Pax3 loss of function delays tumour progression in kRAS-induced zebrafish rhabdomyosarcoma models.

Rhabdomyosarcoma is a soft tissue cancer that arises in skeletal muscle due to mutations in myogenic progenitors that lead to ineffective differentiation and malignant transformation. The transcription factors Pax3 and Pax7 and their downstream target genes are tightly linked with the fusion positive alveolar subtype, whereas the RAS pathway is usually involved in the embryonal, fusion negative variant. Here, we analyse the role of Pax3 in a fusion negative context, by linking alterations in gene expression in pax3a/pax3b double mutant zebrafish with tumour progression in kRAS-induced rhabdomyosarcoma tumours. Several genes in the RAS/MAPK signalling pathway were significantly down-regulated in pax3a/pax3b double mutant zebrafish. Progression of rhabdomyosarcoma tumours was also delayed in the pax3a/pax3b double mutant zebrafish indicating that Pax3 transcription factors have an unappreciated role in mediating malignancy in fusion negative rhabdomyosarcoma.

Alkali-like myosin light chain-1 (myl1) is an early marker for differentiating fast muscle cells in zebrafish.

During myogenesis, muscle precursors become divided into either fast- or slow-twitch fibres, which in the zebrafish occupy distinct domains in the embryo. Genes encoding sarcomeric proteins specific for fast or slow fibres are frequently used as lineage markers. In an attempt to identify and evaluate early definitive markers for cells in the fast-twitch pathway, we analysed genes encoding proteins contributing to the fast sarcomeric structures. The previously uncharacterized zebrafish alkali-like myosin light chain gene (myl1) was found to be expressed exclusively in cells in the fast-twitch pathway initiated at an early stage of fast fibre differentiation. Myl1 was expressed earlier, and in a more fibre type restricted manner, than any of the previously described and frequently used fast myosin light and heavy chain and troponin muscle markers mylz2, mylz3, tnni2, tnnt3a, fMyHC1.3. In summary, this study introduces a novel marker for early differentiating fast muscle cells.

Determination of the expression pattern of the dual promoter of zebrafish fushi tarazu factor-1a following microinjections into zebrafish one cell stage embryos.

The zebrafish fushi tarazu factor-1a (ff1a) is a transcription factor belonging to the NR5A subgroup of nuclear receptors. The NR5A receptors bind DNA as monomers and are considered to be orphans due to their ability to promote transcription of downstream genes without ligands. In zebrafish, four ff1 homologues (Ff1a, Ff1b, Ff1c, and Ff1d) have been identified so far. The gene coding for Ff1a is driven by two separate promoters, and give rise to four splice variants. Ff1a is expressed in the somites and pronephric ducts during somitogenesis and in the brain, liver, and mandibular arch during later embryonic stages. In adults the gene is highly expressed in gonads, liver, and intestine, but can be detected in most tissues. The broad variety of embryonic expression domains indicates several important developmental features. One of the mammalian fushi tarazu factor-1 genes, steroidogenic factor-1 (SF-1), is essential for the development of gonads and adrenals. SF-1 is together with Sox9, WT1, and GATA4 a positive transcriptional regulator of human anti-mullerian hormone (AMH) and thereby linked to the male sex-determining pathway. The zebrafish ff1a dual promoter contains several GATA binding sites and E-boxes, a site for DR4, XFD2, MyoD, Snail, HNF3, S8, and an HMG-box recognition site for Sox9. In a first attempt to dissect the ff1a promoter in vivo we have produced first generation transgenes in order to determine the correlation between the expression of the endogenous ff1a gene and the microinjected ff1a dual promoter coupled to the pEGFP reporter vector. Our results show that the microinjected constructs are expressed in the correct tissues.

Novel steroidogenic factor-1 homolog (ff1d) is coexpressed with anti-Mullerian hormone (AMH) in zebrafish.

ff1d is a novel zebrafish FTZ-F1 gene with sequence characteristics indicating similar basic regulatory mechanisms as the previously characterized ff1 based on the presence of an FTZ-F1 box in the DNA binding domain and an interactive domain (I-Box) and an AF-2 in the ligand binding domain. The highest sequence similarity was found between ff1d and ff1b (NR5A4), a gene previously shown to be a functional homolog to the steroidogenic factor 1 (SF-1). The expression pattern of ff1d was comparable to ff1b both in brain and gonads in adults and in the pituitary and interrenal cells in embryos. SF-1 is crucial in mammalian steroidogenesis and in sex determination by regulating the anti-Mullerian hormone (AMH). In fish, AMH has not been described previously. In this study, we cloned a partial zebrafish AMH. AMH was detected in growing oocytes, the ovarian follicular layer and testicular Sertoli cells, similar to the mammalian pattern, suggesting a conserved role between zebrafish and mammalian AMH. Teleosts lack a gene homolog to SRY, which constitute the universal testis-determining factor in mammalian sex determination. Comparison of sequences and expression patterns indicate that ff1d is a new candidate for sex determination and differentiation in a way similar to SF-1, possibly involving AMH.

Developmental expression patterns of FTZ-F1 homologues in zebrafish (Danio rerio).

The fushi tarazu factor 1 (FTZ-F1) gene family constitutes a subgroup of orphan nuclear receptors which can be divided into two groups (LRH/FTF- and SF-1/Ad4BP-like) based on sequence homology, function, and tissue distribution. Analysis of zebrafish FTZ-F1 homologues (zFF1 and ff1b) during embryogenesis indicated distinct expression patterns for both genes. Besides the previously observed expression in pituitary/hypothalamus and mandibular arch, zFF1 transcripts were also detected in domains corresponding to the pronephric duct, somites, liver, and hindbrain. Additionally, ff1b transcripts were detected at other developmental stages than earlier documented. Comparative sequence analysis showed that zFF1 exhibited higher sequence similarity to the LRH/FTF group than the SF-1/Ad4BP group, whereas ff1b was indistinguishable between the groups. These observations, coupled with obtained expression patterns, indicate that zebrafish FTZ-F1 homologues exhibit characteristics that are indicative of both LRH/FTF- and SF-1/Ad4BP-like genes.

Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development.

In zebrafish (Danio rerio) pigmentation is initiated during embryogenesis and begins in the retinal epithelium and in the melanophores. The pigment cells develop rapidly, and within hours they constitute a prominent feature of the embryo. In order to improve signal detection by whole mount in situ hybridization, confocal microscopy, or expression of GFP, embryos may be treated with 1-phenyl 2-thiourea (PTU) during embryogenesis. PTU inhibits melanogenesis by blocking all tyrosinase-dependent steps in the melanin pathway but can be toxic at high concentrations. The embryos remain transparent as long as the PTU treatment is continued. However, PTU treatment must be initiated before the initial pigmentation because it does not remove already formed pigment. Here we provide a protocol for generating transparent zebrafish while avoiding the toxic and teratogenic effects of PTU treatment.