Interactions between mural cells and endothelial cells stabilize the developing zebrafish dorsal aorta.

Mural cells (vascular smooth muscle cells and pericytes) play an essential role in the development of the vasculature, promoting vascular quiescence and long-term vessel stabilization through their interactions with endothelial cells. However, the mechanistic details of how mural cells stabilize vessels are not fully understood. We have examined the emergence and functional role of mural cells investing the dorsal aorta during early development using the zebrafish. Consistent with previous literature, our data suggest that cells ensheathing the dorsal aorta emerge from a sub-population of cells in the adjacent sclerotome. Inhibition of mural cell recruitment to the dorsal aorta through disruption of pdgfr signaling leads to a reduced vascular basement membrane, which in turn results in enhanced dorsal aorta vessel elasticity and failure to restrict aortic diameter. Our results provide direct in vivo evidence for a functional role for mural cells in patterning and stabilization of the early vasculature through production and maintenance of the vascular basement membrane to prevent abnormal aortic expansion and elasticity.

Rap1 signaling prevents L-type calcium channel-dependent neurotransmitter release.

The small GTPase Rap1 contributes to fear learning and cortico-amygdala plasticity by inhibiting glutamate release from cortical neurons, but mechanisms of this inhibition remain unknown. Conversely, L-type calcium channels (LTCCs) become involved in glutamate release after fear learning and LTP induction. Here, we show that Rap1 deletion in mouse primary cortical neurons increases synaptic vesicle exocytosis without altering endocytosis or vesicle pool size in an LTCC-dependent manner. We identify Erk1/2 as the downstream effector of Rap1 and show that its inhibition increases plasma membrane expression of LTCCs near presynaptic terminals. We propose that the Rap1 signaling enables plasticity and fear learning by regulating LTCCs at cortico-amygdala synapses.

Live imaging of lymphatic development in the zebrafish.

The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes. Progress in understanding the origins and early development of this system, however, has been hampered by difficulties in observing lymphatic cells in vivo and in performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. Here, we show that the optically clear developing zebrafish provides a useful model for imaging and studying lymphatic development, with a lymphatic system that shares many of the morphological, molecular and functional characteristics of the lymphatic vessels found in other vertebrates. Using two-photon time-lapse imaging of transgenic zebrafish, we trace the migration and lineage of individual cells incorporating into the lymphatic endothelium. Our results show lymphatic endothelial cells of the thoracic duct arise from primitive veins through a novel and unexpected pathway.

Ultra-structural identification of interstitial cells of Cajal in the zebrafish Danio rerio.

The interstitial cells of Cajal (ICCs) are important mediators of gastrointestinal (GI) motility because of their role as pacemakers in the GI tract. In addition to their function, ICCs are also structurally distinct cells most easily identified by their ultra-structural features and expression of the tyrosine kinase receptor c-KIT. ICCs have been described in mammals, rodents, birds, reptiles, and amphibians, but there are no reports at the ultra-structural level of ICCs within the GI tract of an organism from the teleost lineage. We describe the presence of cells in the muscularis of the zebrafish intestine; these cells have similar features to ICCs in other vertebrates. The ICC-like cells are associated with the muscularis, are more electron-dense than surrounding smooth muscle cells, possess long cytoplasmic processes and mitochondria, and are situated opposing enteric nervous structures. In addition, immunofluorescent and immunoelectron-microscopic studies with antibodies targeting the zebrafish ortholog of a putative ICC marker, c-KIT (kita), showed c-kit immunoreactivity in zebrafish ICCs. Taken together, these data represent the first ultra-structural characterization of cells in the muscularis of the zebrafish Danio rerio and suggest that ICC differentiation in vertebrate evolution dates back to the teleost lineage.

Endothelial tubes assemble from intracellular vacuoles in vivo.

The formation of epithelial tubes is crucial for the proper development of many different tissues and organs, and occurs by means of a variety of different mechanisms. Morphogenesis of seamless, properly patterned endothelial tubes is essential for the development of a functional vertebrate circulatory system, but the mechanism of vascular lumenization in vivo remains unclear. Evidence dating back more than 100 years has hinted at an important function for endothelial vacuoles in lumen formation. More than 25 years ago, in some of the first endothelial cell culture experiments in vitro, Folkman and Haudenschild described "longitudinal vacuoles" that "appeared to be extruded and connected from one cell to the next", observations confirmed and extended by later studies in vitro showing that intracellular vacuoles arise from integrin-dependent and cdc42/Rac1-dependent pinocytic events downstream of integrin-extracellular-matrix signalling interactions. Despite compelling data supporting a model for the assembly of endothelial tubes in vitro through the formation and fusion of vacuoles, conclusive evidence in vivo has been lacking, primarily because of difficulties associated with imaging the dynamics of subcellular endothelial vacuoles deep within living animals. Here we use high-resolution time-lapse two-photon imaging of transgenic zebrafish to examine how endothelial tubes assemble in vivo, comparing our results with time-lapse imaging of human endothelial-cell tube formation in three-dimensional collagen matrices in vitro. Our results provide strong support for a model in which the formation and intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation.

Palmitoyl protein thioesterase-1 deficiency impairs synaptic vesicle recycling at nerve terminals, contributing to neuropathology in humans and mice.

Neuronal ceroid lipofuscinoses represent the most common childhood neurodegenerative storage disorders. Infantile neuronal ceroid lipofuscinosis (INCL) is caused by palmitoyl protein thioesterase-1 (PPT1) deficiency. Although INCL patients show signs of abnormal neurotransmission, manifested by myoclonus and seizures, the molecular mechanisms by which PPT1 deficiency causes this abnormality remain obscure. Neurotransmission relies on repeated cycles of exo- and endocytosis of the synaptic vesicles (SVs), in which several palmitoylated proteins play critical roles. These proteins facilitate membrane fusion, which is required for neurotransmitter exocytosis, recycling of the fused SV membrane components, and regeneration of fresh vesicles. However, palmitoylated proteins require depalmitoylation for recycling. Using postmortem brain tissues from an INCL patient and tissue from the PPT1-knockout (PPT1-KO) mice that mimic INCL, we report here that PPT1 deficiency caused persistent membrane anchorage of the palmitoylated SV proteins, which hindered the recycling of the vesicle components that normally fuse with the presynaptic plasma membrane during SV exocytosis. Thus, the regeneration of fresh SVs, essential for maintaining the SV pool size at the synapses, was impaired, leading to a progressive loss of readily releasable SVs and abnormal neurotransmission. This abnormality may contribute to INCL neuropathology.

Pde8b haploinsufficiency in mice is associated with modest adrenal defects, impaired steroidogenesis, and male infertility, unaltered by concurrent PKA or Wnt activation.

PDE8B, PRKAR1A and the Wnt/ß-catenin signaling are involved in endocrine disorders. However, how PDEB8B interacts with both Wnt and protein kinase A (PKA) signaling in vivo remains unknown. We created a novel Pde8b knockout mouse line (Pde8b-/-); Pde8b haploinsufficient (Pde8b+/-) mice were then crossed with mice harboring: (1) constitutive beta-catenin activation (Pde8b+/-;ΔCat) and (2) Prkar1a haploinsufficieny (Pde8b+/-;Prkar1a+/-). Adrenals and testes from mice (3-12-mo) were evaluated in addition to plasma corticosterone, aldosterone and Dkk3 concentrations, and the examination of expression of steroidogenesis-, Wnt- and cAMP/PKA-related genes. Pde8b-/- male mice were infertile with down-regulation of the Wnt/ß-catenin pathway which did not change significantly in the Pde8b+/-;ΔCat mice. Prkar1a haploinsufficiency also did not change the phenotype significantly. In vitro studies showed that PDE8B knockdown upregulated the Wnt pathway and increased proliferation in CTNNB1-mutant cells, whereas it downregulated the Wnt pathway in PRKAR1A-mutant cells. These data support an overall weak, if any, role for PDE8B in adrenocortical tumorigenesis, even when co-altered with Wnt signaling or PKA upregulation; on the other hand, PDE8B appears to play a significant role in male fertility.

Transmembrane protein-free membranes fuse into xenopus nuclear envelope and promote assembly of functional pores.

Post-mitotic reassembly of nuclear envelope (NE) and the endoplasmic reticulum (ER) has been reconstituted in a cell-free system based on interphase Xenopus egg extract. To evaluate the relative contributions of cytosolic and transmembrane proteins in NE and ER assembly, we replaced a part of native membrane vesicles with ones either functionally impaired by trypsin or N-ethylmaleimide treatments or with protein-free liposomes. Although neither impaired membrane vesicles nor liposomes formed ER and nuclear membrane, they both supported assembly reactions by fusing with native membrane vesicles. At membrane concentrations insufficient to generate full-sized functional nuclei, addition of liposomes and their fusion with membrane vesicles resulted in an extensive expansion of NE, further chromatin decondensation, restoration of the functionality, and spatial distribution of the nuclear pore complexes (NPCs), and, absent newly delivered transmembrane proteins, an increase in NPC numbers. This rescue of the nuclear assembly by liposomes was inhibited by wheat germ agglutinin and thus required active nuclear transport, similarly to the assembly of full-sized functional NE with membrane vesicles. Mechanism of fusion between liposomes and between liposomes and membrane vesicles was investigated using lipid mixing assay. This fusion required interphase cytosol and, like fusion between native membrane vesicles, was inhibited by guanosine 5'-3-O-(thio)triphosphate, soluble N-ethylmaleimide-sensitive factor attachment protein, and N-ethylmaleimide. Our findings suggest that interphase cytosol contains proteins that mediate the fusion stage of ER and NE reassembly, emphasize an unexpected tolerance of nucleus assembly to changes in concentrations of transmembrane proteins, and reveal the existence of a feedback mechanism that couples NE expansion with NPC assembly.

Imaging the developing lymphatic system using the zebrafish.

The lymphatic system is essential for immune responses, fluid homeostasis, and fat absorption, and is involved in many pathological processes, including tumour metastasis and lymphoedema. Despite its importance, progress in understanding the origins and early development of this system has been hampered by difficulties in observing lymphatic cells in vivo and performing genetic and experimental manipulation of the lymphatic system. These difficulties stem in part from the lack of a model organism combining these features. The zebrafish is a genetically accessible vertebrate with an optically clear embryo permitting high-resolution in vivo imaging, but the existence of a lymphatic vascular system has not been previously reported in this model organism. Using a series of morphological, molecular and functional studies we have visualized and characterized lymphatic vessels in the developing zebrafish. Our studies show that the zebrafish possesses a lymphatic system that shares many of the characteristic features of lymphatic vessels found in other vertebrates. Using multiphoton time-lapse imaging we have carried out in vivo cell tracking experiments to trace the origins of lymphatic endothelial cells. Our data provide conclusive new evidence supporting a venous origin for primitive lymphatic endothelial cells.

Pituitary adenoma with paraganglioma/pheochromocytoma (3PAs) and succinate dehydrogenase defects in humans and mice.

CONTEXT: Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported. DESIGN: We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages. RESULTS: No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex. CONCLUSION: The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.

Dye fills reveal additional olfactory tracts in the protocerebrum of wild-type Drosophila.

The antennal lobe (AL) is the primary olfactory center in insect brains. It receives sensory input from the olfactory sensory neurons (OSNs) and sends, through its projection neurons (PNs), reformatted output to secondary olfactory centers, including the mushroom body (MB) calyx and the lateral horn (LH) in the protocerebrum. By injecting dye into the AL of wild-type Drosophila, we identified previously unknown direct pathways between the AL and the ventrolateral, superior medial, and posterior lateral protocerebra. We found that most of these areas in the protocerebrum are connected with the AL through multiple tracts, suggesting that these areas are sites of convergence for olfactory information. Furthermore, areas such as the superior medial protocerebrum now appear to receive olfactory output both directly from the AL and indirectly from lobes of the MB and the LH, suggesting a degree of functional interaction among these areas. We also analyzed the length and number of fibers in each tract. We compare our results obtained from wild-type flies with recent results from transgenic strains and discuss how information about odorants is distributed to multiple protocerebral areas.

Dual-labeling method for electron microscopy to characterize synaptic connectivity using genetically encoded fluorescent reporters in Drosophila.

Light and electron microscopy (LM and EM) both offer important advantages for characterizing neuronal circuitry in intact brains: LM can reveal the general patterns neurons trace between brain areas, and EM can confirm synaptic connections between identified neurons within a small area. In a few species, genetic labeling with fluorescent proteins has been used with LM to visualize many kinds of neurons and to analyze their morphologies and projection patterns. However, combining these large-scale patterns with the fine detail available in EM analysis has been a technical challenge. To analyze the synaptic connectivity of neurons expressing fluorescent markers with EM, we developed a dual-labeling method for use with pre-embedded brains. In Drosophila expressing genetic labels and also injected with markers we visualized synaptic connections among two populations of neurons in the AL, one of which has been shown to mediate a specific function, odor evoked neural oscillation.

Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation.

Condensins I and II in vertebrates are essential ATP-dependent complexes necessary for chromosome condensation in mitosis. Condensins depletion is known to perturb structure and function of centromeres, however the mechanism of this functional link remains elusive. Depletion of condensin activity is now shown to result in a significant loss of loading of CENP-A, the histone H3 variant found at active centromeres and the proposed epigenetic mark of centromere identity. Absence of condensins and/or CENP-A insufficiency produced a specific kinetochore defect, such that a functional mitotic checkpoint cannot prevent chromosome missegregation resulting from improper attachment of sister kinetochores to spindle microtubules. Spindle microtubule-dependent deformation of both inner kinetochores and the HEC1/Ndc80 microtubule-capturing module, then results in kinetochore separation from the Aurora B pool and ensuing reduced kinase activity at centromeres. Moreover, recovery from mitosis-inhibition by monastrol revealed a high incidence of merotelic attachment that was nearly identical with condensin depletion, Aurora B inactivation, or both, indicating that the Aurora B dysfunction is the key defect leading to chromosome missegregation in condensin-depleted cells. Thus, beyond a requirement for global chromosome condensation, condensins play a pivotal role in centromere assembly, proper spatial positioning of microtubule-capturing modules and positioning complexes of the inner centromere versus kinetochore plates.

Combinatorial interaction between CCM pathway genes precipitates hemorrhagic stroke.

Intracranial hemorrhage (ICH) is a particularly severe form of stroke whose etiology remains poorly understood, with a highly variable appearance and onset of the disease (Felbor et al., 2006; Frizzell, 2005; Lucas et al., 2003). In humans, mutations in any one of three CCM genes causes an autosomal dominant genetic ICH disorder characterized by cerebral cavernous malformations (CCM). Recent evidence highlighting multiple interactions between the three CCM gene products and other proteins regulating endothelial junctional integrity suggests that minor deficits in these other proteins could potentially predispose to, or help to initiate, CCM, and that combinations of otherwise silent genetic deficits in both the CCM and interacting proteins might explain some of the variability in penetrance and expressivity of human ICH disorders. Here, we test this idea by combined knockdown of CCM pathway genes in zebrafish. Reducing the function of rap1b, which encodes a Ras GTPase effector protein for CCM1/Krit1, disrupts endothelial junctions in vivo and in vitro, showing it is a crucial player in the CCM pathway. Importantly, a minor reduction of Rap1b in combination with similar reductions in the products of other CCM pathway genes results in a high incidence of ICH. These findings support the idea that minor polygenic deficits in the CCM pathway can strongly synergize to initiate ICH.

Combinatorial function of ETS transcription factors in the developing vasculature.

Members of the ETS family of transcription factors are among the first genes expressed in the developing vasculature, but loss-of-function experiments for individual ETS factors in mice have not uncovered important early functional roles for these genes. However, multiple ETS factors are expressed in spatially and temporally overlapping patterns in the developing vasculature, suggesting possible functional overlap. We have taken a comprehensive approach to exploring the function of these factors during vascular development by employing the genetic and experimental tools available in the zebrafish to analyze four ETS family members expressed together in the zebrafish vasculature; fli1, fli1b, ets1, and etsrp. We isolated and characterized an ENU-induced mutant with defects in trunk angiogenesis and positionally cloned the defective gene from this mutant, etsrp. Using the etsrp morpholinos targeting each of the four genes, we show that the four ETS factors function combinatorially during vascular and hematopoietic development. Reduction of etsrp or any of the other genes alone results in either partial or no defects in endothelial differentiation, while combined reduction in the function of all four genes causes dramatic loss of endothelial cells. Our results demonstrate that combinatorial ETS factor function is essential for early endothelial specification and differentiation.