Crucial role of zebrafish prox1 in hypothalamic catecholaminergic neurons development

<p>Abstract</p> <p>Background</p> <p><it>Prox1</it>, the vertebrate homolog of <it>prospero </it>in <it>Drosophila melanogaster</it>, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development.</p> <p>Results</p> <p>Here we report that, in zebrafish, <it>prox1 </it>is widely expressed in several districts of the Central Nervous System (CNS). Specifically, we evidenced <it>prox1 </it>expression in a group of neurons, already positive for <it>otp1</it>, located in the hypothalamus at the level of the posterior tuberculum (PT). Prox1 knock-down determines the severe loss of hypothalamic catecholaminergic (CA) neurons, identified by tyrosine hydroxylase (TH) expression, and the synergistic <it>prox1/otp1 </it>overexpression induces the appearance of hypothalamic supernumerary TH-positive neurons and ectopic TH-positive cells on the yolk epitelium.</p> <p>Conclusion</p> <p>Our findings indicate that <it>prox1 </it>activity is crucial for the proper development of the <it>otp1</it>-positive hypothalamic neuronal precursors to their terminal CA phenotype.</p

Full-aperture extended-depth oblique plane microscopy through dynamic remote focusing

Oblique plane microscopy is a method enabling light-sheet fluorescence imaging through a single microscope objective lens by focusing on a tilted plane within the sample. To focus the fluorescence emitted by the oblique plane on a camera, the light is imaged through a pair of remote objective lenses, facing each other at an angle. The aperture mismatch resulting from this configuration limits the effective numerical aperture of the system, reducing image resolution and signal intensity. This manuscript introduces an alternative method to capture the oblique plane on the camera. Instead of relying on angled objective lenses, an electrically tunable lens is employed. This lens adjusts the focal plane of the microscope synchronously with the rolling shutter of a scientific CMOS camera. In this configuration the entire aperture of the objective is effectively employed, increasing the resolution of the system. Moreover, a variety of objective lenses can be employed, enabling the acquisition of wider axial fields of view compared to conventional oblique plane microscopy

Toxin Levels and Profiles in Microalgae from the North-Western Adriatic Sea—15 Years of Studies on Cultured Species

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported

Induced early expression of mrf4 but not myog rescues myogenesis in the myod/myf5 double-morphant zebrafish embryo

Muscle regulatory factors activate myogenesis in all vertebrates, but their role has been studied in great detail only in the mouse embryo, where all but myogenin--Myod, Myf5 and Mrf4--are sufficient to activate (albeit not completely) skeletal myogenesis. In the zebrafish embryo, myod and myf5 are required for induction of myogenesis because their simultaneous ablation prevents muscle development. Here we show that mrf4 but not myog can fully rescue myogenesis in the myod/myf5 double morphant via a selective and robust activation of myod, in keeping with its chromatin-remodelling function in vitro. Rescue does not happen spontaneously, because the gene, unlike that in the mouse embryo, is expressed only at the onset of muscle differentiation, Moreover, because of the transient nature of morpholino inhibition, we were able to investigate how myogenesis occurs in the absence of a myotome. We report that in the complete absence of a myotome, subsequent myogenesis is abolished, whereas myogenesis does proceed, albeit abnormally, when the morpholino inhibition was not complete. Therefore our data also show that the early myotome is essential for subsequent skeletal muscle differentiation and patterning in the zebrafish

CYYR1 gene and Hh-mediated myogenesis during zebrafish development: potential role in rhabdomyosarcoma disease

CYYR1 (Cysteine/tyrosine-rich 1) cloned on HC21 defines a family of highly conserved vertebrate-specific genes. The human locus is characterized by a multitranscript-system including at least six alternative spliced isoforms. To date, the function of the CYYR1 product is still unknown even if original results suggest its possible involvement in myogenesis differentiation with a putative role in the tumorigenic process related to the Hh pathway. Zebrafish cyyr1 orthologue is present in single copy and the predicted protein maintains almost 58% of identity with human protein. By WISH approach, we show a cyyr1 broad expression in central nervous system (CNS), somites and muscles during development. The protein seems to localize in plasma and even nuclear membranes. We perform cyyr1 knock-down with two different approaches: microinjection of morpholino oligos targeting the ATG and the first splice-site of the transcript, and the generation of cyyr1 null fish through CRISPr/Cas9 technique. Defects in heart and muscle development with a significant rescue in embryo co-injected with cyyr1 mRNA, were observed in morphants, while cyyr1 mutants analyses confirmed morphological and molecular weakness in heart. Dysregulation of Nodal and/or Hedgehog (Hh) pathways in zebrafish decreased cyyr1 expression and the injection of cyyr1 mRNA was able to partially rescue Hh-defective phenotype in embryos. In order to verify a putative role of CYYR1 in the process caused by dysfunction of cell differentiation we performed experiments in rhabdomyosarcoma cell lines showing an inverse correlation between CYYR1 expression and the range of differentiating capabilities of these cells. Interestingly, treatments with inhibitors of Hh allow us to confirm a correlation between CYYR1 and this pathway

Crucial role of zebrafish in hypothalamic catecholaminergic neurons development-1

), dorsal view. Eyes or lens have been removed for better lateral viewing. () WISH combined with TH immunohistochemistry. Anti-TH antibody labels the PT and hypothalamic CA neurons at 36 hpf. Colabelling with is evident in a fraction of TH-positive neuroblasts in the hypothalamus (arrowheads), as also confirmed by the longitudinal section of the embryo (). microinjection of MO lowers the number of TH-labelled CA neurons in the hypothalamus in comparison to standard control injected embryos . coinjection of mRNA and MO rescued the morphant phenotype. () Quantitative real time RT-PCR. TH-specific mRNA is almost five-fold decreased following MO injection. The result represents at least three independent experiments, and 18S was used as an internal control. The following abbreviations are used: posterior tuberculum (PT), pituitary (Pit), hypothalamus (Hy), standard control morpholino oligonucleotide (stdr MO). Scale bars indicate 10 μm or 20 μm .<p><b>Copyright information:</b></p><p>Taken from "Crucial role of zebrafish in hypothalamic catecholaminergic neurons development"</p><p>http://www.biomedcentral.com/1471-213X/8/27</p><p>BMC Developmental Biology 2008;8():27-27.</p><p>Published online 10 Mar 2008</p><p>PMCID:PMC2288594.</p><p></p

Crucial role of zebrafish in hypothalamic catecholaminergic neurons development-4

Epiboly (lane 2), 50% epiboly (lane 3), 80% epiboly (lane 4), tail bud (lane 5), 8 somites (lane 6), 15 somites (lane 7), 24 hpf (lane 8), 72 hpf (lane 9), 5 dpf (lane 10) and negative control (lane 11) in the absence of cDNA. ) RT-PCR performed on different adult organs: DNA ladder (L), testis (lane 1), overy (lane 2), gills (lane 3), gut (lane 4), eye (lane 5), brain (lane 6) and liver (lane 7). Arrowhead indicates the size of the -specific PCR product (620 bp). WISH the first signals appeared at 2 s in the otic placode (arrowhead). at 15 s the signal is detected in the lens placode (arrowhead), and somites (inset). at 24 hpf is expressed the hypothalamus (asterisc), the pituitary (black arrowhead), the pretectal segment (prosomere 1) (white arrow), as well as segmentally arranged cells of the hindbrain (black arrow). transverse section through the forebrain of a 24 hpf stage zebrafish embryo shows the signal in the lens (black arrowhead). at 24 hpf additional signals are present in the liver primordium (arrow), and posterior lateral line primordium (arrowheads). later during development, (48 hpf) expression is detected in distinct domains in the liver (arrow) and pancreas (arrowhead), while a further signal appeares in the retina (white arrow). transverse section through the forebrain of a 7 dpf stage zebrafish larva shows the signals in the retina inner nuclear layer (arrow) and in the pretectal nuclei (arrowhead). Lateral views are shown. Frontal view is shown. Dorsal views are shown. Anterior is always to the left. Scale bars indicate 100 μm () or 200 μm ().<p><b>Copyright information:</b></p><p>Taken from "Crucial role of zebrafish in hypothalamic catecholaminergic neurons development"</p><p>http://www.biomedcentral.com/1471-213X/8/27</p><p>BMC Developmental Biology 2008;8():27-27.</p><p>Published online 10 Mar 2008</p><p>PMCID:PMC2288594.</p><p></p

Crucial role of zebrafish in hypothalamic catecholaminergic neurons development-3

Ls in control and overexpressed /embryos at 36 hpf. The most numerous class in the group of GFP mRNA injected control embryos presented 8 CA hypothalamic neurons, and only 3 embryos presented more than 11 TH hypothalamic positive cells (n = 54). The most numerous class in the group of the overexpressed /embryos (n = 64) presented 10 CA neurons, and 20 embryos showed more than 11 TH hypothalamic positive cells. Immunostaining with TH antibody shows ectopic TH positive cells on the yolk surface ectoderm of double injected embryos (arrowheads), while these cells are not present on the yolk of control embryos. Ectopic TH positive cell on the yolk surface ectoderm. Scale bars indicate 50 μm.<p><b>Copyright information:</b></p><p>Taken from "Crucial role of zebrafish in hypothalamic catecholaminergic neurons development"</p><p>http://www.biomedcentral.com/1471-213X/8/27</p><p>BMC Developmental Biology 2008;8():27-27.</p><p>Published online 10 Mar 2008</p><p>PMCID:PMC2288594.</p><p></p

Crucial role of zebrafish in hypothalamic catecholaminergic neurons development-0

Epiboly (lane 2), 50% epiboly (lane 3), 80% epiboly (lane 4), tail bud (lane 5), 8 somites (lane 6), 15 somites (lane 7), 24 hpf (lane 8), 72 hpf (lane 9), 5 dpf (lane 10) and negative control (lane 11) in the absence of cDNA. ) RT-PCR performed on different adult organs: DNA ladder (L), testis (lane 1), overy (lane 2), gills (lane 3), gut (lane 4), eye (lane 5), brain (lane 6) and liver (lane 7). Arrowhead indicates the size of the -specific PCR product (620 bp). WISH the first signals appeared at 2 s in the otic placode (arrowhead). at 15 s the signal is detected in the lens placode (arrowhead), and somites (inset). at 24 hpf is expressed the hypothalamus (asterisc), the pituitary (black arrowhead), the pretectal segment (prosomere 1) (white arrow), as well as segmentally arranged cells of the hindbrain (black arrow). transverse section through the forebrain of a 24 hpf stage zebrafish embryo shows the signal in the lens (black arrowhead). at 24 hpf additional signals are present in the liver primordium (arrow), and posterior lateral line primordium (arrowheads). later during development, (48 hpf) expression is detected in distinct domains in the liver (arrow) and pancreas (arrowhead), while a further signal appeares in the retina (white arrow). transverse section through the forebrain of a 7 dpf stage zebrafish larva shows the signals in the retina inner nuclear layer (arrow) and in the pretectal nuclei (arrowhead). Lateral views are shown. Frontal view is shown. Dorsal views are shown. Anterior is always to the left. Scale bars indicate 100 μm () or 200 μm ().<p><b>Copyright information:</b></p><p>Taken from "Crucial role of zebrafish in hypothalamic catecholaminergic neurons development"</p><p>http://www.biomedcentral.com/1471-213X/8/27</p><p>BMC Developmental Biology 2008;8():27-27.</p><p>Published online 10 Mar 2008</p><p>PMCID:PMC2288594.</p><p></p