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. Author manuscript; available in PMC: 2018 Oct 1.
Published in final edited form as: Dev Dyn. 2017 Aug 30;246(10):759–769. doi: 10.1002/dvdy.24547

tbx6l and tbx16 are redundantly required for posterior paraxial mesoderm formation during zebrafish embryogenesis

Zachary T Morrow 1,*, Adrienne M Maxwell 1,*, Kazuyuki Hoshijima 2, Jared C Talbot 1,3,4, David J Grunwald 2, Sharon L Amacher 1,3,4,5,§
PMCID: PMC5744594  NIHMSID: NIHMS891583  PMID: 28691257

Abstract

Background

T-box genes encode a large transcription factor family implicated in many aspects of development. We are focusing on two related zebrafish T-box genes, tbx6l and tbx16, that are expressed in highly overlapping patterns in embryonic paraxial mesoderm. tbx16 mutants are deficient in trunk, but not tail, somites; we explored whether presence of tail somites in tbx16 mutants was due to compensatory function provided by the tbx6l gene.

Results

We generated two zebrafish tbx6l mutant alleles. Loss of tbx6l has no apparent effect on embryonic development, nor does tbx6l loss enhance the phenotype of two other T-box gene mutants, ta or tbx6, or of the mesp family gene mutant, msgn1. In contrast, loss of tbx6l function dramatically enhances the paraxial mesoderm deficiency of tbx16 mutants.

Conclusion

These data demonstrate that tbx6l and tbx16 genes function redundantly to direct tail somite development. tbx6l single mutants develop normally because tbx16 fully compensates for loss of tbx6l function. However, tbx6l only partially compensates for loss of tbx16 function. These results resolve the question of why loss of function of tbx16 gene, which is expressed throughout the ventral and paraxial mesoderm, profoundly affects somite development in the trunk but not the tail.

Keywords: T-box genes, spadetail, ntl/Brachyury, fss/tbx6, mesogenin, somitogenesis

Introduction

The T-box gene family encodes a large family of transcription factors that are required for development of many different tissue types (Bertolessi, 2015; Papaioannou, 2014). Often, T-box genes are expressed in overlapping patterns and can functionally interact with or substitute for each other, perhaps via co- or redundant regulation of T-box target genes. In this study, we focus on mesodermally-expressed zebrafish T-box genes, a number of which are expressed in overlapping patterns. In some cases, pairs of T-box genes function redundantly, so that single mutant analysis only partially reveals the full scope of gene function. A clear example is the functional redundancy of the zebrafish homologs of the T-box gene family founding member, Brachury (T). In mouse, there is one Brachury (T) gene, but in zebrafish, there are two: ta, originally identified by mutation as no tail (ntl or ntla) (Halpern et al., 1993; Schulte-Merker et al., 1994), and tb, originally named bra, but also called ntlb (Martin and Kimelman, 2008). Whereas mouse embryos that lack Brachyury (T) function exhibit severe depletion of trunk and tail mesoderm, loss of function of individual zebrafish orthologs does not produce such an extreme phenotype. Rather, zebrafish ta mutants lack posterior mesoderm, and tb morphants have no visible phenotype. However the ta mutant; tb morphant combination fully recapitulates the mouse Brachury phenotype (Martin and Kimelman, 2008). In other zebrafish T-box gene mutant combinations, novel and/or synergistic functions are revealed. For example, embryos lacking function of both ta and tbx16 T-box genes completely lack all trunk and tail mesodermal cell types, as well as floor plate, a non-additive, synergistic phenotype not predicted from either single mutant (Amacher et al., 2002; Goering et al., 2003; Payumo et al., 2015).

This study focuses on the functional analysis of zebrafish T-box genes that are co-expressed in paraxial mesoderm, the tissue that gives rise to the embryonic segments, or somites, and other derivatives. For at least two of the genes we examine, the mutant phenotype suggests that the gene does not function uniformly throughout its entire expression domain, but instead has region-specific roles. Such observations have led to the idea that anterior and posterior somite formation are regulated by both shared and distinct pathways in zebrafish (Griffin et al., 1995; Griffin et al., 1998; Julich et al., 2005; Kanki and Ho, 1997; Kimmel et al., 1989; reviewed by Holley, 2007). One example is the zebrafish T-box gene tbx16, originally identified by mutation as spadetail (spt). tbx16 is required for proper specification and morphogenesis of anterior paraxial mesoderm: tbx16 mutants lack trunk somites, but have tail somites (Kimmel et al., 1989; Ho and Kane, 1990; Griffin et al., 1998). Although tail somite formation is relatively normal in tbx16 mutants, double mutant analyses hint that tbx16 functions redundantly with other developmental regulatory genes in posterior paraxial mesoderm and somite formation. For example, double mutant combination of tbx16 with a mutation in the bHLH Mesp family transcription factor gene mesogenin1 (msgn1), a T-box gene target (Garnett et al., 2009; Goering et al., 2003), causes severe tail mesoderm disruption that is not observed in either single mutant (Fior et al., 2012; Manning and Kimelman, 2015; Szeto and Kimelman, 2006). Another zebrafish T-box family gene, tbx6, originally identified by mutation as fused somites (fss), also has region-specific functions. Although tbx6 is required along the entire A-P axis for somite formation, it is required for central dermomyotome formation only in the posterior of the embryo (Nikaido et al, 2002; Windner et al., 2012; Windner et al., 2015).

The prevalence of T-box gene co-expression in embryonic mesoderm and the precedence for complex genetic interactions among tbx genes and their targets highlight the importance of analyzing T-box gene function in the context of an interacting network of genes that function together within a developing tissue (Bertolessi, 2015; Papaioannou, 2014). Here, we focus on the zebrafish tbx6/tbx16 gene family, by creating and characterizing a mutation in the tbx6l gene and analyzing double mutant combinations with other T-box genes and targets. In mouse, the Tbx6 gene is required for paraxial mesoderm specification and proper somite patterning along the entire body axis of the embryo (Chapman et al., 1996; Chapman et al., 1998; Theiler and Varnum, 1985; Watabe-Rudolph et al., 2002; White et al., 2003). In zebrafish, these functions have been proposed to be carried out by three genes belonging to the tbx6/tbx16 T-box transcription factor subfamily: tbx16 (formerly spt), tbx6 (formerly fss and tbx24), and tbx6l (formerly tbx6) (Goering et al., 2003; Griffin et al., 1998). Although well-established functional studies support a role for tbx16 and tbx6, genetic analysis of tbx6l function has not been reported. When initially identified, zebrafish tbx6l was proposed to be the mouse Tbx6 homolog, largely due to sequence similarity and highly overlapping expression pattern: both mouse Tbx6 and zebrafish tbx6l are expressed in the ventrolateral mesoderm of the gastrula and in presomitic mesoderm and tail bud during segmentation stages (Chapman et al., 1996; Hug et al., 1997). However, re-examination of vertebrate T-box gene family sequence similarity, gene structure, and syntenic relationships revealed that the true zebrafish ortholog of mouse Tbx6 was tbx6, not tbx6l (Ahn et al., 2012; Windner et al., 2012), consistent with previous observations that zebrafish tbx6l is more closely related to zebrafish tbx16 than to tetrapod Tbx6 genes (Lardelli, 2003). Here, we show that tbx6l can partially compensate for tbx16 function and suggest that a more appropriate name for tbx6l is tbx16l, which would reflect both the sequence and functional conservation of the genes.

The formation of posterior somites in tbx16 mutants could reflect the region-specific activity of tbx16 in its control of paraxial mesoderm development or the presence of a compensatory gene that operates posteriorly. We favored the latter and hypothesized that tbx6l functions redundantly with tbx16 in tail paraxial mesoderm. Using TALEN-mediated gene targeting, we generated two zebrafish tbx6l mutations and confirmed that tbx6l mutants fail to express Tbx6l protein. Homozygous tbx6l mutants are viable and display no obvious morphological defects during embryogenesis. However, tbx6l; tbx16 double mutants display severe paraxial mesoderm deficiencies along the entire axis, demonstrating that tbx16 and tbx6l function redundantly during tail somitogenesis.

Results and Discussion

Zebrafish tbx6l mutants are homozygous viable and have no overt phenotype

To investigate tbx6l function, we generated zebrafish tbx6l mutants using TALEN-mediated targeted mutagenesis (Huang et al., 2011; Cade et al., 2012; Dahlem et al., 2012) (see Methods). We recovered two alleles, tbx6lz34 and tbx6lz35, which are 8 bp and 7 bp deletions in exon 3 that frameshift the coding sequence and cause the normal 488 amino acid protein to be prematurely truncated, deleting most of the 180 amino acid T-box DNA binding domain (Fig 1A, B). To examine whether Tbx6l protein is depleted in tbx6l mutants, we performed immunohistochemistry and western blotting using a polyclonal antibody raised against a 100 amino acid Tbx6l C-terminal peptide downstream of the T-box domain (Fig 1B; see Methods). During segmentation stages, Tbx6l protein is expressed in the tail bud and posterior presomitic mesoderm in wild-type embryos (Fig 1C), as expected from the previously characterized tbx6l mRNA expression pattern (Hug et al., 1997). In tbx6l mutants, Tbx6l protein is not detected by immunofluorescence (Fig 1D). Western blotting also reveals loss of a prominent ~60 kD band in tbx6l mutant embryos (Fig 1E), providing strong support that full-length protein is not produced in mutants. Despite the lack of Tbx6l protein, tbx6l mutants are homozygous adult viable and morphologically indistinguishable from wild-type siblings (Fig 1F, G). Additionally, mesoderm specification markers, like ta and myoD, are expressed normally in tbx6l mutants (data not shown).

Figure 1. Zebrafish tbx6l mutants do not express Tbx6l protein and are homozygous viable.

Figure 1

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(A) Sequence of tbx6l showing left and right TALEN binding sites (blue) and the spacer region (black), for wild-type, tbx6lz34, and tbx6lz35 alleles. Upper case letters indicate exon 3 coding sequence; lower case letters indicate intron sequence. Dashes indicate deleted nucleotides. A PvuII site (underlined) is deleted in both mutant alleles. (B) Diagram of wild-type Tbx6l protein and predicted truncated Tbx6l proteins encoded by each mutant allele, showing the T-box domain (black box and black text), aberrant sequence caused by the introduced frameshift in mutant alleles (gray box and gray text), and the immunogen peptide (green box). (C, D) Dorsal views of 14–16 somite stage wild-type (C) and tbx6lz34 mutant (D) embryos processed by immunofluorescence with Tbx6l antibody. (E) Western blot of protein extracts prepared from wild-type (WT) and tbx6z35 homozygous mutant embryos, probed sequentially with anti-Tbx6l antibody (left blot), then with anti–αTubulin antibody as a loading control (right blot). The inferred position of Tbx6l, which runs slightly higher than the predicted 55 kD mass, is marked by a single asterisk, and alpha-Tubulin (50 kD) by double asterisks. (F, G) Live images of 24 hpf wild-type (F) and tbx6lz35 mutant (G) embryos. Scale bar in D (for C, D) is 25 µm and in G (for F, G) is 250 µm.

In mouse and Xenopus, Tbx6 orthologs not only promote mesoderm development but also repress neural identity (Chapman et al., 1998; Takemoto et al., 2011; Uchiyama et al., 2001). In mouse Tbx6 mutants, sox2 is improperly expressed in paraxial mesoderm (Takemoto et al., 2011) and ectopic neural tubes form in place of somites (Chapman et al., 1998; Takemoto et al., 2011). We examined sox2 expression in zebrafish tbx6l mutants and found that sox2 is expressed normally (data not shown). Taken together, these data show that tbx6l does not perform the same functions in zebrafish as Tbx6 does in mouse and is not essential for zebrafish development.

Unlike loss of tbx16, loss of tbx6l does not enhance the msgn1 mutant phenotype

In previous work, we showed that mesogenin1 (msgn1), which encodes a Mesp family transcription factor, and tbx6l are both strongly down-regulated in tbx16 mutant embryos during gastrulation and early somitogenesis stages (Garnett et al., 2009; Goering et al., 2003), supporting the hypothesis that both are T-box downstream targets. In the mouse, the Mesogenin1 (Msgn1) gene regulates many aspects of paraxial mesoderm development (Chalamalasetty et al., 2014; Yoon and Wold, 2000). In stark contrast, loss of msgn1 in zebrafish has little effect (Fior et al., 2012). Instead, in zebrafish, msgn1 and tbx16 function together to regulate migration of mesodermal precursor cells out of the tail bud into presomitic mesoderm (Fior et al., 2012; Manning and Kimelman, 2015; Yabe and Takada, 2012). Whereas zebrafish msgn1 mutants have only subtle paraxial mesodermal defects and tbx16 single mutants lack trunk paraxial mesoderm, msgn1; tbx16 double mutants completely lack paraxial mesoderm along the entire anterior-posterior axis, revealing the partially redundant functions of these two genes (Fior et al., 2012; Manning and Kimelman, 2015; Yabe and Takada, 2012). Additionally, the delayed flux of cells out of the msgn1 mutant tail bud leads to smaller, supernumerary tail somites, a phenotype enhanced by tbx16 heterozygosity (Fior et al., 2012). Because tbx6l and tbx16 are closely related in sequence and expression pattern and because tbx6l and msgn1 are both Tbx16 target genes (Garnett et al., 2009; Goering et al., 2003; Morley et al., 2009), we generated the tbx6l; msgn1 double mutant to investigate whether loss of tbx6l, like loss of tbx16, enhances the msgn1 mutant phenotype. Besides a transient enlargement of tail bud, tbx6l; msgn1 double mutants appear overtly normal and there are no obvious differences between tbx6l; msgn1 double mutants and msgn1 single mutants (data not shown). Additionally, tbx6l; msgn1 double mutants do not form more somites than msgn1 single mutants, although both have significantly more somites than wild-type or tbx6l single mutant embryos (Fig 2A–F) (Fior et al., 2012). Thus, although interactions among vertebrate Tbx6 and Mesp transcription factor family members have been shown to regulate paraxial mesoderm segmentation (Dunty et al., 2008; Fior et al., 2012; Hitachi et al., 2008; Kawamura et al., 2008; Oginuma et al., 2008; Takahashi et al., 2010; Yasuhiko et al., 2008), the data presented here show that zebrafish tbx6l and msgn1 do not function redundantly during paraxial mesoderm formation and segmentation.

Figure 2. Loss of tbx6l does not enhance the msgn1 mutant phenotype.

Figure 2

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(A–D) In situ hybridization for the cb1045/xirp2a somite boundary marker gene in 36 hpf wild-type (A), tbx6l mutant (B), msgn1 mutant (C), and tbx6l; msgn1 double mutant (D) embryos, that were raised together at standard temperature and fixed several hours after somitogenesis is normally completed. Lateral views of posterior tail are shown. (E) Graph showing distribution of somite number per embryo for each genotype. (F) Graph showing average somite number per embryo for each genotype. * p < 0.0005, when compared to wild-type.

Loss of tbx6l does not enhance ta or tbx6 T-box gene mutant phenotypes

As mentioned previously, T-box genes can have overlapping functions during embryogenesis that are ‘masked’ in single mutants and revealed in double mutants. In some cases, function of one or both genes may not be apparent until double mutants are made. To examine whether zebrafish tbx6l functions redundantly with other T-box genes, we generated and characterized three double mutant combinations: tbx6l; ta double mutants, tbx6l; tbx6 double mutants, and tbx6l; tbx16 double mutants. For the first two combinations, loss of tbx6l does not enhance the ta or tbx6 single mutant phenotypes (data not shown) (Halpern et al., 1993; Nikaido et al., 2002). In contrast, loss of tbx6l dramatically enhances the tbx16 mutant phenotype, as described below.

Loss of tbx6l dramatically enhances tbx16 mutant paraxial mesoderm defects

As tbx6l and tbx16 are closely related in sequence and expression pattern, we hypothesized they might share overlapping functions during embryogenesis. Additionally, although tbx6l expression is strongly down-regulated in tbx16 mutants during gastrulation and early somitogenesis (Garnett et al., 2009; Goering et al., 2003), tbx6l expression recovers during later stages, roughly coinciding with resumption of somite formation in the tbx16 mutant tail (Griffin et al., 1998). Together, these observations have suggested that tbx6l may functionally substitute for tbx16 in tail, but not trunk, paraxial mesoderm. To directly test this hypothesis, we generated and characterized tbx6l; tbx16 double mutants.

When originally discovered, the tbx16 gene was named spadetail for the prominent tail bud that forms in mutants compared to wild-type siblings (Kimmel et al, 1989). Subsequent work demonstrated that tbx16 mutant cells that should normally contribute to trunk paraxial mesoderm are incorrectly specified and fail to migrate correctly in tbx16 mutants, accumulating instead in the tail bud (Ho and Kane, 1990; Kimmel et al, 1989; Row et al., 2011). Because of this trunk paraxial mesoderm deficit, tbx16 mutants lack trunk somites, but have tail somites (Fig 3A–B’) (Kimmel et al, 1989; Ho and Kane, 1990). If tbx6l functionally substitutes for tbx16 in tail paraxial mesodermal precursors, one would predict that tail bud enlargement would be enhanced and tail somite formation would be disrupted in tbx6l; tbx16 double mutants. When tbx6l+/−; tbx16+/− double heterozygous fish are intercrossed, approximately one-quarter of the clutch have an enlarged tail bud, the ratio expected if tbx16 single mutants and tbx6l; tbx16 double mutants share the “spadetail” phenotype. However, if tbx6l partially compensates for loss of tbx16, one would expect that double mutants would be morphologically distinct from tbx16 single mutants. Indeed, approximately one-quarter of the “spadetail” class embryos have a significantly larger, more necrotic tail bud. The enhanced tail bud phenotype co-segregates with a shorter tail and severely reduced or absent tail somites (Fig 3C, C’). The more severely affected embryos were confirmed by genotyping to be tbx6l; tbx16 double mutants. The enhanced tbx6l; tbx16 double mutant features strongly resemble those in tbx16; msgn1 double mutants.

Figure 3. tbx6l; tbx16 double mutants have an enlarged tail bud and lack tail somites.

Figure 3

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(A–C’) Brightfield images of 24 hpf live wild-type (A, A’), tbx16 (spt) mutant (B, B’), and tbx6l; tbx16 double mutant (C, C’) embryos. tbx6l single mutant embryos are not shown here as they are indistinguishable from wild-type embryos (see Fig 1F, G). Black dots in the magnified views (A’–C’) indicate morphologically visible tail somites. (D–F) In situ hybridization for msgn1 at the 16 somite stage. Scale bar in A (for A–C) is 250 µm, in A’ (for A’–C’) is 100 µm, and in F (for D–F) is 50 µm.

To explore the relationship among functions of tbx6l, tbx16, and msgn1, we examined msgn1 expression in tbx6l; tbx16 double mutants. Previous work has shown that msgn1 is initially barely expressed in tbx16 single mutant gastulae, but expression recovers in mesoderm that will give rise to tail somites (Yabe and Takada, 2012). This observation suggested that msgn1 function may contribute to restored paraxial mesoderm development in the tbx16 single mutant tail, an idea well supported by the observation that tbx16; msgn1 double mutants are deficient in tail paraxial mesoderm (Fior et al., 2012; Manning and Kimelman, 2015; Yabe and Takada, 2012). Because of the striking similarity between tbx6l; tbx16 and tbx16; msgn1 double mutants, we predicted that msgn1 would not be expressed in tail paraxial mesoderm of tbx6l; tbx16 double mutants. Indeed, at mid-segmentation stages, when msgn1 expression is restored in tbx16 single mutants, we observed no msgn1 expression in tbx6l; tbx16 double mutants (Fig 3D–F).

To further characterize tbx6l; tbx16 double mutants, we examined expression of several well-characterized markers. At 24 hours post-fertilization (hpf), we examined ta expression, which is normally expressed in notochord cells and in tail bud mesodermal progenitors (Fig 4A) (Halpern et al., 1993; Schulte-Merker et al., 1994). As expected, the ta-expressing tail bud domain is expanded in tbx16 single mutants (Goering et al., 2003; Griffin and Kimelman, 2002). We found this domain is even further expanded in tbx6l; tbx16 double mutants (Fig 4B, C). The expansion of the tail bud ta expression domain and the increased number of cells in the tail bud are highly specific characteristics that can be used to identify double mutants. We correctly identified 96% (n=22/23) double mutants solely based upon tail bud morphology, general tail length, and ta-expressing tail bud domain size. We also examined expression of the paraxial mesodermal marker tbx6 during mid-somitogenesis (14–16 somite stage), a time at which the unsegmented paraxial mesoderm contains tail somite progenitors and thus is ‘recovering’ in tbx16 single mutants. We observe that tbx6-expressing cell number in the posterior paraxial mesoderm of double mutants is reduced compared to tbx16 single mutants: that is, whereas tbx6 expression begins to recover in tbx16 single mutants as tail progenitors enter the paraxial mesoderm, tbx6 expression remains sparse in tbx6l; tbx16 double mutants (Fig 4D–F).

Figure 4. tbx6l and tbx16 have partially redundant roles in formation of tail paraxial mesoderm.

Figure 4

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(A–R) In situ hybridization for ta (A–C), tbx6 (fss) (D–F), myoD (G–I), pax2a (J–L), fli1a (M–O), and lmo2 (P–R) at 24 hpf (A–C; G–R) and the 14–16 somite stage (D–F). Not shown are tbx6l single mutant embryos as they are indistinguishable from wild-type embryos. In panel J, the row of pax2a-expressing pronephros cells is indicated by an asterisk (*), and the row of pax2a-expressing spinal cord neurons is indicated by a double asterisk (**). Scale bar in C (for A–C) and R (for G–R) is 100 µm, and in F (for D–F) is 50 µm.

To characterize myogenic differentiation, we examined myoD expression at 24 hpf. As previously shown, myoD is expressed throughout the wild-type myotome, but is expressed sparsely in the tbx16 mutant trunk and relatively normally in the tbx16 mutant tail (Fig 4G, H) (Amacher et al., 2002; Amacher and Kimmel, 1998; Weinberg et al., 1996). In contrast, myoD is sparsely expressed along the entire anterior-posterior axis of the tbx6l; tbx16 double mutant (Fig 4I). The patchy expression of tbx6 and myoD in double mutants may reflect that fewer cells are able to exit the tail bud and enter the paraxial mesoderm, or alternatively, that double mutant tail mesodermal progenitors survive poorly or are less proliferative than in wild-type or tbx16 single mutant embryos. Future work will distinguish these possibilities. These data suggest that zebrafish tbx6l and tbx16 share redundant functions in paraxial mesoderm and derivatives.

Loss of tbx6l not does enhance tbx16 mutant defects in intermediate mesoderm

We examined additional markers to assess whether other mesodermal cell types were differentially affected in tbx16 single versus tbx6l; tbx16 double mutant embryos. To characterize cardiac mesoderm, we examined myl7 expression (Yelon et al., 1999) at 24 hpf and observed a similar pattern in single and double mutants, with expression being slightly delayed in mutants compared to wild-type siblings (data not shown). At 36 hpf, both single and double mutant embryos have a beating heart (data not shown).

We also examined markers of intermediate and ventral mesoderm because recent work has shown that these fates are differentially affected in tbx16 mutants, with pronephros being expanded predominantly at the expense of blood and to a lesser extent by vasculature (Warga et al., 2013). Since loss of tbx6l enhances the tbx16 paraxial mesoderm phenotype, we investigated whether the same is true for intermediate mesodermal types. To assess whether pronephros was expanded further in tbx6l; tbx16 double mutants compared to tbx16 single mutants, we examined expression of pax2a, which marks pronephros and spinal cord neurons in the wild-type trunk (* and ** respectively in Fig 4J) (Krauss et al., 1991). At 24 hpf, pax2a staining intensity and cell number appear similarly greater in single and double mutants (Fig 4J–L) compared to wild-type siblings, suggesting that loss of tbx6l does not dramatically enhance pronephric expansion of tbx16 single mutants. To examine whether loss of tbx6l enhances the blood and vasculature phenotype of tbx16 mutants, we examined expression of lmo2, a hemangioblast marker that is expressed in tbx16 single mutants, albeit in a disorganized pattern and in fewer cells, and of fli1a, a vasculature marker that is expressed in tbx16 single mutants in a disorganized pattern compared to wild-type embryos (Thompson et al., 1998; Warga et al., 2013). At 24 hpf, we observed no obvious difference in fli1a or lmo2 expression between tbx16 single and tbx6l; tbx16 double mutant embryos, even though the genotypes can be distinguished by embryo morphology (Fig 4M–R). These data suggest that although zebrafish tbx6l and tbx16 share redundant functions in posterior paraxial mesoderm, the same is not true for intermediate mesoderm. This was not unanticipated considering that fate allocations among pronephros, blood, and vasculature happen early (Warga et al., 2013), prior to the period during which tbx6l function compensates for tbx16 during somitogenesis.

Unlike loss of Tbx6 function in mouse, loss of zebrafish tbx6l and tbx16 does not convert paraxial mesoderm into ectopic neural tube

Because paraxial mesoderm in the mouse Tbx6 mutant transfates into ectopic neural tubes (Chapman et al., 1998; Theiler and Varnum, 1985; Watabe-Rudolph et al., 2002), we examined expression of neural markers in tbx6l; tbx16 double mutants. There is no major difference in pax2a staining intensity or number of pax2a-expressing neurons in the spinal cord of wild-type, tbx6l single (not shown), tbx16 single, and tbx6l; tbx16 double mutants (Fig. 4J–L), although neurons are more disorganized in the latter two genotypes, as previously described for tbx16 mutants (Amacher et al., 2002). We obtained a similar result for expression of isl1, which marks spinal cord motoneurons and sensory neurons at 24 hpf (Appel et al., 1995, Inoue et al., 1994; Tokumoto et al., 1995) (Fig. 5A–C). Previous work had indicated that the number of isl1-expressing neurons is increased in tbx16 mutants (Inoue et al., 1994; Lewis and Eisen, 2004); we also observe increased isl1 staining in tbx16 single and tbx6l; tbx16 double mutants, as well as ectopic isl1 expression at the end of the tail and in the enlarged tail bud. To assess pan-neural development, we examined expression of a well-established, neural marker, sox2 (Okuda et al., 2006). Expression of sox2 within the spinal cord is fairly normal in tbx16 single and tbx6l; tbx16 double mutants, although ventral sox2 expression (in non-neural intermediate mesoderm and hypochord) appears disorganized and expanded (Fig 5D–F). Finally, to assess whether mutant cells expressing early neural markers differentiate, we examined expression of acetylated tubulin, a marker of terminally differentiated neurons. From this analysis, it is clear that tbx6l; tbx16 double mutants form one neural tube (Fig 5G–L).

Figure 5. A single neural tube forms in tbx6l; tbx16 double mutants.

Figure 5

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(A–F) In situ hybridization for isl1 (A–C) and sox2 (D–F) at 24 hpf. (G–L) Confocal projections of embryos labeled for acetylated tubulin (green), fast muscle (F310; red), and myosin heavy chain (A4.1025; blue) in lateral (G–I) and dorsal (J–L) views. Not shown are tbx6l single mutant embryos as they are indistinguishable from wild-type embryos. Scale bar in A (for A–F) and G (for G–I) is 100 µm, and in J (for J–L) is 50 µm.

Our analyses reveal that a single neural tube develops in the zebrafish tbx6l; tbx16 double mutant, contrasting sharply with the dramatic “extra neural tubes” phenotype of the Tbx6 mutant mouse. This functional distinction between mouse Tbx6 and zebrafish tbx6l, together with the functional overlap of zebrafish tbx6l and tbx16 genes and sequence analysis showing that tbx6l is more closely related to tbx16 than to tbx6 (Ahn and Takada, 2012; Windner et al., 2012), suggests that tbx6l might be more appropriately named tbx16l.

Concluding Remarks

We have generated and characterized two zebrafish tbx6l mutant alleles, both of which are null alleles based upon the severity of the predicted protein truncation relative to the T-box DNA binding domain and failure to detect full-length Tbx6l protein via immunofluorescence and western blotting with a Tbx6l polyclonal antibody. Loss of tbx6l alone has no apparent effect on embryonic development or viability, nor does tbx6l loss enhance the phenotype of two additional T-box gene mutants, ta and tbx6, or of the T-box target gene mutant, msgn1. In stark contrast, we show that loss of tbx6l function dramatically enhances the paraxial mesoderm deficiency of tbx16 mutants, demonstrating that these two T-box genes, which are similar in sequence and embryonic expression, function redundantly in the posterior paraxial mesoderm. Whereas tbx16 fully compensates for loss of tbx6l function, tbx6l compensates for tbx16 in tail, but not trunk, paraxial mesoderm. We present a model for these functional interactions in Fig. 6.

Figure 6. Model showing genetic interactions among tbx16, tbx6l, and msgn1 genes during trunk and tail paraxial mesoderm development.

Figure 6

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The model is based largely upon analyses of single and double mutant combinations. In the model, some upstream regulators and downstream paraxial mesoderm genes may be shared among genes or developmental time periods, yet others are distinct; for example, transcriptional regulators of tbx6l are different in the trunk versus the tail (Szeto and Kimelman, 2004). During trunk paraxial mesoderm formation, tbx16 function is critical (large arrow) while loss of tbx6l or msgn1 has little consequence (dotted gray arrows). Later, during tail paraxial mesoderm formation, the ability of tbx6l or msgn1 to partially compensate for tbx16 is revealed in double mutant combinations. However, since tbx6l;msgn1 double mutants are essentially normal and do not phenocopy tbx6l;tbx16 or msgn1;tbx16 double mutants, we propose that tbx16, and/or other regulators and downstream targets, can compensate for combined loss tbx6l and msgn1 function.

Experimental Procedures

Animal Welfare

All experiments were conducted under guidelines approved by The Ohio State University and University of Utah Institutional Animal Care and Use Committees.

Zebrafish Alleles and Husbandry

Zebrafish were raised and maintained at 28.5°C on a 14-hour light/ 10-hour dark cycle. Fish strains used were AB wild-type, tbx16b104 (previously spadetail [spt]; Griffin et al., 1998), tab195 (previously no tail [ntl] or Brachyury a; Schulte-Merker et al., 1994), mesogenin1fh273 (msgn1fh273; Draper et al., 2004; Fior et al., 2012), tbx6te314a (previously fused somites [fss] or tbx24; Nikaido et al., 2002), and tbx6lz34 and tbx6lz35 that we report here for the first time.

To generate tbx6l mutations, we designed TALENs targeting the tbx6l intron2-exon3 junction (see Fig 1A; TALENs described in Dahlem et al., 2012) and generated new alleles as previously described (Dahlem et al., 2012; note in that reference, tbx6l was referred to as tbx6). TALEN sequences are available upon request. Two independent deletion alleles, tbx6lz34 (8 bp deletion) and tbx6lz35 (7 bp deletion), were recovered (Fig 1A). Each causes a frameshift that introduces a premature stop codon, and both alleles appear to encode null proteins. tbx6lz34 is predicted to encode the first 79 amino acids of the Tbx6l protein, including the first 27 amino acids of the 180 amino acid T-box domain, followed by 22 residues of aberrant sequence before termination. tbx6lz35 is predicted to encode the first 80 amino acids of the Tbx6l protein, including the first 28 amino acids of the T-box domain, followed by one aberrant Lysine residue before termination. In the experiments reported here, tbx6lz34 was used to generate double mutants with tab195, tbx6te314a, and msgn1fh273, and tbx6lz34 and tbx6lz35 were used to generate double mutants with tbx16b104.

Embryos were collected from natural spawnings, raised at 25–28.5°C, and staged according to Kimmel et al. (1995). Doubly heterozygous parents were created by crossing homozygous tbx6lz34 adults (and additionally tbx6lz35 adults for the tbx6l; tbx16 combination) to homozygous msgn1fh273 or tbx6te314a adults or to heterozygous tbx16b104 or tab195 carriers. Once genotyped, doubly heterozygous adult carriers were intercrossed to generate double mutant embryos, which were produced at the expected Mendelian frequency of 1:16. In many experiments, expected embryonic genotypes were readily distinguished in the appropriate ratios by morphology and/or gene expression and confirmed by PCR genotyping when necessary (see below). At developmental stages before genotypes are reliably distinguished by morphology, PCR genotyping was used.

Genotyping

High resolution melting analysis (HRMA) (Dahlem et al., 2012) was initially used to identify and genotype tbx6l alleles. Both alleles can also be distinguished by virtue of absence of the PvuII restriction site in the tbx6lz34 and tbx6lz35 alleles (see Fig 1). A 273 bp region of the tbx6l locus (including the 103 bp exon 3) is PCR-amplified using intron 2 forward primer 5’-GGGCTCATGGACTTCACAAT-3’ and intron 3 reverse primer 5’-CAGAAATATGCCCGTGTGTG-3’ and the amplicon is then digested with PvuII. The PCR product amplified from the wild-type allele is cleaved into 115 bp and 158 bp fragments, whereas PCR amplicons from both tbx6l mutant alleles are cleavage-resistant. The size of the amplified product can vary with genetic background due to a variable CA repeat region in intron 3.

tbx6, tbx16, and ta homozygous mutants were genotyped by morphology and heterozygotes were genotyped using standard molecular protocols. Standard molecular genotyping and/or HRMA were used for msgn1 genotyping. Because the msgn1fh273 mutation eliminates a PvuII site, embryos are genotyped via PCR amplification with forward 5’-AGCAGAAGCCGAAAGTGAAG-3’ and reverse 5’-TGGTGTATTTGAGCGTCTGG-3’ primers, followed by diagnostic digest with PvuII. The wild-type PCR amplicon is cut into 98 and 76 bp fragments whereas the mutant amplicon is not cut (174 bp).

In situ hybridization

Whole-mount in situ hybridization was performed as previously described (Thisse et al., 1993; Melby et al., 1997). Embryos were fixed in 4% paraformaldehyde (PFA) for 4 hours at room temperature or overnight at 4°C. Embryos older than 20 somites were treated with Proteinase K and re-fixed in 4% PFA prior to hybridization protocol. Probes were synthesized using linearized plasmid templates for the following markers: ta (Amacher et al., 2002), tbx6 (Fior et al., 2012), myoD (Weinberg et al., 1996), cb1045/xirp2a (Riedel-Kruse et al., 2007), pax2a (Krauss et al., 1991), sox2 (Okuda et al., 2006), isl1 (Appel et al., 1995), msgn1 (Goering et al., 2003), and myl7 (Yelon et al., 1999). Probes for lmo2 and fli1a were synthesized from 3’ UTR sequence amplified by PCR from cDNA using gene-specific primers (with a T7 promoter engineered into the reverse primer). lmo2 primer sequences were: forward (5’-CCACCAGCAACATGAACCTT-3’) and reverse (5’-TTTTTAATACGACTCACTATAGTGCTCTGTTGGCACTTTGAT’-3’). fli1a primer sequences were: forward (5’-TGAGCCTTGAAAGTTGCACC-3’) and reverse (5’- TTTTTAATACGACTCACTATAGAAGCTTTCACATCCTGACGC-3’). Embryos were dehydrated through washes in 1:1 1X PBST:methanol and 100% methanol, rehydrated and cleared in 1:1 Benzyl benzoate: Benzyl alcohol, and mounted in Permount (Fisher Scientific) on bridged slides. For flat mounts, embryos were de-yolked, cleared in glycerol, and imaged in 70% glycerol. For each genotype, at least 10 embryos were scored, and a representative image is shown.

Tbx6l antibody generation and immunofluorescence

A polyclonal antibody against zebrafish Tbx6l was generated by Strategic Diagnostics (SDIX). Briefly, a peptide containing Tbx6l residues 310–409 was used to immunize rabbits. The antiserum from 2 rabbits was collected and affinity purified. For wholemount immunohistochemistry, embryos were fixed in Carnoy’s fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) for 2 hours at room temperature followed by an additional 2 hours at −20°C. Embryos were stored at −20°C until processed for staining. Embryos were removed from Carnoy’s fixative and rehydrated through an ethanol series (90%, 70%, 50%, 25%), followed by washing in PBST. Embryos were incubated 2–3 hours at room temperature in blocking solution (4% normal goat serum, 1% normal sheep serum, 2% bovine serum with or without DMSO) and then in Tbx6l primary antibody (1:250 in blocking solution) for 4 hours. Embryos were washed thoroughly in PBST, re-blocked 2 hours, and then incubated in goat anti-rabbit Alexa Fluor 488 for 2 hours at room temperature. Embryos were washed in PBST for 2 hours before imaging. Embryos were flat-mounted in 70% glycerol and imaged by confocal microscopy.

A different protocol was used to co-label neurons and muscle. Embryos were fixed in 4% PFA and stored overnight in methanol. Embryos were rehydrated, blocked for 4 hours at room temperature, then incubated overnight at 4°C in primary antibody solution containing anti-acetylated tubulin [1:1000; Sigma, T6793], F310 [1:1000; DSHB], and A4.1025 [1:000; DSHB]. After overnight incubation, embryos were thoroughly washed in PBST and incubated at room temperature in secondary antibody. Embryos were mounted in 0.25% agarose in PBST and imaged using an inverted Nikon TiE microscope equipped with an Andor Revolution WD spinning disk confocal system.

Protein extract preparation and Western Blotting

Zebrafish embryos were manually dechorionated at the 18 somite stage and deyolked by rapid pipetting in cold Cell Resuspension Buffer (116 mM NaCl, 2.9 mM KCl, 5.0 mM HEPES, pH 7.2, 0.1 mg/mL Soybean Trypsin Inhibitor, Complete Protease Inhibitor Cocktail [Roche]). Cell lysis was performed under reducing conditions in 2X LDS Buffer (ThermoFisher). Cell extract equivalent to 5 embryos was loaded onto a 4–12% SDS-PAGE gel (Life Technologies), electrophoresed in MES buffer, and transferred to a PVDF membrane (GE Healthcare Life Sciences). The blot was incubated in 10% non-fat milk protein in TBSTw (10mM Tris, 100mM NaCl, 0.1% Tween-20, pH 7.6) for 2 hours at room temperature and then with Tbx6l primary antibody (1:500 in blocking solution) at 4°C overnight. After several TBSTw washes, the blot was blocked again for two hours and incubated with AP-conjugated goat α-rabbit secondary antibody (1:10,000 in blocking solution) for 2 hours. After several TBSTw washes, the blot was washed twice in Assay Buffer (20 mM Tris pH 9.8, 1 mM MgCl), incubated in CDP-STAR chemiluminescent substrate (Sigma) for 5 minutes, and exposed to x-ray film. The blot was then washed two times for 30 minutes in Stripping Buffer (0.2 M Glycine, 0.1% SDS, 1% Tween-20, pH 2.2) and re-probed essentially as described above with 1:2,500 anti-alpha Tubulin primary antibody (12G10; DSHB) and 1:10,000 AP-conjugated goat α-mouse secondary antibody.

Bullet points.

  • Loss of zebrafish tbx6l alone has little to no effect on embryonic development because tbx16, a closely related gene, compensates for loss of tbx6l function.

  • Loss of tbx6l enhances the tbx16 mutant phenotype, revealing that tbx6l functions redundantly with tbx16 during tail paraxial mesoderm development.

  • Double mutant analyses reveal that tbx6l does not appear to genetically interact with ta or tbx6, two other mesodermally-expressed T-box genes. In addition, tbx6l loss does not enhance the supernumerary somite phenotype of mesogenin1 mutants.

Acknowledgments

We thank the Ohio State Zebrafish Facilities staff for excellent zebrafish care, Paula Monsma and the Neurobiology Imaging Core for microscopy assistance and advice, Duy Phan and Nicolas Derr for expert technical assistance, and Amacher lab colleagues for advice and support over the course of the project. We also acknowledge the support of the University of Utah Health Sciences Center Core Facilities.

Grant Sponsors and Numbers:

National Institutes of Health grants R01 GM061952, R01 GM117964, and ARRA supplement (SLA), March of Dimes grant 1-FY09-458 (SLA), NIH Research Supplement for NIH R01 GM061952 to promote diversity in health-related research (AAM), and NIH 1 P01 HD048886 and 1 R01 HD81950 (DJG).

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