Somatic Mutagenesis with a Sleeping Beauty Transposon System Leads to Solid Tumor Formation in Zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers

The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome

<div><p>Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, <i>POLR1C</i> and <i>POLR1D</i> cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that <i>polr1c</i> and <i>polr1d</i> are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, <i>polr1c</i> and <i>polr1d</i> homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that <i>polr1c</i> and <i>polr1d</i> loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of <i>tp53</i> can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in <i>polr1c</i> and <i>polr1d</i> mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for <i>polr1c</i> and <i>polr1d</i> in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established <i>polr1c</i> and <i>polr1d</i> mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention.</p></div

Analysis of NCC development in <i>polr1c</i> and <i>polr1d</i> mutant embryos.

<p>(A-H) <i>sox10</i> expression at 12 hpf and (I-P) <i>foxd3</i> expression at 14 hpf reveal relatively normal patterns of early cranial NCC specification and migration in <i>polr1c</i>^-/- and <i>polr1d</i>^-/- embryos (black arrows). (Q-X) In contrast, <i>dlx2</i> expression at 36 hpf reveals slightly diminished domains of activity in mutant embryos, particularly with respect to the posterior pharyngeal arches, which is suggestive of fewer mature NCC colonizing the pharyngeal arches. White arrows indicate pharyngeal arches 1 and 2. Scale bar = 200 μm.</p

Craniofacial cartilage development is disrupted in <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutant embryos.

<p>(A-C) Alcian blue staining reveals cranial cartilage in 5 dpf <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutant embryos is hypoplastic compared to controls. (D-F) The jaws of mutant embryos are smaller overall, with noticeable differences in the size of Meckel’s cartilage, the palatoquadrate, and ceratohyal elements. (G-I) Staining of the viscerocranium reveals smaller cartilage elements derived from each of the pharyngeal arches in mutant embryos, most notably the ceratobranchials, as well as altered polarity of the ceratohyal. (J-L) Staining of the neurocranium reveals hypoplasia of the ethmoid plate. Abbreviations: M, Meckel’s cartilage; pq, palatoquadrate; ch, ceratohyal; cb, ceratobranchial; ep, ethmoid plate; pch, parachordal. Scale bar = 200 μm.</p

<i>tp53</i> inhibition ameliorates cartilage anomalies in <i>polr1d</i>^-/-mutant embryos in a dosage-dependent manner.

<p>(A-D) Alcian blue staining of cartilage in an allelic series of <i>polr1d</i> and <i>tp53</i> mutant embryos. Dosage-dependent improvement in cartilage development is particularly noticeable in the jaw (E-H), elements of the viscerocranium (I-L), and more specifically the ceratohyal (G-L). Abbreviations: M, Meckel’s cartilage; pq, palatoquadrate; ch, ceratohyal; cb, ceratobranchial. Scale bar = 200 μm.</p

Mutations in <i>polr1c</i> and <i>polr1d</i> disrupt craniofacial development in zebrafish embryos.

<p>24 hpf (A-D), 3 dpf (E-G) and 5 dpf (H-J) <i>polr1c</i>^-/- and <i>polr1d</i>^-/- zebrafish exhibit craniofacial defects, including smaller eyes, a disrupted midbrain-hindbrain boundary and cranial necrosis compared to controls. At 3 dpf, distinct craniofacial anomalies such as a smaller jaw and eyes become apparent. By 5 dpf, <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutants are distinguished from their control siblings by their smaller head, microphthalmia, jaw hypoplasia, and failure to inflate their swim bladder (E-G). Abbreviations: e, eye; mbhb, midbrain hindbrain boundary; j, jaw; h, heart; sb, swim bladder. Scale bar = 200 μm (A-G) and 500 μm (H-J).</p

<i>polr1c</i> and <i>polr1d</i> are dynamically expressed during zebrafish embryogenesis.

<p><i>polr1c</i> and <i>polr1d</i> are maternally expressed at early stages (A,B, arrows) and ubiquitously expressed at 6 hpf (C,D) and 11 hpf (E,F) when the embryo surrounds the yolk (dashed lines). At 24 hpf, expression becomes enriched in regions such as the eye and midbrain-hindbrain boundary (G,H). Elevated levels of expression are evident in the pharyngeal arches (adjacent to curved line) at 36 hpf (I,J) whereas lower levels are observed throughout the embryo at 48 hpf and beyond (K,L) and beyond. Abbreviations: e, eye; mbhb, midbrain-hindbrain boundary; pa, pharyngeal arches; l, lens; t, tectum. Scale bar = 200 μm.</p

Ribosome biogenesis is disrupted in <i>polr1c</i> and <i>polr1d</i> mutant embryos.

<p>(A, B) qPCR quantification of 47S rRNA production. (A) <i>polr1c</i>^-/- mutants exhibit reduced levels of 5’ETS (39%), ITS2 (23%) and 18S rRNA (58%) compared to controls. (B) <i>polr1d</i>^-/- mutants similarly exhibit reduced levels of 5’ETS (25%), ITS2 (39%), and 18S rRNA (32%) compared to controls. (C, D) Polysome profiling shows decreased 80S and polysome peaks in <i>polr1c</i>^-/- (C) and <i>polr1d</i>^-/- (D) mutant embryos.</p

Pharyngeal arch size is reduced in <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutants.

<p>(A-C) Immunostaining of 36hpf <i>fli1a</i>:<i>egfp</i> labeled control, <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutant embryos with Zn-8 (red), which labels the endodermal pouches, revealed comparatively normal pharyngeal arch and pharyngeal pouch patterning. Pharyngeal arches 1–5 are indicated. (D-F) <i>fli1a</i>:<i>egfp</i> labeling of post-migratory NCC illustrates an overall reduction in pharyngeal arch size in <i>polr1c</i>^-/- and <i>polr1d</i>^-/- mutants. Pharyngeal arches 1 and 2 are outlined in red. (G-H) Quantification of pharyngeal arches 1 & 2 (red arrows, D-F) volume revealed a reduction in <i>polr1c</i>^-/- (G) and <i>polr1d</i>^-/- (H) mutants. Scale bar = 100 μm. * = p < 0.05 and error bars represent 95% confidence intervals.</p

Mef2c-F10N enhancer driven β-galactosidase (LacZ) and Cre recombinase mice facilitate analyses of gene function and lineage fate in neural crest cells

Neural crest cells (NCC) comprise a multipotent, migratory stem cell and progenitor population that gives rise to numerous cell and tissue types within a developing embryo, including craniofacial bone and cartilage, neurons and glia of the peripheral nervous system, and melanocytes within the skin. Here we describe two novel stable transgenic mouse lines suitable for lineage tracing and analysis of gene function in NCC. Firstly, using the F10N enhancer of the Mef2c gene (Mef2c-F10N) linked to LacZ, we generated transgenic mice (Mef2c-F10N-LacZ) that express LacZ in the majority, if not all migrating NCC that delaminate from the neural tube. Mef2c-F10N-LacZ then continues to be expressed primarily in neurogenic, gliogenic and melanocytic NCC and their derivatives, but not in ectomesenchymal derivatives. Secondly, we used the same Mef2c-F10N enhancer together with Cre recombinase to generate transgenic mice (Mef2c-F10N-Cre) that can be used to indelibly label, or alter gene function in, migrating NCC and their derivatives. At early stages of development, Mef2c-F10N-LacZ and Mef2c-F10N-Cre label NCC in a pattern similar to Wnt1-Cre mice, with the exception that Mef2c-F10N-LacZ and Mef2c-F10N-Cre specifically label NCC that have delaminated from the neural plate, while premigratory NCC are not labeled. Thus, our Mef2c-F10N-LacZ and Mef2c-F10N-Cre transgenic mice provide new resources for tracing migratory NCC and analyzing gene function in migrating and differentiating NCC independently of NCC formation