cyrano is evolutionarily conserved lncRNA and based on morpholino studies, has been suggested to have essential functions during zebrafish embryogenesis (Ulitsky et al., 2011) and brain morphogenesis (Sarangdhar et al., 2018). cyrano has also been suggested to act as a sponge (decoy-factor) for HuR during neuronal proliferation (Kim et al., 2016a), regulate miR-7 mediated embryonic stem cell differentiation (Smith et al., 2017), and control the level of miR-7 in the adult mouse brain (Kleaveland et al., 2018). We generated two mutant alleles that removed the TSS (cyrano^a171) or the gene (cyrano^a172), including the highly conserved miR-7 binding-site (Figure 2A,B). The expression level of the nearby gene (oip5) was not affected in either of these mutants (Figure 1—figure supplement 1–4). In contrast to previous morpholino studies in zebrafish (Ulitsky et al., 2011) but in support of recent findings in mouse (Kleaveland et al., 2018), cyrano mutants developed normally and were viable and fertile.

The difference between morphant (Ulitsky et al., 2011) and mutant phenotypes might be caused by compensation in the mutants (Rossi et al., 2015; El-Brolosy and Stainier, 2017). To test this possibility, we injected the previously used morpholinos targeting the first exon-intron boundary (e1i1) or the conserved miR-7 binding site (CMiBS) into wild type and homozygous deletion mutants. The TSS-mutant allele lacked the e1i1 morpholino-binding site and the gene deletion allele lacked the CMiBS morpholino-binding site (Figure 2A). The previously reported phenotypes, including small heads and eyes, heart edema, and kinked tails were found in both wild type and mutants (Figure 2C), demonstrating that the morpholino-induced phenotypes were non-specific. These results reveal that cyrano transcripts or their evolutionarily conserved miR-7-binding site, are not required for embryogenesis, viability or fertility.

gas5 is an evolutionarily conserved lncRNA (growth-arrest specific 5) (Coccia et al., 1992) that is highly expressed in early development (Figure 3B) and hosts several snoRNAs implicated in zebrafish development (Higa-Nakamine et al., 2012). Knockdown and knockout studies in cell culture (Ma et al., 2016) have indicated that gas5 might act as a tumor suppressor (Pickard and Williams, 2015) and exert effects at distant genomic sites (Schneider et al., 1988). However, the role of this lncRNA in development has not been studied in any vertebrate. Our gas5^a173 mutant allele removed the sequences containing the TSS (−169 to +127) (Figure 3A) and resulted in complete elimination of its expression (Figure 3B and D). Expression of the neighboring gene osbpl9, encoding a lipid-binding protein, was increased by 50% (Figure 3D). Previous studies have shown that gas5 lncRNA can act in trans to affect pten expression (ptena and ptenb in zebrafish) by sequestering specific microRNAs (Li et al., 2017; Zhang et al., 2018; Liu et al., 2018). Additionally, gas5 transcript can mimic Glucocorticoid Response Element and act as a decoy factor (riborepressor) for the Glucocorticoid Receptor (nr3c1)-mediated transcription (Kino et al., 2010). We analyzed the expression level changes of these genes in MZgas5^a173 embryos (at 1-dpf) and found significant upregulation for ptena in MZgas5^a173 mutants (Figure 3E). Despite these changes in gene expression, gas5^a173 mutants were indistinguishable from wild type (Figure 3C), reached adulthood and were fertile.

Lnc-setd1ba is the zebrafish orthologue of human LIMT (Sas-Chen et al., 2016) (LncRNA Inhibiting Metastasis), which has been implicated in basal-like breast cancers. It is expressed from a shared promoter region that also drives the expression of the histone methyltransferase setd1ba in opposite direction (Figure 4A). Evolutionary conservation in vertebrates and proximity to setd1ba, whose mouse homolog is essential for embryonic development (Eymery et al., 2016; Kim et al., 2016b) prompted us to investigate the function of this lncRNA in zebrafish. We removed the gene of lnc-setd1ba downstream of its TSS (3137 bp deletion) (lnc-setd1ba^a174). In situ hybridization and qRT-PCR revealed absence of lncRNA expression (Figure 4C and E) and strong upregulation of setd1ba (Figure 4D and E) during cleavage stages and slight upregulation of setd1ba and the other neighboring gene rhoF at one-day post fertilization (1-dpf) (Figure 4E). Despite these changes, maternal-zygotic lnc-setd1ba^a174 mutants were indistinguishable from wild type (Figure 4B), reached adulthood and produced normal progeny.

Squint encodes a Nodal ligand involved in mesendoderm specification (Pei et al., 2007; Heisenberg and Nüsslein-Volhard, 1997). The previously studied squint insertion mutant alleles (squint^{Hi975Tg 50} and squint^{cz35 51}) lead to delayed mesendoderm specification and partially penetrant cyclopia (Dougan et al., 2003). Morpholino and misexpression studies have suggested an additional, non-coding role for maternally provided squint, wherein the squint 3'UTR mediates dorsal localization of squint mRNA, induces the expression of dorsal mesoderm genes, and is required for the development of dorsal structures (Gore et al., 2005; Lim et al., 2012). This mode of activity assigns squint to the cncRNA family - RNAs with both protein-coding and non-coding roles (Sampath and Ephrussi, 2016). To investigate the non-coding roles of squint mRNA we generated a deletion allele (squint^a175) that lacked most of the protein coding region and the 3’UTR, including the Dorsal Localization Element (DLE) implicated in maternal squint RNA localization (Gilligan et al., 2011) (Figure 5A). In this allele 525 bp (178 bp 5’UTR, 280 bp first exon and 67 bp of second exon) out of the 1592bp-long mature transcript remain in the genome (Figure 5A). In situ hybridization (Figure 5B) and qRT-PCR (Figure 5C) showed that the level of remaining squint transcript was greatly reduced (~90%). MZsquint ^a175 embryos displayed partially penetrant cyclopia, similar to existing protein-disrupting squint alleles (Figure 5D) (Pei et al., 2007; Heisenberg and Nüsslein-Volhard, 1997; Golling et al., 2002), but the defects proposed to be caused by interference with squint non-coding activity (Gore et al., 2005) were not detected.

To further test whether squint mRNA might have non-coding roles, we injected wild-type and MZsquint ^a175 embryos with either control RNA, full-length squint mRNA, a non-coding version of squint mRNA, or the putative transcript produced in squint ^a175 (Figure 5—figure supplement 5–S1). We found that in contrast to wild-type squint mRNA, control RNA, non-protein coding squint RNA or squint ^a175 RNA did not cause any phenotypes and did not rescue MZsquint ^a175 mutants. These results indicate that squint 3’UTR does not have the previously proposed non-coding functions and that the squint transcript may not be a member of the cncRNA family.

Lnc-phox2bb neighbors phox2bb and smtnl1. Phox2bb is a transcription factor implicated in the development of the sympathetic nervous system (Pei et al., 2013), (Moreira et al., 2016; Tolbert et al., 2017), while smtnl1 has been implicated in smooth muscle contraction (Borman et al., 2009). Whole-gene deletion of lnc-phox2bb (lnc-phox2bb^a177) (Figure 6A) led to jaw deformation and failure to inflate the swim-bladder (Figure 6B), and no homozygous mutant fish survived to adulthood. Like the whole-gene deletion allele, the TSS-deletion allele (lnc-phox2bb^a176) lacked lnc-phox2bb RNA (Figure 6E), but in contrast to the whole-gene deletion mutants, TSS-deletion mutants developed normally and gave rise to fertile adults. To determine the cause of this difference, we analyzed the expression level and pattern of neighboring genes. We found that the anterior expression domain of phox2bb in the hindbrain was absent in the whole-gene deletion allele (Figure 6D). This finding is consistent with the observation that the deleted region contains enhancer elements for phox2bb (McGaughey et al., 2008), conserved non-coding elements (CNEs) (Hiller et al., 2013) (Figure 6C), and histone marks related to enhancer regions (H3K4me1 and H3K27Ac) (Bogdanovic et al., 2012). We also found that the expression level of smtnl1 increased in gene deletion mutants relative to the TSS-deletion mutant and wild type (Figure 6E). These results indicate that lnc-phox2bb RNA is not required for normal development but that the lnc-phox2bb overlaps with regulatory elements required for proper expression of phox2bb and smtnl1 (Figure 6E).

In summary, our systematic mutant studies indicate that none of the 25 lncRNAs analyzed here are essential for embryogenesis, viability or fertility, including the prominent lncRNAs cyrano, gas5, and lnc-setd1ba. Additionally, they refute the proposed non-coding function of squint RNA. Our phenotypic screen does not exclude more subtle phenotypes; for example in behavior or brain activity (Rihel et al., 2010; Randlett et al., 2015; Summer et al., 2018). This mutant collection can now be analyzed for subtle, context specific or redundant functions, but extrapolation suggests that most individual zebrafish lncRNAs are not required for embryogenesis, viability or fertility.

TL/AB zebrafish (Danio rerio) were used as wild-type fish in this study. Fish were maintained on daily 14 hr (light): 10 hr (dark) cycle at 28°C. All animal works were performed at the facilities of Harvard University, Faculty of Arts and Sciences (HU/FAS). This study was approved by the Harvard University/Faculty of Arts and Sciences Standing Committee on the Use of Animals in Research and Teaching (IACUC; Protocol #25–08)

Guide RNAs (gRNAs) were designed using CHOPCHOP (Montague et al., 2014) and synthesized in pool for each candidate as previously described (Gagnon et al., 2014). (See supplementary file 1 for the gRNA sequences). gRNAs were combined with Cas9 protein (50 μM) and co-injected (~1 nL) into the one-cell stage TL/AB wild-type embryos. Genomic DNA from 10 injected and 10 un-injected siblings was extracted (Meeker et al., 2007) and screened for the difference in amplified band pattern from the targeted region (See supplementary file 1 for the genotyping primer sequences). The rest of injected embryos were raised to adulthood, crossed to wild-type fish and screened for passing the mutant allele to the next generation. Founder fish with desirable mutations were selected and confirmed by Sanger sequencing of the amplified mutant allele. Heterozygous mutants were crossed together to generate homozygous mutants. At least 15 adult homozygous mutant pairs per allele were crossed to test fertility of mutants and to generate maternal and zygotic mutants (MZ) devoid of maternally and zygotic lncRNA activity.

Visual assessment of live embryos and larvae performed (Driever et al., 1996; Haffter et al., 1996) to identify mutant phenotypes, ranging from gastrulation movements and axis formation to the formation of brain, spinal cord, floor plate, notochord, somites, eyes, ears, heart, blood, pigmentation, vessels, kidney, pharyngeal arches, head skeleton, liver, and gut.

At day 5, formation of swim bladder and overall appearance of the embryos were checked again (at any stage 60–100 embryos were scored). Sixty to hundred fish from heterozygous mutant crosses were grown to adulthood and genotyped to identify the viability of adult homozygous fish. Validated homozygous mutant fish were further crossed together to test for potential fertility phenotypes or putative maternal functions of candidate lncRNAs.

Antisense probes for in situ hybridization were transcribed using the DIG RNA labeling kit (Roche). All RNAs were purified using EZNA Total RNA kits (Omega Biotek). Embryos were fixed in 4% formaldehyde overnight at 4°C (embryos younger than 50% epiboly fixed for 2 days). In situ hybridizations were performed according to standard protocols (Thisse and Thisse, 2008). NBT/BCIP/Alkaline phosphatase-stained embryos were dehydrated in methanol and imaged in benzyl benzoate:benzyl alcohol (BBBA) using a Zeiss Axio Imager.Z1 microscope.

Total RNA was isolated from three individuals or sets of 10–20 embryos per condition using EZNA Total RNA kits (Omega Biotek). cDNA was generated using iScript cDNA Synthesis kit (Bio-Rad). qPCR was conducted using iTaq Universal SYBR Green Supermix (Bio-Rad) on a CFX96 (Bio-Rad). Gene expression levels were calculated relative to a reference gene, ef1a. Three technical replicates were used per condition. The qPCR primer sequences are listed in supplementary file 1.

Embryos were anesthetized in Tricaine (Sigma) and mounted in 1% low melting temperature agarose (Sigma) with Tricaine, then imaged using a Zeiss SteREO Discovery.V12 microscope or Zeiss Axio Imager.Z1 microscope. Images were processed in FIJI/ImageJ (Schindelin et al., 2012). Brightness, contrast and color balance was applied uniformly to images.

The sequences for the wild-type squint mRNA, non-protein coding squint transcript (One Adenine base was added after eight in-frame ATG codons, and the 3’UTR sequence kept unchanged) and the squint^a175 transcript were synthesized as gBlocks (IDT) containing 5’ XhoI cut site and 3’ NotI site. Fragments were digested and inserted the pCS2 plasmid. Positive colonies were selected, and sanger sequenced to assure the accuracy of the gene synthesis process. Sequences of the constructs are provided in supplementary file 1. mRNA was in vitro transcribed by mMessage mMachine (Ambion) and purified by EZNA Total RNA kits (Omega Biotek). h2b-gfp was used as control mRNA. Each injection mix contained 30 ng/ul of squint or control mRNA). 1 nl of mRNA mix was injected into the yolk of one-cell stage embryos.

Morpholinos were ordered from Gene Tools and injected based on Ulitsky et al. (2011).