Abstract
In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, but neither loss nor gain of hmx3a function was sufficient to generate ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.
Introduction
The otic placode—precursor of the vertebrate inner ear—has the remarkable ability to generate a mirror-image organ with duplicate structures under some experimental conditions in fish and amphibians, as originally described by R. G. Harrison over eighty years ago (reviewed in (Whitfield and Hammond, 2007)). Understanding the generation of such duplicated structures can give us fundamental insights into mechanisms of organ patterning, tissue polarity and symmetry-breaking during embryogenesis. During normal development in the zebrafish, anteroposterior asymmetries in otic gene expression are evident as early as the 4-somite stage (11.5 hours post fertilisation (hpf)), when expression of the transcription factor gene hmx3a appears at the anterior of the otic placode (Feng and Xu, 2010). Additional genes with predominantly anterior patterns of expression in the otic placode or vesicle begin to be expressed over the next 10 hours, including the transcription factor genes hmx2 and pax5 (Feng and Xu, 2010; Kwak et al., 2006), together with the fibroblast growth factor (Fgf) family genes fgf3, fgf8a and fgf10a (Léger and Brand, 2002; McCarroll and Nechiporuk, 2013). Later, at otic vesicle stages (24 hpf onwards), the size and position of the otoliths, together with the position, shape and planar polarity patterns of the sensory maculae, provide landmarks for distinguishing anterior and posterior structures in the ear (Hammond and Whitfield, 2011) (Fig. 1). In addition, a few markers begin to be expressed specifically in posterior otic tissue (pou3f3b, bmp7a and fsta) at otic vesicle stages (Kwak et al., 2006; Mowbray et al., 2001; Schmid et al., 2000), but these are not reliable posterior markers at earlier otic placode stages.
Concomitant with the appearance of anteroposterior asymmetry in the zebrafish otic domain, other early patterning events occur that are symmetrical about the anteroposterior axis. Of relevance for our study, a single sensory-competent domain, marked by the expression of atoh1b, splits into two domains, one at each pole of the ear, by 12 hpf. This process is dependent on Notch signalling and atoh1b function, and defines differences between the poles of the otic placode and a central zone (Millimaki et al., 2007). The two poles express various markers symmetrically, including atoh1a and deltaD, between 14–18 hpf (Millimaki et al., 2007), presaging the appearance of pairs of myo7aa-positive sensory hair cells (tether cells) at each pole by 18–24 hpf (Ernest et al., 2000). Thus, by the completion of otic induction at 14 hpf (10 somites), the otic domain has two clear poles defined by the symmetric expression of atoh1a and deltaD, with the anterior pole distinguished from the posterior by the asymmetric expression of hmx3a.
Although anteroposterior asymmetries in otic gene expression are already apparent by 12 hpf in the zebrafish, these can be disrupted by interfering with extrinsic signalling pathway activity after this time. For example, manipulations of either Fibroblast growth factor (Fgf) or Hedgehog (Hh) signalling between 14-19 hpf can result in striking double-anterior or double-posterior mirror-image ears. Fgf signalling is both required and sufficient to act as an anteriorising cue, whereas Hh signalling is both required and sufficient for the acquisition of posterior otic identity (Hammond et al., 2003; Hammond et al., 2010; Hammond and Whitfield, 2011). In these studies, we showed that transient fgf3 mis-expression at 14 hpf or Hh pathway loss-of-function result in the loss of posterior-specific expression domains of fsta at 30 hpf and otx1b at 45–48 hpf, and the gain of anterior-specific gene expression in the posterior of the ear (hmx2 and pax5 at 24 hpf after fgf3 mis-expression; hmx3a at 30 hpf in Hh loss-of-function mutants). These findings suggest that Fgf and Hh signalling normally act to establish and determine the asymmetric expression of marker genes within the otic epithelium. However, the details of their temporal mode of action in the duplication of anterior otic fates have not been explored.
In this study, we have compared the dynamics of the transcriptional responses that precede the acquisition of a duplicated anterior otic fate in an Fgf gain-of-signalling or a Hh loss-of-signalling context. Although the final duplicated ear structures appear similar after each manipulation, the early transcriptional responses differ for each signalling pathway, progressing in distinct ways to give rise to the double-anterior pattern at larval stages. One gene that shows an early transcriptional response in the zebrafish otic placode to disruption of either Fgf or Hh signalling is the Hmx family homeobox gene hmx3a. We have examined the effects of both loss-of-function and gain-of-function of hmx3a on inner ear patterning. Our data suggest that hmx3a is a key early target for the otic anteriorising activity of Fgf signalling, and that the function of hmx3a is required for the anterior-specific otic expression of fgf3 and pax5, together with correct positioning and development of the sensory maculae. However, unlike high Fgf levels or low Hh pathway activity, mis-expression of hmx3a was unable to generate full duplications of anterior character at the posterior of the ear. A mathematical model based on our experimental findings can recapitulate both the wild-type and duplicated anterior pattern, allowing us to explore the dynamical principles underlying the generation of a mirror-image duplicated organ system.
Results
Early mis-expression of fgf3, but not fgf8a, can generate complete double-anterior ear duplications
To establish optimal conditions for generating a double-anterior ear in the zebrafish embryo, we compared otic phenotypes in transgenic lines for two different fgf genes, fgf3 and fgf8a, with systemic transgene expression driven under the control of the hsp70 heat-shock promoter (Lecaudey et al., 2008; Millimaki et al., 2010). Previously, we showed that a 2-hour heat shock in the Tg(hsp70:fgf3) line at 14 hpf (10-somite stage) resulted in a robust duplication of anterior otic structures (Hammond and Whitfield, 2011). We chose this time point to avoid any interference with otic placode induction, which is also Fgf-dependent, but is complete by 14 hpf (Kimmel et al., 1995; Phillips et al., 2001). The 14 hpf time point is also after completion of the Notch-dependent signalling event that distinguishes the otic poles from a central zone of epithelium (Millimaki et al., 2007). For the treatments described here, we reduced the time of heat shock to 30 minutes at 39°C. This shorter heat shock still results in a full ear duplication, but should minimise effects of Fgf mis-expression on other developing organ systems. After heat shock, embryos were then cultured at 33°C for a further 30 minutes, to reduce the incubation temperature gradually, before being returned to 28.5°C and incubated until 3 days post fertilisation (dpf) for processing and analysis (Fig. 1). This stepwise reduction in temperature is thought to extend transgene activation and reduce cell death following heat shock (Padanad et al., 2012; Zou et al., 1998). Non-transgenic sibling embryos, subjected to the same heat-shock treatment, served as controls (Fig. 1—Supplemental file 1).
In Tg(hsp70:fgf3) embryos, a 30-minute heat shock at 14 hpf gave a robust and complete duplication of anterior otic patterning at 72 hpf, as indicated by the mis-positioning or fusion of the posterior otolith, loss of posterior elements of the saccular macula, and a duplication of anterior (utricular)-like sensory elements on the posteroventral floor of the ear (Fig. 1B, G,G’). The phenotypes seen after mis-expression of fgf8a (30-minute heat shock at 14 hpf) were milder and more variable than those for fgf3, and included a split and mis-positioned saccular macula rather than a complete duplication of anterior elements (Fig. 1C, H), and a normal complement of three cristae (Fig. 1H’). A 30-minute heat shock of either transgenic line at a later stage (18 hpf) resulted in only mild effects on ear size and shape, and otolith position (Fig. 1—Supplemental file 2A–D) (Hammond and Whitfield, 2011). We therefore chose to use the Tg(hsp70:fgf3) line, with a 30-minute heat shock (39°C) at 14 hpf followed by 30 minutes at 33°C, in subsequent heat-shock experiments.
Genetic or pharmacological inhibition of Hh signalling can also result in complete double-anterior ear duplications
To optimise our protocols for generating double-anterior duplicated ears through inhibition of Hh signalling, we first examined the ear phenotype in smohi1640Tg/hi1640Tg mutants. The hi1640Tg allele (a transgenic insertion in the smoothened gene, and a likely null (Chen et al., 2001)) is thought to result in a stronger reduction in Hh signalling than the point mutation alleles smob641 and smob577, both of which predict single amino acid substitutions (Varga et al., 2001), and which we used in previous studies (Hammond et al., 2003; Hammond and Whitfield, 2011). The smohi1640Tg/hi1640Tg mutants showed a fully penetrant double-anterior duplicated ear phenotype, with two similar-sized small otoliths located ventrally, complete loss of the posterior (saccular) macula, and duplication of the anterior (utricular) macula at the posterior of the ear, with anterior and posterior elements sometimes present as a contiguous patch of hair cells covering the ventral floor (Fig. 1D,I). Four cristae, rather than the usual three, were present in all (8/8) mutant ears imaged (Fig. 1I’). (For comparison, four cristae were present in only about 50% of ears of smob641/b641 mutant embryos (Hammond et al., 2003).)
Pharmacological inhibition of the transducer of the Hh pathway, Smoothened, using the small molecule cyclopamine, can also produce double-anterior ear duplications (Hammond et al., 2010; Sapède and Pujades, 2010). This approach enables a conditional inhibition of Hh signalling over a defined time window. For the experiments described here, we treated wild-type embryos with 100 µM cyclopamine from 14–22.5 hpf. To examine later stages, we washed out the cyclopamine at 22.5 hpf and allowed embryos to develop further until 3 dpf (72 hpf), when they were fixed for staining and imaging. Stage-matched sibling embryos—either untreated, or treated with vehicle (ethanol) only—served as controls. This cyclopamine treatment regime was sufficient to generate the double-anterior ear phenotype, characterised by two ventrally-positioned, small (utricular-like) otoliths, loss of the posterior (saccular) macula, and a clear duplication of the anterior (utricular) macula (Fig. 1E,J). Ears in 4/8 treated embryos had four cristae (Fig. 1J’); the remaining 4/8 had the normal complement of three cristae. The size and shape of the ear were less affected than in the smohi1640Tg/hi1640Tg mutant embryos, presumably due to the transient nature of the cyclopamine treatment. Taken together, these data show that either genetic or pharmacological inhibition of Hh signalling in wild-type zebrafish embryos between 14–22.5 hpf results in a robust and reproducible double-anterior ear phenotype at 3 dpf.
Following early fgf3 mis-expression, otic expression of anterior markers is initially broad, with pax5 resolving into two discrete domains
One of the most striking transcriptional changes in response to fgf3 mis-expression is the expansion or duplication of the expression of the anterior otic markers hmx2 and pax5 by 24 hpf (Hammond and Whitfield, 2011). To examine the temporal dynamics of this transcriptional response, we assayed for expression of these and additional anterior otic marker genes following our optimised ‘early’ heat-shock regime (14 hpf, 30 min, 39°C) at three different time points: 16 hpf (2 hours post heat shock, to examine any rapid response), 22.5 hpf (8.5 hours post heat shock, when anterior otic expression of hmx2 and pax5 is strongly established in wild-type embryos) and 36 hpf (22 hours post heat shock, to examine whether any disruption to the expression pattern resolves or changes over time). For hmx2 and pax5, which showed dynamic expression changes, we subsequently included two additional time points (25.5 hpf and 30 hpf) to capture these changes in more detail.
We first tested expression of three genes coding for transcription factors (hmx3a, hmx2 and pax5; Fig. 2). At the earliest time point (16 hpf, two hours after heat shock), hmx3a showed the strongest response: expression had already expanded to cover the entire anteroposterior extent of the otic placode (Fig. 2A,B). The anterior markers hmx2 and pax5, not normally expressed at this stage in wild-type embryos, were expressed at very low levels in the anterior of the otic placode of heat-shocked embryos (Fig. 2C–F). We were also able to detect widespread and robust up-regulation of the Fgf target gene etv4 (formerly pea3) in transgenic embryos at this time point (Fig. 2—Supplemental file 1). By 22.5 hpf, all three transcription factor genes were strongly expressed in a broad zone across the entire anteroposterior axis of the otic vesicle in heat-shocked embryos, on the medial side, as can be seen in a dorsal view (Fig. 2G–L). Note that the overall size and shape of these otic vesicles were relatively normal. Although there was some variability (including between both ears of the same fish), the vesicles were oval in shape, indicating that otic induction had not been compromised (compare with the small, rounded vesicles of fgf8ati282/ti282 mutants, in which otic induction is disrupted (Léger and Brand, 2002)). By 36 hpf, wild-type otic expression of hmx genes was more complex, but a clear difference between anterior expressing and posterior non-expressing regions was evident in ventral otic epithelium (Fig. 2M, M’,O,O’). By contrast, in heat-shocked embryos, expression of hmx3a remained strong across the entire anteroposterior axis of the ear in ventral regions (Fig. 2N, N’); expression of hmx2 weakened in central regions during intermediate stages, but at 36 hpf was present in a contiguous ventral domain (Fig. 2P, P’, Fig. 2—Supplemental File 2), while expression of pax5 was lost in central regions, resolving into two discrete ventral domains at the anterior and posterior poles by 25.5 hpf (Fig. 2R, R’, Fig. 2—Supplemental File 3).
To test whether the milder ear phenotype caused by a later heat shock reflects a failure to establish the early transcriptional responses described above, we also examined expression of anterior markers in Tg(hsp70:fgf3) embryos after heat shock at 18 hpf (30 min, 39°C). Unexpectedly, we found that the otic expression of hmx3a and pax5 was very similar to that following an early (14 hpf) heat shock, with a broad band of ectopic expression extending across the entire anteroposterior axis of the otic vesicle by 22.5 hpf (Fig. 1—Supplemental file 2E–H’). This suggests that the loss of competence to generate a complete double-anterior ear after a late heat shock is not due to an inability to express hmx3a and pax5 ectopically throughout the otic epithelium at otic vesicle stages.
Inhibition of Hh signalling results in a slower and spatially distinct transcriptional response in the otic vesicle
To compare the transcriptional response after fgf3 mis-expression at 14 hpf with that following conditional Hh pathway inhibition from the same time point, we examined otic expression of anterior marker genes after treatment of wild-type embryos with cyclopamine (100 µM, 14–22.5 hpf; Fig. 3). To confirm the efficacy of cyclopamine treatment, we also examined the expression of ptch2, a known target of Hh signalling. Expression of ptch2 was down-regulated throughout the embryo at 22.5 hpf, but not abolished (Fig. 3—Supplemental file 1). (By contrast, ptch2 expression is almost entirely lost at 24 hpf in smohi1640Tg/hi1640Tg mutants (Chen et al., 2001)). We also checked expression of etv4 following cyclopamine treatment, but found no major changes in expression at 22.5 hpf (Fig. 3—Supplemental file 1). This result confirmed that there are no strong direct effects of the transient inhibition of the Hh pathway on Fgf signalling activity in the ear, in line with our previous findings after genetic abrogation of Hh signalling (Hammond and Whitfield, 2011).
We examined otic marker genes at two different time points following cyclopamine treatment (22.5 hpf and 36 hpf). Otic expression of both hmx3a and hmx2 was expanded posteriorly on the medial side of the otic vesicle at 22.5 hpf, 8.5 hours after the start of the treatment (Fig. 3A-D’; Fig. 3—Supplemental file 2). Expanded otic hmx gene expression was also present by 23 hpf in the smohi1640Tg/hi1640Tg mutant, in which the Hh pathway is constitutively inactive (Fig. 3—Supplemental file 2). Importantly, there was no significant difference in the expansion of hmx3a expression in the ear at 22.5–23 hpf between the smo mutants and cyclopamine-treated embryos, indicating that our cyclopamine treatment regime is effective at suppressing Hh signalling relevant to otic patterning at this stage (Fig. 3—Supplemental file 2).
The spatial pattern of hmx expansion in response to Hh inhibition was different to that seen after mis-expression of fgf3. Specifically, in cyclopamine-treated embryos or smohi1640Tg/hi1640Tg mutants, hmx3a and hmx2 were expressed in a graded fashion across the ear at 22.5 hpf (8.5 hours after treatment), with higher levels anteriorly, rather than in a uniform broad band (compare Fig. 3B with Fig. 2H). To examine later time points, treated embryos were transferred to fresh medium at 22.5 hpf without cyclopamine, as described above. By 36 hpf (13.5 hours post wash), expression of hmx genes had expanded further posteriorly to cover most of the ventral floor of the otic vesicle in cyclopamine-treated embryos (Fig. 3G-J’), as observed previously at 30 hpf in contf18b/tf18b and smob641/b641 mutants, both of which have a strong reduction in Hh signalling (Hammond et al., 2003).
Expression of pax5 was slower to respond following cyclopamine-mediated inhibition of Hh signalling, with no apparent expansion of the expression domain within the otic vesicle at 22.5 hpf (Fig. 3E-F’). These results corroborate our previous observations in contf18b/tf18b and smob641/b641 mutants, where there was little change in the otic expression of pax5 at 24 hpf (Hammond et al., 2003). However, by the later time point (36 hpf; 13.5 hours post wash), a new, discrete domain of pax5 expression appeared within posteromedial otic epithelium of cyclopamine-treated embryos (Fig. 3K, L). Anteroposterior asymmetry in treated ears was still evident at this stage: the posterior domain of expression was weaker, and in a more medial position, than the anterior expression domain (Fig. 3K, L). However, the epithelium in posteroventral regions was thicker than normal, indicating development of a duplicate domain of sensory tissue (Fig. 3K’, L’). Taken together, our data indicate that the duplicated anterior domain resulting from either an Fgf gain-of-function or Hh loss-of-function includes a duplication of pax5 expression, but that otic patterning progresses through completely different intermediate states to achieve this duplicated pattern, depending on the signalling pathway that has been disrupted.
Expression of Fgf family genes in the otic epithelium following fgf3 mis-expression or Hh inhibition
Expression of fgf3 is itself a marker of anterior otic epithelium from 21 hpf (Millimaki et al., 2007), and so can also be used to indicate the presence of a duplicated anterior otic pattern. We therefore examined the expression of fgf genes to provide additional confirmation of anterior character in the duplicated ears. To distinguish between expression of the fgf3 transgene and endogenous fgf3 expression, we used a probe generated from the fgf3 3’ UTR, which is not included in the transgenic construct. In Tg(hsp70:fgf3) embryos after early (14 hpf) heat shock, expression of endogenous fgf3 now appeared in a new domain at the posterior of the otic vesicle at 22.5 hpf (Fig. 4A–B’, arrowheads). Importantly, expression was not found across the entire anteroposterior axis, but was only present at the poles. Expression of endogenous fgf3 in pharyngeal endoderm beneath the ear was reduced or missing in heat-shocked transgenic embryos (Fig. 4A–B’, asterisks). We also examined the otic expression of fgf8a and fgf10a (Fig. 4C–F’). These genes are also normally expressed in the anterior of the otic vesicle, but show a less restricted pattern of expression than that of fgf3, with weaker expression also normally found in posterior regions (Léger and Brand, 2002; McCarroll and Nechiporuk, 2013; Thisse and Thisse, 2004). Following early heat shock of Tg(hsp70:fgf3) embryos, there was little change in the expression of fgf8a in the otic epithelium, whereas expression of fgf10a was strengthened at both anterior and posterior poles (Fig. 4C–F’).
We also examined the otic expression of fgf genes after pharmacological inhibition of Hh signalling. At 22.5 hpf, following cyclopamine treatment from 14 hpf, there was little change in the expression domain or levels of fgf3 or fgf8a in the otic epithelium (Fig. 4G–J’), although there was loss of an fgf3 expression domain in pharyngeal pouch endoderm ventral to the ear (Fig. 4H’, asterisk). Otic expression of fgf10a was strengthened in about 50% of cyclopamine-treated embryos (n=17/29) at this early time point, especially at the anterior otic pole (Fig. 4K–L’). At 36 hpf (13.5 hours after cyclopamine wash-out), new discrete domains of fgf3 and fgf8a had appeared at the posterior of the ear, indicating a duplication of anterior otic character (Fig. 4M–P’, arrowheads). By 48 hpf, the duplicated expression domain of fgf8a persisted (Fig. 4Q’, arrowhead), and loss of the thickened epithelium characteristic of the posterior macula on the medial wall of the otic vesicle was also apparent (Fig. 4Q, Q’, brackets). These data demonstrate that in both Fgf gain-of-function and Hh loss-of-function contexts, the duplicated anterior otic character includes expression of fgf genes.
Loss of hmx3a function results in a fusion of sensory maculae and otoliths, and a reduction in anterior otic character
Given the early anterior-specific otic expression of hmx3a (Feng and Xu, 2010), the dependence of this expression on Fgf signalling (Adamska et al., 2000; Hammond and Whitfield, 2011; Kwak et al., 2002), and the rapid change in otic hmx3a expression after mis-expression of fgf3 or Hh inhibition (this work), we hypothesised that hmx3a is required for normal otic anterior development. A previous study using morpholino-mediated knockdown suggested a requirement for both hmx3a and hmx2 in acquisition of anterior otic identity and expression of pax5 (Feng and Xu, 2010). However, the effects of individual gene knockdown or mutation were not reported. To test the individual requirement for hmx3a function in the acquisition of otic anterior identity, we examined the ear phenotype in homozygous mutants for a recessive truncating allele lacking the homeodomain, hmx3aSU3, which we generated using CRISPR/Cas9 technology (Fig. 5A; Materials and Methods). In homozygous hmx3aSU3/SU3 mutants, the otoliths were positioned close together at 33 hpf, were side by side at 48 hpf, had started to fuse at 66 hpf and had fully fused by 4 dpf (Fig. 5B, C and data not shown). This phenotype appeared to be fully penetrant (38/143 embryos from a cross between heterozygous parents; 26.6%). Semicircular canal pillars and the dorsolateral septum were present in the ears of mutant embryos, although formation of the ventral pillar was delayed. Overall, the ear shape appeared more symmetrical than in wild-type siblings (Fig. 5B, C). We imaged ears from three mutant embryos at 3 dpf to analyse sensory patch formation (Fig. 5D–E’ and Fig. 5—Supplemental file 1). In all three ears imaged, the two maculae appeared fused or closely juxtaposed. Although the anterior and posterior elements of the fused macula were not obviously distinct, the overall shape retained some anteroposterior asymmetry. Hair cells of the anterior (utricular) macula were displaced medially, and in one of three ears imaged, were reduced in number. In the two other examples, however, normal numbers of hair cells were present (Fig. 5—Supplemental file 1). The posterior macula was misshapen, and lacked the anterior extension present in the wild type. All three cristae were present (n=3 ears; Fig. 5D’, E’ and Fig. 5—Supplemental file 1).
To understand the basis of the hmx3aSU3/SU3 mutant otic phenotype at 3–4 dpf, we examined expression of markers at earlier (otic vesicle) stages. At 24 hpf, expression of both hmx3a and hmx2 was reduced in intensity within the otic epithelium. On the medial side of the ear, the spatial extent of hmx3a and hmx2 expression was unchanged (Fig. 5F–I, black arrowheads), but levels were reduced (white arrowheads); anteroventrally, there was a reduction in hmx3a expression in presumed neuroblasts (Fig. 5F’, G’, blue and light blue arrowheads), and a mild posterior expansion of the spatial extent of expression for both genes in ventral otic epithelium (Fig. 5G’, I’, red arrowheads). Expression of the anterior markers pax5 and fgf3 was drastically reduced within anterior otic epithelium in hmx3aSU3/SU3 mutants at 24 hpf (Fig. 5J–M’, arrowheads), although expression of fgf3 in pharyngeal pouch endoderm ventral to the ear was unaffected (Fig. 5M, M’, doubleheaded white arrows). Expression of the same markers in hmx3aSU3/SU3 mutants at 27 hpf was similar, but otic expression of hmx2 was more strongly reduced than that of hmx3a, especially in the anterior pole in the area corresponding to the normal expression domain of fgf3 and pax5 (Fig. 5—Supplemental file 2). Expression of the posterior marker fsta at 30 hpf did not reveal any significant duplication of expression in anterior otic epithelium in hmx3aSU3/SU3 mutant ears (Fig. 5—Supplemental file 2). Taken together, the results suggest that a loss of hmx3a function results in a similar phenotype to that of fgf3-/- (liat21142/t21142) mutants (Hammond and Whitfield, 2011; Kwak et al., 2006; Maier and Whitfield, 2014). Although some anteroposterior asymmetry has been lost, the phenotype is not as strong as the double-posterior duplications that result from inhibition of all Fgf signalling or over-activity of Hh signalling, which show a complete loss of the anterior macula and lateral crista, and duplication of elements of the posterior macula (Hammond et al., 2010; Hammond and Whitfield, 2011). We conclude that hmx3a function is required for normal anterior otic expression of pax5 and fgf3. However, loss of hmx3a function is not sufficient to result in a complete loss of anterior character and duplication of posterior structures at the anterior of the ear.
Mis-expression of hmx3a is not sufficient to result in a duplication of anterior otic identity
As otic expression of the anterior markers pax5 and fgf3 is strongly reduced in hmx3aSU3/SU3 single mutants, and because expression of hmx3a is an early transcriptional response to manipulations of both Fgf and Hh signalling, we hypothesised that mis-expression of hmx3a alone would be sufficient to drive the expression of pax5 and fgf3 in the posterior of the otic placode and to give rise to a double-anterior duplication, bypassing the requirement for Fgf or Hh pathway manipulation. To test this idea, we created a transgenic line driving expression of the hmx3a coding sequence under the control of the hsp70 heat-shock promoter. A 60-minute heat shock of Tg(hsp70:hmx3a) embryos at 12 hpf resulted in a robust and widespread expression of the hmx3a transgene two hours later (Fig. 6A–D). To avoid any disruption of otic placode induction, and to be comparable to the fgf3 heat-shock experiments, we heat-shocked Tg(hsp70:hmx3a) embryos at 14 hpf to induce systemic mis-expression of hmx3a. After 30 minutes at 39°C, heat-shocked embryos were incubated at 33°C for 30 minutes before being returned to 28.5°C and incubated until 22.5 hpf, when they were fixed for processing and analysis, or until 3 dpf for assessment of any ear duplication (Fig. 6E–Q’).
Despite robust expression of the hmx3a transgene, the ears of Tg(hsp70:hmx3a) embryos heat-shocked for 30 minutes at 14 hpf did not recapitulate the duplicated double-anterior otic phenotype seen in Tg(hsp70:fgf3) embryos. Position and number of the otoliths, morphology of the semicircular canal pillars and position of the sensory patches in heat-shocked embryos were normal at 3 dpf (Fig. 6E–H’; compare with Fig. 1B). At 22.5 hpf, otic vesicles were slightly smaller and rounder in heat-shocked transgenic embryos than those in heat-shocked non-transgenic siblings, but markers were expressed normally in most cases (Fig. 6I–Q’). Otic expression of hmx2 was mildly up-regulated in a graded fashion (higher at the anterior) in 5/16 transgenic embryos at 22.5 hpf (Fig. 6I–K’), similar to the de-repression of hmx expression seen after Hh inhibition in wild-type embryos. There was also a mild up-regulation of pax5 expression in posterior otic epithelium at 22.5 hpf in 3/20 embryos (Fig. 6L–N’), but pax5 was never expressed in a broad zone as in the Tg(hsp70:fgf3) embryos. A weak patch of fgf3 expression appeared in posterior otic epithelium at 22.5 hpf, similar to the duplicated zone of endogenous fgf3 expression in Tg(hsp70:fgf3) embryos, but in only 2/15 embryos (Fig. 6O–Q’).
To check that the hmx3a transgene was functional, we sequenced it from genomic DNA of transgenic embryos, which indicated that the open reading frame was intact (data not shown). We also examined the phenotype of transgenic embryos after an even earlier heat shock, during otic placode induction (8–9 hpf and 10–11 hpf). Here, we saw a range of otic abnormalities in 80% of transgenic embryos (n=106), including missing otoliths, but some embryos also had small heads and eyes (Fig. 6—Supplemental file 1). Ear patterning appeared normal in about 20% of transgenic embryos heat-shocked at these earlier stages. Longer (1- or 2-hour) heat shocks at 14–15 hpf also resulted in normal otic patterning (n=49; Fig. 6—Supplemental file 1). We conclude that the hmx3a transgene is likely to be functional, but that its mis-expression alone during otic placode stages (14–15 hpf, which should result in strong systemic expression until at least 17 hpf) cannot substitute for Fgf mis-expression or Hh inhibition in the generation of a double-anterior duplicated ear. Up-regulation of hmx3a in the ear at later stages, beyond 18 hpf, was not sufficient either, as our late fgf3 heat shock experiments demonstrated (Fig. 1—Supplemental File 2).
A dynamical model of anteroposterior patterning in the zebrafish ear
Taken together, our data and those from previously-published studies suggest a temporal hierarchy of events for otic anteroposterior patterning dependent on extrinsic sources of Fgf and Hh signalling (Fig. 7). To assess whether this network of inferred genetic regulatory interactions can account for the dynamic expression patterns we observe, we developed a mathematical model of otic anteroposterior patterning in the wild-type ear and following manipulation of the Fgf and Hh signalling pathways. The model is based on a set of differential equations describing the genetic interactions in the otic epithelium outlined in Figure 7A. In addition, patterning in the model is dependent on the existence of two sources of spatial information. First, we assume that otic competence to express fgf genes in response to Fgf and Hmx3a protein is localised to the two poles of the developing otic vesicle. This is necessary in the model to ensure that induced endogenous fgf mRNA expression in the otic epithelium (fgfi) is restricted to the poles, even when fgf3 is expressed uniformly throughout the tissue following heat shock. Second, we represent the effect of fgf mRNA expression in rhombomere 4 as an anterior-to-posterior gradient of extrinsic Fgf (Fgfe) protein, present at high levels up to 30% of the otic vesicle length (corresponding to the position of the rhombomere 4/5 boundary), and forming a decreasing spatial gradient across the remainder of the otic axis (Fig. 7A and Fig. 8—Supplemental File 1). Although we do not have a measure of actual Fgf protein concentration, our assumption is supported by measurements of fluorescence across the otic anteroposterior axis in the Tg(dusp6:d2EGFP) reporter line, which expresses a destabilised GFP variant as an indirect readout of Fgf activity (Molina et al., 2007) (Fig. 7—Supplemental file 1).
We assume a spatially uniform level of Hh signalling throughout the otic epithelium (see discussion in (Hammond et al., 2003), and that Hh signalling antagonises the effects of Fgf signalling on otic anterior marker genes (fgfi, hmx3a and pax5) by increasing their response threshold for Fgf-induced expression. This functional attenuation is unlikely to be at the level of an immediate target of Fgf signalling such as etv4, as Hh inhibition did not result in major changes to etv4 expression ((Hammond and Whitfield, 2011); this work). One possibility is that it could reflect integration of activity of the two signalling pathways at the level of binding sites in the promoters of the otic anterior genes. In addition, we propose that Hmx3a, together with Fgf and Hh, regulates its own expression and that of other genes in the network. Currently, our data do not distinguish whether these regulatory relationships are direct or indirect.
The dynamic behaviour of the model is presented in Figure 8 (for full details, see Fig. 8—Supplemental File 1). In wild-type embryos (Fig. 8, left-hand column), expression of hmx3a and pax5 is triggered in anterior otic tissue. The extent of expression is determined by the spatial reach of the extrinsic Fgf protein (Fgfe) gradient from rhombomere 4. Although all cells in the model are competent to express the anterior markers hmx3a and pax5, they do not receive sufficient Fgfe to do so at the posterior otic pole in a wild-type embryo. After transient heat shock-induced systemic mis-expression of fgf3 at 14 hpf (Fig. 8, middle column), expression of both hmx3a and pax5 is induced across the entire anteroposterior axis. However, the ability of heat shock-induced fgf3 mis-expression to trigger endogenous intrinsic fgf (fgfi) expression requires the coincidence of both Fgf protein and competence to express fgfi at the poles, and so fgfi is not induced in the middle of the otic axis. After decay of heat shock-induced Fgf protein, expression of pax5 is lost from central regions, but is maintained at the poles by Fgf signalling from fgfi expression. By contrast, expression of hmx3a is maintained in central regions due to its autoregulation. De-repression of anterior markers after Hh pathway inhibition (Fig. 8, right-hand column) results from a lowering of the threshold for response to Fgf signalling, establishing duplicate expression domains of pax5 and fgf3 at the posterior pole. Thus, although both heat shock-driven mis-expression of fgf3 and inhibition of Hh signalling result in anterior duplications (Fig. 8; compare the patterns at the 36 hpf time point), the transient dynamics exhibited by the model at earlier time points are distinct.
Discussion
Different transcriptional dynamics in the otic vesicle in response to manipulations of Fgf and Hh signalling
The zebrafish otic placode is a convenient system in which to understand the gene network dynamics that lead to asymmetries along the axis of a developing organ. Asymmetries in gene expression are evident from early (otic placode) stages, but the system is clearly equipotential, since either a gain of Fgf signalling or a loss of Hh pathway activity at otic placode stages can produce remarkably similar double-anterior zebrafish ears at 3 dpf (Hammond et al., 2003; Hammond and Whitfield, 2011); this work. Interestingly, we have shown here that this final duplicated pattern arises via very different intermediate states in terms of gene expression patterns, depending on the signalling pathway that has been disrupted. Mis-expression of fgf3 at 14 hpf leads to a rapid loss of asymmetry, with broad expansion of anterior otic markers across the entire anteroposterior axis of the ear within a few hours of heat shock-driven mis-expression. Expression of pax5, which is required for normal development of the anterior (utricular) macula (Kwak et al., 2006), later resolves into two discrete domains. By contrast, initial asymmetries in gene expression persist for several hours after inhibition of Hh pathway activity, with new duplicate expression domains of anterior markers (pax5, fgf3 and fgf8a) only appearing nearly a day later at the posterior otic pole. We have identified hmx3a as an early otic transcriptional response to manipulations of both signalling pathways. However, although a loss of hmx3a demonstrates its requirement for normal otic patterning, this does not result in a complete double-posterior duplication, and mis-expression of hmx3a does not appear to be sufficient to drive the formation of a double-anterior ear.
Our data and mathematical model suggest that the Fgf/Hh system is sufficient to pattern the anteroposterior axis of the ear. In our scheme, there is only one input (extrinsic Fgf activity) that has a graded distribution across the otic anteroposterior axis. Notably, there is no need to infer an opposing graded input of extrinsic signalling activity that is high at the posterior of the ear. Although Retinoic Acid (RA) is thought to form such a gradient, and contributes to anteroposterior patterning in both the chick and zebrafish ear (Bok et al., 2011; Radosevic et al., 2011), its activity can clearly be over-ridden by manipulations of Fgf or Hh signalling in generating either double-anterior or double-posterior zebrafish ears. Our model therefore differs from other models of axial patterning, for example in generation of dorsoventral pattern in the vertebrate neural tube. Here, information from two anti-parallel noisy gradients is integrated and refined by cross-repressing interactions between target genes, providing precise positional information along the axis (Briscoe and Small, 2015; Zagorski et al., 2017). However, the sufficiency of our network and model does not necessarily rule out a contribution from the RA gradient in generating correct anteroposterior patterning in the wild-type ear.
At present, we do not have a full mechanistic explanation for the differences in response dynamics after manipulations of the Fgf and Hh signalling pathways. Although hmx3a responds rapidly to manipulation of Fgf signalling, its regulation may well be indirect; a recent study identified only one gene, Etv5, as a direct up-regulated target of Fgf signalling during induction of otic-epibranchial precursor cells in the chick (Anwar et al., 2017). In zebrafish, transcription of etv4 and spry4 is known to be an early response to Fgf signalling (Raible and Brand, 2001; Roehl and Nüsslein-Volhard, 2001; Scholpp and Brand, 2004), with spry4 expression appearing within one hour of implantation of a bead coated with Fgf8 protein during epiboly stages (Scholpp and Brand, 2004). Our work here shows that robust, systemic expression of etv4 occurs within two hours of the onset of heat shock in Tg(hsp70:fgf3) embryos; we had previously shown strong expression of etv4 in the otic placode four hours after heat shock (Hammond and Whitfield, 2011). Thus, Etv4 is a good candidate for an immediate early transcriptional effector of Fgf signalling in our proposed genetic network. However, as etv4 mRNA expression is not strongly perturbed by Hh pathway inhibition ((Hammond and Whitfield, 2011); this work), effects of Hh and Fgf on hmx3a expression are likely to be integrated further downstream, for example at the level of the hmx3a promoter. The slower response of hmx3a transcription to Hh inhibition might reflect the persistence of Hh pathway effectors, such as Gli activator proteins, which must be degraded before the effect of inhibiting Smoothened with cyclopamine can take effect.
As the otic vesicle develops, additional levels of regulation are likely to contribute to the regulation of hmx3a and other genes in the network. For example, in the chick, regulation of Hmx3 expression in the dorsolateral otocyst has recently been shown to be influenced by both Shh and non-canonical BMP signalling through PKA and GLI3R (Ohta et al., 2016). In addition, negative feedback on otic fgf expression via sprouty genes (Léger and Brand, 2002) is likely to help to restrict gene expression to the poles and sharpen expression domain boundaries within the otic epithelium.
Requirement for intrinsic factors at the otic anterior and posterior poles in establishing a duplicate pattern after signalling pathway manipulation
One of the intriguing features of the double-anterior ears is that systemic mis-expression of an anteriorising factor (fgf3) gives rise to two defined and separate anterior maculae with mirror-image symmetry, rather than establishing uniform anterior identity across the entire medial otic domain. The final duplicate pattern develops despite the initial broad expression of anterior markers after heat shock, which demonstrates that the entire medial side of the otic placode and vesicle is competent to express hmx genes and pax5 in response to Fgf signalling. However, a day after heat shock, expression of pax5 is lost from the centre of this domain and only maintained at the anterior and posterior poles of the otic vesicle, suggesting either that expression is subsequently repressed in the central domain, or that an intrinsic factor or factors is required to maintain expression at the poles. Attractive candidates for the latter role include atoh1a, which is expressed in discrete domains at the anterior and posterior otic poles at 14 hpf (Millimaki et al., 2007). Atoh1a is thought to act in a positive feedback loop together with Fgf signalling in the zebrafish ear (Millimaki et al., 2007; Sweet et al., 2011). Fgf pathway activity is also observed at both poles of the otic vesicle at 24 hpf (this work), 28 hpf and 50 hpf using a destabilised fluorescent transgenic reporter, Tg(dusp6:d2EGFP) (Molina et al., 2007). We hypothesise that a positive feedback loop involving a pole-specific factor and all three fgf genes contributes to the maintenance of anterior-specific gene expression and generation of the double-anterior pattern. This builds on previous feedback models for anterior otic patterning and the regulation of otic pax5 expression (Feng and Xu, 2010; Kwak et al., 2006).
A similar broad medial expansion of hmx3a and pax5 has been recently reported to result from systemic mis-expression of sox2 or sox3 at 12.5 hpf (Gou et al., 2018). However, this early mis-expression results in a smaller and mis-shapen otic vesicle (most likely due to a disruption of otic induction), and phenotypes were not followed beyond 30 hpf. It will be interesting to see whether a duplicated anterior pattern results from these manipulations.
Comparison of the hmx3aSU3/SU3 and fgf3t21142/t21142 mutant otic phenotypes in the zebrafish
The otic phenotype of hmx3aSU3/SU3 single mutants closely resembles that of hmx3a/hmx2 double morphants (Feng and Xu, 2010), and of fgf3t21142/t21142 mutants (Hammond and Whitfield, 2011; Kwak et al., 2006; Maier and Whitfield, 2014). The similarity of the fgf3 and hmx3a otic mutant phenotypes suggests that a major role for the extrinsic Fgf3 signal is to activate hmx3a expression in anterior otic epithelium. Note that pharmacological inhibition of all Fgf signalling (Hammond and Whitfield, 2011) or over-activity of the Hh pathway (Hammond et al., 2010) both result in a stronger otic phenotype than in hmx3aSU3/SU3 mutants. The retention of some anteroposterior asymmetries in gene expression and the fused sensory macula in hmx3aSU3/SU3 mutants, together with the presence of the lateral crista, suggest that the hmx3aSU3/SU3 otic phenotype, like that of fgf3t21142/t21142 mutants, does not represent a complete double-posterior duplication. We also failed to see strong ectopic expression of the posterior marker fsta at the anterior of the ear in hmx3aSU3/SU3 mutants, although this is a less reliable indicator of posterior duplication; it is expressed at both poles of the ear following strong Fgf inhibition (Hammond and Whitfield, 2011), but lost altogether in the extreme double-posterior ears that can result from elevated Hh signalling (Hammond et al., 2010).
Despite the similarities between the loss-of-function phenotypes for fgf3 and hmx3a in the zebrafish ear, the gain-of-function effects for each of the two genes are strikingly different. Whereas mis-expression of fgf3 at 14 hpf reliably generates a complete double-anterior ear, mis-expression of hmx3a at the same time point had very little effect on otic development. It is remarkable just how robust the embryo is to this kind of perturbation, considering that the systemic high levels of transgene expression must be energetically expensive to support. Indeed, there is usually some transient developmental delay after heat shock, but gross patterning of the ear at 3 dpf appeared normal in Tg(hsp70:hmx3a) heat-shocked embryos.
Why, then, is hmx3a ineffective in establishing duplicate anterior development when mis-expressed? It is possible that it needs to be delivered together with hmx2; the two genes are tightly linked on zebrafish chromosome 17 (Wotton et al., 2009), spatially co-expressed in the zebrafish otic vesicle (although with different temporal onset) (Feng and Xu, 2010), and are known to have partially overlapping roles in the mouse ear (Wang et al., 2004). A predicted hmx3b gene (RefSeq XM_017358610.2) is also present in the zebrafish genome on chromosome 12, although it does not appear to be expressed in the ear (S. England and K. Lewis, unpublished). If a second Hmx family protein or other binding partner was limiting, this might explain the lack of activity of the mis-expressed hmx3a transcript. Alternatively, Hmx3a could act as a competence factor, only functioning in the context of high Fgf or low Hh signalling to initiate duplicate anterior otic development. It is also possible that Fgf signalling abrogates an unidentified negative regulator of the otic anterior gene network at the same time as activating the expression of hmx3a. In the presence of such an inhibitor, systemic over-expression of hmx3a would be ineffective at activating the expression of genes such as hmx2, pax5 and fgf3 in posterior otic domains.
Comparison of the effects of loss of Hmx3 function on otic development between zebrafish and amniotes
Anterior-specific otic expression of Hmx3 and Hmx2, including their temporal order of expression onset in the ear, is conserved between zebrafish, mouse and chick (Feng and Xu, 2010; Herbrand et al., 1998; Rinkwitz-Brandt et al., 1995; Wang et al., 1998). Loss of Hmx3 function in the mouse causes a range of reported otic defects with variable penetrance and expressivity, which depend on the nature of the targeted mutant allele. A homozygous targeted deletion of exon 1 and part of exon 2 of Hmx3 resulted in a variable disruption of the lateral (horizontal) and posterior semicircular canal ducts, and loss of the lateral (horizontal) crista (Hadrys et al., 1998). A weaker phenotype was seen after disruption of the homeodomain in exon 3 of Hmx3; in these mutants, all three semicircular canal ducts were present, but the lateral (horizontal) ampulla and crista were missing. The utricular and saccular maculae were juxtaposed in a common utriculosaccular chamber (Wang et al., 2004; Wang et al., 1998), as we found in the zebrafish hmx3aSU3/SU3 mutant. A notable difference between the mouse and zebrafish mutants is the presence of all three cristae, including the lateral crista, in the zebrafish hmx3aSU3/SU3 mutants. Formation of the ventral pillar for the lateral canal was also present, although delayed. It will be interesting to see whether mutations in hmx2 (not currently available) affect morphogenesis of the zebrafish semicircular canal system; in the mouse, targeted disruption of Hmx2 results in a loss of all three semicircular canal ducts, with partial or complete loss of some ampullae and cristae, in addition to a fused utriculosaccular chamber (Wang et al., 2001). In humans, HMX3 and HMX2 are located together, close to FGFR4, on chromosome 10; hemizygous microdeletions that remove all three genes are thought to be causative for syndromes characterised by inner ear morphological anomalies, vestibular dysfunction and sensorineural hearing loss (Miller et al., 2009; Sangu et al., 2016).
In conclusion, our study demonstrates that although Fgf gain-of-signalling and Hh loss-of-signalling produce similar morphological duplications of the zebrafish ear, they do so via distinct dynamical patterns of gene expression, providing valuable insights into normal anterior otic development. In addition, we determine that hmx3a, a gene expressed as an early transcriptional response to both Fgf and Hh manipulation, has a conserved role in correct separation of the sensory maculae within the otic vesicle, and is required—but not sufficient—for normal anterior otic development. We have also shown that our proposed genetic network for zebrafish otic anterior development can be recapitulated with a mathematical model that assumes interactions between a graded extrinsic source of Fgf, a uniform inhibitory influence of Hh, and equipotential competence to adopt an anterior identity at the otic poles. Interactions between these inputs and their downstream targets within the otic tissue (hmx3a, hmx2, pax5 and fgf genes) lead to correct anteroposterior patterning in the developing zebrafish ear. The model will be a useful framework for further elucidation and functional validation of the proposed gene regulatory network required for the acquisition of anterior otic identity in the zebrafish.
Materials and Methods
Animals
Adult zebrafish (Danio rerio) were kept in circulating water at 28.5°C with a 14-hour light/10-hour dark cycle. The wild-type line used was AB; mutant alleles were hmx3aSU3 (this work; see below for details), and smo hi1640Tg (Chen et al., 2001); transgenic lines were Tg(dusp6:d2EGFP) (Molina et al., 2007), Tg(hsp70:fgf3) (Lecaudey et al., 2008), Tg(hsp70:fgf8a)×17 (Millimaki et al., 2010) and Tg(hsp70:hmx3a) (this work; see below for details). The Tg(hsp70:fgf3) line was maintained on a mitfaw2/w2 background to reduce pigmentation. Embryos were staged as described (Kimmel et al., 1995) and incubated at 28.5°C in E3 (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.0001% methylene blue), unless otherwise indicated.
Heat shock
Embryos were cultured in E3 at 28.5°C prior to heat shock. For heat shock, embryos from either a cross between two hemizygous transgenic carriers, or an outcross between a transgenic carrier and a wild-type, were transferred to 25 ml of preheated E3 in a Falcon tube and incubated at 39°C for 30 minutes, unless otherwise indicated. Embryos were then returned to their original plates of E3, which had been preheated to 33°C during the heat shock, and incubated for a further 30 minutes at 33°C. Plates were then returned to 28.5°C and incubated until embryos reached the desired stage for fixation. In heat-shock experiments with mixed batches of transgenic and non-transgenic embryos, a transgenic genotype was confirmed by expression of tdTomato in Tg(hsp70:hmx3a) embryos or abnormal shape of the yolk extension in Tg(hsp70:fgf3) embryos, in addition to analysis of the phenotypes described in the text.
Cyclopamine treatment
Embryos were treated in 12-well plates (3 ml total volume; ≤30 embryos per well) at 28.5°C with InSolution Cyclopamine, V. californicum (Calbiochem). Chorions were punctured with a sterile hypodermic needle prior to treatment to improve compound penetration. After treatment, embryos were washed twice in E3 before either being fixed or incubated in E3 before fixation later. Vehicle-only controls consisted of a volume of the solvent (ethanol) equivalent to that used in the highest experimental treatment concentration. Embryos from the same batch (siblings) were randomly allocated into control and treatment groups.
In situ hybridisation
Embryos were dechorionated and fixed in 4% paraformaldehyde overnight at 4°C. In situ hybridisation was carried out as described (Thisse and Thisse, 2008). For most experiments, at least 25 embryos (biological replicates) were stained in any given batch. Where relevant, numbers of embryos with the phenotype of interest and total number in the batch (e.g. 29/30) are shown directly on the figure panels (see figure legends for details). Analysis of gene expression via in situ hybridisation is not quantitative, but we have chosen markers that give a clear and robust qualitative response to changes in signalling pathway activity. We have used information from these spatial expression patterns to infer parameters for the mathematical model (see below). Where appropriate, we have measured the spatial extent of expression along the medial side of the otic vesicle in a dorsal view using ImageJ.
Generation of a template for the fgf3 3’ UTR-specific in situ hybridisation probe
The 3’ UTR of fgf3 was amplified from wild-type (AB strain) genomic DNA in a nested PCR, incorporating the T7 promoter, using the following primers: F1 TCTCTTGACACAGATGGAGATCC, R1 AATATACAAAGTACTCCTGATTGCA; F2 AAGGCCACTGAGAGTCCAAAA, T7-R2 TAATACGACTCACTATAGGGCAGTAGCCTATCACATGTACGT. Each PCR was run for 30 cycles with an annealing temperature of 53°C.
Generation of the hmx3aSU3 mutant allele
The single guide RNA (sgRNA) targeting hmx3a was designed using CHOPCHOP (Labun et al., 2016; Montague et al., 2014). The sgRNA DNA template was generated using the cloning-free method of Gagnon and colleagues (Gagnon et al., 2014). The template was transcribed and purified using the standard protocols of the MEGAshortscript T7 kit (AM1354, Thermo Fisher Scientific). sgRNA was resuspended in 40 µl of sterile water and the concentration and purity measured using spectrophotometry, before aliquoting for storage at −80°C. To make Cas9 mRNA, pCS2-nls-zCas9-nls plasmid DNA (Jao et al., 2013) was digested with NotI and purified by phenol:chloroform extraction, before being transcribed and purified using standard protocols of the mMESSAGE mMACHINE SP6 kit (AM1340, Thermo Fisher Scientific). The resultant mRNA was resuspended, assayed and stored as for the sgRNA. The single cell of one-cell stage AB wild-type embryos was injected with 2 nl of a mixture of 200 ng/µl sgRNA + 600 ng/µl nls-ZCas9-nls mRNA. Founders were identified by high resolution melt analysis, using the following primers: PMA F: CGAATGCTAATTTGGCCTCTATTACT and PMA R: TTTTGTTGTCGTCTTCATCGTCC, and Precision Melt Supermix for High Resolution Melt (HRM) Analysis (172-5112, Bio-Rad), performed on a CFX96 Touch System (1855195, Bio-Rad), equipped with Precision Melt Analysis Software (1845025, Bio-Rad). Amplification data were generated using the following program: 95.0°C for 3 minutes, followed by 45 cycles of 95.0°C for 15 seconds, 60.0°C for 20 seconds and 70.0°C for 20 seconds. Melt data were generated using the following program: 65.0°C for 30 seconds, 65.0°C–95.0°C at an incremental rate change of 0.2°C, held for 5 seconds each step, 95.0°C for 15 seconds. Stable F1 heterozygous fish were confirmed by sequencing. All subsequent genotyping was performed by PCR, using the primers F: TGGCAAAGTGACACGACCAG and R: GAGAACACCGTGCGAGTTTTC, Taq DNA Polymerase (M0320S, NEB) and the PCR program: (94.0°C for 2 minutes, 35 cycles of: 94.0°C for 30 seconds, 64.9°C for 30 seconds and 72.0°C for 30 seconds, followed by a final extension at 72.0°C for 2 minutes). The hmx3aSU3 allele is a 69 bp insertion, flanked on either side by 2-base mismatches. The insertion introduces a premature stop codon at nucleotides 352-354 of the edited coding sequence. The insertion in the mutant allele can be distinguished by performing gel electrophoresis on a 2% TBE agarose gel (100V for 40 minutes). The wild-type allele generates a 331 bp product, compared to the 400 bp mutant allele product.
Generation of the Tg(hsp70:hmx3a) line
The zebrafish hmx3a cDNA sequence (RefSeq NM_131634.2), including the complete open reading frame, endogenous Kozak sequence and 15 bp of 3’ UTR, was cloned into a Tol2-containing ubi:tdtomato destination vector, flanked by a 5’ hsp70 promoter and a 3’ SV40 late polyadenylation signal sequence, using the Tol2kit (Kwan et al., 2007) (Invitrogen). 50 ng of this construct were injected into one-cell stage embryos together with 50 ng of in vitro-transcribed transposase RNA. Injected embryos (G0) were raised to adulthood, and their progeny (F1) screened for expression of the tdTomato marker. F1 embryos with positive expression were raised to adulthood to generate a stable Tg(hsp70:hmx3a) transgenic line. Progeny were tested by in situ hybridisation after heat shock to check for misexpression of the hmx3a transgene.
Phalloidin staining
Embryos were fixed in 4% PFA overnight, washed in PBS (3×10 minutes) and permeabilised in 2% Triton-X100 (Sigma) for 3-4 days at 4°C. Following further washes in PBS (3×5 minutes), embryos were stained with FITC-phalloidin (1:20; Sigma) or Alexa Fluor 647-phalloidin (1:100; Thermo) in PBS (overnight, 4°C). Embryos were washed in PBS (3×60 minutes), dissected in PBS and mounted in Vectashield (Vectorlabs) prior to confocal imaging.
Microscopy, photography and image processing
Live and fixed embryos were imaged on either an Olympus BX51 or a Zeiss Axio Imager M1 compound microscope using brightfield, DIC and epifluorescence optics as appropriate, and CellB or Axiovision image acquisition software, respectively. For confocal imaging, either a Nikon A1 or a Zeiss LSM 710 confocal microscope were used. For fluorescent imaging requiring large fields of view, a Zeiss Axio Zoom.V16 stereomicroscope with Zen acquisition software was used. The movie and associated still images shown in the supplementary material for Fig. 7 were acquired with a Zeiss Z.1 light-sheet microscope. Sample drift was corrected using the Manual Drift Correction plugin within FIJI (Fiji Is Just ImageJ) (Schindelin et al., 2012). FIJI was used for all image processing. Figure panels were assembled using Adobe Photoshop 2015.5.0. All dorsal views (except in the supplementary material for Fig. 7) are shown with anterior to the top; lateral views show anterior to the left.
Statistical analysis
Statistical analyses were performed using GraphPad Prism version 7.0c for Mac OSX (GraphPad software, La Jolla California USA, www.graphpad.com). See figure legends for details.
Mathematical model
Full information for generation of the mathematical model, including a list of parameters used, is given in Figure 8—Supplemental File 1.
Author contributions
RDH: Conceptualisation, investigation, formal analysis, methodology, validation, visualisation, preparation of figures, contribution to writing the manuscript
SJE, KEL: Generation and analysis of the hmx3aSU3 mutant allele, preparation of figures, contribution to writing the manuscript
SB, MM, NvH: investigation, visualisation
NAMM: Supervision, design and generation of the mathematical model, preparation of figures, contribution to writing the manuscript
TTW: Conceptualisation, funding acquisition, project administration, supervision, investigation, validation, formal analysis, visualisation, preparation of figures, writing the manuscript
Funding
RDH was funded by a BBSRC White Rose Doctoral Training Partnership Award in Mechanistic Biology (BB/J014443/1). Work in the Whitfield lab was funded by the BBSRC (BB/M01021X/1). The Sheffield zebrafish aquarium facilities were supported by the MRC (G0700091). Fluorescent imaging was carried out in the Sheffield Wolfson Light Microscopy Facility, supported by a BBSRC ALERT14 award for light-sheet microscopy (BB/M012522/1) to TTW and SB. Research conducted in the Lewis Lab was funded by NIH NINDS R01 NS077947 awarded to KEL. The Lewis Lab aquarium facilities were also supported by HFSP RGP0063, NSF IOS 1257583 and New York State Spinal Cord Injury Fund (KEL).
Statement on ethical issues
All animal work in the Whitfield lab was covered by licencing from the UK Home Office. All zebrafish experiments conducted in the Lewis lab were approved by the Syracuse University Institutional Animal Care and Use Committee (IACUC).
Supplemental movie 1
Time-lapse movie of a representative Tg(dusp6:d2EGFP) embryo from the 8-somite stage to the 16-somite stage, used to generate panels A–D’ in Figure 7—Supplemental file 1. Images were captured by light-sheet microscopy every 5 minutes for 4 hours (from the 8- to the 16-somite stage). Individual time points were manually drift corrected. An average intensity projection of the fluorescent signal is shown (produced from 20 z-slices encompassing the otic region). A dorsal view is shown; anterior is to the top left. Scale bar, 50 µm.
Figure 8—Supplemental file 1. Details of the mathematical model. See appendix.
Details of the mathematical model
Representation of otic tissue
For the purposes of modelling gene expression in the developing otic tissue between 14 and 36 hours post fertilisation (hpf), we represent the medial side of the otic tissue as a one-dimensional array of cells. Distance along this array—represented by the variable x—is measured in percentage length along the anterior-posterior (AP) axis. At the stages studied, the length of the medial side is approximately 100 µm.
Competence to express fgf
Our data suggest that competence to express fgf in the otic tissue (fgfi) begins at around 14 hpf (see Fig. 7B) and is strongest at the poles. We therefore assume that this competence can be represented by a function of the form where 0 ≤ x ≤ 100 is percentage length along the AP axis, xc is a measure of the extent of the polar competence regions, and m is a measure of the sharpness of the boundaries between regions of high and low competence. In our model simulations, we assume xc = 20% AP length and m = 5, giving the competence profile shown in Fig. S1A.
Extrinsic Fgf expression
We assume that rhombomere 4 of the hindbrain acts as the main source of extrinsic Fgf signalling; both fgf3 and fgf8a are expressed here at the time of initial otic anteroposterior patterning (Maves et al., 2002). Further support is provided by analysis of mutants for mafba (Kwak et al., 2002) and hnf1ba (Lecaudey et al., 2007). These two genes code for transcription factors expressed in the hindbrain and are required for restriction of fgf3 expression to rhombomere 4. In the mafba−/− and hnf1ba−/− mutants, posterior expansions of fgf3 expression in the hindbrain correlate with expansions or duplications of otic anterior markers similar to those we see in Tg(hsp70:fgf3) embryos after heat shock.
We assume that the extrinsic Fgf3 protein spreads away from the cells in which it is produced, resulting in a graded expression profile in the neighbouring otic tissue. In support of this, we show in Fig. 7 – Supplemental File 1 that a reporter of Fgf signalling activity is expressed in a decreasing gradient in the otic region, with highest activity in the tissue neighbouring rhombomere 4. Up to time t = 18 hpf, we assume that the distribution of rhombomeric Fgf protein in the otic tissue is given by where Fr (x) is the Fgf protein concentration (in arbitrary units) in the otic tissue at AP position x, 0 ≤ x ≤ x0 is the region of overlap between rhombomere 4 and the otic tissue, F0 is the maximum protein concentration, and λf is the effective “diffusion wavelength” of Fgf. The resulting concentration profile is shown in Fig. S1B for F0 = 18.5, x0 = 30% AP axis, and λf = 15% AP axis (approximately 15 µm at the stages studied). fgf expression in rhombomere 4 decreases significantly at around 18 hpf (Maves et al., 2002). Up until this time, the Fgf concentration profile in the otic tissue is given by Eq. (2). At times later than 18 hpf, we assume linear degradation of the Fgf protein. The Fgf concentration at time t is therefore given by where τe is the half-life (in hours) of the Fgf protein.
Regulated gene expression in the otic tissue
Fgf protein originating from rhombomere 4 initiates a temporal sequence of spatially patterned gene expression in the otic tissue. Based on the inferred interactions summarised in Fig. 7A, we represent transcription and translation using a system of coupled differential equations as follows: where represents the total amount of Fgf protein in the otic tissue and the transcription regulation functions are given by increasing sigmoid (Hill) functions of the general form:
In these functions, the parameter θ represents the activation threshold — the concentration of activator required to achieve a half-maximal rate of transcription. The effect of Hh attenuation is to increase the threshold in Eq. (14) by an amount ρR, where R is a measure of the amount of Hh signalling in the otic tissue, and ρ is the relative attenuation strength for each gene. Hh attenuation thus reduces the rate of transcription resulting from a given concentration of Fgf.
The meaning of all model variables and parameters is summarised in Tables S1–S3.
In the model, expression of all genes is activated by the total amount of Fgf protein (both extrinsic and that produced within the otic tissue) and attenuated by Hh protein. Expression of hmx3a, pax5, fgf3, fgf8a and fgf10a is additionally activated by Hmx3a protein.
Simulations of the model were performed with the parameter values listed in Tables S2 and S3 on a discrete spatial domain comprising 100 spatial cells (with the diffusion terms in Eqs. (10)–(12) represented by a simple finite difference scheme), with zero-flux boundary conditions at the anterior and posterior poles of the otic vesicle. Simulations covered the time period 10–36 hpf, with initial values of all intrinsic mRNA and protein variables set to zero. The resulting spatiotemporal mRNA expression patterns are shown in Fig. 8, for three conditions: wild type (1st column), heat shock induction of fgf3 (2nd column), and inhibition of Hh signalling by cyclopamine treatment (3rd column). The simulation protocols for the latter two conditions are described below. Fig. S2 shows the spatial profiles of mRNA and total Fgf protein expression for the three conditions at 22.5 hpf and 36 hpf.
Transcription and translation rates for all endogenous genes and proteins have been set to 1, so all expression levels are expressed in arbitrary units. Half-lives of mRNA and protein have been set to reflect the observed dynamics of expression patterns. For example, the pax5 mRNA half life is set to be low (0.5 hrs) to reflect the fact that pax5 mRNA expression induced in the middle part of the otic vesicle by heat shock Fgf3 protein is lost by 36 hpf. The hmx3a mRNA half life is also set to be low (0.5 hrs) to reflect the early onset of hmx3a expression. In contrast, the fgf mRNA half lives are set to be higher to reflect the later onset of their expression. The Fgf protein diffusion coefficient is set to be low in order to avoid Fgf protein produced in the anterior and posterior poles “flooding” the otic vesicle. The short half lives of the Fgf proteins also contribute to the restriction of Fgf proteins to the poles. Indeed, Fgf protein diffusion can be omitted from the model without affecting the dynamics of the mRNA expression patterns.
The transcription regulation parameters, which reflect the level of expression at which regulating proteins effect regulation of their targets, were chosen with reference to the expression levels achieved by each protein in the model (shown in Fig. S3). For example, the threshold for regulation of hmx3a expression by Fgf protein (θ1) is the primary determinant of the extent of the anterior expression domain of hmx3a mRNA.
Simulation of heat shock induction of fgf3a expression and cyclopamine treatment
To simulate heat shock induction of fgf3 expression, we include additional variables to represent fgf3 mRNA and Fgf3 protein produced from the fgf3 transgene. We assume that transcription from the transgene starts at 14 hpf and terminates at 14.5 hpf. Because of the time taken for the resulting mRNA and protein to decay, the effects of the heat shock on target gene expression extend beyond 14.5 hpf.
To simulate treatment of embryos with cyclopamine (a chemical inhibitor of Hh signalling) at 14 hpf, we assume that a consequent reduction in the Hh-dependent antagonism of Fgf-dependent transcription (the variable R in the model equations) does not begin until 15 hpf. In this way, we represent the time taken for a reduction in Hh signalling to feed through to a reduction in the intracellular effectors of Hh signalling. We further assume that the inhibitory term R decays exponentially for t > 15 hpf, with a half-life of 3 hrs.
Acknowledgements
We thank the Gilmour lab for the Tg(hsp70:fgf3) line, the Riley lab for the Tg(hsp70:fgf8a) line, and Michael Tsang and aquarium staff at University College London for the Tg(dusp6:d2EGFP) line. We are grateful to Whitfield lab members for discussion, Sarah Burbridge, Montserrat Garcia Romero, Ginny Grieb and Leslie Vogt for excellent technical support, and the Sheffield and Lewis lab Aquarium Teams for expert fish care.