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Introduction: Heart regeneration has been proposed as a treatment for heart failure. However, the challenge of detecting bona fide heart regeneration has complicated the validation of potential regenerative factors. We sought to develop a method for quantifying cardiomyogenesis in the mouse heart using multicolor lineage tracing. Hypothesis: We hypothesized that multicolor lineage tracing in combination with modeling of nearest neighbor distances could be used to quantify clonal expansion of cardiomyocytes (CMs) during growth and regeneration. Methods: Myh6- MerCreMer ; R26R-Rainbow bitransgenic mice underwent cryoinjury (CI) or sham injury on postnatal day 1 (P1). Tamoxifen was then administered such that limited numbers of CMs were randomly labeled with mCerulean, mOrange, or mCherry fluorophores. Hearts were collected at 21 days post CI. We analyzed a total of 81 sections, across 6 hearts (3 with sham injury and 3 with cryoinury). Images of entire cardiac sections were manually segmented to generate positional information for each labeled CM. Results: We used the nearest neighbor distributions of CMs carrying the same fluorophore and CMs carrying different fluorophores to develop a Bayesian model for estimating the probability that two CMs are clonally related. We found that, across a range of recombination efficiency, CMs carrying the same fluorophore within 30 μm are likely to be clonally related. By classifying CMs based on this distance threshold, we were able to confirm that clonally related CMs are smaller than non-clonally related CMs (165.20 ± 13.79 μm 2 vs 252.37 ± 20.51 μm 2 , p = 0.007, Welch t-test). We also confirmed that rates of cardiomyogenesis in cryoinjured and sham injured hearts are approximately the same (8.57 ± 2.69 % in cryoinjured hearts vs 9.45 ± 1.23 % in sham hearts, p=0.788). Conclusions: We have developed a robust and objective approach for quantifying cardiomyogenesis in the mouse heart. This method can be applied to studies of heart regeneration and can also be adapted to other tissues.

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting bona fide heart regeneration limits the validation of potential regenerative factors. One method to detect new cardiomyocytes is multicolor lineage tracing with clonal analysis. Clonal analysis experiments can be difficult to undertake, because labeling conditions that are too sparse lack sensitivity for rare events such as cardiomyocyte proliferation, and diffuse labeling limits the ability to resolve clones. Presented here is a protocol to undertake clonal analysis of the neonatal mouse heart by using statistical modeling of nearest neighbor distributions to resolve cardiomyocyte clones. This approach enables resolution of clones over a range of labeling conditions and provides a robust analytical approach for quantifying cardiomyocyte proliferation and regeneration. This protocol can be adapted to other tissues and can be broadly used to study tissue regeneration.

Metastatic clear-cell renal cell carcinoma (ccRCC) affects thousands of patients worldwide each year. Antiangiogenic therapy has been shown to have beneficial effects initially, but resistance is eventually developed. Therefore, it is important to accurately track the response of cancer to different therapeutics in order to appropriately adjust the therapy to maximize efficacy. Change in tumor volume is the current gold standard for determining efficacy of treatment. However, functional variations can occur much earlier than measurable volume changes. Contrast-enhanced ultrasound (CEUS) is an important tool for assessing tumor progression and response to therapy, since it can monitor functional changes in the physiology. In this study, we demonstrate how ultrasound molecular imaging (USMI) can accurately track the evolution of the disease and molecular response to treatment. Methods A cohort of NSG (NOD/scid/gamma) mice was injected with ccRCC cells and treated with either the VEGF inhibitor SU (Sunitinib malate, Selleckchem, TX, USA) or the Notch pathway inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY, USA), or started on SU and later switched to GSI (Switch group). The therapies used in the study focus on disrupting angiogenesis and proper vessel development. SU inhibits signaling of vascular endothelial growth factor (VEGF), which is responsible for the sprouting of new vasculature, and GSI inhibits the Notch pathway, which is a key factor in the correct maturation of newly formed vasculature. Microbubble contrast agents targeted to VEGFR-2 (VEGF Receptor) were delivered as a bolus, and the bound agents were imaged in 3D after the free-flowing contrast was cleared from the body. Additionally, the tumors were harvested at the end of the study and stained for CD31. Results The results show that MI can detect changes in VEGFR-2 expression in the group treated with SU within a week of the start of treatment, while differences in volume only become apparent after the mice have been treated for three weeks. Furthermore, USMI can detect response to therapy in 92% of cases after 1 week of treatment, while the detection rate is only 40% for volume measurements. The amount of targeting for the GSI and Control groups was high throughout the duration of the study, while that of the SU and Switch groups remained low. However, the amount of targeting in the Switch group increased to levels similar to those of the Control group after the treatment was switched to GSI. CD31 staining indicates significantly lower levels of patent vasculature for the SU group compared to the Control and GSI groups. Therefore, the results parallel the expected physiological changes in the tumor, since GSI promotes angiogenesis through the VEGF pathway, while SU inhibits it. Conclusion This study demonstrates that MI can track disease progression and assess functional changes in tumors before changes in volume are apparent, and thus, CEUS can be a valuable tool for assessing response to therapy in disease. Future work is required to determine whether levels of VEGFR-2 targeting correlate with eventual survival outcomes.

Supplemental Digital Content is available in the text., Objective— Wound healing is accompanied by neoangiogenesis, and new vessels are thought to originate primarily from the microcirculation; however, how these vessels form and resolve during wound healing is poorly understood. Here, we investigated properties of the smallest capillaries during wound healing to determine their spatial organization and the kinetics of formation and resolution. Approach and Results— We used intravital imaging and high-resolution microscopy to identify a new type of vessel in wounds, called tortuous microvessels. Longitudinal studies showed that tortuous microvessels increased in frequency after injury, normalized as the wound healed, and were closely associated with the wound site. Tortuous microvessels had aberrant cell shapes, increased permeability, and distinct interactions with circulating microspheres, suggesting altered flow dynamics. Moreover, tortuous microvessels disproportionately contributed to wound angiogenesis by sprouting exuberantly and significantly more frequently than nearby normal capillaries. Conclusions— A new type of transient wound vessel, tortuous microvessels, sprout dynamically and disproportionately contribute to wound-healing neoangiogenesis, likely as a result of altered properties downstream of flow disturbances. These new findings suggest entry points for therapeutic intervention.

The BMP pathway regulates developmental processes including angiogenesis, yet its signaling outputs are complex and context-dependent. Recently, we showed that SMAD6, an intracellular BMP inhibitor expressed in endothelial cells, decreases vessel sprouting and branching both in vitro and in zebrafish. Genetic deletion of SMAD6 in mice results in poorly characterized cardiovascular defects and lethality. Here, we analyzed the effects of SMAD6 loss on vascular function during murine development. SMAD6 was expressed in a subset of blood vessels throughout development, primarily in arteries, while expression outside of the vasculature was largely confined to developing cardiac valves with no obvious embryonic phenotype. Mice deficient in SMAD6 died during late gestation and early stages of postnatal development, and this lethality was associated with vessel hemorrhage. Mice that survived past birth had increased branching and sprouting of developing postnatal retinal vessels and disorganized tight and adherens junctions. In vitro, knockdown of SMAD6 led to abnormal endothelial cell adherens junctions and increased VE-cadherin endocytosis, indicative of activated endothelium. Thus, SMAD6 is essential for proper blood vessel function during murine development, where it appears to stabilize endothelial junctions to prevent hemorrhage and aberrant angiogenesis.

Background: Functional and molecular changes often precede gross anatomical changes, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. Objective: Our intent was to test the hypothesis that an ultrasound microvascular imaging approach might provide indications of response to therapy prior to assessment of tumor size. Methods: Mice bearing clear-cell renal cell carcinoma xenograft tumors were treated with antiangiogenic and Notch inhibition therapies. An ultrasound measurement of microvascular density was used to serially track the tumor response to therapy. Results: Data indicated that ultrasound-derived microvascular density can indicate response to therapy a week prior to changes in tumor volume and is strongly correlated with physiological characteristics of the tumors as measured by histology ( $\rho = {\text{0.75}}$ ). Furthermore, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups with high sensitivity and specificity. Conclusion/Significance: Results suggests that future applications utilizing ultrasound imaging to monitor tumor response to therapy may be able to provide earlier insight into tumor behavior from metrics of microvascular density rather than anatomical tumor size measurements.

The vertebrate pancreas is comprised of a highly branched tubular epithelium, which is intimately associated with an extensive and specialized vasculature. While we know a great deal about basic vascular anatomy of the adult pancreas, as well as islet capillaries, surprisingly little is known about the ontogeny of its blood vessels. Here, we analyze development of the pancreatic vasculature in the mouse embryo. We show that pancreatic epithelial branches intercalate with the fine capillary plexus of the surrounding pancreatic mesenchyme. Endothelial cells (ECs) within this mesenchyme are heterogeneous from the onset of organogenesis. Pancreatic arteries take shape before veins, in a manner analogous to early embryonic vessels. The main central artery forms during mid-gestation, as a result of vessel coalescence and remodeling of a vascular plexus. In addition, we show that vessels in the forming pancreas display a predictable architecture that is dependent on VEGF signaling. Over-expression of VEGF disrupts vascular patterning and arteriovenous differentiation within the developing pancreas. This study constitutes a first-time in-depth cellular and molecular characterization of pancreatic blood vessels, as they coordinately grow along with the pancreatic epithelium.

Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation., The mechanism underlying endothelial cell responses to BMP signals is unknown. Here, the authors show that the endothelial response to pro-angiogenic BMP ligands is regulated by Notch via its effect on SMAD6, a known inhibitor of BMP intracellular signaling cascade.

SummaryCardiovascular function depends on patent blood vessel formation by endothelial cells (ECs). However, the mechanisms underlying vascular “tubulogenesis” are only beginning to be unraveled. We show that endothelial tubulogenesis requires the Ras interacting protein 1, Rasip1, and its binding partner, the RhoGAP Arhgap29. Mice lacking Rasip1 fail to form patent lumens in all blood vessels, including the early endocardial tube. Rasipl null angioblasts fail to properly localize the polarity determinant Par3 and display defective cell polarity, resulting in mislocalized junctional complexes and loss of adhesion to extracellular matrix (ECM). Similarly, depletion of either Rasip1 or Arhgap29 in cultured ECs blocks in vitro lumen formation, fundamentally alters the cytoskeleton, and reduces integrin-dependent adhesion to ECM. These defects result from increased RhoA/ROCK/myosin II activity and blockade of Cdc42 and Rac1 signaling. This study identifies Rasip1 as a unique, endothelial-specific regulator of Rho GTPase signaling, which is essential for blood vessel morphogenesis.

During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial-haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells.

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.

Arteriovenous (AV) differentiation is a critical step during blood vessel formation and stabilization. Defects in arterial or venous fate lead to inappropriate fusion of vessels, resulting in damaging arteriovenous shunts. While many studies have unraveled the molecular underpinnings that drive AV fate, surprisingly, the spatiotemporal emergence of arteries and veins in mammalian embryos remains unknown. Here, we examine artery and vein specification and differentiation during vasculogenesis. We show that the first intraembryonic vessels formed are arteries, which differentiate in a stepwise manner. By contrast, veins emerge later, progressively forming after embryonic turning. In addition, we demonstrate that hemodynamic flow is not required for arterial specification, but is required for maintenance of select arterial markers. Together, our results provide a first spatiotemporal analysis of mammalian AV cell fate establishment and anatomy, as well as a delineation of a molecular toolkit for analysis of arteries and veins during early vessel development.

The mammalian pancreas is a highly branched gland, essential for both digestion and glucose homeostasis. Pancreatic branching, however, is poorly understood, both at the ultrastructural and cellular levels. In this article, we characterize the morphogenesis of pancreatic branches, from gross anatomy to the dynamics of their epithelial organization. We identify trends in pancreatic branch morphology and introduce a novel mechanism for branch formation, which involves transient epithelial stratification and partial loss of cell polarity, changes in cell shape and cell rearrangements, de novo tubulogenesis and epithelial tubule remodeling. In contrast to the classical epithelial budding and tube extension observed in other organs, a pancreatic branch takes shape as a multi-lumen tubular plexus coordinately extends and remodels into a ramifying, single-lumen ductal system. Moreover, our studies identify a role for EphB signaling in epithelial remodeling during pancreatic branching. Overall, these results illustrate distinct, step-wise cellular mechanisms by which pancreatic epithelium shapes itself to create a functional branching organ.

Cell–cell communication is critical for regulating embryonic organ growth and differentiation. The Bone Morphogenetic Protein (BMP) family of transforming growth factor β (TGFβ) molecules represents one class of such cell–cell signaling molecules that regulate the morphogenesis of several organs. Due to high redundancy between the myriad BMP ligands and receptors in certain tissues, it has been challenging to address the role of BMP signaling using targeting of single Bmp genes in mouse models. Here, we present a detailed study of the developmental expression profiles of three BMP ligands (Bmp2, Bmp4, Bmp7) and three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (noggin), in the early embryo during the initial steps of murine organogenesis. In particular, we focus on the expression of Bmp family members in the first organs and tissues that take shape during embryogenesis, such as the heart, vascular system, lungs, liver, stomach, nervous system, somites and limbs. Using in situ hybridization, we identify domains where ligand(s) and receptor(s) are either singly or co-expressed in specific tissues. In addition, we identify a previously unnoticed asymmetric expression of Bmp4 in the gut mesogastrium, which initiates just prior to gut turning and the establishment of organ asymmetry in the gastrointestinal tract. Our studies will aid in the future design and/or interpretation of targeted deletion of individual Bmp or Bmpr genes, since this study identifies organs and tissues where redundant BMP signaling pathways are likely to occur.

Ras proteins are small GTPases that regulate cellular growth and differentiation. Components of the Ras signaling pathway have been shown to be important during embryonic vasculogenesis and angiogenesis. Here, we report that Rasip1, which encodes a novel Ras-interacting protein, is strongly expressed in vascular endothelial cells throughout development, in both mouse and frog. Similar to the well-characterized vascular markers VEGFR2 and PECAM, Rasip1 is specifically expressed in angioblasts prior to vessel formation, in the initial embryonic vascular plexus, in the growing blood vessels during angiogenesis and in the endothelium of mature blood vessels into the postnatal period. Rasip1 expression is undetectable in VEGFR2 null embryos, which lack endothelial cells, suggesting that Rasip1 is endothelial specific. siRNA-mediated reduction of Rasip1 severely impairs angiogenesis and motility in endothelial cell cultures, and morpholino knockdown experiments in frog embryos demonstrate that Rasip1 is required for embryonic vessel formation in vivo. Together, these data identify Rasip1 as a novel endothelial factor that plays an essential role in vascular development.

Ngn3 is a bHLH transcription factor critical for the specification of endocrine cells in the pancreatic Islets of Langerhans. Previous studies in mouse embryos have reported transient expression of Ngn3 in scattered cells within the developing pancreatic epithelium during midgestation (Schwitzgebel et al. [2000] Development 127:3533–3542). Specifically, these Ngn3-expressing cells have been shown to be progenitor cells fated to give rise to islet endocrine cells (Gradwohl et al. [2000] Proc Natl Acad Sci USA 97:1607–1611). Here, we characterize the expression of Ngn3 transcripts and protein throughout pancreatic development. Interestingly, we identify and define a dramatic and previously unnoticed gap in developmental Ngn3 expression. We show that both Ngn3 transcript and protein expression occur in two distinct temporal waves, the first occurring early from approximately E8.5 to E11.0, and the second initiating at approximately E12.0. Strikingly, this observed biphasic expression correlates with the “first” and “second” transitions, which encompass two distinct waves of embryonic endocrine differentiation. In addition, our studies demonstrate that Ngn3 transcripts are markedly more widespread in the pancreatic epithelium than NGN3 protein, indicating that post-transcriptional regulation is likely to play a critical role during endocrine differentiation. Developmental Dynamics 237:3270–3279, 2008. © 2008 Wiley-Liss, Inc.