Generation of mice with a conditional allele for Trim33

Trim33 (Tif1Γ, ectodermin, moonshine), a member of the TIF1 family of transcriptional coactivators and corepressors, is a large nuclear protein that contains an N-terminal tripartite (Trim) domain composed of a RING domain, two B-box domains, and a coiled coil domain. It has been suggested that Trim33 (Ectodermin) mediates ectodermal induction in the Xenopus by functioning as a Smad4 ubiquitin ligase, while in the zebrafish Trim33 (moonshine) has been reported to act as a R-Smad binding protein in induction of erythroid differentiation. Since the developmental role of Trim33 in mammals is currently unknown, we generated mice carrying the conditional Trim33 ( Trim33 FX ) allele by flanking exons 2–4 encoding most of the functionally critical N-terminal tripartite domain by loxP sites. We confirmed the null genotype by using the EIIa-Cre transgenic approach to create mice that lack exons 2–4. Embryos deficient in Trim33 die during early somitogenesis, demonstrating that Trim33 plays an important nonredundant role in mammalian embryonic development. genesis 46:329–333, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60227/1/20401_ftp.pd

Smad4 and Trim33/Tif1γ Redundantly Regulate Neural Stem Cells in the Developing Cortex

During central nervous system (CNS) development, proliferation and differentiation of neural stem cells (NSCs) have to be regulated in a spatio-temporal fashion. Here, we report different branches of the transforming growth factor β (TGFβ) signaling pathway to be required for the brain area-specific control of NSCs. In the midbrain, canonical TGFβ signaling via Smad4 regulates the balance between proliferation and differentiation of NSCs. Accordingly, Smad4 deletion resulted in horizontal expansion of NSCs due to increased proliferation, decreased differentiation, and decreased cell cycle exit. In the developing cortex, however, ablation of Smad4 alone did not have any effect on proliferation and differentiation of NSCs. In contrast, concomitant mutation of both Smad4 and Trim33 led to an increase in proliferative cells in the ventricular zone due to decreased cell cycle exit, revealing a functional redundancy of Smad4 and Trim33. Furthermore, in Smad4-Trim33 double mutant embryos, cortical NSCs generated an excess of deep layer neurons concurrent with a delayed and reduced production of upper layer neurons and, in addition, failed to undergo the neurogenic to gliogenic switch at the right developmental stage. Thus, our data disclose that in different regions of the developing CNS different aspects of the TGFβ signaling pathway are required to ensure proper developmen

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

BACKGROUND: Congenital 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. RESULTS: We 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. CONCLUSION: Our results demonstrate that ALK5-mediated signaling in neural crest cells plays an essential cell-autonomous role in the pharyngeal and cardiac outflow tract development

Growth factor signaling in lung morphogenetic centers: automaticity, stereotypy and symmetry

Lung morphogenesis is stereotypic, both for lobation and for the first several generations of airways, implying mechanistic control by a well conserved, genetically hardwired developmental program. This program is not only directed by transcriptional factors and peptide growth factor signaling, but also co-opts and is modulated by physical forces. Peptide growth factors signal within repeating epithelial-mesenchymal temporospatial patterns that constitute morphogenetic centers, automatically directing millions of repetitive events during both stereotypic branching and nonstereotypic branching as well as alveolar surface expansion phases of lung development. Transduction of peptide growth factor signaling within these centers is finely regulated at multiple levels. These may include ligand expression, proteolytic activation of latent ligand, ligand bioavailability, ligand binding proteins and receptor affinity and presentation, receptor complex assembly and kinase activation, phosphorylation and activation of adapter and messenger protein complexes as well as downstream events and cross-talk both inside and outside the nucleus. Herein we review the critical Sonic Hedgehog, Fibroblast Growth Factor, Bone Morphogenetic Protein, Vascular Endothelial Growth Factor and Transforming Growth Factorβ signaling pathways and propose how they may be functionally coordinated within compound, highly regulated morphogenetic gradients that drive first stereotypic and then non-stereotypic, automatically repetitive, symmetrical as well as asymmetrical branching events in the lung

Generation of mice with a conditional allele for the p75 NTR neurotrophin receptor gene

The p75 NTR neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75 NTR , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75 NTR (p75 NTR‐FX ) allele made by flanking Exons 4–6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest‐specific p75 NTR /Wnt1‐Cre mutants and conventional p75 NTR null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75 NTR in neural crest‐derived cells causes a peripheral neuropathy similar to that seen in conventional p75 NTR mutants. This novel conditional p75 NTR allele will offer new opportunities to investigate the role of p75 NTR in specific tissues and cells. genesis 49:862–869, 2011. © 2011 Wiley‐Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88029/1/20747_ftp.pd

Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart

AbstractDevelopmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-β superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-β superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-β Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis

TiF1-Gamma Plays an Essential Role in Murine Hematopoiesis and Regulates Transcriptional Elongation of Erythroid Genes

Transcriptional regulators play critical roles in the regulation of cell fate during hematopoiesis. Previous studies in zebrafish have identified an essential role for the transcriptional intermediary factor TIF1γ in erythropoiesis by regulating the transcription elongation of erythroid genes. To study if TIF1γ plays a similar role in murine erythropoiesis and to assess its function in other blood lineages, we generated mouse models with hematopoietic deletion of TIF1γ. Our results showed a block in erythroid maturation in the bone marrow following tif1γ deletion that was compensated with enhanced spleen erythropoiesis. Further analyses revealed a defect in transcription elongation of erythroid genes in the bone marrow. In addition, loss of TIF1γ resulted in defects in other blood compartments, including a profound loss of B cells, a dramatic expansion of granulocytes and decreased HSC function. TIF1γ exerts its functions in a cell-autonomous manner as revealed by competitive transplantation experiments. Our study therefore demonstrates that TIF1γ plays essential roles in multiple murine blood lineages and that its function in transcription elongation is evolutionally conserved.Stem Cell and Regenerative Biolog

Glycosaparaginase from human leukocytes. Inactivation and covalent modification with diazo-oxonorvaline

The apparent active site of human leukocyte glycoasparaginase (N4-(beta-acetylglucosaminyl)-L-asparaginase EC 3.5.1.26) has been studied by labeling with an asparagine analogue, 5-diazo-4-oxo-L-norvaline. Glycoasparaginase was purified 4,600-fold from human leukocytes with an overall recovery of 12%. The purified enzyme has a Km of 110 microM, a Vmax of 34 mumol x l^-1 x min^-1, and a specific activity of 2.2 units/mg protein with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. The carbohydrate content of the enzyme is 15%, and it exhibits a broad pH maximum between 7 and 9. The 88-kDa native enzyme is composed of 19- kDa light (L) chains and 25-kDa heavy (H) chains and it has a heterotetrameric structure of L2H2-type. The glycoasparaginase activity decreases rapidly and irreversibly in the presence of 5-diazo-4-oxo-L- norvaline. At any one concentration of the compound, the inactivation of the enzyme is pseudo-first-order with time. The inhibitory constant, K1, is 80 microM and the second-order rate constant 1.25 x 10^(3) M^-1 min^-1 at pH 7.5. The enzyme activity is competitively protected against this inactivation by its natural substrate, aspartylglucosamine, indicating that this inhibitor binds to the active site or very close to it. The covalent incorporation of [5-14C]diazo-4-oxo-L-norvaline paralleled the loss of the enzymatic activity and one inhibitor binding site was localized to each L-subunit of the heterotetrameric enzyme. Four peptides with the radioactive label were generated, purified by high performance liquid chromatography, and sequenced by Edman degradation. The sequences were overlapping and all contained the amino-terminal tripeptide of the L-chain. By mass spectrometry, the reacting group of 5-diazo-4-oxo-L-norvaline was characterized as 4-oxo-L- norvaline that was bound through an alpha-ketone ether linkage to the hydroxyl group of the amino-terminal amino acid threonine

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein–Induced Retinal AngiogenesisHighlights

OBJECTIVE: Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2, and Alk3 in mouse retinal vessels. APPROACH AND RESULTS: Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2. Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. CONCLUSIONS: Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels

The induction and function of the anti-inflammatory fate of TH17 cells

TH17 cells exemplify environmental immune adaptation: they can acquire both a pathogenic and an anti-inflammatory fate. However, it is not known whether the anti-inflammatory fate is merely a vestigial trait, or whether it serves to preserve the integrity of the host tissues. Here we show that the capacity of TH17 cells to acquire an anti-inflammatory fate is necessary to sustain immunological tolerance, yet it impairs immune protection against S. aureus. Additionally, we find that TGF-β signalling via Smad3/Smad4 is sufficient for the expression of the anti-inflammatory cytokine, IL-10, in TH17 cells. Our data thus indicate a key function of TH17 cell plasticity in maintaining immune homeostasis, and dissect the molecular mechanisms explaining the functional flexibility of TH17 cells with regard to environmental changes.Fil: Xu, Hao. University of Yale. School of Medicine; Estados UnidosFil: Agalioti, Theodora. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Zhao, Jun. University of Yale. School of Medicine; Estados UnidosFil: Steglich, Babett. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Wahib, Ramez. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Amezcua Vesely, Maria Carolina. University of Yale. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Bielecki, Piotr. University of Yale. School of Medicine; Estados UnidosFil: Bailis, Will. University of Yale. School of Medicine; Estados UnidosFil: Jackson, Ruaidhri. University of Yale. School of Medicine; Estados UnidosFil: Perez, Daniel. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Izbicki, Jakob. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Licona-Limón, Paula. University of Yale. School of Medicine; Estados UnidosFil: Kaartinen, Vesa. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Geginat, Jens. University Medical Center Hamburg-Eppendorf; AlemaniaFil: Esplugues, Enric. University of Yale. School of Medicine; Estados UnidosFil: Tolosa, Eva. University of Yale. School of Medicine; Estados UnidosFil: Huber, Samuel. University of Yale. School of Medicine; Estados UnidosFil: Flavell, Richard A.. University of Yale. School of Medicine; Estados UnidosFil: Gagliani, Nicola. University Medical Center Hamburg-Eppendorf; Alemani