Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator.

The protein EP300 and its paralog CREBBP (CREB-binding protein) are ubiquitously expressed transcriptional co-activators and histone acetyl transferases. The gene EP300 is essential for normal cardiac and neural development, whereas CREBBP is essential for neurulation, hematopoietic differentiation, angiogenesis and skeletal and cardiac development. Mutations in CREBBP cause Rubinstein-Taybi syndrome, which is characterized by mental retardation, skeletal abnormalities and congenital cardiac defects. The CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) binds EP300 and CREBBP with high affinity and regulates gene transcription. Here we show that Cited2-/- embryos die with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly. The cardiac defects include atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle, persistent truncus arteriosus and right-sided aortic arches. We find increased apoptosis in the midbrain region and a marked reduction in ErbB3-expressing neural crest cells in mid-embryogenesis. We show that CITED2 interacts with and co-activates all isoforms of transcription factor AP-2 (TFAP2). Transactivation by TFAP2 isoforms is defective in Cited2-/- embryonic fibroblasts and is rescued by ectopically expressed CITED2. As certain Tfap2 isoforms are essential in neural crest, neural tube and cardiac development, we propose that abnormal embryogenesis in mice lacking Cited2 results, at least in part, from its role as a Tfap2 co-activator.

Development and tissue origins of the mammalian cranial base.

The vertebrate cranial base is a complex structure composed of bone, cartilage and other connective tissues underlying the brain; it is intimately connected with development of the face and cranial vault. Despite its central importance in craniofacial development, morphogenesis and tissue origins of the cranial base have not been studied in detail in the mouse, an important model organism. We describe here the location and time of appearance of the cartilages of the chondrocranium. We also examine the tissue origins of the mouse cranial base using a neural crest cell lineage cell marker, Wnt1-Cre/R26R, and a mesoderm lineage cell marker, Mesp1-Cre/R26R. The chondrocranium develops between E11 and E16 in the mouse, beginning with development of the caudal (occipital) chondrocranium, followed by chondrogenesis rostrally to form the nasal capsule, and finally fusion of these two parts via the midline central stem and the lateral struts of the vault cartilages. X-Gal staining of transgenic mice from E8.0 to 10 days post-natal showed that neural crest cells contribute to all of the cartilages that form the ethmoid, presphenoid, and basisphenoid bones with the exception of the hypochiasmatic cartilages. The basioccipital bone and non-squamous parts of the temporal bones are mesoderm derived. Therefore the prechordal head is mostly composed of neural crest-derived tissues, as predicted by the New Head Hypothesis. However, the anterior location of the mesoderm-derived hypochiasmatic cartilages, which are closely linked with the extra-ocular muscles, suggests that some tissues associated with the visual apparatus may have evolved independently of the rest of the "New Head".

Ultrastructural analysis of interleukin-1 beta-induced leukocyte recruitment to the rat retina.

PURPOSE: To examine, by ultrastructural analysis, the effect of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) on the integrity of the rat retina and blood-retinal barrier (BRB) and to investigate the site of barrier leakage and the path of leukocyte infiltration into the retina. METHODS: After a single injection of IL-1 beta into the vitreous of the Lewis rat, leukocyte recruitment and retinal disease was assessed immediately and at frequent periods up to 14 days after injection by light and electron microscopy and by immunohistochemistry. The integrity of the BRB and the site of barrier disruption were evaluated using the large molecular weight tracer horseradish peroxidase. The phenotype of recruited leukocytes to the retina was assessed by ultrastructural morphology and immunohistochemistry. RESULTS: At 4 hours after the intravitreal administration of IL-1 beta, leukocytes were observed infiltrating the retina. Leukocyte infiltration increased gradually and peaked between 24 and 48 hours after injection. Associated with this infiltrate were edema and fibrin leakage, indicative of a breakdown in the BRB. This was confirmed by the demonstration of horseradish peroxidase extravasation across the retinal vascular bed, with leakage of the tracer through disrupted endothelial tight junctions. Using immunohistochemical and morphologic criteria, the majority of infiltrating cells were identified as monocytes-macrophages and neutrophils; occasionally, T cells were found. Ultrastructural analysis showed that the majority of cells entered the retina by migrating through retinal endothelial cells and that there was a smaller contribution from the ciliary body. CONCLUSIONS: The administration of IL-1 beta to the vitreous of the Lewis rat causes an acute, reversible retinal inflammatory response that is accompanied by breakdown of the vascular BRB. Interleukin-1 beta induces the recruitment of mononuclear and polymorphonuclear leukocytes that enter the retina predominantly through the retinal vasculature and that appear to migrate through retinal endothelial cells. These results suggest that IL-1 beta may be an important factor in the pathogenesis of human retinal inflammation.

Amnion rupture sequence in previable fetuses.

Amnion rupture sequence is considered an uncommon, sporadic condition among liveborn infants. We have examined 1,010 previable fetuses (9-20 weeks developmental age) to determine the incidence and nature of amnion rupture sequence at this stage of development. We found 18 affected fetuses (15 spontaneous and 3 induced abortions) with the incidence of 1:56. Eleven fetuses had limb constrictions and amputations only; 7 fetuses also had nonlimb involvement, including disruptions of the craniofacial region mimicking encephalocele, unusual facial clefts, and abdominal defects. In 6 pregnancies, constrictions of the umbilical cord by amniotic bands were the cause of fetal intrauterine death.

Two pairs of male monozygotic twins discordant for Wiedemann-Beckwith syndrome.

Wiedemann-Beckwith syndrome (WBS) is a congenital anomaly syndrome which classically consists of exomphalos, macroglossia, and gigantism. The syndrome is also associated with a variety of minor anomalies and affected individuals have an increased risk of developing rare embryonal cell tumors. To date, 15 monozygotic (MZ) twin pairs have been reported of which 13 are discordant for WBS. All except one pair of the discordant WBS twin pairs have been female. We report two pairs of male MZ twins, each discordant for WBS.

Protist biogeography.

Autecology (cellular organelles and secretions, encystment and dispersal abilities), environmental conditions (physiological tolerances and interaction with other organisms), and evolutionary history contribute to protist biogeography, which manifests ecological and historical aspects. Ecological biogeography is seen in the influence of geochemistry on the distribution of fresh-water phytoflagellates and algae, and of moisture and vegetation type on soil-litter protists. A temporal feature is often present because many protists encyst and respond only to certain ranges of temperature and organic content. Historical biogeography has occurred by radiative host evolution on symbiotic protozoa (e.g., termite flagellates and rumen ciliates), and by the isolating effects of water currents, temperature, and density gradients on oceanic protists (coccoliths, dinoflagellates, foraminifera, radiolaria, and tintinnines). These two aspects combine in protists living on animal surfaces. Humans affect protist biogeography by increasing parasite ranges through human migrations and by water pollution. They can diminish these situations by disease control and exploiting appropriate ciliates in sewage disposal. Many free-living protozoa appear to be cosmopolitan, but mating types and isoenzyme studies suggest that speciation with its geographic connotations may be more widespread than presently appreciated.

Spina bifida and hydrocephalus: a population study over a 35-year period.

The records of the B.C. Health Surveillance Registry were used to analyze all live births with spina bifida and hydrocephalus (SBHC) in British Columbia between 1952 and 1986 inclusive. A total of 479 cases (218 males and 261 females) occurred during this period in 1,298,267 consecutive live births, for an incidence of 3.7/10,000. There were more females, with the sex ratio being significantly different from that of the general population born over this period. No significant seasonal differences were observed over the time period. A comparison of life expectancy for individuals born 1962-1970 and 1970-1986 showed significant improvement in the probability of surviving to the age of 1 year for the latter group. There was also a small but statistically significant increased chance of surviving to age 7 years in the latter group but no difference in the probability of surviving from 7 years to 16 years. Life expectancy figures are shown in a format practical for counseling with regard to prognosis for affected individuals. Additional malformations not attributable to SBHC were observed in 6% (27 cases). These included renal anomalies, cleft lip and/or palate, tracheoesophageal fistula, and diaphragmatic hernia. The incidence of each defect was significantly greater than in the general population of births.

Research ethics in Canada: experience of a group operating a human embryo and fetal tissue bank.

A Canadian research group is establishing a human embryo and fetal tissue bank. Its purpose is to provide researchers with frozen or fixed tissue specimens for use in protein and gene expression studies. Several legal and ethical issues have arisen, including questions about consent, use of these rare tissues, cost recovery, and profit-making. These issues are discussed here in light of the present lack of legislation in Canada. We make recommendations in these areas, and suggest that the bank's operations could legally fall under the jurisdiction of the Human Tissue Gift Act.

The effect of TNF-alpha and IL-6 on the permeability of the rat blood-retinal barrier in vivo.

The blood-retinal barrier (BRB), which is formed by the retinal vascular endothelium and the retinal pigment epithelium, is responsible for controlling the passage of cells and molecules into the neuroretina. During ocular inflammatory diseases, however, this selective control is altered due to changes in BRB function such as increased permeability and leucocyte recruitment. The causative factors leading to barrier breakdown are not entirely understood although cytokines have recently been implicated. We have investigated the effect of the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) upon the integrity of the rat BRB. Lewis rats received a dose of each cytokine by intravitreal injection and the permeability of the BRB was assessed using the smaller molecular weight vascular tracer 14C mannitol. A significant opening of the barrier to mannitol was detected following an intravitreal injection of 2 x 10(4) U of TNF-alpha which persisted from day 1 to day 5 post-injection (PI). The permeability of the BRB returned to normal values by day 7 PI. Only occasional mononuclear inflammatory cells were seen in the retina and vitreous of the TNF-alpha-treated eyes although they remained in evidence up to day 5 PI. In the TNF-alpha-infected eye there was immunohistological evidence of activation of tissue-resident cells, particularly in the inner plexiform layer. Of particular interest was the observation that the BRB of the non-injected contralateral eye also exhibited increased permeability over a similar time-course but without any evidence of cellular infiltration or activation of tissue-resident cells. Unlike TNF-alpha, the administration of 1 x 10(3) U of IL-6 into the vitreous caused no measurable increase in BRB permeability despite inducing a small infiltration of inflammatory cells.

Interleukin-1 beta-induced disruption of the retinal vascular barrier of the central nervous system is mediated through leukocyte recruitment and histamine.

The vascular barriers of the central nervous system form a selective cellular interface between the blood and the neural parenchyma and restrict the transfer of both molecules and hematogenous cells. During immune-mediated diseases, leukocyte infiltration becomes dramatically up-regulated and the permeability of these barriers increases, leading to edema formation. The etiology of this damage remains largely unresolved although inflammatory cytokines have been implicated in the process. The effect of the proinflammatory cytokine interleukin (IL)-1 beta on the integrity of the rat blood-retinal barrier (BRB) was investigated up to 14 days after an intravitreal injection. The permeability of the BRB was evaluated quantitatively using the low molecular weight tracer [14C]mannitol. After IL-1 beta administration, a biphasic opening of the BRB to [14C]mannitol was recorded, peaking at 4 to 8 hours and 24 to 48 hours post-injection (PI). The early disruption coincided with the appearance of both polymorphonuclear and mononuclear leukocytes within the retina. By 12 hours PI, BRB permeability had returned to control values despite a continued increase in the number of infiltrating leukocytes. The second, more pronounced increase in barrier permeability detected at 24 to 48 hours PI corresponded with maximal leukocyte infiltration. Barrier dysfunction had resolved by 72 hours, and by 7 days the leukocyte infiltrate had disappeared. The IL-1 beta-induced increase in permeability could be completely abrogated at 4 and 24 hours PI by treating the animals with the histamine H2-receptor antagonist ranitidine, which also reduced leukocyte infiltration by 47.2%. The ability of histamine to disrupt the BRB was demonstrated by intravitreal and intravascular administration, which caused a rapid and significant increase in BRB permeability. Treatment of the animals with the cyclo-oxygenase inhibitor indomethacin had no effect on IL-1 beta-induced disruption of the BRB at 4 hours PI, but by 24 hours PI a significant reduction in permeability was observed that coincided with a 75.2% reduction in the leukocyte infiltrate. The depletion of circulating leukocytes to < 2% of control levels reduced the retinal leukocyte recruitment induced by IL-1 beta by 73.0% and decreased BRB permeability at both 4 and 24 hours after IL-1 beta injection. These data demonstrate that intravitreal IL-1 beta in the rat induces a biphasic opening of the BRB that appears to be mediated through recruited leukocytes and release of the vasoactive amine histamine.

Congenital cutis laxa with retardation of growth and development.

Seven patients with congenital cutis laxa are presented. The associated features include developmental delay, joint laxity, wide anterior fontanelle, growth retardation, dental caries, and osteopenia. The heterogeneity and inheritance of congenital cutis laxa are discussed. This particular syndrome appears distinct and is likely to be autosomal recessive in view of the two brother-sister sib pairs in this report.

Marfan syndrome: exclusion of genetic linkage to the COL1A2 gene.

Marfan Syndrome is a genetic disorder of the connective tissue. Individuals from one large family with this disorder were genotyped for COL1A2 gene associated RFLPs. Our results demonstrated that the COL1A2 gene, encoding the proa2(I) collagen chain, segregated independently of the phenotype and it is therefore excluded as the mutant locus in this family.

Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors.

Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway. Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs.

A dominant mutant of occludin disrupts tight junction structure and function.

The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.

Congenital rubella syndrome associated with calcific epiphyseal stippling and peroxisomal dysfunction.

An infant girl had the clinical and immunologic findings of congenital rubella syndrome but also had arthrogryposis multiplex and calcific epiphyseal stippling. Spastic quadriparesis developed, and both physical and behavioral development were slow. Increased spasticity of the legs at 5 1/2 years was related not to progressive rubella encephalomyelopathy but to spinal cord compression by abnormal cartilaginous tissue. The presence of a peroxisomal disorder was demonstrated by a greatly increased level of phytanic acid and slightly increased levels of hexacosanoate in serum and by reduced activity of peroxisomal dihydroxyacetone phosphate acyltransferase and a slightly increased ratio of cytosolic to peroxisomal catalase activity in cultured fibroblasts. A reduction in the number and size of peroxisomes was demonstrated in cultured fibroblasts, and a needle biopsy specimen of the liver also showed the peroxisomes to have a smaller diameter than usual. We recommend that any child with epiphyseal stippling be assessed for peroxisomal disease and that the potential for spinal cord compression by dysplastic bone or cartilage be recognized. The association of peroxisomal dysfunction with congenital rubella has not been described previously. The interaction between rubella virus infection and peroxisomal function may need further investigation.