muscle around aortic arch arteries is deficient. Moreover, in Alk2 mutants, the distal outflow tract fails to express Msx1, one of the major effectors of BMP signaling. Thus, the type I BMP receptor ALK2 plays an essential cell-autonomous role in the development of the cardiac outflow tract and aortic arch derivatives.

Craniofacial defects in mice lacking BMP type I receptor Alk2 in neural crest cells

Neural crest cells (NCCs) are pluripotent migratory cells that contribute to the development of various craniofacial structures. Many signaling molecules have been implicated in the formation, migration and differentiation of NCCs including bone morphogenetic proteins (BMPs). BMPs signal through a receptor complex composed of type I and type II receptors. Type I receptors (Alk2, Alk3 and Alk6) are the primary determinants of signaling specificity and therefore understanding their function is important in revealing the developmental roles of molecular pathways regulated by BMPs. Here we used a Cre/loxP system for neural crest specific deletion of Alk2. Our results show that mice lacking Alk2 in the neural crest display multiple craniofacial defects including cleft palate and a hypotrophic mandible. Based on the present results we conclude that signaling via Alk2 receptors is non-redundant and regulates normal development of a restricted set of structures derived from the cranial neural crest.

Modulation of morphogenesis by noncanonical Wnt signaling requires ATF/CREB family-mediated transcriptional activation of TGFβ2

Transcriptional readout downstream of canonical Wnt signaling is known to be mediated by β-catenin activation of well-described targets, but potential transcriptional readout in response to noncanonical Wnt signaling remains poorly understood. Here, we define a transcriptional pathway important in noncanonical Wnt signaling. We have found that Wnt11 is a direct target of a canonical β-catenin pathway in developing heart and that Wnt11 mutants show cardiac outflow tract defects. We provide genetic and biochemical evidence thatWnt11 signaling affects extracellular matrix composition, cytoskeletal rearrangements and polarized cell movement required for morphogenesis of the cardiac outflow tract. Notably, transforming growth factor β2 (TGFβ2), a key effector of organ morphogenesis, is regulated by Wnt11-mediated noncanonical signaling in developing heart and somites via one or more activating transcription factor (ATF)/cyclic AMP response element binding protein (CREB) family members. Thus, we propose that transcriptional readout mediated at least in part by a Wnt11 → ATF/CREB → TGFβ2 pathway is critical in regulating morphogenesis in response to noncanonical Wnt signaling.

Transforming growth factor‐β3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane

Studies on transforming growth factor β3 (TGF‐β3) deficient mice have shown that TGF‐β3 plays a critical role in palatogenesis. These null mutant mice have clefting of the secondary palate, caused by a defect in the process of fusion of the palatal shelves. A critical step in mammalian palatal fusion is removal of the medial edge epithelial cells from the midline seam and formation of continuous mesenchyme. To determine in more detail the role of TGF‐β3 in palatogenesis, we cultured TGF‐β3 null mutant and wild‐type control palatal shelves in an organ culture system. The fate of the medial edge epithelial cells was studied in vitro using vital cell labeling and immunohistochemical techniques. Despite clear adherence, the null mutant palatal shelves did not fuse in vitro, but instead the medial edge epithelial cells survived at the midline position, and the basement membrane was resistant towards degradation. Supplementation of the culture medium with the mature form of TGF‐β3 was able to fully correct the defective fusion in the null mutant specimens. Our results demonstrate that the reason for the defective palatal fusion in TGF‐β3 (−/−) samples is not impaired adhesion. Our data define a specific role for TGF‐β3 in the events that control transdifferentiation of the medial edge epithelial cells including degradation of the underlying basement membrane. Dev. Dyn. 209:255–260, 1997.

Fibrillin controls TGF-β activation

Secreted transforming growth factor-βs (TGF-βs) are rendered biologically inactive by binding proteins that also target and concentrate them to the extracellular matrix. Specific, but still poorly understood, activation is required for disassembly of the extracellular matrix–bound protein complex to liberate the mature growth factor and to obtain a correct biological effect. A new study shows that fibrillin-1, the protein defective in Marfan syndrome, has a biologically important role in controlling TGF-β activation in the lung.

Signaling via the Tgf-beta type I receptor Alk5 in heart development.

Trophic factors secreted both from the endocardium and epicardium regulate appropriate growth of the myocardium during cardiac development. Epicardially-derived cells play also a key role in development of the coronary vasculature. This process involves transformation of epithelial (epicardial) cells to mesenchymal cells (EMT). Similarly, a subset of endocardial cells undergoes EMT to form the mesenchyme of endocardial cushions, which function as primordia for developing valves and septa. While it has been suggested that transforming growth factor-betas (Tgf-beta) play an important role in induction of EMT in the avian epi- and endocardium, the function of Tgf-betas in corresponding mammalian tissues is still poorly understood. In this study, we have ablated the Tgf-beta type I receptor Alk5 in endo-, myo- and epicardial lineages using the Tie2-Cre, Nkx2.5-Cre, and Gata5-Cre driver lines, respectively. We show that while Alk5-mediated signaling does not play a major role in the myocardium during mouse cardiac development, it is critically important in the endocardium for induction of EMT both in vitro and in vivo. Moreover, loss of epicardial Alk5-mediated signaling leads to disruption of cell-cell interactions between the epicardium and myocardium resulting in a thinned myocardium. Furthermore, epicardial cells lacking Alk5 fail to undergo Tgf-beta-induced EMT in vitro. Late term mutant embryos lacking epicardial Alk5 display defective formation of a smooth muscle cell layer around coronary arteries, and aberrant formation of capillary vessels in the myocardium suggesting that Alk5 is controlling vascular homeostasis during cardiogenesis. To conclude, Tgf-beta signaling via Alk5 is not required in myocardial cells during mammalian cardiac development, but plays an irreplaceable cell-autonomous role regulating cellular communication, differentiation and proliferation in endocardial and epicardial cells.

Formation and Differentiation of Multiple Mesenchymal Lineages during Lung Development Is Regulated by β-catenin Signaling

Background The role of ß-catenin signaling in mesodermal lineage formation and differentiation has been elusive. Methodology To define the role of ß-catenin signaling in these processes, we used a Dermo1(Twist2)Cre/+ line to target a floxed β-catenin allele, throughout the embryonic mesenchyme. Strikingly, the Dermo1Cre/+; β-cateninf/− conditional Knock Out embryos largely phenocopy Pitx1−/−/Pitx2−/− double knockout embryos, suggesting that ß-catenin signaling in the mesenchyme depends mostly on the PITX family of transcription factors. We have dissected this relationship further in the developing lungs and find that mesenchymal deletion of β-catenin differentially affects two major mesenchymal lineages. The amplification but not differentiation of Fgf10-expressing parabronchial smooth muscle progenitor cells is drastically reduced. In the angioblast-endothelial lineage, however, only differentiation into mature endothelial cells is impaired. Conclusion Taken together these findings reveal a hierarchy of gene activity involving ß-catenin and PITX, as important regulators of mesenchymal cell proliferation and differentiation.

Defective ALK5 signaling in the neural crest leads to increased postmigratory neural crest cell apoptosis and severe outflow tract defects.

BackgroundCongenital cardiovascular diseases are the most common form of birth defects in humans. A substantial portion of these defects has been associated with inappropriate induction, migration, differentiation and patterning of pluripotent cardiac neural crest stem cells. While TGF-β-superfamily signaling has been strongly implicated in neural crest cell development, the detailed molecular signaling mechanisms in vivo are still poorly understood.ResultsWe deleted the TGF-β type I receptor Alk5 specifically in the mouse neural crest cell lineage. Failure in signaling via ALK5 leads to severe cardiovascular and pharyngeal defects, including inappropriate remodeling of pharyngeal arch arteries, abnormal aortic sac development, failure in pharyngeal organ migration and persistent truncus arteriosus. While ALK5 is not required for neural crest cell migration, our results demonstrate that it plays an important role in the survival of post-migratory cardiac neural crest cells.ConclusionOur results demonstrate that ALK5-mediated signaling in neural crest cells plays an essential cell-autonomous role in the pharyngeal and cardiac outflow tract development.

The type I BMP receptors, Bmpr1a and Acvr1, activate multiple signaling pathways to regulate lens formation

BMPs play multiple roles in development and BMP signaling is essential for lens formation. However, the mechanisms by which BMP receptors function in vertebrate development are incompletely understood. To determine the downstream effectors of BMP signaling and their functions in the ectoderm that will form the lens, we deleted the genes encoding the type I BMP receptors, Bmpr1a and Acvr1, and the canonical transducers of BMP signaling, Smad4, Smad1 and Smad5. Bmpr1a and Acvr1 regulated cell survival and proliferation, respectively. Absence of both receptors interfered with the expression of proteins involved in normal lens development and prevented lens formation, demonstrating that BMPs induce lens formation by acting directly on the prospective lens ectoderm. Remarkably, the canonical Smad signaling pathway was not needed for most of these processes. Lens formation, placode cell proliferation, the expression of FoxE3, a lens-specific transcription factor, and the lens protein, alphaA-crystallin were regulated by BMP receptors in a Smad-independent manner. Placode cell survival was promoted by R-Smad signaling, but in a manner that did not involve Smad4. Of the responses tested, only maintaining a high level of Sox2 protein, a transcription factor expressed early in placode formation, required the canonical Smad pathway. A key function of Smad-independent BMP receptor signaling may be reorganization of actin cytoskeleton to drive lens invagination.