Erratum to "Evolutionary conserved role of ptf1a in the specification of exocrine pancreatic fates" [Dev. Biol. 268 (2004) 174–184]

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Structure and functional analysis of a tilapia (Oreochromis mossambicus) growth hormone gene: activation and repression by pituitary transcription factor Pit-1

A gene encoding the Tilapia mossambica (Oreochromis mossambicus) growth hormone (tiGH) was isolated and sequenced. The gene spans 5.6 kb, including 3.7 kb of 5' and 0.2 kb of 3' flanking sequences and a 1.7-kb transcription unit comprised of six exons and five introns. The gene and the 5' flanking region contain several potential binding sites for Pit-1, a key transcription activator of mammalian GH genes. One of these (-57/-42) is highly conserved in fish GH genes. It activates transcription in pituitary cells and binds Pit-1. Transfection of luciferase reporter plasmids containing either the -3602/+19 tiGH sequence or one of its 5' deletion mutants (-2863/, -1292/, and -463/+19) resulted in strong activity in Pit-1-producing rat pituitary GC cells. A dose-dependent activation of the tiGH promoter was achieved in nonpituitary fish EPC and monkey COS cells cotransfected with a rat Pit-1 expression vector, demonstrating the crucial role played by Pit-1 as an activator of the tiGH gene. Fusion of the tiGH promoter with the beta-galactosidase gene led to transient expression specifically in the nervous system of microinjected zebrafish embryos. The activity of the tiGH promoter in GC and EPC cells was strongly repressed by extending its 3' end from +19 to +40, a sequence in which a Pit-1-binding site was identified using gel retardation assays. Point mutations of the site that suppressed Pit-1 binding in vitro restored full tiGH promoter activity. Thus, a Pit-1-binding site located in the 5' untranslated region mediates Pit-1-dependent repression of the tiGH gene

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers ( foxb1.2/mariposa , krox20 , pax2.1 and pax6.1 ), including anteriorly expressed Hox genes ( hoxb1a , hoxa2 and hoxb2 ), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1 , but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development

Mutageneze v Danio rerio pomocí CRISPR technologie

CRISPR/Cas9 systém je nástroj genového inženýrství umožňující sekvenčně specifické editace genomu. Tato technologie byla využita za účelem studia funkce transkripčních faktorů s DNA vazebnou TALE homeodoménou (TALE - three amino acids loop extension) v průběhu vývoje buněk neurální lišty a její derivátů. Hlavními proteiny zájmu této práce jsou Meis1 transkripční faktory, které se v genomu Dania vyskytují v podobě dvou paralogních genů meis1a a meis1b. Funkce jednotlivých proteinů byla analyzována prostřednictvím mutageneze TALE homeodomény za účelem narušení schopnosti transkripčního faktoru vázat DNA a tím narušit regulaci podřízených genů. Tvorba a následná analýza fenotypu mutantních ryby by mohla odhalit potenciální roli Meis1 proteinů v regulaci vývoje buněk neurální lišty, popřípadě poukázat na důležitost homeodomény v regulační funkci těchto proteinů. Současně byl proveden knock-down experiment pomocí morpholino oligonucleotidů k předběžné analýze funkce jednotlivých meis1 genů a odhadu vzájemné funkční komplementarity. Předběžné výsledky poukazují na důležitost Meis1b proteinu v regulaci vývoje buněk neurální lišty a funkční důležitost jeho DNA vazebné domény. Snížení exprese Meis1a ukázalo, že i tento protein má podíl na regulaci kraniofaciálního vývoje, přičemž detailní popis jeho funkce...CRISPR/Cas9 technology is currently one of the most important tools of genome engineering. This technology allows a precise site-specific gene editing and such ability was applied to study the role of TALE (TALE - three amino acids loop extension) homeodomain transcription factors during neural crest cells development. The main genes of interest, belonging to sub-family of TALE proteins, are Meis1 transcription factors that are present in the zebrafish genome as two paralogous genes, meis1a and meis1b. Their function was assessed by mutating their DNA-binding domain - homeodomain to abrogate the ability of transcription factor to bind DNA and by that disturb regulatory network, in which Meis1 proteins operate in. Phenotype analysis of mutant fish would reveal a potential role of Meis1 proteins in regulation of neural crest cells development and outline the functional significance of the homeodomain in regulatory operations. To determine the regulatory relationship between meis1a and meis1b genes morpholino-based knock-down of the genes was performed. Preliminary results suggest a dominant role of Meis1b in neural crest cells regulation and importance of its homeodomain. Furthermore, knock-down of Meis1a indicates its contribution to regulation of craniofacial development. However, a detailed...Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult

The evolution of the neural crest from an annelid perspective: conserved cell types and signaling pathways in Platynereis dumerilii

The neural crest is indisputably one of the major vertebrate innovations. Neural crest arises at the neural plate border and is the source of many cell types, such as those of the peripheral nervous system (sensory, autonomic neurons and supporting cells), pigments and cartilage. This region of the neural plate also gives rise to Rohon Beard cells (RBc, primary sensory neurons) that differentiate from the same precursor cells of the neural crest (Rossi, Kaji, & Artinger, 2009), (Jacobson, 1981). Despite the recent proposal for neural crest-like cells in basal chordates (Jeffery, Strickler, & Yamamoto, 2004), and the postulation of the origin of neural crest from migrating Rohon Beard cells -like cells, the evolution of the neural crest remains obscure. The aim of my PhD was to shed light on the evolution of such a special cell population in bilaterians. Using classical whole mount in situs, Edu pulse experiments, live imaging and drug treatments I studied the development of the pax3/7+ lateral neuroectoderm of the marine worm Platynereis dumerilii. I used Platynereis because it is a protostome that retains ancestral features and it has been successfully used in previous studies to investigate cell type evolution. I investigated the lateral trunk region because it has been recently proposed that this domain corresponds topologically and molecularly to the dorsal neural tube, where the vertebrate neural crest originates (Denes et al., 2007) I found that the pax3/7+ territory is set very early in development and expresses Rohon Beard cells and neural crest specific genes, such as prdm1-a , msx, ap-2 and snail. Furthermore, I found that canonical Wnt signaling controls the patterning of the annelid lateral neuroectdoderm, as in vertebrates. Next, I analyzed the fate of the cells emerging from this lateral territory. I found that sensory differentiation genes are turned on in ngn+ precursor neurons in a temporal sequence, similar to the one occurring in the neural crest derived sensory neurons (Marmigère & Ernfors, 2007), (Lallemend & Ernfors, 2012). The annelid neurons that arise from the lateral pax3/7+ domain have molecular features of the Rohon Beard-like cells and visceral sensory neurons. I found that also putative supporting cells ensheathing the axons arise peripherally. Next, I asked whether the other typical cell types that are neural crest-derived in vertebrates are present in Platynereis. I found that MitF + melanoblasts , putative enteric neurons as well as collagenous skeleton are also present in Platynereis, but apparently do not arise from the lateral domain. The development, survival and axon-pathfinding of the neural crest derived-sensory neurons depends on the neurotrophic signaling (Davies, 1994), (Gershon, 1994), (Tessarollo, 1998), (Sieber-Blum, 1998),(Ernsberger, 2009) Furthermore, the evolution of the neural crest has been associated with the emergence of this pathway, considered for long time a vertebrate innovation (Wittbrodt, 2007). This prompted me to search for the neurotrophic molecules in Platynereis dumerilii. I found that all the molecules of the canonical neurotrophic signaling are present in the worm and show vertebrate-like molecular features. They are widely expressed in the nervous system, therefore they likely act during neuronal development. This finding refutes the belief that neurotrophic signaling is a chordate novelty: a hypothesis based on a lack of conservation in other protostomes such as Drosophila (Pulido, Campuzano, Koda, Modolell, & Barbacid, 1992)and Lymnea (Beck et al., 2003) . Collectively, these annelid data suggest that the formation of Rohon Beard-like sensory neurons, putative visceral sensory neurons and supporting cells were already a feature of the cells emerging from the lateral neuroectoderm (a neural plate-like territory) at the dawn of bilaterians. A gradual co-option of genetic modules acting in other tissues into the neural plate-like territory might have driven the evolution of bona fine neural crest

Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo

Adult pancreatic islets comprise four cell types, alpha, beta, delta and PP, expressing glucagon, insulin, somatostatin and pancreatic-polypeptide, respectively, arising from cell lineages whose relationships during endocrine pancreas differentiation are still uncertain [Edlund, 1998. Diabetes 47, 1817-1823]. As zebrafish (Danio rerio) represents an attractive vertebrate model to study mutants affecting pancreatic organogenesis [Pack et al., 1996. Development 123, 321-328], we have investigated the expression patterns of islet hormones in zebrafish embryos, from the 16-somite (17 h) to 48-h stages, by whole-mount in situ hybridization and immunofluorescence. Results showed that in the zebrafish pancreatic primordium (a) insulin is the first hormone gene to be expressed, and (b) somatostatin colocalizes with insulin while glucagon-expressing cells, since their appearance, are distinct from insulin- or insulin/somatostatin-expressing cells. Notably, both somatostatin and glucagon, but not insulin, are first expressed in extrapancreatic regions

Differential expression of two somatostatin genes during zebrafish embryonic development

We have identified the cDNAs of two new zebrafish preprosomatostatins, PPSS1 and PPSS3, in addition to the previously cloned PPSS2 (Argenton et al., 1999). PPSS1 is the orthologue of mammalian PPSSs, with a conserved C-terminal SS-14 sequence, PPSS2 is a divergent SS precursor and PPSS3 is a cortistatin-like prohormone. Using whole-mount in situ hybridisation, we have analysed the expression of PPSS1 and PPSS2 in zebrafish embryos up to 5 days post fertilisation. PPSS1 was expressed in the developing pancreas and central nervous system (CNS), whereas PPSS2 expression was exclusively pancreatic. In the CNS, PPSS1 was detected in several areas, in particular in the vagal motor nucleus and in cells that pioneer the tract of the postoptic commissure. PPSS1 was also expressed transiently in the telencephalon and spinal motor neurons. In all areas but the telencephalon PPSS1 was coexpressed with islet-1