Osteoblast maturation occurs in overlapping proximal‐distal compartments during fin regeneration in zebrafish

During fin regeneration, osteoblasts must continually differentiate for outgrowth of the bony fin rays. Bone maturity increases in a distal‐proximal manner, and osteoblast maturation can be detected similarly when following gene expression. We find that early markers for osteoblast differentiation are expressed in a discrete domain at the distal end of the fin, just proximal to the adjacent germinal compartment of dividing cells. Matrix genes, required at later stages developmentally, are expressed in a population of cells proximally to the early genes. A marker for mature osteoblasts is expressed in cells further proximal. These domains of gene expression are partially overlapping, perhaps revealing additional levels of osteoblast maturity. We suggest a model for growth where new cells are continually added to the distal‐most osteoblast compartment, while osteoblasts in more proximal locations differentiate, thus translating developmental time to location on the proximal‐distal axis. Developmental Dynamics 238:2922–2928, 2009. © 2009 Wiley‐Liss, Inc.     

Hematopoietic growth factors: the scenario in zebrafish

Humoral regulation by ligand/receptor interactions is a fundamental feature of vertebrate hematopoiesis. Zebrafish are an established vertebrate animal model of hematopoiesis, sharing with mammals conserved genetic, molecular and cell biological regulatory mechanisms. This comprehensive review considers zebrafish hematopoiesis from the perspective of the hematopoietic growth factors (HGFs), their receptors and their actions. Zebrafish possess multiple HGFs: CSF1 (M-CSF) and CSF3 (G-CSF), kit ligand (KL, SCF), erythropoietin (EPO), thrombopoietin (THPO/TPO), and the interleukins IL6, IL11, and IL34. Some ligands and/or receptor components have been duplicated by various mechanisms including the teleost whole genome duplication, adding complexity to the ligand/receptor interactions possible, but also providing examples of several different outcomes of ligand and receptor subfunctionalization or neofunctionalization. CSF2 (GM-CSF), IL3 and IL5 and their receptors are absent from zebrafish. Overall the humoral regulation of hematopoiesis in zebrafish displays considerable similarity with mammals, which can be applied in biological and disease modelling research.     

斑马鱼类胰岛素生长因子信号途径及作用机制研究进展

斑马鱼是研究早期发育中类胰岛素生长因子（insulin-like growth factors, IGF）信号途径的模式生物,斑马鱼IGF信号系统主要包括IGF配体、受体、结合蛋白（IGFBPs）。IGF配体包括IGF-1,IGF-2。受体为IGF-1R,该受体有两种全长结构（igf-1ra和igf-1rb）。结合蛋白包括IGFBP-1,IGFBP-2,IGFBP-3,IGFBP-5和 IGFBP-6,它们的结构特征已经阐明,从斑马鱼模式动物中获得的信息不仅可以为斑马鱼胚胎发育生物学提供新的观点,还可以进一步加深我们对一般意义上的脊椎动物的生长和发育的理解。本文就近年来斑马鱼IGF系统的进展作一综述。     

Maternal stress suppresses cell proliferation in the forebrain of zebrafish larvae

Exposure to early life stress (ELS) can increase vulnerability to various psychiatric disorders. Although ELS has been shown to alter structure and functions of the hippocampus, amygdala and prefrontal cortex in the adult mammalian brain, it remains largely unclear whether ELS also affects embryonic or early‐stage brain development. In this study, I investigated the effects of a maternal stress (maternal starvation for 4 days) of adult zebrafish on offspring's larval brain development. Although maternal starvation did not largely affect proliferation rate in the midbrain and hindbrain, it significantly decreased that in the forebrain of larvae at 5 days post‐fertilization (dpf). I also found that embryos at 10 hr post‐fertilization (hpf) born from a starved mother showed elevated cortisol levels compared to those born from a control mother. Furthermore, cortisol treatment was sufficient to decrease proliferating cells in the forebrain of 5 dpf larvae. Our findings thus demonstrate for the first time that maternal starvation induces neurodevelopmental changes in the forebrain of zebrafish larvae and points to a possible role of maternal cortisol in mediating this effect of maternal stress to offsprings.     

Midkine-b regulates cell specification at the neural plate border in zebrafish.

Midkines compose a family of secreted, heparin-binding growth factors with neurotrophic activity in vitro, but largely unknown functions in mammals in vivo. Here we show that one member of this family, Midkine-b (Mdkb), is responsible for establishment of the neural plate border in zebrafish. We propose that MdkB acts downstream of several signaling factors, most notably retinoic acid, implicated in neural crest cell (ncc) induction and refines a zone of competence for ncc and Rohon-Beard (RB) sensory neuron formation. Overexpression of Mdkb expands the cell numbers of various ncc precursor subtypes and results in a significant increase in the number of RB neurons at the neural plate border. On the other hand, Morpholino-mediated knockdown of Mdkb leads to a dramatic reduction of ncc and a loss of sensory neurons. Our results imply that Mdkb is required for the earliest steps of cell specification at the neural plate border in zebrafish.     

Many ribosomal protein mutations are associated with growth impairment and tumor predisposition in zebrafish

We have characterized 28 zebrafish lines with heterozygous mutations in ribosomal protein (rp) genes, and found that 17 of these are prone to develop zebrafish malignant peripheral nerve sheath tumors (zMPNST). Heterozygotes from the vast majority of tumor‐prone rp lines were found to be growth‐impaired, though not all growth‐impaired rp lines were tumor‐prone. Significantly, however, the rp lines with the greatest incidence of zMPNSTs all displayed a growth impairment. Furthermore, heterozygous cells from one tumor‐prone rp line were out‐competed by wild‐type cells in chimeric embryos. The growth impairment resulting from heterozygosity for many rp genes suggests that a global defect in protein translation exists in these lines, raising the possibility that a translation defect that precedes tumor development is predictive of tumorigenesis. Developmental Dynamics 238:76–85, 2009. © 2008 Wiley‐Liss, Inc.     

Gene expression analysis on sections of zebrafish regenerating fins reveals limitations in the whole-mount in situ hybridization method.

The caudal fin of adult zebrafish is used to study the molecular mechanisms that govern regeneration processes. Most reports of gene expression in regenerating caudal fins rely on in situ hybridization (ISH) on whole-mount samples followed by sectioning of the samples. In such reports, expression is mostly confined to cells other than those located between the dense collagenous structures that are the actinotrichia and lepidotrichia. Here, we re-examined the expression of genes by performing ISH directly on cryo-sections of regenerates. We detected expression of some of these genes in cell types that appeared to be non-expressing when ISH was performed on whole-mount samples. These results demonstrate that ISH reagents have a limited capacity to penetrate between the regenerating skeletal matrices and suggest that ISH performed directly on fin sections is a preferable method to study gene expression in fin regenerates.     

Development of Cortical Bone Geometry in the Human Femoral and Tibial Diaphysis

Ontogenetic growth processes in human long bones are key elements, determining the variability of adult bone structure. This study seeks to identify and describe the interaction between ontogenetic growth periods and changes in femoral and tibial diaphyseal shape. Femora and tibiae (n = 46) ranging developmentally from neonate to skeletally mature were obtained from the Norris Farms No. 36 archeological skeletal series. High‐resolution X‐ray computed tomography scans were collected. Whole‐diaphysis cortical bone drift patterns and relative bone envelope modeling activity across ages were assessed in five cross‐sections per bone (total bone length: 20%, 35%, 50%, 65%, and 80%) by measuring the distance from the section centroid to the endosteal and periosteal margins in eight sectors using ImageJ. Pearson correlations were performed to document and interpret the relationship between the cross‐sectional shape (I_max/I_min), total subperiosteal area, cortical area, and medullary cavity area for each slice location and age for both the femur and the tibia. Differences in cross‐sectional shape between age groups at each cross‐sectional position were assessed using nonparametric Mann‐Whitney U tests. The data reveal that the femoral and tibial midshaft shape are relatively conserved throughout growth; yet, conversely, the proximal and distal femoral diaphysis and proximal tibial diaphysis appear more sensitive to developmentally induced changes in mechanical loading. Two time periods of accelerated change are identified: early childhood and prepuberty/adolescence. Anat Rec, 296:774–787, 2013. © 2013 Wiley Periodicals, Inc.     

The zebrafish embryo as a dynamic model of anoxia tolerance.

Developing organisms depend upon a delicate balance in the supply and demand of energy to adapt to variable oxygen availability, although the essential mechanisms determining such adaptation remain elusive. In this study, we examine reversible anoxic arrest and dynamic bioenergetic transitions during zebrafish development. Our data reveal that the duration of anoxic viability corresponds to the developmental stage and anaerobic metabolic rate. Diverse chemical inhibitors of mitochondrial oxidative phosphorylation induce a similar arrest in normoxic embryos, suggesting a pathway responsive to perturbations in aerobic energy production rather than molecular oxygen. Consistent with this concept, arrest is accompanied by rapid activation of the energy-sensing AMP-activated protein kinase pathway, demonstrating a potential link between the sensing of energy status and adaptation to oxygen availability. These observations permit mechanistic insight into energy homeostasis during development that now enable genetic and small molecule screens in this vertebrate model of anoxia tolerance.     

Valproic acid,a histone deacetylase inhibitor,regulates cell proliferation in the adult zebrafish optic tectum

Background: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Several reports have demonstrated that VPA functions as a histone deacetylase (HDAC) inhibitor. While VPA is known to cause teratogenic changes in the embryonic zebrafish brain, its effects on neural stem cells (NSCs) in both the embryonic and adult zebrafish are not well understood. Results: In this study, we observed a proliferative effect of VPA on NSCs in the embryonic hindbrain. In contrast, VPA reduced cell proliferation in the adult zebrafish optic tectum. Treatment with HDAC inhibitors showed a similar inhibitory effect on cell proliferation in the adult zebrafish optic tectum, suggesting that VPA reduces cell proliferation through HDAC inhibition. Cell cycle progression was also suppressed in the optic tectum of the adult zebrafish brain because of HDAC inhibition. Recent studies have demonstrated that HDAC inhibits the Notch signaling pathway; hence, adult zebrafish were treated with a Notch inhibitor. This increased the number of proliferating cells in the adult zebrafish optic tectum with down‐regulated expression of her4, a target of Notch signaling. Conclusions: These results suggest that VPA inhibits HDAC activity and upregulates Notch signaling to reduce cell proliferation in the optic tectum of adult zebrafish. Developmental Dynamics 243:1401–1415, 2014. © 2014 Wiley Periodicals, Inc.     

Ontogenetic relationships between in vivo strain environment, bone histomorphometry and growth in the goat radius

Vertebrate long bone form, at both the gross and the microstructural level, is the result of many interrelated influences. One factor that is considered to have a significant effect on bone form is the mechanical environment experienced by the bone during growth. The work presented here examines the possible relationships between in vivo bone strains, bone geometry and histomorphology in the radii of three age/size groups of domestic goats. In vivo bone strain data were collected from the radii of galloping goats, and the regional cortical distribution of peak axial strain magnitudes, radial and circumferential strain gradients, and longitudinal strain rates related to regional patterns in cortical growth, porosity, remodelling and collagen fibre orientation. Although porosity and remodelling decreased and increased with age, respectively, these features showed no significant regional differences and did not correspond to regional patterns in the mechanical environment. Thicker regions of the radius's cortex were significantly related to high strain levels and higher rates of periosteal, but not endosteal, growth. However, cortical growth and strain environment were not significantly related. Collagen fibre orientation varied regionally, with a higher percentage of transverse fibres in the caudal region of the radius and primarily longitudinal fibres elsewhere, and, although consistent through growth, also did not generally correspond to regional strain patterns. Although strain magnitudes increased during ontogeny and regional strain patterns were variable over the course of a stride, mean regional strain patterns were generally consistent with growth, suggesting that regional growth patterns and histomorphology, in combination with external loads, may play some role in producing a relatively 'predictable' strain environment within the radius. It is further hypothesized that the absence of correlation between regional histomorphometric patterns and the measured strain environments is the result of the variable mechanical environment. However, the potential effects of other physiological and mechanical factors, such as skeletal metabolism and adjacent muscle insertions, that can influence the gross and microstructural morphology of the radius during ontogeny, cannot be ignored.     

Transgenic analysis of the anterior eye-specific enhancers of the zebrafish gelsolin-like 1 (gsnl1) gene.

The anterior segment of the eye includes such structures as the cornea, lens, iris, and ciliary body and is essential for many visual and physiological functions of the eye. The zebrafish gelsolin-like 1 (gsnl1) gene encodes an actin regulatory protein and is expressed in the anterior segment of the eye. We report the transgenic analyses of the gsnl1 promoter and enhancer that are required for expression in the anterior segment of the eye. A 6.4-kb genomic fragment upstream from the translation initiation site (ATG) was capable of driving green fluorescent protein (GFP) expression in transient transgenic embryos and stable transgenic adult fish, which mimics the endogenous gsnl1 expression. The GFP expression was localized in the corneal epithelium (CE) and the annular ligament (AL) at the iridocorneal angle. A unique enhancer for each of these two tissues was identified at 3.7-kb upstream from the ATG. The 60-bp AL and 25-bp CE enhancers were separated by 100-bp and functioned independently from each other. Deletion analysis indicated that the proximal promoter was located 1.6-kb upstream from the ATG. Stable GFP transgenic lines were established for future studies of genetic regulation in the anterior segment of the fish eye.     

Dynamic analysis of BMP‐responsive smad activity in live zebrafish embryos

Bone morphogenetic proteins (BMPs) are critical players in development and disease, regulating such diverse processes as dorsoventral patterning, palate formation, and ossification. These ligands are classically considered to signal via BMP receptor‐specific Smad proteins 1, 5, and 8. To determine the spatiotemporal pattern of Smad1/5/8 activity and thus canonical BMP signaling in the developing zebrafish embryo, we generated a transgenic line expressing EGFP under the control of a BMP‐responsive element. EGFP is expressed in many established BMP signaling domains and is responsive to alterations in BMP type I receptor activity and smad1 and smad5 expression. This transgenic Smad1/5/8 reporter line will be useful for determining ligand and receptor requirements for specific domains of BMP activity, as well as for genetic and pharmacological screens aimed at identifying enhancers or suppressors of canonical BMP signaling. Developmental Dynamics 240:682–694, 2011. © 2011 Wiley‐Liss, Inc.     

Gateway compatible vectors for analysis of gene function in the zebrafish.

The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos.     

Alendronate rescued osteoporotic phenotype in a model of glucocorticoid‐induced osteoporosis in adult zebrafish scale

Long‐term effects of glucocorticoid treatment in humans induce bone loss and increase the risk of fracture in the skeleton. The pathogenic mechanisms of glucocorticoid‐induced osteoporosis (GIOP) are still unclear. The GIOP and its effects have been reproduced in several animal models including Danio rerio (zebrafish) embryo. The treatment of adult fish with prednisolone (PN) has shown a dose‐dependent decrease of mineralized matrix in the scales. Large resorption lacunae are characterized by single TRAP‐positive cells which migrate to the margin of the scale merging into a multinucleated structures. The treatment with PN of cultured scales did not increase TRAP activity suggesting that the massive presence of osteoclasts in the resorption sites could be likely the result of a systemic recruitment of monocyte–macrophage precursors. We observed that treatment with PN induced a significant decrease of the alkaline phosphatase (ALP) activity in scale scleroblasts if compared with untreated controls. Then, we investigated the total mineral balance under prednisolone treatment using a time‐dependent double live staining. The untreated fish fully repaired the resorption lacuna induced by prednisolone, whereas treated fish failed. The presence of osteoclast resorption fingerprints on new matrix suggested that the osteoclast activity counterbalances the osteodepositive activity exerted by scleroblasts. The treatment with PN in association with alendronate (AL) has surprisingly resulted in a significant decrease of TRAP activity and increase of ALP compared to PN‐treated fish in biochemical and histological assays confirming the action of alendronate against GIOP in fish as well in humans.     

Nerve growth factor is expressed and stored in central neurons of adult zebrafish

Nerve growth factor (NGF), a member of the neurotrophin family, was initially described as neuronal survival and growth factor, but successively has emerged as an active mediator in many essential functions in the central nervous system of mammals. NGF is synthesized as a precursor pro‐NGF and is cleaved intracellularly into mature NGF. However, recent evidence demonstrates that pro‐NGF is not a simple inactive precursor, but is also secreted outside the cells and can exert multiple roles. Despite the vast literature present in mammals, studies devoted to NGF in the brain of other vertebrate models are scarce. Zebrafish is a teleost fish widely known for developmental genetic studies and is well established as model for translational neuroscience research. Genomic organization of zebrafish and mouse NGF is highly similar, and zebrafish NGF protein has been reported in mature and two‐precursors forms. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the NGF mRNA and protein distribution in the adult zebrafish brain and to characterize the phenotype of NGF‐positive cells. NGF mRNA was visualized by in situ hybridization on whole‐mount brains. NGF protein distribution was assessed on microtomic sections by using an antiserum against NGF, able to recognize pro‐NGF in adult zebrafish brain as demonstrated also in previous studies. To characterize NGF‐positive cells, anti‐NGF was employed on microtomic slides of aromatase B transgenic zebrafish (where radial glial cells appeared fluorescent) and by means of double‐immunolabeling against NGF/proliferative cell nuclear antigen (PCNA; proliferation marker) and NGF/microtube‐associated protein2 (MAP2; neuronal marker). NGF mRNA and protein were widely distributed in the brain of adult zebrafish, and their pattern of distribution of positive perikaryal was overlapping, both in males and females, with few slight differences. Specifically, the immunoreactivity to the protein was observed in fibers over the entire encephalon. MAP2 immunoreactivity was present in the majority of NGF‐positive cells, throughout the zebrafish brain. PCNA and aromatase B cells were not positive to NGF, but they were closely intermingled with NGF cells. In conclusion, our study demonstrated that mature neurons in the zebrafish brain express NGF mRNA and store pro‐NGF.     

Epidermal growth factor receptor signalling contributes to osteoblastic stromal cell proliferation, osteoclastogenesis and disease progression in giant cell tumour of bone

Balla P, Moskovszky L, Sapi Z, Forsyth R, Knowles H, Athanasou N A, Szendroi M, Kopper L, Rajnai H, Pinter F, Petak I, Benassi M S, Picci P, Conti A & Krenacs T
(2011) Histopathology 59 , 376–389 Epidermal growth factor receptor signalling contributes to osteoblastic stromal cell proliferation, osteoclastogenesis and disease progression in giant cell tumour of bone Aims: Epidermal growth factor receptor (EGFR) is implicated in bone remodelling. The aim was to determine whether EGFR protein expression contributes to the aggressiveness and recurrence potential of giant cell tumour of bone (GCTB), an osteolytic primary bone tumour that can exhibit markedly variable clinical behaviour. Methods and results: Immunohistochemical analysis on tissue microarrays (TMA) of 231 primary, 97 recurrent, 17 metastatic and 26 malignant GCTBs was performed using TMA analysis software and whole digital slides allowing validated scoring. EGFR expression was restricted to neoplastic stromal cells and was significantly more frequent in recurrent (71 of 92; 77%) than in non‐recurrent GCTBs (86 of 162; 53%) (P = 0.002); and in clinicoradiologically aggressive (31 of 43; 72%) than latent (27 of 54; 50%) cases (P = 0.034). Detecting phosphotyrosine epitopes pY1068 and ‐pY1173 indicated active EGFR signalling, and finding EGFR ligands EGF and transforming growth factor‐α restricted to cells of the monocytic lineage suggested paracrine EGFR activation in stromal cells. In functional studies EGF supported proliferation of GCTB stromal cells, and the addition of EGF and macrophage‐colony stimulating factor promoted osteoclastogenesis. Conclusion: In GCTB, EGFR signalling in neoplastic stromal cells may contribute to disease progression through promoting stromal cell proliferation and osteoclastogenesis.     

fgf1 is required for normal differentiation of erythrocytes in zebrafish primitive hematopoiesis.

Hematopoiesis in vertebrate development involves an embryonic, primitive wave and a later, definitive wave in which embryonic blood cells are replaced with adult blood cells. We here show that zebrafish fgf1 is involved in vivo in primitive hematopoiesis. Fibroblast growth factor-1 (FGF1) morpholino knockdown leads to abnormal accumulation of blood cells in the posterior intermediate cell mass at 32 hr postfertilization. Expression of the erythroid markers gata1 and ika, normally diminishing in differentiating erythrocytes at this stage, is maintained at abnormally high levels in primitive blood cells. The onset of erythrocyte differentiation as assessed by o-dianisidine staining is severely delayed. Most fgf1 morphants later recover to wild-type appearance, and primitive erythrocytes eventually differentiate. Zebrafish fgf1 is syntenic to human FGF1, which maps to a critically deleted region in human del(5q) syndrome posing an increased risk of leukemia to patients. As its knockdown in zebrafish changes expression of gata1, a gene involved in hematopoietic stem cell decisions, FGF1 should be considered to play a role in the pathogenesis of del(5q) syndrome.