WNT signaling affects gene expression in the ventral diencephalon and pituitary gland growth

We examined the role of WNT signaling in pituitary development by characterizing the pituitary phenotype of three WNT knockout mice and assessing the expression of WNT pathway components. Wnt5a mutants have expanded domains of Fgf10 and bone morphogenetic protein expression in the ventral diencephalon and a reduced domain of LHX3 expression in Rathke's pouch. Wnt4 mutants have mildly reduced cell differentiation, reduced POU1F1 expression, and mild anterior lobe hypoplasia. Wnt4 , Wnt5a double mutants exhibit an additive pituitary phenotype of dysmorphology and mild hypoplasia. Wnt6 mutants have no obvious pituitary phenotype. We surveyed WNT expression and identified transcripts for numerous Wnts , Frizzleds , and downstream pathway members in the pituitary and ventral diencephalon. These findings support the emerging model that WNT signaling affects the pituitary gland via effects on ventral diencephalon signaling, and suggest additional Wnt genes that are worthy of functional studies. Developmental Dynamics 237:1006–1020, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58083/1/21511_ftp.pd

A 3D iPSC-differentiation model identifies interleukin-3 as a regulator of early human hematopoietic specification

Hematopoietic development is spatiotemporally tightly regulated by defined cell-intrinsic and extrinsic modifiers. The role of cytokines has been intensively studied in adult hematopoiesis; however, their role in embryonic hematopoietic specification remains largely unexplored. Here, we used induced pluripotent stem cell (iPSC) technology and established a 3-dimensional, organoid-like differentiation system (hemanoid) maintaining the structural cellular integrity to evaluate the effect of cytokines on embryonic hematopoietic development. We show, that defined stages of early human hematopoietic development were recapitulated within the generated hemanoids. We identified KDR+/CD34high/CD144+/CD43-/CD45- hemato-endothelial progenitor cells (HEPs) forming organized, vasculature-like structures and giving rise to CD34low/CD144-/CD43+/CD45+ hematopoietic progenitor cells. We demonstrate that the endothelial to hematopoietic transition of HEPs is dependent on the presence of interleukin 3 (IL-3). Inhibition of IL-3 signalling blocked hematopoietic differentiation and arrested the cells in the HEP stage. Thus, our data suggest an important role for IL-3 in early human hematopoiesis by supporting the endothelial to hematopoietic transition of hemato-endothelial progenitor cells and highlight the potential of a hemanoid-based model to study human hematopoietic development

Genomic Targets of Brachyury (T) in Differentiating Mouse Embryonic Stem Cells

The T-box transcription factor Brachyury (T) is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse.Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5'-TCACACCT-3' in the vicinity of most Brachyury target genes. Rather, we have identified an (AC)(n) repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents.Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species

Brachyury and Related Tbx Proteins Interact with the Mixl1 Homeodomain Protein and Negatively Regulate Mixl1 Transcriptional Activity

Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity

Influence of antenatal physical exercise on haemodynamics in pregnant women: a flexible randomisation approach

Background: Normal pregnancy is associated with marked changes in haemodynamic function, however theinfluence and potential benefits of antenatal physical exercise at different stages of pregnancy and postpartumremain unclear. The aim of this study was therefore to characterise the influence of regular physical exercise onhaemodynamic variables at different stages of pregnancy and also in the postpartum period.Methods: Fifty healthy pregnant women were recruited and randomly assigned (2 × 2 × 2 design) to a land orwater-based exercise group or a control group. Exercising groups attended weekly classes from the 20th week ofpregnancy onwards. Haemodynamic assessments (heart rate, cardiac output, stroke volume, total peripheralresistance, systolic and diastolic blood pressure and end diastolic index) were performed using the Task Forcehaemodynamic monitor at 12–16, 26–28, 34–36 and 12 weeks following birth, during a protocol including posturalmanoeurvres (supine and standing) and light exercise.Results: In response to an acute bout of exercise in the postpartum period, stroke volume and end diastolic indexwere greater in the exercise group than the non-exercising control group (p = 0.041 and p = 0.028 respectively).Total peripheral resistance and diastolic blood pressure were also lower (p = 0.015 and p = 0.007, respectively) in theexercise group. Diastolic blood pressure was lower in the exercise group during the second trimester (p = 0.030).Conclusions: Antenatal exercise does not appear to substantially alter maternal physiology with advancinggestation, speculating that the already vast changes in maternal physiology mask the influences of antenatalexercise, however it does appear to result in an improvement in a woman’s haemodynamic function (enhancedventricular ejection performance and reduced blood pressure) following the end of pregnancy

Primordial germ cells: the first cell lineage or the last cells standing?

Embryos of many animal models express germ line determinants that suppress transcription and mediate early germ line commitment, which occurs before the somatic cell lineages are established. However, not all animals segregate their germ line in this manner. The ‘last cell standing’ model describes primordial germ cell (PGC) development in axolotls, in which PGCs are maintained by an extracellular signalling niche, and germ line commitment occurs after gastrulation. Here, we propose that this ‘stochastic’ mode of PGC specification is conserved in vertebrates, including non-rodent mammals. We postulate that early germ line segregation liberates genetic regulatory networks for somatic development to evolve, and that it therefore emerged repeatedly in the animal kingdom in response to natural selection

TROP2 Expressed in the Trunk of the Ureteric Duct Regulates Branching Morphogenesis during Kidney Development

TROP2, a cell surface protein structurally related to EpCAM, is expressed in various carcinomas, though its function remains largely unknown. We examined the expression of TROP2 and EpCAM in fetal mouse tissues, and found distinct patterns in the ureteric bud of the fetal kidney, which forms a tree-like structure. The tip cells in the ureteric bud proliferate to form branches, whereas the trunk cells differentiate to form a polarized ductal structure. EpCAM was expressed throughout the ureteric bud, whereas TROP2 expression was strongest at the trunk but diminished towards the tips, indicating the distinct cell populations in the ureteric bud. The cells highly expressing TROP2 (TROP2high) were negative for Ki67, a proliferating cell marker, and TROP2 and collagen-I were co-localized to the basal membrane of the trunk cells. TROP2high cells isolated from the fetal kidney failed to attach and spread on collagen-coated plates. Using MDCK cells, a well-established model for studying the branching morphogenesis of the ureteric bud, TROP2 was shown to inhibit cell spreading and motility on collagen-coated plates, and also branching in collagen-gel cultures, which mimic the ureteric bud's microenvironment. These results together suggest that TROP2 modulates the interaction between the cells and matrix and regulates the formation of the ureteric duct by suppressing branching from the trunk during kidney development

Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

<p>Abstract</p> <p>Background</p> <p>β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured <it>in vitro </it>using His-tag purified proteins and <it>in vivo </it>in a β-carotene-accumulating <it>E. coli </it>system.</p> <p>Results</p> <p>Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our <it>ab initio </it>model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326.</p> <p>Conclusions</p> <p>Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.</p

The T-box transcription factor Tbx18 maintains the separation of anterior and posterior somite compartments

The compartmentalization of somites along their anterior–posterior (AP) axis is pivotal to the segmental organization of the vertebrate axial skeleton and the peripheral nervous system. Anterior and posterior somite halves contribute to different vertebral elements. They are also characterized by different proliferation rates and properties with respect to neural crest cell migration and spinal nerve passage. AP-somite polarity is generated in the anterior presomitic mesoderm by Mesp2 and Delta/Notch signaling. Here, we demonstrate that maintenance of AP-somite polarity is mediated by the T-box transcription factor Tbx18. Mice deficient for Tbx18 show expansion of pedicles with transverse processes and proximal ribs, elements derived from the posterior lateral sclerotome. AP-somite polarity is established in Tbx18 mutant embryos but is not maintained. During somite maturation, posterior somite compartments expand most likely because of posterior cells invading the anterior somite half. In the anterior lateral sclerotome, Tbx18 acts as an antiapoptotic factor. Ectopic expression experiments suggest that Tbx18 can promote anterior at the expense of posterior somite compartments. In summary, Tbx18 appears to act downstream of Mesp2 and Delta/Notch signaling to maintain the separation of anterior and posterior somite compartments