Mcrs1 is required for branchial arch and cranial cartilage development.

Mcrs1 is a multifunctional protein that is critical for many cellular processes in a wide range of cell types. Previously, we showed that Mcrs1 binds to the Six1 transcription factor and reduces the ability of the Six1-Eya1 complex to upregulate transcription, and that Mcrs1 loss-of-function leads to the expansion of several neural plate genes, reduction of neural border and pre-placodal ectoderm (PPR) genes, and pleiotropic effects on various neural crest (NC) genes. Because the affected embryonic structures give rise to several of the cranial tissues affected in Branchio-otic/Branchio-oto-renal (BOR) syndrome, herein we tested whether these gene expression changes subsequently alter the development of the proximate precursors of BOR affected structures - the otic vesicles (OV) and branchial arches (BA). We found that Mcrs1 is required for the expression of several OV genes involved in inner ear formation, patterning and otic capsule cartilage formation. Mcrs1 knockdown also reduced the expression domains of many genes expressed in the larval BA, derived from either NC or PPR, except for emx2, which was expanded. Reduced Mcrs1 also diminished the length of the expression domain of tbx1 in BA1 and BA2 and interfered with cranial NC migration from the dorsal neural tube; this subsequently resulted in defects in the morphology of lower jaw cartilages derived from BA1 and BA2, including the infrarostral, Meckel's, and ceratohyal as well as the otic capsule. These results demonstrate that Mcrs1 plays an important role in processes that lead to the formation of craniofacial cartilages and its loss results in phenotypes consistent with reduced Six1 activity associated with BOR.

Biallelic disruption of PKDCC is associated with a skeletal disorder characterised by rhizomelic shortening of extremities and dysmorphic features.

BACKGROUND: During mouse embryonic development the protein kinase domain containing, cytoplasmic (Pkdcc) gene, also known as Vlk, is expressed in several tissues including the ventral midbrain, with particularly strong expression in branchial arches and limb buds. Homozygous Pkdcc knockout mice have dysmorphic features and shortened long bones as the most obvious morphological abnormalities. The human PKDCC gene has currently not been associated with any disorders. OBJECTIVE: To use clinical diagnostic exome sequencing (DES) for providing genetic diagnoses to two apparently unrelated patients with similar skeletal abnormalities comprising rhizomelic shortening of limbs and dysmorphic features. METHODS: Patient-parents trio DES was carried out and the identified candidate variants were confirmed by Sanger sequencing. RESULTS: Each patient had a homozygous gene disrupting variant in PKDCC considered to explain the skeletal phenotypes shared by both. The first patient was homozygous for the nonsense variant p.(Tyr217*) (NM_1 38 370 c.651C>A) expected to result in nonsense-mediated decay of the mutant transcripts, whereas the second patient was homozygous for the splice donor variant c.639+1G>T predicted to abolish the donor splice site by three in silico splice prediction algorithms. CONCLUSIONS: Biallelic gene disrupting variants in PKDCC in humans, just like in mice, cause dysmorphic features and rhizomelic shortening of limbs.

In vivo effects of 17α-ethinylestradiol, 17ß-estradiol and 4-nonylphenol on insulin-like growth-factor binding proteins (igfbps) in Atlantic salmon.

Feminizing endocrine disrupting compounds (EDCs) affect the growth and development of teleost fishes. The major regulator of growth performance, the growth hormone (Gh)/insulin-like growth-factor (Igf) system, is sensitive to estrogenic compounds and mediates certain physiological and potentially behavioral consequences of EDC exposure. Igf binding proteins (Igfbps) are key modulators of Igf activity, but their alteration by EDCs has not been examined. We investigated two life-stages (fry and smolts) of Atlantic salmon (Salmo salar), and characterized how the Gh/Igf/Igfbp system responded to waterborne 17α-ethinylestradiol (EE2), 17ß-estradiol (E2) and 4-nonylphenol (NP). Fry exposed to EE2 and NP for 21 days had increased hepatic vitellogenin (vtg) mRNA levels while hepatic estrogen receptor α (erα), gh receptor (ghr), igf1 and igf2 mRNA levels were decreased. NP-exposed fry had reduced body mass and total length compared to controls. EE2 and NP reduced hepatic igfbp1b1, -2a, -2b1, -4, -5b2 and -6b1, and stimulated igfbp5a. In smolts, hepatic vtg mRNA levels were induced following 4-day exposures to all three EDCs, while erα only responded to EE2 and E2. EDC exposures did not affect body mass or fork length; however, EE2 diminished plasma Gh and Igf1 levels in parallel with reductions in hepatic ghr and igf1. In smolts, EE2 and E2 diminished hepatic igfbp1b1, -4 and -6b1, and stimulated igfbp5a. There were no signs of compromised ionoregulation in smolts, as indicated by unchanged branchial ion pump/transporter mRNA levels. We conclude that hepatic igfbps respond (directly and/or indirectly) to environmental estrogens during two key life-stages of Atlantic salmon, and thus may modulate the growth and development of exposed individuals.

Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes.

European sea bass Dicentrarchus labrax undertake seasonal migrations to estuaries and lagoons that are characterized by fluctuations in environmental conditions. Their ability to cope with these unstable habitats is undeniable, but it is still not clear how and to what extent salinity acclimation mechanisms are affected at temperatures higher than in the sea. In this study, juvenile sea bass were pre-acclimated to seawater (SW) at 18°C (temperate) or 24°C (warm) for 2weeks and then transferred to fresh water (FW) or SW at the respective temperature. Transfer to FW for two weeks resulted in decreased blood osmolalities and plasma Cl- at both temperatures. In FW warm conditions, plasma Na+ was ~15% lower and Cl- was ~32% higher than in the temperate-water group. Branchial Na+/K+-ATPase (NKA) activity measured at the acclimation temperature (Vapparent) did not change according to the conditions. Branchial Na+/K+-ATPase activity measured at 37°C (Vmax) was lower in warm conditions and increased in FW compared to SW conditions whatever the considered temperature. Mitochondrion-rich cell (MRC) density increased in FW, notably due to the appearance of lamellar MRCs, but this increase was less pronounced in warm conditions where MRC's size was lower. In SW warm conditions, pavement cell apical microridges are less developed than in other conditions. Overall gill morphometrical parameters (filament thickness, lamellar length and width) differ between fish that have been pre-acclimated to different temperatures. This study shows that a thermal change affects gill plasticity affecting whole-organism ion balance two weeks after salinity transfer.

Extrinsic nerves are not involved in branchial 5-HT dynamics or pulsatile urea excretion in Gulf toadfish, Opsanus beta.

Gulf toadfish (Opsanus beta) can switch from continuously excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses. Previous studies have shown that the neurotransmitter serotonin (5-HT) is involved in controlling this process, but it is unknown if 5-HT availability is under central nervous control or if the 5-HT signal originates from a peripheral source. Following up on a previous study, cranial nerves IX (glossopharyngeal) and X (vagus) were sectioned to further characterize their role in controlling pulsatile urea excretion and 5-HT release within the gill. In contrast to an earlier study, nerve sectioning did not result in a change in urea pulse frequency. Total urea excretion, average pulse size, total nitrogen excretion, and percent ureotely were reduced the first day post-surgery in nerve-sectioned fish but recovered by 72h post-surgery. Nerve sectioning also had no effect on toadfish urea transporter (tUT), 5-HT transporter (SERT), or 5-HT2A receptor mRNA expression or 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) abundance in the gill, all of which were found consistently across the three gill arches except 5-HIAA, which was undetectable in the first gill arch. Our findings indicate that the central nervous system does not directly control pulsatile urea excretion or local changes in gill 5-HT and 5-HIAA abundance.

Identification of the Long-Sought Leptin in Chicken and Duck: Expression Pattern of the Highly GC-Rich Avian leptin Fits an Autocrine/Paracrine Rather Than Endocrine Function.

More than 20 years after characterization of the key regulator of mammalian energy balance, leptin, we identified the leptin (LEP) genes of chicken (Gallus gallus) and duck (Anas platyrhynchos). The extreme guanine-cytosine content (∼70%), the location in a genomic region with low-complexity repetitive and palindromic sequence elements, the relatively low sequence conservation, and low level of expression have hampered the identification of these genes until now. In vitro-expressed chicken and duck leptins specifically activated signaling through the chicken leptin receptor in cell culture. In situ hybridization demonstrated expression of LEP mRNA in granular and Purkinje cells of the cerebellum, anterior pituitary, and in embryonic limb buds, somites, and branchial arches, suggesting roles in adult brain control of energy balance and during embryonic development. The expression patterns of LEP and the leptin receptor (LEPR) were explored in chicken, duck, and quail (Coturnix japonica) using RNA-sequencing experiments available in the Short Read Archive and by quantitative RT-PCR. In adipose tissue, LEP and LEPR were scarcely transcribed, and the expression level was not correlated to adiposity. Our identification of the leptin genes in chicken and duck genomes resolves a long lasting controversy regarding the existence of leptin genes in these species. This identification was confirmed by sequence and structural similarity, conserved exon-intron boundaries, detection in numerous genomic, and transcriptomic datasets and characterization by PCR, quantitative RT-PCR, in situ hybridization, and bioassays. Our results point to an autocrine/paracrine mode of action for bird leptin instead of being a circulating hormone as in mammals.

An Essential Role for Parathyroid Hormone in Gill Formation and Differentiation of Ion-Transporting Cells in Developing Zebrafish.

In vertebrates, parathyroid hormone (PTH) is important for skeletogenesis and Ca(2+) homeostasis. However, little is known about the molecular mechanisms by which PTH regulates skeleton formation and Ca(2+) balance during early development. Using larval zebrafish as an in vivo model system, we determined that PTH1 regulates the differentiation of epithelial cells and the development of craniofacial cartilage. We demonstrated that translational gene knockdown of PTH1 decreased Ca(2+) uptake at 4 days after fertilization. We also observed that PTH1-deficient fish exhibited reduced numbers of epithelial Ca(2+) channel (ecac)-expressing cells, Na(+)/K(+)-ATPase-rich cells, and H(+)-ATPase-rich cells. Additionally, the density of epidermal stem cells was decreased substantially in the fish experiencing PTH1 knockdown. Knockdown of PTH1 caused a shortening of the jaw and impeded the development of branchial arches. Results from in situ hybridization suggested that the expression of collagen 2a1a (marker for proliferating chondrocytes) was substantially reduced in the cartilage that forms the jaw and branchial aches. Disorganization of chondrocytes in craniofacial cartilage also was observed in PTH1-deficient fish. The results of real-time PCR demonstrated that PTH1 morphants failed to express the transcription factor glial cell missing 2 (gcm2). Coinjection of PTH1 morpholino with gcm2 capped RNA rescued the phenotypes observed in the PTH1 morphants, suggesting that the defects in PTH1-deficient fish were caused, at least in part, by the suppression of gcm2. Taken together, the results of the present study reveal critical roles for PTH1 in promoting the differentiation of epidermal stem cells into mature ionocytes and cartilage formation during development.

Hoxa2 selectively enhances Meis binding to change a branchial arch ground state.

Hox transcription factors (TFs) are essential for vertebrate development, but how these evolutionary conserved proteins function in vivo remains unclear. Because Hox proteins have notoriously low binding specificity, they are believed to bind with cofactors, mainly homeodomain TFs Pbx and Meis, to select their specific targets. We mapped binding of Meis, Pbx, and Hoxa2 in the branchial arches, a series of segments in the developing vertebrate head. Meis occupancy is largely similar in Hox-positive and -negative arches. Hoxa2, which specifies second arch (IIBA) identity, recognizes a subset of Meis prebound sites that contain Hox motifs. Importantly, at these sites Meis binding is strongly increased. This enhanced Meis binding coincides with active enhancers, which are linked to genes highly expressed in the IIBA and regulated by Hoxa2. These findings show that Hoxa2 operates as a tissue-specific cofactor, enhancing Meis binding to specific sites that provide the IIBA with its anatomical identity.

Sonic hedgehog from pharyngeal arch 1 epithelium is necessary for early mandibular arch cell survival and later cartilage condensation differentiation.

BACKGROUND: Morphogenesis of vertebrate craniofacial skeletal elements is dependent on a key cell population, the cranial neural crest cells (NCC). Cranial NCC are formed dorsally in the cranial neural tube and migrate ventrally to form craniofacial skeletal elements as well as other tissues. Multiple extracellular signaling pathways regulate the migration, survival, proliferation, and differentiation of NCC. RESULTS: In this study, we demonstrate that Shh expression in the oral ectoderm and pharyngeal endoderm is essential for mandibular development. We show that a loss of Shh in these domains results in increased mesenchymal cell death in pharyngeal arch 1 (PA1) after NCC migration. This increased cell death can be rescued in utero by pharmacological inhibition of p53. Furthermore, we show that epithelial SHH is necessary for the early differentiation of mandibular cartilage condensations and, therefore, the subsequent development of Meckel's cartilage, around which the dentary bone forms. Nonetheless, a rescue of the cell death phenotype does not rescue the defect in cartilage condensation formation. CONCLUSIONS: Our results show that SHH produced by the PA1 epithelium is necessary for the survival of post-migratory NCC, and suggests a key role in the subsequent differentiation of chondrocytes to form Meckel's cartilage.

Novel variants in GNAI3 associated with auriculocondylar syndrome strengthen a common dominant negative effect.

Auriculocondylar syndrome is a rare craniofacial disorder comprising core features of micrognathia, condyle dysplasia and question mark ear. Causative variants have been identified in PLCB4, GNAI3 and EDN1, which are predicted to function within the EDN1-EDNRA pathway during early pharyngeal arch patterning. To date, two GNAI3 variants in three families have been reported. Here we report three novel GNAI3 variants, one segregating with affected members in a family previously linked to 1p21.1-q23.3 and two de novo variants in simplex cases. Two variants occur in known functional motifs, the G1 and G4 boxes, and the third variant is one amino acid outside of the G1 box. Structural modeling shows that all five altered GNAI3 residues identified to date cluster in a region involved in GDP/GTP binding. We hypothesize that all GNAI3 variants lead to dominant negative effects.

Sonic Hedgehog promotes the survival of neural crest cells by limiting apoptosis induced by the dependence receptor CDON during branchial arch development.

Cell-adhesion molecule-related/Downregulated by Oncogenes (CDO or CDON) was identified as a receptor for the classic morphogen Sonic Hedgehog (SHH). It has been shown that, in cell culture, CDO also behaves as a SHH dependence receptor: CDO actively triggers apoptosis in absence of SHH via a proteolytic cleavage in CDO intracellular domain. We present evidence that CDO is also pro-apoptotic in the developing neural tube where SHH is known to act as a survival factor. SHH, produced by the ventral foregut endoderm, was shown to promote survival of facial neural crest cells (NCCs) that colonize the first branchial arch (BA1). We show here that the survival activity of SHH on neural crest cells is due to SHH-mediated inhibition of CDO pro-apoptotic activity. Silencing of CDO rescued NCCs from apoptosis observed upon SHH inhibition in the ventral foregut endoderm. Thus, the pair SHH/dependence receptor CDO may play an important role in neural crest cell survival during the formation of the first branchial arch.

Comparative expression analysis of cysteine-rich intestinal protein family members crip1, 2 and 3 during Xenopus laevis embryogenesis.

Members of the cysteine-rich intestinal protein (Crip) family belong to the group 2 LIM proteins. Crip proteins are widely expressed in adult mammals but their expression profile and function during embryonic development are still mostly unknown. In this study, we have described for the first time the spatio-temporal expression pattern of the three family members crip1, crip2 and crip3 during Xenopus laevis embryogenesis by RT-PCR and whole mount in situ hybridization approaches. We observed that all three genes are expressed in the pronephros, branchial arches and the eye. Furthermore, crip1 transcripts could be visualized in the developing cranial ganglia and neural tube. In contrast, crip2 could be detected in the cardiovascular system, the brain and the neural tube while crip3 was expressed in the cranial ganglions and the heart. Based on these findings, we suggest that each crip family member may play an important role during embryonic development.

Morpho-functional response of Nile tilapia (Oreochromis niloticus) to a homeopathic complex.

BACKGROUND: This study evaluated the performance, prevalence of ectoparasites and morpho-functional response of the liver and the branchiae of Nile tilapia (Oreochromis niloticus) raised on fish meal with added of the homeopathic complex Homeopatila 100(®) at different concentrations. METHODS: Post-reversed juvenile Nile tilapia (O. niloticus) of the GIFT (Genetic Improvement of Farmed Tilapia) strain were used in this study. The performance, ectoparasite prevalence and parasite load in the branchiae and skin as well as the liver and branchial histology. Fish were randomly assigned to receive one of four treatments: control, 20 mL hydroalcoholic solution (alcohol 30° GL); 20 mL Homeopatila 100(®) per kg of meal; 40 mL Homeopatila 100(®) per kg of meal; or 60 mL of Homeopatila 100(®) per kg of meal, compared to control with out the addition of the complex. There were four replications per treatment type (16 experimental units total) at a density of 40 fish per m(3) over a period of 57 days. The Kruskal-Wallis H test (p < 0.05) was employed to analyse the physical and chemical parameters of water as well as for parasite prevalence; whereas analysis of variance was used for liver performance. If the values were significant (p < 0.05), they were compared by Tukey's test. Multiple comparisons of averages were performed using Student's t test (p < 0.05). RESULTS: There were no significant between the physical and chemical parameters of the water between the different groups at the end of the experiment. Significant differences (p < 0.05) in the mixed parasite conditions were found within the different Homeopatila 100(®) treatments. The hepatosomatic ratio of fish treated with Homeopatila 100(®) was significantly lower than that of fish from the control group. The best results in the liver and branchiae occurred in fish receiving Homeopatila 100(®) at 40 mL/kg in terms of the number of hepatocytes/mm(2), the intercellular glycogenic behaviour, the rates of histological changes (hyperplasia, lamella fusion and telangiectasia) and the percentage of neutral and acidic mucin-producing cells. CONCLUSION: The addition of Homeopatila 100(®) at a concentration 40 mL per kg/meal to the diet of juvenile Nile tilapias resulted in improved hepatocytes and intracellular glycogen levels as well as the lowest mean rate of branchial histological changes with an increase in acidic mucin-producing cells compared to neutral mucin-producing cells, compared to control.

Effect of low pH exposure on Na(+) regulation in two cichlid fish species of the Amazon.

We evaluated the effects of acute exposure to low pH on Na(+) regulation in two Amazon cichlids collected from natural ion-poor "blackwaters", angelfish (Pterophyllum scalare) and discus (Symphysodon discus). Na(+) uptake kinetic parameters, unidirectional Na(+) fluxes, and net Cl(-) fluxes were determined at pH6.0 and 3.6. At pH6.0, both species presented low unidirectional Na(+) flux rates, with kinetics showing a relatively low affinity for Na(+) (angelfish Km=79, discus Km=268µmolL(-1)), with similar maximum transport capacities (Jmax~535nmolg(-1)h(-1)). Overall, there appeared to be high sensitivity to inhibition by low pH, yet low intrinsic branchial permeability limiting diffusive ion effluxes, resulting in relatively low net loss rates of Na(+), the same strategy as seen previously in other blackwater cichlids, and very different from the strategy of blackwater characids. At low pH, Na(+) uptake in angelfish was inhibited competitively (increased Km=166µmolL(-1)) and non-competitively (decreased Jmax=106nmolg(-1)h(-1)), whereas in discus, only a decrease in Jmax (112nmolg(-1)h(-1)) was statistically significant. An acute reduction in H(+)-ATPase activity, but not in Na(+)/K(+)-ATPase activity, in the gills of angelfish suggests a possible mechanism for this non-competitive inhibition at low pH. Discus fish were more tolerant to low pH than angelfish, as seen by lesser effects of exposure to pH3.6 on unidirectional Na(+) uptake and efflux rates and net Na(+) and Cl(-) loss rates. Overall, discus are better than angelfish in maintaining ionic balance under acidic, ion-poor conditions.

Hes1 is required for the development of pharyngeal organs and survival of neural crest-derived mesenchymal cells in pharyngeal arches.

Hes genes are required to maintain diverse progenitor cell populations during embryonic development. Loss of Hes1 results in a spectrum of malformations of pharyngeal endoderm-derived organs, including the ultimobranchial body (progenitor of C cells), parathyroid, thymus and thyroid glands, together with highly penetrant C-cell aplasia (81%) and parathyroid aplasia (28%). The hypoplastic parathyroid and thymus are mostly located around the pharyngeal cavity, even at embryonic day (E) 15.5 to E18.5, indicating the failure of migration of the organs. To clarify the relationship between these phenotypes and neural crest cells, we examine fate mapping of neural crest cells colonized in pharyngeal arches in Hes1 null mutants by using the Wnt1-Cre/R26R reporter system. In null mutants, the number of neural crest cells labeled by X-gal staining is markedly decreased in the pharyngeal mesenchyme at E12.5 when the primordia of the thymus, parathyroid and ultimobranchial body migrate toward their destinations. Furthermore, phospho-Histone-H3-positive proliferating cells are reduced in number in the pharyngeal mesenchyme at this stage. Our data indicate that the development of pharyngeal organs and survival of neural-crest-derived mesenchyme in pharyngeal arches are critically dependent on Hes1. We propose that the defective survival of neural-crest-derived mesenchymal cells in pharyngeal arches directly or indirectly leads to deficiencies of pharyngeal organs.

Emerging paradigms for complex iron-sulfur cofactor assembly and insertion.

[FeFe]-hydrogenses and molybdenum (Mo)-nitrogenase are evolutionarily unrelated enzymes with unique complex iron-sulfur cofactors at their active sites. The H cluster of [FeFe]-hydrogenases and the FeMo cofactor of Mo-nitrogenase require specific maturation machinery for their proper synthesis and insertion into the structural enzymes. Recent insights reveal striking similarities in the biosynthetic pathways of these complex cofactors. For both systems, simple iron-sulfur cluster precursors are modified on assembly scaffolds by the activity of radical S-adenosylmethionine (SAM) enzymes. Radical SAM enzymes are responsible for the synthesis and insertion of the unique nonprotein ligands presumed to be key structural determinants for their respective catalytic activities. Maturation culminates in the transfer of the intact cluster assemblies to a cofactor-less structural protein recipient. Required roles for nucleotide binding and hydrolysis have been implicated in both systems, but the specific role for these requirements remain unclear. In this review, we highlight the progress on [FeFe]-hydrogenase H cluster and nitrogenase FeMo-cofactor assembly in the context of these emerging paradigms.

Effects of low environmental salinity on the cellular profiles and expression of Na+, K+-ATPase and Na+, K+, 2Cl- cotransporter 1 of branchial mitochondrion-rich cells in the juvenile marine fish Monodactylus argenteus.

The goal of this study was to determine the osmoregulatory ability of a juvenile marine fish, silver moony (Monodactylus argenteus), for the purpose of developing a new experimental species for ecophysiological research. In this study, M. argenteus was acclimated to freshwater (FW), brackish water (BW), or seawater (SW). The salinity tolerance of this euryhaline species was effective, and the fish survived well upon osmotic challenges. The largest apical surface of mitochondrion-rich cells was found in the FW individuals. Immunohistochemical staining revealed that Na(+), K(+)-ATPase immunoreactive (NKA-IR) cells were distributed in the interlamellar region of the gill filaments of the silver moony in all experimental groups. In addition to the filaments, NKA-IR cells were also found in the lamellae of the FW individuals. The number of NKA-IR cells in the gills of the FW individuals exceeded that of the BW and SW individuals. The NKA-IR cells of FW and SW individuals exhibited bigger size than that of BW fish. The NKA activities and protein expression of the NKA α-subunit in the gills of the FW individuals were significantly higher than in the BW and SW groups. Additionally, the relative amounts of Na(+), K(+), 2Cl(-) cotransporter 1 (NKCC1) were salinity-dependent in the gills. Immunofluorescent signals of NKCC1 were localized to the basolateral membrane of NKA-IR cells in all groups. In the gills of the FW individuals, however, some NKA-IR cells did not exhibit a basolateral NKCC1 signal. In conclusion, the present study illustrated the osmoregulatory mechanisms of this easy- and economic-to-rear marine teleost with euryhaline capacity and proved the silver moony to be a good experimental animal.

Chromobox protein homolog 3 is essential for stem cell differentiation to smooth muscles in vitro and in embryonic arteriogenesis.

OBJECTIVE: Smooth muscle cell (SMC) differentiation is a critical process during cardiovascular formation and development, but the underlying molecular mechanism remains unclear. METHODS AND RESULTS: Here we demonstrated that chromobox protein homolog 3 (Cbx3) is crucial for SMC differentiation from stem cells and that the chromodomain and chromoshadow domain of Cbx3 are responsible for Cbx3-induced SMC differentiation. Moreover, we identified that 4 amino acids (165 to 168) within the chromoshadow domain of Cbx3 are key elements for Cbx3 interaction with Dia-1- and Cbx3-induced SMC differentiation. Mechanistically, we found that Cbx3 mediates SMC differentiation through modulating serum response factor (SRF) recruitment to the promoters of SMC genes, in which the interaction between Cbx3 and Dia-1/SRF plays a crucial role in this process. Moreover, our in vivo study demonstrated that the misexpression of Cbx3 within neural crest cells of chick embryos resulted in the death of chick embryos at early stages because of the maldevelopment of branchial arch arteries. CONCLUSIONS: Our findings suggest that the interaction between Cbx3 and Dia-1/SRF is essential for SMC differentiation from stem cells and for the development of functional cardiovascular system.

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia.

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL177 and PRL188; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na+/Cl- cotransporter and a Na+/H+ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na+/K+/2Cl- cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.

Zebrafish wnt9a is expressed in pharyngeal ectoderm and is required for palate and lower jaw development.

Wnt activity is critical in craniofacial morphogenesis. Dysregulation of Wnt/ß-catenin signaling results in significant alterations in the facial form, and has been implicated in cleft palate phenotypes in mouse and man. In zebrafish, we show that wnt9a is expressed in the pharyngeal arch, oropharyngeal epithelium that circumscribes the ethmoid plate, and ectodermal cells superficial to the lower jaw structures. Alcian blue staining of morpholino-mediated knockdown of wnt9a results in loss of the ethmoid plate, absence of lateral and posterior parachordals, and significant abrogation of the lower jaw structures. Analysis of cranial neural crest cells in the sox10:eGFP transgenic demonstrates that the wnt9a is required early during pharyngeal development, and confirms that the absence of Alcian blue staining is due to absence of neural crest derived chondrocytes. Molecular analysis of genes regulating cranial neural crest migration and chondrogenic differentiation suggest that wnt9a is dispensable for early cranial neural crest migration, but is required for chondrogenic development of major craniofacial structures. Taken together, these data corroborate the central role for Wnt signaling in vertebrate craniofacial development, and reveal that wnt9a provides the signal from the pharyngeal epithelium to support craniofacial chondrogenic morphogenesis in zebrafish.