FOXO1 represses sprouty 2 and sprouty 4 expression to promote arterial specification and vascular remodeling in the mouse yolk sac.

Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.

The effects of reduced hemodynamic loading on morphogenesis of the mouse embryonic heart.

Development of the embryonic heart involves an intricate network of biochemical and genetic cues to ensure its proper growth and morphogenesis. However, studies from avian and teleost models reveal that biomechanical force, namely hemodynamic loading (blood pressure and shear stress), plays a significant role in regulating heart development. To study how hemodynamic loading impacts development of the mammalian embryonic heart, we utilized mouse embryo culture and manipulation techniques and performed optical projection tomography imaging followed by morphometric analysis to determine how reduced-loading affects heart volume, myocardial thickness, trabeculation and looping. Our results reveal that hemodynamic loading can regulate these features at different thresholds. Intermediate levels of hemodynamic loading are sufficient to promote proper myocardial growth and heart size, but insufficient to promote looping and trabeculation. Whereas, low levels of hemodynamic loading fails to promote proper growth of the myocardium and heart size. These results reveal that the regulation of heart development by biomechanical force is conserved across many vertebrate classes, and this study begins to elucidate how these specific forces regulate development of the mammalian heart.

Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy.

Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as post-implantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.

Dynamic responses of endothelial cells to changes in blood flow during vascular remodeling of the mouse yolk sac.

Despite extensive work showing the importance of blood flow in angiogenesis and vessel remodeling, very little is known about how changes in vessel diameter are orchestrated at the cellular level in response to mechanical forces. To define the cellular changes necessary for remodeling, we performed live confocal imaging of cultured mouse embryos during vessel remodeling. Our data revealed that vessel diameter increase occurs via two distinct processes that are dependent on normal blood flow: vessel fusions and directed endothelial cell migrations. Vessel fusions resulted in a rapid change in vessel diameter and were restricted to regions that experience the highest flow near the vitelline artery and vein. Directed cell migrations induced by blood flow resulted in the recruitment of endothelial cells to larger vessels from smaller capillaries and were observed in larger artery segments as they expanded. The dynamic and specific endothelial cell behaviors captured in this study reveal how sensitive endothelial cells are to changes in blood flow and how such responses drive vascular remodeling.

Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway.

Proliferation and apoptosis must be precisely regulated to form organs with appropriate cell numbers and to avoid tumour growth. Here we show that Hippo (Hpo), the Drosophila homologue of the mammalian Ste20-like kinases, MST1/2, promotes proper termination of cell proliferation and stimulates apoptosis during development. hpo mutant tissues are larger than normal because mutant cells continue to proliferate beyond normal tissue size and are resistant to apoptotic stimuli that usually eliminate extra cells. Hpo negatively regulates expression of Cyclin E to restrict cell proliferation, downregulates the Drosophila inhibitor of apoptosis protein DIAP1, and induces the proapoptotic gene head involution defective (hid) to promote apoptosis. The mutant phenotypes of hpo are similar to those of warts (wts), which encodes a serine/threonine kinase of the myotonic dystrophy protein kinase family, and salvador (sav), which encodes a WW domain protein that binds to Wts. We find that Sav binds to a regulatory domain of Hpo that is essential for its function, indicating that Hpo acts together with Sav and Wts in a signalling module that coordinately regulates cell proliferation and apoptosis.

Visualizing Blood Vessel Development in Cultured Mouse Embryos Using Lightsheet Microscopy.

Lightsheet microscopy is a form of fluorescence microscopy that can be used to visualize specimen with high resolution, a large depth-of-field, and minimal photodamage and photobleaching as compared to traditional confocal microscopy. As this technology becomes much more readily available, it will be useful in revealing new findings in the cardiovascular development field that may be hidden or difficult to image. In this manuscript, we describe an approach for mounting and culturing postimplantation mouse embryos to visualize blood vessel development with a lightsheet microscope.

Hemodynamic force is required for vascular smooth muscle cell recruitment to blood vessels during mouse embryonic development.

Blood vessel maturation, which is characterized by the investment of vascular smooth muscle cells (vSMCs) around developing blood vessels, begins when vessels remodel into a hierarchy of proximal arteries and proximal veins that branch into smaller distal capillaries. The ultimate result of maturation is formation of the tunica media-the middlemost layer of a vessel that is composed of vSMCs and acts to control vessel integrity and vascular tone. Though many studies have implicated the role of various signaling molecules in regulating maturation, no studies have determined a role for hemodynamic force in the regulation of maturation in the mouse. In the current study, we provide evidence that a hemodynamic force-dependent mechanism occurs in the mouse because reduced blood flow mouse embryos exhibited a diminished or absent coverage of vSMCs around vessels, and in normal-flow embryos, extent of coverage correlated to the amount of blood flow that vessels were exposed to. We also determine that the cellular mechanism of force-induced maturation was not by promoting vSMC differentiation/proliferation, but instead involved the recruitment of vSMCs away from neighboring low-flow distal capillaries towards high-flow vessels. Finally, we hypothesize that hemodynamic force may regulate expression of specific signaling molecules to control vSMC recruitment to high-flow vessels, as reduction of flow results in the misexpression of Semaphorin 3A, 3F, 3G, and the Notch target gene Hey1, all of which are implicated in controlling vessel maturation. This study reveals another role for hemodynamic force in regulating blood vessel development of the mouse, and opens up a new model to begin elucidating mechanotransduction pathways regulating vascular maturation.

Cardiovascular Patterning as Determined by Hemodynamic Forces and Blood Vessel Genetics.

BACKGROUND: Vascular patterning depends on coordinated timing of arteriovenous specification of endothelial cells and the concomitant hemodynamic forces supplied by the onset of cardiac function. Using a combination of 3D imaging by OPT and embryo registration techniques, we sought to identify structural differences between three different mouse models of cardiovascular perturbation. RESULTS: Endoglin mutant mice shared a high degree of similarity to Mlc2a mutant mice, which have been shown to have a primary developmental heart defect causing secondary vessel remodeling failures. Dll4 mutant mice, which have well-characterized arterial blood vessel specification defects, showed distinct differences in vascular patterning when compared to the disruptions seen in Mlc2a-/- and Eng-/- models. While Mlc2a-/- and Eng-/- embryos exhibited significantly larger atria than wild-type, Dll4-/- embryos had significantly smaller hearts than wild-type, but this quantitative volume decrease was not limited to the developing atrium. Dll4-/- embryos also had atretic dorsal aortae and smaller trunks, suggesting that the cardiac abnormalities were secondary to primary arterial blood vessel specification defects. CONCLUSIONS: The similarities in Eng-/- and Mlc2a-/- embryos suggest that Eng-/- mice may suffer from a primary heart developmental defect and secondary defects in vessel patterning, while defects in Dll4-/- embryos are consistent with primary defects in vessel patterning.

Understanding vascular development.

The vasculature of an organism has the daunting task of connecting all the organ systems to nourish tissue and sustain life. This complex network of vessels and associated cells must maintain blood flow, but constantly adapt to acute and chronic changes within tissues. While the vasculature has been studied for over a century, we are just beginning to understand the processes that regulate its formation and how genetic hierarchies are influenced by mechanical and metabolic cues to refine vessel structure and optimize efficiency. As we gain insights into the developmental mechanisms, it is clear that the processes that regulate blood vessel development can also enable the adult to adapt to changes in tissues that can be elicited by exercise, aging, injury, or pathology. Thus, research in vessel development has provided tremendous insights into therapies for vascular diseases and disorders, cancer interventions, wound repair and tissue engineering, and in turn, these models have clearly impacted our understanding of development. Here we provide an overview of the development of the vascular system, highlighting several areas of active investigation and key questions that remain to be answered.

The ebb and flow of lymphatic valve formation.

Recently, substantial advances have been made in understanding the formation and remodeling of the lymphatic vasculature. In this issue, Sabine et al. (2012) further define the mechanisms of lymphatic valve formation and implicate oscillatory shear stress in regulating the molecular events that control valve morphogenesis.

Transcription factor FoxO1 is essential for enamel biomineralization.

The Transforming growth factor ß (Tgf-ß) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-ß signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta.

Imaging mouse embryonic development.

For the past three decades, methods for culturing mouse embryos ex vivo have been optimized in order to improve embryo viability and physiology throughout critical stages of embryogenesis. Combining advances made in the production of transgenic animals and in the development of different varieties of fluorescent proteins (FPs), time-lapse imaging is becoming more and more popular in the analysis of dynamic events during mouse development. Targeting FPs to specific cell types or subcellular compartments has enabled researchers to study cell proliferation, apoptosis, migration, and changes in cell morphology in living mouse embryos in real time. Here we provide a guide for time-lapse imaging of early stages of mouse embryo development.

Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.

The Hippo pathway plays a key role in organ size control by regulating cell proliferation and apoptosis in Drosophila. Although recent genetic studies have shown that the Hippo pathway is regulated by the NF2 and Fat tumor suppressors, the physiological regulations of this pathway are unknown. Here we show that in mammalian cells, the transcription coactivator YAP (Yes-associated protein), is inhibited by cell density via the Hippo pathway. Phosphorylation by the Lats tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP oncoprotein. Furthermore, attenuation of this phosphorylation of YAP or Yorkie (Yki), the Drosophila homolog of YAP, potentiates their growth-promoting function in vivo. Moreover, YAP overexpression regulates gene expression in a manner opposite to cell density, and is able to overcome cell contact inhibition. Inhibition of YAP function restores contact inhibition in a human cancer cell line bearing deletion of Salvador (Sav), a Hippo pathway component. Interestingly, we observed that YAP protein is elevated and nuclear localized in some human liver and prostate cancers. Our observations demonstrate that YAP plays a key role in the Hippo pathway to control cell proliferation in response to cell contact.