Ximena Soto

Spib is required for primitive myeloid development in Xenopus

Vertebrate blood formation occurs in 2 spatially and temporally distinct waves, so-called primitive and definitive hematopoiesis. Although definitive hematopoiesis has been extensively studied, the development of primitive myeloid blood has received far less attention. In Xenopus, primitive myeloid cells originate in the anterior ventral blood islands, the equivalent of the mammalian yolk sac, and migrate out to colonize the embryo. Using fluorescence time-lapse video microscopy, we recorded the migratory behavior of primitive myeloid cells from their birth. We show that these cells are the first blood cells to differentiate in the embryo and that they are efficiently recruited to embryonic wounds, well before the establishment of a functional vasculature. Furthermore, we isolated spib, an ETS transcription factor, specifically expressed in primitive myeloid precursors. Using spib antisense morpholino knockdown experiments, we show that spib is required for myeloid specification, and, in its absence, primitive myeloid cells retain hemangioblast-like characteristics and fail to migrate. Thus, we conclude that spib sits at the top of the known genetic hierarchy that leads to the specification of primitive myeloid cells in amphibians.

C/EBPα initiates primitive myelopoiesis in pluripotent embryonic cells

The molecular mechanisms that underlie the development of primitive myeloid cells in vertebrate embryos are not well understood. Here we characterize the role of cebpa during primitive myeloid cell development in Xenopus. We show that cebpa is one of the first known hematopoietic genes expressed in the embryo. Loss- and gain-of-function studies show that it is both necessary and sufficient for the development of functional myeloid cells. In addition, we show that cebpa misexpression leads to the precocious induction of myeloid cell markers in pluripotent prospective ectodermal cells, without the cells transitioning through a general mesodermal state. Finally, we use live imaging to show that cebpa-expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds and phagocytose bacteria, and the ability to enter the circulation. Thus, C/EPBalpha is the first known single factor capable of initiating an entire myelopoiesis pathway in pluripotent cells in the embryo.

A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis

The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences.

Inositol kinase and its product accelerate wound healing by modulating calcium levels, Rho GTPases, and F-actin assembly

Wound healing is essential for survival. We took advantage of the Xenopus embryo, which exhibits remarkable capacities to repair wounds quickly and efficiently, to investigate the mechanisms responsible for wound healing. Previous work has shown that injury triggers a rapid calcium response, followed by the activation of Ras homolog (Rho) family guanosine triphosphatases (GTPases), which regulate the formation and contraction of an F-actin purse string around the wound margin. How these processes are coordinated following wounding remained unclear. Here we show that inositol-trisphosphate 3-kinase B (Itpkb) via its enzymatic product inositol 1,3,4,5-tetrakisphosphate (InsP4) plays an essential role during wound healing by modulating the activity of Rho family GTPases and F-actin ring assembly. Furthermore, we show that Itpkb and InsP4 modulate the speed of the calcium wave, which propagates from the site of injury into neighboring uninjured cells. Strikingly, both overexpression of itpkb and exogenous application of InsP4 accelerate the speed of wound closure, a finding that has potential implications in our quest to find treatments that improve wound healing in patients with acute or chronic wounds.

09-P045 C/EBP alpha initiates primitive myelopoiesis in pluripotent embryonic cells

The Prdm1/Blimp1 protein acts as a transcriptional repressor by recruiting co-repressors leading to direct repression and indirect activation of gene expression. Prdm1 is dynamically expressed during development and, strikingly, has been shown to control specific differentiation programs in each of the different domains studied to date. During heart formation, Prdm1 is expressed in the second heart field (SHF) that will contribute to the outflow tract (OFT) formation. In this context, Prdm1 is also expressed in the adjacent endoderm where it is required to support the growth of the mesenchymal cells of the branchial arches 2–6. As a result, Prdm1 mutant embryos display heart septation defects. In order to analyse a cell-autonomous function in the SHF, we carried out a conditional deletion of Prdm1 using two Cre lines: MesP1Cre (early cardiac mesoderm) and Mef2cCre (anterior heart field). Mesp1Cre conditional mutants die at birth and display defects in structures of the distal OFT, derived from the SHF: uneven semilunar valves, misalignment of the great arteries and aortic arch defects. Mef2cCre conditional mutants are viable, but display a retrooesophageal subclavian artery, a defect also observed in Mesp1Cre mutants. Examination of pharyngeal arch artery (PAA) remodelling demonstrates that these phenotypes are linked with a defect in the formation of the PAA4. The difference in phenotypic severity between the two Cre lines used in this study suggests that Prdm1 plays an essential early regulatory role in mesodermal progenitor cells of the SHF that contribute to morphogenesis of the arterial pole of the heart.

13-P088 Study: The role and regulation of the cytoskeleton during myeloid chemotaxis

Breakdowns of the blood–brain barrier (BBB) are frequently associated with various central nervous system (CNS) diseases. Cerebrovascular endothelial cells (ECs) play important roles in maintaining a stable microenvironment for the CNS by interacting with pericytes. However, the genetic mechanisms controlling the functions of cerebral ECs are still largely unknown. Here, we report that disruption of Smad4, the central intracellular mediator of transforming growth factor-b (TGF-b) signaling, specifically in the cerebral ECs, results in intracranial hemorrhage and BBB breakdown due to defective endothelium–pericyte interaction. The molecular mechanisms underlying the function of Smad4-mediated TGF-b signals in stabilizing the cerebrovascular EC–pericyte interactions had been investigated. Our findings provide a new mechanistic link between TGF-b and Notch signaling pathways in an important physiological system, and have important implications for various CNS diseases related with cerebrovascular dysfunction. The cerebrovascular EC-specific Smad4 knockout mouse could serve as a unique and valuable model for further investigating the potential mechanisms of HHT pathology in brain.

19-P041 The role of IP3 signalling during embryonic wound healing in Xenopus

A major goal in regenerative medicine is to understand and ultimately facilitate our body’s ability to repair itself following injury. As a first step toward this goal, we have begun to investigate the molecular and cellular basis of embryonic wound healing, given that embryos have the capacity to heal wounds quickly and completely. Tissue repair resembles embryo morphogenesis in several ways, including cell migration, proliferation and differentiation. In a screen for genes involved in morphogenesis in Xenopus, we identified an Ins(1,4,5)P3 phosphatase (IPP), which impairs embryonic wound healing, suggesting that IP3 signalling plays a role in this process. This finding has motivated us to investigate the role of IP3 signalling during embryonic wound healing, using Xenopus as a model system. Two sets of enzymes regulate this pathway, the IP3 kinases and IPPs. IPP, such as Ins(1,4,5)P3-5 phosphatase (IPP5), promote Ins(1,4)P2 generation, which is an inactive product, while IP3 kinases generate Ins(1,3,4,5)P4, which is involved in calcium release modulation. We have cloned IP3 kinase-B and IPP5-A from Xenopus tropicalis and have begun to investigate theirs effects during embryonic wound healing. By misexpressing IPP5-A we inhibited wound healing while IP3 kinase-B accelerated it during early development in embryonic wound healing assays. Opposite to this effect knocking down IP3 kinase-B we inhibited wound healing. Finally, we are investigating the consequence of these manipulations on the organization of the cytoskeleton such as analyzing microtubules, tau2, or F-actin, moesin and phalloidin-rhodamine, and investigating which pathways are modulated by IP3 signalling during wound healing.