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. Author manuscript; available in PMC: 2017 Aug 1.
Published in final edited form as: Dev Dyn. 2016 May 27;245(8):874–880. doi: 10.1002/dvdy.24417

A novel cold-sensitive mutant of ntla reveals temporal roles of Brachyury in zebrafish

David Kimelman 1
PMCID: PMC4947019  NIHMSID: NIHMS785048  PMID: 27153483

Abstract

Background

With the exception of the head, the vertebrate embryonic body is formed progressively in an anterior-posterior direction, originating from a posteriorly located bipotential neural-mesodermal progenitor population. The T-box transcription factor Brachyury is expressed within the progenitors, and is essential for the formation of the posterior mesoderm. A novel cold-sensitive mutant of zebrafish Brachyury (ntlacs) is described that allows exploration of the temporal role of this key factor.

Results

The ntlacs mutant is used to show that Ntla has an essential role during early gastrulation, but as gastrulation proceeds the importance of Ntla declines as Ntlb acquires a capacity to form the posterior mesoderm. Remarkably, ntlacs embryos held at the non-permissive temperature just during the gastrula stages show recovery of normal levels of mesodermal gene expression, demonstrating the plasticity of the posterior progenitors.

Conclusion

ntlacs is a valuable tool for exploring the processes forming the posterior body since it allows temporally specific activation and inactivation of Brachyury function. It is used here to show the changing roles of Ntla during early development, and the dynamics of the neuromesdermal progenitors.

Keywords: Brachyury, neuromesodermal progenitors, T-box genes, early vertebrate development

INTRODUCTION

The brachyury gene is one of the major players involved in forming the early vertebrate embryonic body (reviewed in Kimelman, 2016; Wardle and Papaioannou, 2008). Mice lacking brachyury (also known as T) lack a notochord and have an almost complete absence of somites (Chesley, 1935; Gluecksohn-Schoenheimer, 1944). A brachyury mutant called no tail (ntl), identified in a mutant screen in zebrafish, has normal trunk somites but lacks a notochord and tail somites, suggesting that brachyury has a less important role in the lower vertebrates (Halpern et al., 1993; Schulte-Merker et al., 1994). However, a second ntl gene was later discovered, and loss of both alleles recapitulates the mouse phenotype, demonstrating a conserved ancestral role for brachyury (Martin and Kimelman, 2008). The original ntl is now called ntla (also ta) and the second one is ntlb (tb).

Brachyury is expressed in the progenitors found at the most posterior end of the growing vertebrate embryo, together with the essential neural factor sox2 (Garriock et al., 2015; Martin and Kimelman, 2012; Olivera-Martinez et al., 2012; Tsakiridis et al., 2014). Cells exiting this progenitor zone can either become mesodermal (brachyury+/sox2−) or neural (brachyury−/sox2+) as they begin to differentiate, thus producing the somites and neural tube (Bouldin et al., 2015; Garriock et al., 2015; Gouti et al., 2014; Jurberg et al., 2014; Martin and Kimelman, 2012; Tsakiridis et al., 2014; Tzouanacou et al., 2009; Wymeersch et al., 2016). The cells in the progenitor zone are therefore referred to as the bipotent neuromesodermal progenitor cells (reviewed in Gouti et al., 2014; Henrique et al., 2015; Kimelman, 2016).

Brachyury is a transcription factor with many embryonic targets in the notochord and mesoderm (Evans et al., 2012; Garnett et al., 2009; Gentsch et al., 2013; Morley et al., 2009). Surprisingly, cells lacking both ntla and ntlb develop normally into muscle cells when transplanted into wild-type zebrafish embryos, demonstrating that the essential role of brachyury in the somites is non-cell autonomous (Martin and Kimelman, 2008). Importantly, Brachyury activates the expression of wnt genes, which are essential for sustaining the neuromesodermal progenitors and forming the mesoderm (reviewed in Kimelman, 2016; Wymeersch et al., 2016). In zebrafish, we showed that transgenic embryos containing a heat inducible specific inhibitor of the Wnt signaling pathway rapidly cease forming the posterior body soon after the embryos are heat shocked anytime during the gastrula or somite-forming stages (Martin and Kimelman, 2008). Based on this, and results showing that Wnts activate brachyury expression (Arnold et al., 2000; Vonica and Gumbiner, 2002; Yamaguchi et al., 1999), we proposed that Wnt and Brachyury exist in an autoregulatory loop that begins in gastrulation and runs through the somitogenesis stages (Martin and Kimelman, 2008). However, because there is no completely specific inhibitor of Brachyury we were unable to analyze the temporal role of Brachyury in forming the embryo.

I report here a novel cold-sensitive mutant of ntla, ntlacs, that arises from two conservative missense mutations in the C-terminus of the protein. ntlacs embryos maintained at the non-permissive temperature have a phenotype and gene expression profile that looks very similar to ntla null mutants. Using timed temperature shifts I show that Ntla is essential during gastrulation but its role diminishes as embryos enter somitogenesis since Ntlb provides an increasingly important role. Importantly, embryos held at the non-permissive temperature only during gastrulation produce normal levels of posterior mesodermal gene expression and significantly well developed bodies compared to embryos maintained at the non-permissive temperature, revealing the inherent plasticity of the neuromesodermal progenitors.

RESULTS and DISCUSSION

Identification of a cold-sensitive mutation in ntla

In order to study zebrafish embryos during the very early stages of somitogenesis we routinely grow them at the standard temperature of ~29°C until the start of gastrulation (shield stage), place them in room temperature embryo media, and then put them in a 17°C incubator overnight, returning them to 29°C the next morning. Under these conditions, the embryos grow completely normally with no loss of viability. While crossing pairs of fish from one family, I observed that approximately one-quarter of the embryos had a variably truncated tail (Fig. 1C, D). When the same adults were crossed and the embryos were raised at 29°C, the embryos showed no phenotypic abnormalities and could be raised to the adult stage (see below).

Figure 1.

Figure 1

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Identification of a cold-sensitive mutation in ntla. (A–D) A cross from one family of fish produced embryos with a truncated tail phenotype (C, D) as well as wild-type (wt) embryos after being raised overnight at 17°C from shield stage. The phenotype is less severe than seen with the ntlab195 mutant (B, ntla−/−). (E–H) Staining with the muscle antibody MF20 shows that the embryos with the truncated tails have less somitic tissue (G, H), although with less reduction than seen with ntla−/− (F). (I) The mutant embryos have two base pair changes in the C-terminal domain of the Ntla coding region. The numbers indicate the base pair where the change occurred relative to the first base of the start codon. (J, K) A cross of ntla−/+ and ntlacs/+ adults produced phenotypically wild-type and mutant embryos after being raised overnight at 17°C from shield stage. The genotypes of both are indicated on the figure. All embryos are shown at the equivalent of two days post-fertilization.

As shown by staining with a muscle marker, the embryos grown in the cold condition had a variably reduced number of somites compared to wild-type embryos (Fig. 1E, G, H). This phenotype appeared similar to that seen in ntla hypomorphs ntlats260, ntlam550 and ntlab487 (Doyon et al., 2008; Odenthal et al., 1996; Stemple et al., 1996) but less severe than seen in the ntla null mutant, ntlab195 (Fig. 1B, F, Halpern et al., 1993), suggesting that the mutation might be in ntla or an interacting factor. DNA sequence analysis of ntla cDNA isolated from embryos with a mutant phenotype showed two conservative amino acid changes in the C-terminal region of Ntla (Fig. 1I). Based on these changes, primers for both High Resolution Melt Analysis (Talbot and Amacher, 2014) and dCAPS (Neff et al., 2002) were designed and used to test embryos. In every case, embryos that showed the mutant phenotype also had the C-terminal mutations (n=32). Based on these results, this cold-sensitive mutation of ntla was designated ntlacs (the official name of this line is ntlaw181). As a final test, ntlacs adults were crossed to ntlab195 null mutants (ntla−/−) and placed at 17°C at shield stage. 21% (26/126) of the embryos had a truncated tail-phenotype. When the wild-type and mutant embryos were genotyped (n=16), all of the mutant embryos were ntlacs/−, whereas the phenotypically wild-type embryos were wild-type, ntlacs/+ or ntla−/+ (Fig. 1J, K), further confirming that ntlacs is a novel cold-sensitive allele of ntla.

Of the two amino acid mutations (Fig. 1I), the alanine that changes to valine is a reside that is conserved among fish species including Spotted gar, Fugu, Stickleback and Medaka, although not in Coelacanth. In contrast, the methionine that changes to valine is found as a valine in some fish species such as Spotted gar and Coelacanth, although it is deleted in other fish species such as Medaka, Stickleback and Fugu. Thus, of all these fish species, Spotted gar is the only one likely to acquire the same cold-sensitive mutation, if the equivalent alanine were to change to a valine.

Ntla is required during the gastrula stages

With a temperature regulated Ntla it was now possible to determine when it is required during embryogenesis without the need for specialized photo-regulated morpholino oligonucleotides, which have been used previously to study notochord and floor plate formation (Shestopalov et al., 2012; Shestopalov et al., 2007; Tallafuss et al., 2012). Because the ntlacs embryos are completely viable it was possible to isolate homozygous adults, which were used for the remainder of the experiments presented here since 100% of the progeny of a cross of two homozygous adults are mutant, obviating the need to genotype the resulting embryos. Embryos raised from a cross of homozygous ntlacs adults were placed overnight at 17°C at the start of gastrulation (shield stage), halfway through gastrulation (75% epiboly) or at the end of gastrulation (bud stage). The next morning the embryos were placed at 29°C and raised at this temperature until they reached the equivalent stage that wild-type embryos reach at two days post-fertilization (dpf). All embryos treated this way showed at least some minor defect. The embryos were then categorized as severe/moderate or mild (Fig. 2A–E). While cold treatment at the start of gastrulation resulted in embryos that were mostly severe and moderate (n=44), this effect was gradually lost during gastrulation (Fig. 2F). Cold treatment at bud stage produced almost exclusively mild embryos that had only a mild kink in the tail or often just minor defects at the most posterior end, which were recognizable by slight morphological aberrations in the most posterior tail fin (Fig. 2G–I; n=44). Thus, the major role of Ntla in forming the posterior body occurs during the gastrula stages.

Figure 2.

Figure 2

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Ntla is essential during the gastrula stages. (A–E) ntlacs/cs embryos were categorized as severe, moderate or mild. Mild embryos had either a kinked tail (E) or an essentially normal body axis with minor tail fin defects at the most posterior end (D). (F) Quantification of the results from placing embryos in the cold at the indicated stages. Severe and moderate phenotypes were grouped together since embryos were a continuum between the phenotypes shown in panels B and C. (G–I) Close up images showing the posterior tail fin defects observed in the mild embryos.

Ntla continues to function post-gastrulation

Ntlacs produces a less severe phenotype at the non-permissive temperature than is seen with the ntla null allele (Fig. 1A–H). The same effect was also seen when the expression of two mesodermal genes downstream of Ntla, tbx16/spadetail (Griffin et al., 1998; Ruvinsky et al., 1998) and tbx6l (Goering et al., 2003; Hug et al., 1997), were examined at the mid-somitogenesis stage (Fig. 3A–F), demonstrating that the increased number of somites in ntlcs mutants is due to continued production of new mesoderm during later stages of somitogenesis. These results suggested that the missense mutations in ntlacs might be unable to produce a null phenotype and instead only cause a partial loss of function. Alternatively, since the embryos placed at 17°C overnight were returned to 29°C the next morning (when they have reached the end of gastrulation and just begun somitogenesis), it was possible that when the embryos were returned to the permissive temperature the accumulated Ntlacs protein was able to provide some degree of rescue. To test this, embryos were maintained at 17°C until they had reached the equivalent of two days post-fertilization. Under this procedure, wild-type embryos developed mostly normally although the body was often curved (Fig. 4A). In contrast, ntlacs mutant embryos phenotypically resembled ntla null mutants (Fig. 4B, compare to Fig. 1B–D; 100%, n=55). Similarly, analysis of molecular markers at mid-somitogenesis revealed highly diminished expression of posterior mesodermal gene expression, similar to that seen in the ntla null mutants (Fig. 3L, N compare to B, E). Similarly, when the notochord marker col9a2 (Mangos et al., 2010) was examined, ntla null mutants showed no expression of this marker whereas ntlacs embryos raised at 29°C after an overnight 17°C incubation had strong posterior expression of col9a2 (Fig. 3G–J), indicating recovery of notochordal gene expression. In contrast, ntlacs embryos kept at 17°C had almost no col9a2 expression (Fig. 3P). These results show that ntlacs produces an essentially null phenotype when kept at 17°C, whereas shifting back to the permissive temperature allows complete recovery of the posterior mesoderm from the neuromesodermal progenitor population. In addition, these results provide further support for the model that the notochord in the fish, as in higher vertebrates, is continuously induced from a progenitor population in the tailbud during the post-gastrula stages and is not induced only during the gastrula stages (Row et al., 2016)

Figure 3.

Figure 3

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Analysis of gene expression in ntla mutant embryos. (A–J) Wild-type, ntla−/− and ntlacs/cs embryos were placed at 17°C at shield stage, then incubated at 29°C the next morning (when the embryos were at bud stage) and raised to the 14 somite stage. Whereas ntla−/− embryos had essentially no expression of the presomitic genes tbx16 and tbx6l or the notochordal gene col9a2, ntlacs/cs embryos had strong expression of all three genes. All these embryos were hybridized with the indicated probe at the same time and for the same length of time. (K–P) In contrast, when ntlacs/cs embryos were kept continuously at 17°C, expression of all three genes was considerably diminished. Arrows show accumulation of col9a2 in the posterior of ntlacs/cs embryos.

Figure 4.

Figure 4

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Maintaining ntlacs/cs embryos at 17°C enhances the mutant phenotype. Wild-type and ntlacs/cs embryos were maintained at 17°C from shield stage until the equivalent of 2 dpf. Whereas wild-type embryos formed a normal body (although often with a curved tail), ntlacs/cs embryos appeared morphologically the same as ntla−/− embryos (see Figure 1).

Ntlb is essential for forming the posterior body during the post-gastrula stages

We previously showed that whereas ntla null mutant embryos produce approximately 18 well formed somites of the trunk (Halpern et al., 1993), embryos lacking both Ntla and Ntlb form only 8–12 poorly formed somites (Martin and Kimelman, 2008). These results suggested that Ntlb might function largely during formation of the trunk even though it is also expressed in the neuromesodermal progenitors during the stages when the tail somites form. Since ntlacs embryos placed at 17° at the end of gastrulation (bud stage) develop virtually normally (Figure 2), this provided an opportunity to examine the later role of Ntlb. To test the role of Ntlb, its translation was inhibited with a morpholino oligonucleotide (MO) as previously described (Martin and Kimelman, 2008). Whereas wild-type embryos injected with the ntlb MO and placed at 17° at bud stage develop normally (Fig. 5A), ntlacs embryos containing the ntlb MO and treated the same way have a truncated axis (Fig. 5B; 91%, n=22). The same effects were observed when the embryos were placed at 17° during the early somitogenesis stages (Fig. 5C, D; ntlacs 100%, n=23), with milder defects when the embryos were placed at 17° at mid-somitogenesis (Fig. 5E, F; ntlacs 82%, n=65). These results demonstrate that Ntla can continue to function in the post-gastrula stages but that its role is obscured by Ntlb, which is co-expressed with Ntla.

Figure 5.

Figure 5

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Ntla is required for posterior development in the absence of Ntlb. Wild-type or homozygous ntlacs/cs embryos were injected with a ntlb MO and then placed at 17°C overnight at bud stage (A, B), 6 somite stage (C, D) or 15 somite stage (E, F). The embryos were then grown to the equivalent of 2 dpf at 30°C. Interfering with Ntlb function causes a truncated tail phenotype in ntlacs embryos.

Conclusions

The cold-sensitive ntla mutant described here will be a valuable tool for studies of this essential protein. Whereas light-controlled MOs have been useful for the study of the role of ntla in notochord and floor plate formation (Shestopalov et al., 2012; Shestopalov et al., 2007; Tallafuss et al., 2012), once the MO is light activated it will irreversibly activate or eliminate Ntla function. In contrast, a temperature sensitive allele can be variably inactivated and activated just by shifting the temperature of the growth medium. Thus, ntlacs is used here to show that Ntla plays an essential role during the gastrula stages, but if Ntla function is restored at the end of gastrulation, the neuromesodermal progenitors will return to producing normal levels of mesoderm as long as Ntlb is present. Thus Ntla is essential because it provides a “kick start” to get the system running, which Ntlb alone cannot provide. It is interesting to note that all the bony fish sequenced, including Coelacanth and Spotted Gar that predate the teleost genome duplication (Amemiya et al., 2013; Braasch et al., 2016), have two copies of the brachyury gene whereas other vertebrates have only one. Since, at least in zebrafish, the role of Ntlb in forming both the trunk and tail is unnecessary as long as Ntla is present, it remains an interesting mystery why all these species maintain two copies of brachyury.

EXPERIMENTAL PROCEDURES

Fish lines

All fish are Hybrid WIK/AB. Genetic analysis used either High Resolution Melt Analysis (Talbot and Amacher, 2014) or dCAPS (Neff et al., 2002). Since the dCAPS was more useful in segregating wild-type, heterozygous and homozygous genotypes, that procedure is provided here. Genomic DNA was amplified with 5’-AGTTCCTACGCGGTTCATCG-3’ and 5’-CAGTAGCTCTGAGCCACAGG-3’ using standard PCR conditions with GoTaq Flexi buffer (Promega). After amplification the PCR reaction was digested with ClaI and separated on a 2.5% agarose gel using a LiCl buffer. ClaI cuts the wild-type allele but not the ntlacs allele.

In situ hybridization, immunofluorescence and morpholino

Alkaline phosphatase in situs were performed as described (Griffin et al., 1995). The MF20 monoclonal antibody (Developmental Studies Hybridoma Bank) was used at a 1:50 dilution. The ntlb translation blocking morpholino oligonucleotide was previously described (MO2, Martin and Kimelman, 2008) and used at 2.5 ng along with 0.5 ng of a p53 morpholino (Robu et al., 2007).

Acknowledgments

I am indebted to Ben Martin for providing bra MO, Iain Drummond for providing the col9a2 plasmid, and the reviewers for their valuable contributions. D.K. was supported by a National Institutes of Health grant (RO1GM079203).

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