Continuous measurements of ammonia, nitrous oxide and methane from air scrubbers at pig housing facilities.

Ammonia, largely emitted by agriculture, involves a great risk for eutrophication and acidification leading to biodiversity loss. Air scrubbers are widely applied to reduce ammonia emission from pig and poultry housing facilities, but it is not always clear whether their performance meets the requirements. Besides, there is a growing international concern for the livestock related greenhouse gases methane and nitrous oxide but hardly any data concerning their fate in air scrubbers are available. This contribution presents the results from measurement campaigns conducted at a chemical, a biological and a two-stage biological air scrubber installed at pig housing facilities in Flanders. Ammonia, nitrous oxide and methane at the inlet and outlet of the air scrubbers were monitored on-line during one week using a photoacoustic gas monitor, which allowed to investigate diurnal fluctuations in the removal performance of air scrubbers. Additionally, the homogeneity of the air scrubbers, normally checked by gas detection tubes, was investigated in more detail using the continuous data. The biological air scrubber with extra nitrification tank performed well in terms of ammonia removal (86 ± 6%), while the two-stage air scrubber suffered from nitrifying bacteria inhibition. In the chemical air scrubber the pH was not kept constant, lowering the ammonia removal efficiency. A lower ammonia removal efficiency was found during the day, when the ventilation rate was the highest. Nitrous oxide was produced inside the biological and two-stage scrubber, resulting in an increased outlet concentration of more than 200%. Methane could not be removed in the different air scrubbers because of its low water solubility.

Expression of the dlx gene family during formation of the cranial bones in the zebrafish (Danio rerio): differential involvement in the visceral skeleton and braincase.

We have used dlx genes to test the hypothesis of a separate developmental program for dermal and cartilage bones within the neuro- and splanchnocranium by comparing expression patterns of all eight dlx genes during cranial bone formation in zebrafish from 1 day postfertilization (dPF) to 15 dPF. dlx genes are expressed in the visceral skeleton but not during the formation of dermal or cartilage bones of the braincase. The spatiotemporal expression pattern of all the members of the dlx gene family, support the view that dlx genes impart cellular identity to the different arches, required to make arch-specific dermal bones. Expression patterns seemingly associated with cartilage (perichondral) bones of the arches, in contrast, are probably related to ongoing differentiation of the underlying cartilage rather than with differentiation of perichondral bones themselves. Whether dlx genes originally functioned in the visceral skeleton only, and whether their involvement in the formation of neurocranial bones (as in mammals) is secondary, awaits clarification.

Expression of Dlx genes during the development of the zebrafish pharyngeal dentition: evolutionary implications.

In order to investigate similarities and differences in genetic control of development among teeth within and between species, we determined the expression pattern of all eight Dlx genes of the zebrafish during development of the pharyngeal dentition and compared these data with that reported for mouse molar tooth development. We found that (i) dlx1a and dlx6a are not expressed in teeth, in contrast to their murine orthologs, Dlx1 and Dlx6; (ii) the expression of the six other zebrafish Dlx genes overlaps in time and space, particularly during early morphogenesis; (iii) teeth in different locations and generations within the zebrafish dentition differ in the number of genes expressed; (iv) expression similarities and differences between zebrafish Dlx genes do not clearly follow phylogenetic and linkage relationships; and (v) similarities and differences exist in the expression of zebrafish and mouse Dlx orthologs. Taken together, these results indicate that the Dlx gene family, despite having been involved in vertebrate tooth development for over 400 million years, has undergone extensive diversification of expression of individual genes both within and between dentitions. The latter type of difference may reflect the highly specialized dentition of the mouse relative to that of the zebrafish, and/or genome duplication in the zebrafish lineage facilitating a redistribution of Dlx gene function during odontogenesis.

Tooth development in vitro in two teleost fish, the cichlid Hemichromis bimaculatus and the cyprinid Danio rerio.

A technique for organotypic in vitro culture with serum-free medium was tested for its appropriateness to mimic normal odontogenesis in the cichlid fish Hemichromis bimaculatus and the zebrafish Danio rerio. Serial semithin sections were observed by light microscopy to collect data on tooth patterning and transmission electron microscopy was used to compare cellular and extracellular features of tooth germs developing in vitro with the situation in vivo. Head explants of H. bimaculatus from 120 h post-fertilization (hPF) to 8.5 days post-fertilization (dPF) and of zebrafish from 45 hPF to 79 hPF and adults kept in culture for 3, 4 or 7 days revealed that tooth germs developed in vitro from explants in which the buccal or pharyngeal epithelium was apparently undifferentiated and, when present at the time of explantation, they continued their development up to a stage of attachment. In addition, the medium allowed the morphogenesis and cytodifferentiation of the tooth germs similar to that observed in vivo and the establishment of a dental pattern (place and order of tooth appearance and of attachment) that mimicked that in vivo. Organotypic culture in serum-free conditions thus provides us with the means of studying epithelial-mesenchymal interactions during tooth development in teleost fish and of analysing the genetic control of either mandibular or pharyngeal tooth development and replacement in these polyphyodont species. Importantly, it allows heads from embryonically lethal (zebrafish) mutants or from early lethal knockdown experiments to develop beyond the point at which the embryos normally die. Such organotypic culture in serum-free conditions could therefore become a powerful tool in developmental studies and open new perspectives for craniofacial research.

Fish dentitions as paradigms for odontogenic questions.

Bony fish, and in particular teleosts, represent a morphologically extremely diverse group of vertebrates, well suited to study certain problems in odontogenesis. In this article we address some questions that can benefit much from the use of fish dentitions as paradigms, such as endodermal participation in tooth formation and epithelial primacy in initiation events. Next, we highlight some results recently obtained in our laboratory with respect to two models, the zebrafish (Cyprinidae), and selected species of cichlids (Cichlidae). Finally, we pinpoint some questions that lend themselves admirably to be examined using fish models, such as the factors that control renewed initiation of teeth, and the relationship (or absence thereof) between Hox genes and tooth formation.

Tooth succession in the zebrafish (Danio rerio).

To test whether the formation of replacement teeth in arbitrarily chosen zebrafish follows the same pattern as described for larval and young zebrafish of known age, dentitions of more than 90 animals of different ages and standard lengths were observed under the stereomicroscope. Only the teeth of the ventral tooth row (1V-5V) were considered. Statistical results (G-tests) suggested that tooth replacement does not occur randomly. The most common order of replacement can be represented as the formula 5V-2V-3V-1V-4V and counts for approximately 70% of the observed patterns. Initiation of replacement teeth at positions 4V and 5V is separated by a larger time interval than between any other combination. It is hypothesized that in older juveniles and adults, replacement teeth may be formed during three odontogenic waves.

A quantitative analysis of pharyngeal tooth shape in the zebrafish (Danio rerio, Teleostei, Cyprinidae).

To test whether successive replacement cycles in the pharyngeal dentition of the zebrafish, a polyphyodont vertebrate model organism, entail overall shape changes in the teeth, a qualitative and quantitative analysis was made of size and shape variables in the five ventral teeth. The following measurements were defined: tooth length, tooth height, neck-crown angle, cusp depth, and crown curvature. Ontogenetic changes in fish, ranging between 6 and 29 mm standard length (SL), were analysed by linear regressions on to SL. The teeth became significantly larger with growth of the fish, through successive replacements and cusp depth also increased over time. Neck--crown angle and crown curvature did not change over time. Position-dependent differences were analysed by Friedman ANOVA and Kendall concordance tests. Measurements differed significantly according to tooth position in the pharyngeal jaws. Tooth 1V was always the smallest, 3V the largest. The neck--crown angle and curvature of the crown increased from 1V to 5V. Cusp depth increased from 1V to 3V, and then decreased again. These results indicate that successive replacement cycles entail a size increase accompanied by shape changes apparently restricted to the crown. These quantitative data lay the basis for further descriptive and experimental studies of tooth shape in this model-species.

Development and fine structure of pharyngeal replacement teeth in juvenile zebrafish (Danio rerio) (Teleostei, Cyprinidae).

Teeth are commonly used model systems for the study of epithelial-mesenchymal interactions during organogenesis. We describe here the ultrastructural characteristics of developing pharyngeal replacement teeth in juvenile zebrafish, an increasingly important model organism for vertebrate development. Replacement teeth develop in close association with the dental organ of a functional tooth. Morphogenesis is well advanced prior to the start of cytodifferentiation. Fibrillar enameloid matrix is formed first, followed by the deposition of predentine. Initial mineralization of the enameloid proceeds quickly; maturation involves the presence of ruffled-bordered ameloblasts. Dentine mineralization is inotropic and is mediated by matrix vesicles. Woven-fibred attachment bone matrix is deposited before completion of dentine mineralization. Eruption of fully ankylosed teeth is a fast process and may involve degenerative changes in the pharyngeal epithelium. Mononucleated osteoclasts and clastic cells located in the pulp cavity intervene in tooth resorption prior to shedding. Structural differences with larval, first-generation zebrafish teeth include the presence of dentinal tubules and the absence of an electron-dense covering membrane. Part of these differences may relate to size differences of the teeth. Others, like the site of the replacement tooth bud, suggest that initiation may take place in already committed epithelium from the first initiation event in the larval stage.

Dynamics of tooth formation and replacement in the zebrafish (Danio rerio) (Teleostei, Cyprinidae).

We have used three-dimensional reconstructions from serial sections as well as cleared and stained specimens to infer patterning of the pharyngeal dentition throughout ontogeny in the zebrafish. Each pharyngeal tooth has been monitored from its initiation to its complete disappearance (resorption and shedding). We have identified tooth families and have studied the persistence of the pattern through successive replacements. Teeth arise in two seemingly independent clusters, a ventral and a dorsal cluster, with differing patterning features. The ventral cluster consists of one row of five teeth in which a tooth is first initiated in position four, and subsequent teeth in adjacent positions, posterior and anterior to it. Replacement teeth in odd and even positions are initiated simultaneously during successive odontogenic waves but differ in generation number according to the timing of appearance of the first-generation tooth, i.e., the founder of the tooth family. Up to four teeth of a single tooth family are simultaneously present in early juveniles of which two are usually "co-functional." The number of teeth per tooth family is reduced in older juveniles and adults, reflecting a slowing down of the replacement rate. The consistent way in which the pattern is established and maintained during ontogeny calls for research of the presence of specific molecular controls.

Early development of the zebrafish (Danio rerio) pharyngeal dentition (Teleostei, Cyprinidae).

In order to build a reference system to assess ongoing in vitro and in situ hybridisation experiments on epithelial-mesenchymal interactions governing odontogenesis in the zebrafish, we describe here the generation of the pharyngeal dentition, and the histological development of teeth up to fourteen days post-fertilization, using serial semithin sections, handmade and computer-assisted reconstructions and transmission electron microscopy. The tooth pattern in larval zebrafish is generated in a predictable, and bilaterally symmetrical manner from shortly before hatching onwards. Characteristics related to tooth development and structure differ considerably from those seen in juvenile specimens and those described for other bony fishes. Particular features related to the cyprinid condition include the complex epithelial connectivity and the mode of attachment of the teeth.