Andre Nagy

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.

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.

Vestibular dysgenesis in mice lacking Abr and Bcr Cdc42/RacGAPs

The inner ear develops from a simple epithelium (otic placode) into the complex structures specialized for balance (vestibule) and sound (cochlea) detection. Abnormal vestibular and cochlear development is associated with many birth defects. During recent years, considerable progress has been made in understanding the molecular bases of these conditions. To determine the biological function of two closely related GTPase activating proteins for the Cdc42/Rac GTPases, Abr and Bcr, we generated a mouse strain deficient in both of these proteins. Double null mutant mice exhibit hyperactivity, persistent circling, and are unable to swim. These phenotypes are typically found in mice with vestibular defects. Indeed, adult double null mutants display abnormal dysmorphic structures of both the saccule and utricle. Moreover, a total loss of otoconia can be seen in the utricle, whereas in the saccule, otoconia are either missing or their number is drastically decreased and they are abnormally large. Interestingly, both the cochlea and semicircular canals are normal and the double null mutant mice are not deaf. These data demonstrate that Abr and Bcr play important complementary roles during vestibular morphogenesis and that a function of Cdc42/RacGAPs and, therefore, that of the small Rho‐related GTPases is critically important for balance and motor coordination.

Targeted Expression of Activated Rac3 in Mammary Epithelium Leads to Defective Postlactational Involution and Benign Mammary Gland Lesions

Rac3, a novel member of the Rho subfamily of the small GTPases, is frequently activated in cultured breast cancer cells and has been shown to mediate its effect via the p21-activated kinase (Pak) pathway. In order to evaluate these findings in vivo, we generated transgenic mice that express human constitutively active V12Rac under the control of the mouse mammary tumor virus (MMTV) promoter, which targets the transgene expression to the mammary epithelium. V12Rac3 expression could be detected during the first pregnancy, and the transgenic mammary gland tissues displayed an elevated Pak1 phosphorylation. Although milk proteins, β-casein and whey acidic protein were expressed and milk fat globules accumulated normally during pregnancy, 60% of transgenic mothers failed to nurse their pups. Surprisingly, although full lactational differentiation was never achieved in transgenic mice, gland involution was incomplete. For 5 days after weaning, involution was normal, but thereafter, epithelial islands characteristic of this early stage of involution persisted for months. The apoptotic index decreased after 5 days, and these glands were associated with increased p38 MAPK phosphorylation. Nine months postpartum, the transgenic mammary glands still demonstrated a large amount of persistent epithelial islands and abnormally large ducts with lymphocyte infiltration, whereas the tissues of non-transgenic controls had returned to their normal ‘virgin-like’ phenotype. These data show that sustained activation of Rac3 in the mammary epithelium leads to impaired mammary gland physiology and results in the formation of mammary gland lesions.

BMP type I receptor ALK2 is essential for proper patterning at late gastrulation during mouse embryogenesis.

Bone morphogenetic proteins (BMPs) have multiple functions during vertebrate development. Previously, it was shown that BMP type I receptor ALK2 (also known as ACVRI, ActRI, or ActRIA) was important for normal mouse gastrulation by deleting exon 4 or exon 5 of Alk2. Recently, flanking exon 7 by loxP sites generated a conditional allele for Alk2. To assess whether the deletion of exon 7 causes functional null of ALK2, and does not produce a dominant negative form or a partially functional form of ALK2, we performed a comparative analysis between Alk2 homozygous mutant embryos with an exon 5 deletion (Alk2Δ5/Δ5) and embryos with an exon 7 deletion (Alk2Δ7/Δ7). Both Alk2Δ5/Δ5 and Alk2Δ7/Δ7 mutants showed identical morphological gastrulation defects. Histological examinations and molecular marker analyses revealed identical abnormal gastrulation phenotypes in Alk2Δ5/Δ5 and Alk2Δ7/Δ7 mutants. Although Fgf8 was expressed in the primitive streak of Alk2Δ5/Δ5 and Alk2Δ7/Δ7 mutants, Brachyury, Wnt3a, and Tbx6 were dramatically downregulated in Alk2Δ5/Δ5 and Alk2Δ7/Δ7 mutants. These results indicate that deletion of exon 7 for Alk2 leads to a functionally null mutation in vivo, and Alk2 is crucial for sustaining the proper gastrulation events in early mouse embryogenesis. Developmental Dynamics 236:512–517, 2007. Published 2006 Wiley‐Liss, Inc.

ABSTRACTS (pp 58–112)

Background: Gastrointestinal blood loss accounts for a significant part of chronic recurrent anemia. Conventional diagnostic work up includes esophagogastroduodenoscopy (EGD), colonoscopy and small bowel follow-through (SBF). Aim: To determine the diagnostic yield of CE in patients with chronic recurrent anemia without active gastrointestinal bleeding referred for CE in a tertiary center in Switzerland. Methods: CE is a minuscule color videocamera, light source, battery, transmitter and antenna all embedded in a swallowable capsule (M2A capsuleâ). Pictures are received through electrodes attached to the abdomen and stored on hard disk. Data are then transferred to a workstation and analysed (RAPID workstation). All pts referred to our institution for CE are enrolled in a prospective study evaluating acceptance, diagnostic yield and possible complications of the method. Pts referred for evaluation of chronic recurrent anemia were eligible. All patients had a normal SBF and no bleeding source detected in EGD and colonoscopy. Patients with known active bleeding were excluded. Other exclusion criteria were small bowel stenosis in SBF and patients with pacemakers or ICD. Results: 32 pts underwent CE from May 2002 until May 2003. Of those, 8 pts were referred for chronic anemia and included. There were 4 men (50%) and 4 women (50%). The mean age was 60 years (range 40– 78 years). The mean hemoglobin was 94 g/l (range 65–161 g/l), the mean MCV was 85 fl (range 76–93 fl). In 4 of these 8 pts pathologies were found in the small bowel accounting for the anemia. There were 2 pts with angiodysplasias, 1 pt with an ulcer in the ileum and 1 pt with multiple erosions in the distal small bowel. All of these lesions were deemed to be potential bleeding sources. The mean hemoglobin in those pts with bleeding sources detected by CE was 84 g/l compared to 105 g/l in those pts with normal CE. The diagnostic yield of CE in this group of pts with chronic recurrent anemia and a normal SBF was 50%. Conclusions: CE provides a meaningful diagnostic yield in patients with chronic recurrent anemia and normal EGD and colonoscopy and a normal SBF. The diagnostic yield seems to increase in pts with a lower hemoglobin.