Conotruncal myocardium arises from a secondary heart field.

The primary heart tube is an endocardial tube, ensheathed by myocardial cells, that develops from bilateral primary heart fields located in the lateral plate mesoderm. Earlier mapping studies of the heart fields performed in whole embryo cultures indicate that all of the myocardium of the developed heart originates from the primary heart fields. In contrast, marking experiments in ovo suggest that the atrioventricular canal, atria and conotruncus are added secondarily to the straight heart tube during looping. The results we present resolve this issue by showing that the heart tube elongates during looping, concomitant with accretion of new myocardium. The atria are added progressively from the caudal primary heart fields bilaterally, while the myocardium of the conotruncus is elongated from a midline secondary heart field of splanchnic mesoderm beneath the floor of the foregut. Cells in the secondary heart field express Nkx2.5 and Gata-4, as do the cells of the primary heart fields. Induction of myocardium appears to be unnecessary at the inflow pole, while it occurs at the outflow pole of the heart. Accretion of myocardium at the junction of the inflow myocardium with dorsal mesocardium is completed at stage 12 and later (stage 18) from the secondary heart field just caudal to the outflow tract. Induction of myocardium appears to move in a caudal direction as the outflow tract translocates caudally relative to the pharyngeal arches. As the cells in the secondary heart field begin to move into the outflow or inflow myocardium, they express HNK-1 initially and then MF-20, a marker for myosin heavy chain. FGF-8 and BMP-2 are present in the ventral pharynx and secondary heart field/outflow myocardium, respectively, and appear to effect induction of the cells in a manner that mimics induction of the primary myocardium from the primary heart fields. Neither FGF-8 nor BMP-2 is present as inflow myocardium is added from the primary heart fields. The addition of a secondary myocardium to the primary heart tube provides a new framework for understanding several null mutations in mice that cause defective heart development.

FGF-8 in the ventral pharynx alters development of myocardial calcium transients after neural crest ablation.

Cardiac neural crest ablation results in depressed myocardial calcium transients and elevated proliferation in myocardium at a stage when cardiac neural crest cells are not in contact with the myocardium. To test the hypothesis that cardiac neural crest-derived cells, which migrate into the caudal, ventral pharynx at stage 14, block a signal from the ventral pharynx, we cultured stage 12 chick heart tube or myocardial strips in the presence or absence of ventral pharynx. We found that myocardium cultured with ventral pharynx that had not yet contacted neural crest cells had significantly reduced calcium transients and an increased rate of proliferation. Ventral pharynx from intact embryos at a stage when neural crest-derived cells had reached the pharynx had no effect on myocardial calcium transients. Ventral pharynx from neural crest-ablated embryos continued to suppress myocardial calcium transients at this later stage. Myocardium cultured with FGF-2 also showed a significant reduction in calcium transients. An FGF-2-neutralizing Ab reversed the deleterious effect of the ventral pharynx on myocardial calcium transients and proliferation. We therefore examined the expression of FGF-2 and similar FGFs in the ventral pharynx. Only FGF-8 was expressed in a temporospatial pattern that made it a viable candidate for altering the myocardial calcium transient during stages 14-18. In explant cultures, neutralizing Ab for FGF-8 rescued development of the myocardial calcium transient in neural crest-ablated chick embryos.

A novel role for cardiac neural crest in heart development.

Ablation of premigratory cardiac neural crest results in defective development of the cardiac outflow tract. The purpose of the present study was to correlate the earliest functional and morphological changes in heart development after cardiac neural crest ablation. Within 24 hours after neural crest ablation, the external morphology of the hearts showed straight outflow limbs, tighter heart loops, and variable dilations. Incorporation of bromodeoxyuridine in myocytes, an indication of proliferation, was doubled after cardiac neural crest ablation. The myocardial calcium transients, which are a measure of excitation-contraction coupling, were depressed by 50% in both the inflow and outflow portions of the looped heart tube. The myocardial transients could be rescued by replacing the cardiac neural crest. The cardiac jelly produced by the myocardium was distributed in an uneven, rather than uniform, pattern. An extreme variability in external morphology could be attributed to the uneven distribution of cardiac jelly. In the absence of cardiac neural crest, the myocardium was characterized by somewhat disorganized myofibrils that may be a result of abnormally elevated proliferation. In contrast, endocardial development appeared normal, as evidenced by normal expression of fibrillin-2 protein (JB3 antigen) and normal formation of cushion mesenchyme and trabeculae. The signs of abnormal myocardial development coincident with normal endocardium suggest that the presence of cardiac neural crest cells is necessary for normal differentiation and function of the myocardium during early heart development. These results indicate a novel role for neural crest cells in myocardial maturation.

Use of a repetitive mouse B2 element to identify transplanted mouse cells in mouse-chick chimeras.

Monitoring the migrations of cells during embryonic development requires a system in which cells can be identified in situ during locomotion. One promising system involves the generation of chimeras by transplanting mouse cells into chick embryos in ovo to exploit the wealth of mouse genetic variants. The success of this technique relies on the ability to detect individual mouse cells in a chick environment with high specificity. The murine B2 family of short interspersed elements is present in the mouse genome at copy numbers in excess of 10(5), whereas this sequence is absent in the chick genome based on hybridization techniques. This differential of five orders of magnitude produces signals in mouse cells that are easily identified, even in an environment that is predominantly chick. Thus, the B2 repeat probe is highly effective for the purpose of identifying mouse cells in mouse-chick chimeras.

HIRA, a DiGeorge syndrome candidate gene, is required for cardiac outflow tract septation.

DiGeorge syndrome (DGS) is a congenital disease characterized by defects in organs and tissues that depend on contributions by cell populations derived from neural crest for proper development. A number of candidate genes that lie within the q11 region of chromosome 22 commonly deleted in DGS patients have been identified. Orthologues of the DGS candidate gene HIRA are expressed in the neural crest and in neural crest-derived tissues in both chick and mouse embryos. By exposing a portion of the premigratory chick neural crest to phosphorothioate end-protected antisense oligonucleotides, ex ovo, followed by orthotopic backtransplantation to the untreated embryos, we have shown that the functional attenuation of cHIRA in the chick cardiac neural crest results in a significantly increased incidence of persistent truncus arteriosus, a phenotypic change characteristic of DGS, but does not affect the repatterning aortic arch arteries, the ventricular function, or the alignment of the outflow tract.

Compensatory responses and development of the nodose ganglion following ablation of placodal precursors in the embryonic chick (Gallus domesticus).

The nodose ganglion is the distal cranial ganglion of the vagus nerve which provides sensory innervation to the heart and other viscera. In this study, removal of the neuronal precursors which normally populate the right nodose ganglion was accomplished by ablating the right nodose placode in stage 9 chick embryos. Subsequent histological evaluation showed that in 54% of lesioned embryos surviving to day 6, the right ganglion was absent. Most embryos surviving to day 12, however, had identifiable right ganglia. In day 12 embryos, the right ganglion which developed was abnormal, with ganglion volume and ganglion cell diameter reduced by 50% and 20%, respectively, compared to control ganglia. To investigate the source of the neuron population in the regenerated ganglion, we combined nodose placode ablation with bilateral replacement of chick with quail "cardiac" neural crest (from mid-otic placode to somite 3). These cells normally provide only non-neuronal cells to the nodose ganglion, but produce neurons in other regions. At day 9, quail-derived neurons were identified in the right nodose ganglia of these chimeras, indicating that cardiac neural crest cells can generate neurons in the ganglion when placode-derived neurons are absent or reduced in number. On the other hand, we found that "sympathetic" neural crest (from somites 10 to 20) does not support ganglion development, suggesting that only neural crest cells normally present in the ganglion participate in reconstituting its neuronal population. Our previous work has shown that right nodose placode ablation produces abnormal cardiac function, which mimics a life-threatening human heart condition known as long QT syndrome. The present results suggest that the presence of neural crest-derived neurons in the developing right nodose ganglion may contribute to the functional abnormality in long QT syndrome.

Differential expression of the L10 ribosomal protein during heart development.

Neural crest cells originating from the posterior rhombencephalon migrate to the cardiac outflow tract and participate in division of the aorta and pulmonary trunk. Ablation of this region of premigratory neural crest in the chick embryos results in nondivision of the outflow vessels. Subtractive hybridization was used to identify messages in the outflow tract of embryos with cardiac neural crest cells versus those lacking the cardiac neural crest cells. The chick L10 ribosomal protein was found to be differentially expressed in the outflow tract of embryos with cardiac neural crest. Several conditions have been identified that involve differential expression of ribosomal proteins, but this is the first report of differential expression in eukaryotic embryonic development.

Developmental characteristics of the chick nodose ganglion.

Morphometric studies were carried out on the chick nodose ganglion between day 5 of incubation and 2 weeks after hatching. Previous findings showed that ablation of the nodose placode, the locus of precursor cells of nodose ganglion sensory neurons, results in abnormal cardiac function, and that these precursors can be induced to migrate abnormally to the heart and express abnormal phenotypes there, following cardiac neural crest ablation. These results prompted us to investigate further the normal development of nodose ganglion neurons. We find that the major period of neuron generation from placodal precursor cells in the ganglion occurs prior to day 5 of incubation. The loss of more than half of these neurons takes place between embryonic days 5 and 20, while neuron and ganglion sizes increase dramatically. Myelination is not seen at day 12 of incubation, but is present on day 15. Neurons continue to develop after hatching (day 21), reaching their adult size by 2 weeks after hatching. Unexpectedly, we found that the number of neurons increases after hatching, reaching the adult level of 62% more than embryonic day-20 numbers by 2 weeks after hatching. The mechanisms underlying the increase in neuron number after hatching are unexplained and require further investigation.

[A new measure for degree of urbanization: the address density of the surrounding area]. / Een nieuwe maatstaf voor stedelijkheid: de omgevingsadressendichtheid.

A new method of measuring the degree of urbanization of a given area, developed by the Dutch Central Bureau of Statistics, is introduced. "The measure classifies areas according to what is called address density of the surroundings: the average address density within a radius of 1 km of an address in the area." The advantages are discussed, and the measure is used to classify the 647 municipalities in the Netherlands into five groups according to urbanization level. (SUMMARY IN ENG)

[The institutional population: trends]. / De institutionele bevolking: omvang en ontwikkeling.

"The institutional population includes the population in homes for the aged, nursing homes, psychiatric hospitals, prisons and educational and religious institutions. On 1 January 1990 there were 278,000 people in this population group in the Netherlands, which amounts to 2% of the total population. During the period 1960-1990 the institutional population showed a smaller increase than the total population (6% vs. 30%); during the eighties the number has decreased. In this population the share of people of 65 or older has increased from 29% in 1960 to 67% in 1987." (SUMMARY IN ENG)

Electrocardiographic QT prolongation after ablation of the nodose placode in the chick embryo: a developmental model of the idiopathic long QT syndrome.

Electrocardiographic abnormalities characteristic of the idiopathic long QT syndrome are thought to be caused by an imbalance of sympathetic activity in the heart. Recent evidence indicates that autonomic and sensory innervation density in the end-organ is modulated by reciprocal interactions. Ablation of one neuronal population allows reciprocal increases in growth of the remaining nerves. To test whether QT prolongation could be produced in chick embryos by altering sensory innervation to the heart, microcautery was used to ablate premigratory areas of the right nodose placode, a coalescence of cells in the embryonic ectoderm that generates neurons providing sensory innervation to the heart via the inferior ganglion of the vagus (nodose ganglion). After functional autonomic innervation was established, three-lead ECG were obtained in embryos with the right nodose placode ablated (experimental) and in sham-operated controls (sham) at incubation days 17-20 in a controlled temperature environment. Electrocardiograms were analyzed for RR and QT intervals. The QT interval was corrected for heart rate using the formula QTc = QT/(RR)1/2 using an average of ten complexes. Compared with shams (n = 8), experimental embryos (n = 7) had significantly longer QTc (0.339 +/- 0.005 versus 0.318 +/- 0.004), and slower heart rates (RR = 0.29 +/- 0.005 versus 0.27 +/- 0.007). These findings mimic those in children with the idiopathic long QT syndrome. Experimental manipulation of the sensory innervation to the heart in the chick embryo via the nodose placode may provide an animal model to improve understanding of the pathogenesis of the idiopathic long QT syndrome.

Large chopping secondary mirror for the 15-m submillimeter James Clerk Maxwell telescope.

A 75-cm diam chopping secondary mirror has been developed for the 15-m diam James Clerk Maxwell telescope. The large focal ratio (D/F = 1:0.36) requires a highly convex secondary mirror, which was manufactured out of a solid slab of high-grade aluminum, consisting of a 3-mm thick shell with a support ring along the edge. The mirror surface approaches a predescribed hyperboloid with a rms precision better than 9 microm, which is adequate for use in the submillimeter wavelength region. The design aspects, the performance of a two-axis chopping mechanism, and the construction of the mirror are described.

High speed frequency modulation of far infrared lasers using the Stark effect.

Electronic frequency tuning of an optically pumped far infrared waveguide laser has been achieved by using the Stark effect. Frequency modulation with a 50-kHz modulation frequency and an index greater than 1 has been observed as well as a maximum modulation frequency of 300 kHz.