Characterization of the zebrafish matrix metalloproteinase 9 gene and its developmental expression pattern

Members of the matrix metalloproteinase (MMP) family are important for the remodeling of the extracellular matrix in a number of biological processes including a variety of immune responses. Two members of the family, MMP2 and MMP9, are highly expressed in specific myeloid cell populations in which they play a role in the innate immune response. To further expand the repertoire of molecular reagents available to study zebrafish myeloid cell development, the matrix metalloproteinase 9 (mmp9) gene from this organism has been identified and characterized. The encoded protein is 680 amino acids with high homology to known MMP9 proteins, particularly those of other teleost fish. Maternal transcripts of mmp9 are deposited in the oocyte and dispersed throughout the early embryo. These are replaced by specific zygotic transcripts in the notochord from 12 h post fertilization (hpf) and also transiently in the anterior mesoderm from 14 to 16 h post fertilization. From 24 h post fertilization, mmp9 expression was detected in a population of circulating white blood cells that are distinct from macrophages, and which migrate to the site of trauma, and so likely represent zebrafish heterophils. In the adult, mmp9 expression was most prominent in the splenic cords, a site occupied by mature myeloid cells in other species. These results suggest that mmp9 will serve as a useful marker of mature myeloid cells in the zebrafish.

The influence of age on reproductive success and diet in Australasian gannets (Morus Serrator) breeding at Pope's Eye, Port Phillip Bay, Victoria

The influence of age on reproductive success and diet was examined in ‘old’ (experienced; 12 years and older) and ‘young’ (5–8 years of age) Australasian gannets (Morus serrator) breeding at Pope’s Eye, Port Phillip Bay, Victoria during the 2002–2003 breeding period. Although food availability, as indicated by commercial fish catches, throughout this breeding period was low, there were no significant differences in breeding success or chick growth between groups. Nevertheless, old birds tended to have higher reproductive success, replacing more lost eggs and fledging chicks of a greater mass. However, old birds also laid more eggs that failed to hatch. Five fish species, including jack mackerel (Trachurus declivis), barracouta (Thyrsites atun), redbait (Emmelichthys nitidus), anchovy (Engraulis australis) and red mullet (Upeneichthys vlamingii), were important in the gannet diet during this breeding period. There were no significant differences in dietary parameters, including range of species and size of prey, between old and young gannets, nor were there any differences between those of the chicks and their parents, suggesting that adults do not forage selectively for their chicks. This study showed that even during a period of presumed low food availability, when experienced (older) birds might be expected to have enhanced success, the differences between these and less experienced (younger) birds may not be apparent.

Genomic analyses and cloning of novel chicken natriuretic peptide genes reveal new insights into natriuretic peptide evolution

The natriuretic peptide (NP) family consists of multiple subtypes in teleosts, including atrial, B-type, ventricular, and C-type NPs (ANP, BNP, VNP, CNP-1–4, respectively), but only ANP, BNP, CNP-3, and CNP-4 have been identified in tetrapods. As part of understanding the molecular evolution of NPs in the tetrapod lineage, we identified NP genes in the chicken genome. Previously, only BNP and CNP-3 have been identified in birds, but we characterized two new chicken NP genes by cDNA cloning, synteny and phylogenetic analyses. One gene is an orthologue of CNP-1, which has only ever been reported in teleostei and bichir. The second gene could not be assigned to a particular NP subtype because of high sequence divergence and was named renal NP (RNP) due to its predominant expression in the kidney. CNP-1 mRNA was only detected in brain, while CNP-3 mRNA was expressed in kidney, heart, and brain. In the developing embryo, BNP and RNP transcripts were most abundant 24 h post-fertilization, while CNP mRNA increased in a stage-dependant manner. Synthetic chicken RNP stimulated an increase in cGMP production above basal level in chicken kidney membrane preparations and caused a potent dose-dependant vasodilation of pre-constricted dorsal aortic rings. From conserved chromosomal synteny, we propose that the CNP-4 and ANP genes have been lost in chicken, and that RNP may have evolved from a VNP-like gene. Furthermore, we have demonstrated for the first time that CNP-1 is retained in the tetrapod lineage.

Preliminary investigation of the costs of incubation in the Australasian Gannet (Morus serrator) breeding in Port Phillip Bay, Victoria

To optimise lifetime reproductive success, individuals must balance current reproductive effort against future reproductive prospects. In birds, incubation and chick-rearing must involve costs, and manipulation of the length of incubation offers an insight into some costs affecting adults. An experiment was conducted at a colony of Australasian Gannets in Port Phillip Bay, Victoria, in which length of incubation was manipulated so that some adults experienced short (10–20 days duration), long (70–80 days) or normal (~45 days) incubation periods. Adults with a manipulated incubation period did not show significant differences in weight change (taken here to reflect cost) during incubation or chick-rearing compared with controls. Manipulation of length of incubation did not significantly affect the hatching success or the growth rate of chicks involved and is not, therefore considered to impose an increased reproductive cost. This suggests that the Australasian Gannet has the capacity to maintain body condition and successfully rear young despite modified duration of incubation.

Zebrafish SPI-1 marks a site of myeloid development independent of primitive erythropoiesis: implications for axial patterning.

The mammalian transcription factor SPI-1 (synonyms: SPI1, PU.1, or Sfpi1) plays a critical role in myeloid development. To examine early myeloid commitment in the zebrafish embryo, we isolated a gene from zebrafish that is a SPI-1 orthologue on the basis of homology and phylogenetic considerations. The zebrafish spi1 (pu1) gene was first expressed at 12 h postfertilization in rostral lateral plate mesoderm (LPM), anatomically isolated from erythroid development in caudal lateral plate mesoderm. Fate-mapping traced rostral LPM cells from the region of initial spi1 expression to a myeloid fate. spi1 expression was lost in the bloodless mutant cloche, but rostral spi1 expression and myeloid development were preserved in the mutant spadetail, despite its complete erythropoietic failure. This dissociation of myeloid and erythroid development was further explored in studies of embryos overexpressing BMP-4, or chordin, in bmp-deficient swirl and snailhouse mutants, and chordin-deficient chordino mutants. These studies demonstrate that, in zebrafish, spi1 marks a rostral population of LPM cells committed to a myeloid fate anatomically separated from and developmentally independent of erythroid commitment in the caudal LPM. Such complete anatomical and developmental dissociation of two hematopoietic lineages adds an interesting complexity to the understanding of vertebrate hematopoietic development and presents significant implications for the mechanisms regulating axial patterning.

Using word problems in Malaysian mathematics education: looking beneath the surface

This paper reports on aspects of a project that investigated the influence of Chinese Malaysian students’ schooling in a tradition of abstract, technical mathematics and rote learning on ways that they responded to mathematical word problems. Data from an action research project are reported. Supposedly “shallow” and “ deep” learning are shown to be interlinked, and assumptions frequently made by Western educators about modelling and practice are questioned.

Planning and teaching mathematics lessons as a dynamic, interactive process

We are researching actions that teachers can take to improve mathematics learning for all students, with particular attention to specific groups of students who might experience difficulty. After identifying possible barriers to learning, we offered teachers mathematics lessons structured in a particular way. Teachers’ use of the model outlined in this paper seemed productive and their resulting planning and teaching proved to be dynamic and interactive. This paper uses excerpts from a conversation between two teachers to illustrate specific aspects of the model.

Extruded products with Fenugreek (Trigonella foenum-graecium) chickpea and rice: Physical properties, sensory acceptability and glycaemic index

The present study investigated the effects of fenugreek flour (Trigonella foenum-graecum) and debittered fenugreek polysaccharide (FenuLife^®) inclusion on the physical and sensory quality characteristics, and glycaemic index (GI) of chickpea–rice based extruded products. Based on preliminary evaluation with different proportions of chick pea and rice, a blend of 70:30 chickpea and rice was chosen as the control for further studies. The control blend, replaced with fenugreek flour at 2%, 5% and 10%, or fenugreek polysaccharide at 5%, 10%, 15% and 20%, was extruded at the optimum processing conditions as specified in the detailed study. The extruded products were evaluated for their physical (moisture retention, expansion, hardness, water solubility index (WSI) and water absorption index (WAI)), sensory (flavor, texture, color and overall acceptability) characteristics and in vitro GI to evaluate their suitability as extruded snack products.

Due to the distinct bitter taste, inclusion of fenugreek flour was not acceptable at levels more than 2% in extruded chickpea based products. Addition of fenugreek polysaccharide resulted in slight reduction in radial expansion (P < 0.05), while longitudinal expansion increased. WAI increased while WSI decreased compared to the control (P < 0.05). The mean scores of sensory evaluation indicated that all products containing fenugreek polysaccharide up to 15% were within the acceptable range. There were no significant differences (P > 0.05) between products containing 5–15% fenugreek polysaccharide in their color, flavor, texture and overall quality.

Fenugreek, in the form of debittered polysaccharide (FenuLife^®) could be incorporated up to a level of 15% in a chickpea–rice blend to develop snack products of acceptable physical and sensory properties with low GI Index.

Begging and provisioning of thin-billed prions, Pachyptila belcheri, are related to testosterone and corticosterone

Vigorous begging is usually seen as an expression of parent–offspring conflict over limited resources. Chicks signal need by begging, but the evolution of honest signals requires the signals to be costly. Although some possible costs have been identified, the cost-inducing mechanisms underlying this widely distributed signalling system remain unclear. Because hormones associated with stress and hunger (corticosterone) and aggressive behaviour (testosterone) have deleterious side-effects, signalling costs may be coupled to the expression of such hormones, if they are closely associated with the signal. We tested whether begging in chicks of thin-billed prions (Aves, Procellariiformes) is associated with secretion of corticosterone and testosterone. Prion chicks honestly signalled their nutritional state. Begging increased with decreased body condition, both within and between chicks. Adults responded to more intense begging by delivering larger meals. Chick testosterone levels were positively correlated with measures of begging intensity and the mean body condition of chicks was correlated positively with testosterone and negatively with corticosterone. In a cross-fostering experiment, the change in testosterone and corticosterone between control and experimental periods was positively correlated with the change in begging intensity. This is the first experimental evidence that the control of chick begging by endogenously produced testosterone and corticosterone may form a mechanism controlling parental provisioning in birds, and that chick behaviour can help to explain the variation in growth patterns between individual birds.

Is sex-specific mass gain in Cory's shearwaters Calonectris diomedea related to begging and steroid hormone expression?

Mass differences between the sexes of dimorphic bird species often appear early in the nestling development. But how do adults know how much to feed a chick in a sexually dimorphic species? Do chicks of the heavier sex beg more? We studied begging in Cory’s shearwaters Calonectris diomedea, a species with heavier adult and juvenile males than females. We found that begging rates and call numbers were not different between male and female chicks, but parameters of begging intensity differed between the sexes in their relationship to chick body condition. For the same body condition, males had significantly higher begging call numbers and rates. Acoustical parameters, which were analysed semi-automatically, included the lengths of call and silence intervals, the minimum, mean and maximum frequency in a call and the number of frequency peaks within a call. We found no consistent differences of acoustic begging call elements between the sexes. Male and female chicks did not differ in the levels of the steroid hormones testosterone or corticosterone in the second quarter of the nestling period, and the mechanism leading to sex-related differences in begging rates for a given body condition remains unknown.

Reproductive biology of the eastern shovelnose stingaree Trygonoptera imitata from south-eastern Australia

Abstract. In applying a quantitative approach to the reproduction of Trygonoptera imitata, the present study contributes to understanding the wide diversity in the reproductive biology of the family Urolophidae and provides insights to help determine phylogenetic relationships. This localised species is taken as bycatch in several inshore fisheries and potentially impacted by a range of other anthropogenic pressures, including introduced species, particularly in shallow-water pupping areas.T. imitata can be characterised as a species of comparatively lowmatrotrophic histotrophy with an extended period of relatively large eggs in utero (5–8 months) followed by rapid growth of the embryos (4–6 months). The reproductive cycle is annual with parturition occurring during late-February–April, followed immediately by ovulation. Mean size-at-birth is ~225mm total length and there is a ~1000% gain in mean wet mass from egg (15 g) to full-term embryo in utero (150 g), the lowest reported for any viviparous batoid. Litter size increases with maternal length, reaching a maximum of seven, and sex ratio of embryos is 1 : 1. Maximum length and estimates of the maturity–ogive parameters l₅₀ and l₉₅ are similar for females and males.

Prolactin, body condition and the cost of good parenting: an interyear study in a long-lived seabird, Gould's Petrel (Pterodroma leucoptera)

1. The pituitary hormone prolactin is thought to play an important role in the promotion of parental care in birds and mammals. The level of care parents provide is, however, likely to be influenced by additional factors, such as their physiological condition at the time of breeding.

2. We examined relationships between parental body condition, plasma prolactin levels and reproductive performance in Gould's Petrels (Pterodroma leucoptera), a long-lived seabird. We predicted that parental body condition would correlate positively with both prolactin level and parenting intensity, as measured by the quality of the chick they produced. We also examined the effects of parenting intensity on parental body condition and reproductive success in the subsequent breeding season.

3. Body condition of male parents positively correlated with prolactin levels at the start of their second protracted incubation bout. The body condition of both parents correlated positively with the body condition of their chick at its peak mass. However, producing a good-quality chick did not negatively affect parental body condition or reproductive success the following year.

4. These results suggest that prolactin reinforces parental behaviour in parents in good body condition, which facilitates production of good-quality chicks. Moreover, good-quality parents consistently produce good-quality chicks with no apparent trade-off to their physical condition.

Differential rates of offspring provisioning in Gould’s petrels: are better feeders better breeders?

Procellariiformes (albatrosses and petrels) must accumulate substantial energy reserves to sustain them while incubating their single egg. They then produce a chick that is often more than 130% of their own body mass. Thus, despite the variable nature of resource availability in the marine environment, successful reproduction requires a considerable increase in foraging rates. Birds that are better foragers are, therefore, likely to be better parents. As surrogates of foraging ability, we assessed two parental traits that are separated temporally over the breeding season, body condition during incubation and provisioning performance, in Gould’s petrel (Pterodroma leucoptera). Although parental condition did not influence hatching success, we found significant positive correlations between the average body condition of a breeding pair and both the growth rate of chicks (g day^–1) and the body condition of chicks at peak mass. Provisioning rate also correlated positively with chick condition. Chick condition was positively correlated with haemoglobin concentration [Hb] at peak mass, which was positively correlated with [Hb] at fledging. Because the probability of survival after fledging may be influenced by chick body condition and [Hb], the ability of parents to acquire additional resources for breeding is likely to be an important determinant of reproductive success.

A novel zebrafish jak2a V581F model shared features of human JAK2 V617F polycythemia vera

Objective : The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2^V617F) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2^V617F (referred herewith jak2a^V581F) by site-directed mutagenesis and examined its relevance as a model of human PV.

Materials and Methods : Zebrafish embryos at one-cell stage were injected with jak2a^V581F mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites.

Results : Injection with jak2a^V581F mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp+ population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% ± 0.08%; jak2a^V581F injected: 5.71% ± 0.07%, coinjecting jak2a^V581F mRNA and stat5.1 morpholino: 4.66% ± 0.13%; p < 0.01). jak2a^V581F mRNA also upregulated gata1 (1.83 ± 0.08 fold; p = 0.005), embryonic α-hemoglobin (1.61 ± 0.12 fold; p = 0.049), and β-hemoglobin gene expression (1.65 ± 0.13–fold; p = 0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a^V581F mRNA significantly reduced erythropoietin gene (0.24 ± 0.03 fold; p = 0.006) and protein expression (control: 0.633 ± 0.11; jak2a^V581F mRNA: 0.222 ± 0.07 mIU/mL; p = 0.019).

Conclusion : The zebrafish jak2a^V581F model shared many features with human PV and might provide us with mechanistic insights of this disease.