Keywords:barx1,hand2, Cartilage, Joint, Skeleton, Zebrafish

Zebrafish mutants and transgenic constructs

Fish were raised and staged as described previously (Kimmel et al., 1995). Mutant alleles ofbarx1 were generated by TILLING (targeting induced local lesions in genomes) (Draper et al., 2004). Bothbarx1 alleles behaved in a Mendelian recessive fashion with complete penetrance and larval lethality. Genotyping was carried out forbarx1^fh330 with primers CTTCTGTTTTTCAAATCGTTTTTCT and GGCTAACACACACTCTTATTTCATTC, and digestion withMseI.barx1^fh331 was genotyped using ATAGATCTGGCAGAGTCCTTAAGCT and ACCTTCAAAACAATGATAGGTTTACATTT, and digestion withHindIII. Thehand2:Crimson reporter line was generated by crossing two independently generated stable transgenic linesTg(hand2:GAL4VP16)b1228 andTg(UAS:E2Crimson;cmlc2:EGFP)b1229. We generated thehand2:GAL4VP16 construct with the 2573 nucleotides upstream of thehand2 initiating methionine codon and the Tol2 kit (Kwan et al., 2007). This construct contains a conservedhand2 pharyngeal arch enhancer (Charité et al., 2001), and a control construct lacking this enhancer did not drive pharyngeal arch expression (data not shown). TheUAS:E2Crimson;cmlc2:EGFP construct was generated withE2Crimson (Clontech) and the Tol2 kit. Thefli1a:NLSmCherry;cmlc2:EGFP construct was generated with the p5E-fli1a-F-hsp70I 5′ entry vector (Das and Crump, 2012) and the Tol2 kit. Thefli1a:barx1;cmlc2:EGFP construct was created by first amplifying full-lengthbarx1 cDNA (Open Biosystems) and inserting the product into p221 to generate pME-barx1, which was then recombined with p5E-fli1a-F-hsp70I and the Tol2kit to generate the injection construct. The following lines have been described previously:Df(chr01:hand2)s6 (Yelon et al., 2000),furina^tg419 (Walker et al., 2006),sox9a^zc81Tg (DeLaurier et al., 2012),trps1^j127aGt (Talbot et al., 2010),Tg(sox10:mRFP)vu234 (Kirby et al., 2006) andTg(fli1a:EGFP)y1 (Lawson and Weinstein, 2002).

Tissue labeling

Alcian Blue and Alizarin Red stains on fixed animals and vital staining with Alizarin Red were performed as described previously (Kimmel et al., 2010;Walker and Kimmel, 2007). Staining with fluorescently labeled phalloidin (Invitrogen) was performed overnight. Staining with anti-col2 (II-II6B3-DSHB) and GaM Alexa546 followed treatment with trypsin and hyaluronidase. Staining with SYTO 59 was carried out as described previously (Huycke et al., 2012).

Whole-mountin situ hybridization was carried out as previously described (Talbot et al., 2010) with either fluorescent or immunohistochemical detection. The followingin situ probes have been previously described:barx1 (Walker et al., 2006),sox9a (Yan et al., 2002),chd (Schulte-Merker et al., 1997),hand2 (Angelo et al., 2000) andnkx3.2 (Miller et al., 2003).

Microscopy

Skeletal preparations and immunohistochemicalin situ stained animals were imaged on a Zeiss Axiophot 2. Static confocal images were captured on either a Zeiss LSM 5 Pascal confocal or a Leica SD6000 spinning disk confocal. Images were assembled in ImageJ and Photoshop, with any adjustments applied to all panels. For time-lapse movies, animals were imaged as described previously (Huycke et al., 2012). Movies were assembled using Metamorph (Molecular Devices) and ImageJ.

barx1 mutant zebrafish exhibit gaps within segmentally homologous pharyngeal cartilages

To identify null alleles ofbarx1, we searched for mutations by screening genomic DNA from a library of ENU mutagenized zebrafish (Draper et al., 2004). We found 29 mutations in thebarx1 gene, including two recovered putative null nonsense mutations conferring early stop codons in the homeobox at L150 (barx1^fh330) and L174 (barx1^fh331).barx1 homozygous mutant larvae are readily identified by a prominent indentation in their lower jaw, which is visible by light microscopy (Fig. 1B, arrow). Staining the skeleton with Alcian Blue and Alizarin Red to mark cartilage and bone revealed this indentation is due to a prominent cartilage loss, or gap that divides Meckel’s cartilage in mutants (Fig. 1D, arrow). Following dissection and close examination of Alcian Blue Alizarin Red stained skeletons, we discovered a number of remarkable skeletal defects, including gaps dividing Meckel’s cartilage, the ceratohyal cartilage (Fig. 1F, mj, chj) and all five of the ceratobranchial cartilages (supplementary material Fig. S1D, cbj), all of which are considered to be segmentally homologous (Kimmel et al., 2001). Additionally, the dorsal arch 1 pterygoid process is shortened (Fig. 1F, ptp) (supplementary material Table S1) and the dorsal arch 2 hyosymplectic cartilage is variably reduced or notched inbarx1 mutants (Fig. 1F, hs) (supplementary material Fig. S1H-J; Table S1). The phenotypes are recessive; heterozygotes appear phenotypically wild type. Importantly, the two putative null alleles demonstrate like phenotypes (supplementary material Table S1) and fail to complement (supplementary material Fig. S1A-D), providing compelling genetic evidence that the phenotypes we observe in these two lines are due to mutations in thebarx1 gene. For consistency, we show results with only thebarx1^fh331 allele in all subsequent figures.

To examine chondrocyte arrangement, and the orientation of cartilage elements in live animals, we used the transgenic linesox9a:EGFP, which labels all chondrocytes (Talbot et al., 2012). These studies revealed gaps insox9a-expressing cells at the jaw joint, as well as the Meckel’s and ceratohyal cartilage gaps in mutants (Fig. 1G-J, jj, mj, chj). In addition, the distalmost segments of the ceratohyals near the midline are reversed along the anterior-posterior axis. Live Alizarin Red staining of larvae revealed a suite of bone defects inbarx1 mutants. The arch 1 ventrally and dorsally derived dentary and maxillary dermal bones are dysmorphic, while the arch 1 dorsally derived entopterygoid bone is absent (Fig. 1G-J, d, mx, en). Of note, these dermal bones all associate with the cartilage skeleton near sites of cartilage defects, suggesting the bone defects are secondary consequences of dysmorphic cartilage.

barx1 is required for tooth and musculoskeletal attachment

barx1 is required for proper oral tooth development in mice (Miletich et al., 2011), andbarx1 is expressed in the pharyngeal dentition in cichlids and zebrafish (Fraser et al., 2009;Sperber and Dawid, 2008). Zebrafish pharyngeal tooth development progresses through initiation, morphogenesis, matrix deposition and attachment (Van der Heyden and Huysseune, 2000). We find that teeth inbarx1 mutants progress thorough matrix deposition normally, yet do not undergo attachment (Fig. 2A,B). Interestingly, at the site where the teeth would normally ankylose to the ceratobranchial 5 bones (Fig. 2B, arrowhead), we observe an ectopic gap (Fig. 2B, arrow). These findings suggestbarx1 is not required for tooth initiation and morphogenesis but is required to specify the bone of attachment.

Craniofacial muscles also attach to the larval skeleton (Schilling and Kimmel, 1997). We tested the hypothesis that, like teeth, muscles requirebarx1 for proper association with the skeletal scaffold by labeling craniofacial muscles with phalloidin infli1a:EGFP larvae. These analyses revealed several muscle groups are out of place inbarx1 mutants (Fig. 2C,D). The intramandibular posterior muscles, which in wild types attach to Meckel’s cartilages (Fig. 2C, arrowhead) at or near the site that forms the cartilage gap in mutants, are disorganized and invade the gap (Fig. 2D, arrow). Inbarx1 mutants, the sternohyal muscles are shorter and fail to attach in the manner we see in wild type (Fig. 2C,D, sh). The hyohyal muscles reverse their orientation along the anterior-posterior axis, similar to the ceratohyal cartilage (Fig. 2C,D, hh). Our data suggest pharyngeal teeth and cranial muscles are properly specified but association with their skeletal scaffold is disrupted inbarx1 mutants.

The reported morpholino phenotype (Sperber and Dawid, 2008) very poorly represents loss of function ofbarx1. None of the mutant phenotypes described above - focal gaps in ventral cartilages, loss of tooth and muscle attachments, and dermal bone phenotypes - were reported in the morpholino knockdown study. Conversely, in our studies withbarx1-null mutants we did not see any of the phenotypes of the morpholino-injected animals. For example, we never observed a general loss in Alcian Blue staining, joint fusions or gaps between the hyomandibular and symplectic cartilages inbarx1 mutants. Although we do observe a range of phenotypic severities (supplementary material Fig. S1E-P; Table S1), loss of cartilage occurs consistently at discrete locations, at variance with the morpholino knockdown study.

barx1 is expressed in an arch 1 subdomain during chondrocyte differentiation

How does expression ofbarx1, which is known to be restricted to dorsal and ventral cartilage-forming domains, and absent from the joint-forming intermediate domains (Walker et al., 2006), explain the presence of gaps in the ventral cartilages when its function is lost? To help identify its possible function in chondrocyte differentiation, we monitoredbarx1 expression along with the transcription factorsox9a, which is expressed in both early pharyngeal arches and differentiating chondrocytes (Talbot et al., 2010;Yan et al., 2002). In the pharyngula period (monitored at 30 hpf), broad weaksox9a expression is detected in the ectomesenchyme of the pharyngeal arches, while a strongbarx1 expression domain is present ventral to stomodeum (Fig. 3A, arrowhead). During the hatching period (48 hpf) overt cartilage differentiation occurs, evidenced by strongly upregulatedsox9a in differentiating cartilage cells of individual skeletal elements (Fig. 3B, mc, pq) relative to weakly labeled joint regions (Fig. 3B, jj). At this stage of differentiation, the strongestbarx1 expression domain resolves into a transverse stripe across the developing Meckel’s cartilage distal to the developing retroarticular process near the site where the cartilage gap would develop inbarx1 mutants (Fig. 3B, arrowhead). Later in the hatching period (60 hpf),barx1 remains undetectable in the arch 1 joint region. Strongbarx1 expression is maintained distal to the joint in cells sheathing a region of Meckel’s cartilage (Fig. 3C, arrowhead).

Previous works have identified markers,nkx3.2 (formerlybapx1) andhand2, as functional regulators expressed in the intermediate and ventral domains, respectively (Miller et al., 2003;Talbot et al., 2010). Based on these studies, we were surprised to discover thathand2 andnkx3.2 do not directly juxtapose in ventral arch 1 at 31 hpf. Instead,barx1 is expressed in the region betweenhand2 andnkx3.2 (Fig. 3D, arrowhead), in addition to partially overlapping withhand2 (Fig. 3D, arrow).barx1 expression does not extend all the way to the ventral-most aspect of the first arch, ashand2 does (Fig. 3D, asterisk). By 48 hpf,nkx3.2, barx1 andhand2 expression domains have mostly resolved to be exclusive of one another (Fig. 3E). These expression results reveal the presence of a previously not recognized ‘subintermediate’ domain in the ventral first arch, between the intermediate and ventral domains. We identify the subintermediate domain by expression ofbarx1 in the absence of bothnkx3.2 andhand2.

Meckel’s cartilage gaps exhibit hallmarks of joint identity

Loss ofbarx1 expression in the first arch subintermediate domain might explain the presence of the Meckel’s cartilage gap at what would seem to be the corresponding location inbarx1 mutants after skeletal differentiation. In this scenario, the loss of subintermediate expression would mimic the absence ofbarx1 expression in the wild-type intermediate domain and confer joint rather than cartilage identity, yielding a ‘gap’ that may in fact be an ectopic joint. We queried several aspects of cartilage and joint cellular identity during late hatching and early larval periods to examine this hypothesis. Initially, we examined the morphology of cells near the jaw joint by fluorescently labeling all cells with the nucleic acid stain SYTO59, in combination with the transgenic chondrocyte markersox9a:EGFP at 4 days post-fertilization. Inspection of wild-type animals revealed large chondrocytes labeled with EGFP, bounded by flat perichondrial cells brightly labeled with the nucleic acid stain. In addition, a population of densely packed, flattened cells populates the interface between Meckel’s and palatoquadrate cartilages in wild type (Fig. 4A, jj). Adhering to our previous definition of the mesenchyme connecting early larval skeletal elements (Talbot et al., 2010), we refer to these as joint cells. The morphology of these cells is similar to that described for appendicular (limb) interzone joint precursor cells in other vertebrates (Hartmann and Tabin, 2001;Mitrovic, 1977). We have observed direct kinematic evidence that the larval jaw joint is freely mobile in zebrafish at the time of this analysis (4 dpf,supplementary material Movie 1). Inbarx1 mutant animals, we find an additional population of flattened joint cells at the Meckel’s gap, supporting that the gap is an ectopic Meckel’s joint (Fig. 4B, mj), and kinematic studies in non-anesthetized fish demonstrate some mobility mediated by the ectopic joint (supplementary material Movie 1).

Close examination of wild types revealed that Meckel’s cartilage chondrocytes can be divided into two distinct populations based on morphology. The jaw joint-adjacent population of chondrocytes consists of rounded interlocking cells, whereas cells more distal to the joint exhibit a columnar and stacked morphology. Of particular interest, the boundary between these two populations (Fig. 4A, arrowhead) corresponds to the location of the ectopic Meckel’s joint inbarx1 mutants.

Late in the hatching period in wild-type fish, chondrocytes express high levels ofsox9a and joint cells express high levels ofchordin (Fig. 4C). Hence, these markers reveal cells executing either the cartilage or joint program. At the site of the ectopic Meckel’s joint in mutants,sox9a expression is minimally detected, consistent with our findings withsox9a:EGFP. Meanwhile, ectopicchordin expression is detected in mutants in and around the ectopic Meckel’s joint (Fig. 4D, mj). Comparison with wild-type controls suggests cells that would expresssox9a in wild types expresschordin in mutants.

The transgenic linesox10:mRFP marks cartilage cell membranes with red-fluorescent protein. Conversely, cells in the wild-type jaw joint region are labeled in the transgenic linetrps1:EGFP. Imaging live double transgenic wild-type animals during the larval period revealed the brightesttrps1 expressing cells to be joint cells populating the interface between Meckel’s and palatoquadrate cartilages (Fig. 4E, jj). In transgenicbarx1 mutants,sox10:mRFP expression is reduced in conjunction with increasedtrps1:EGFP expression, when compared with wild type (Fig. 4F, mj).trps1:EGFP also weakly labels wild-type chondrocytes proximal to the joint (for example, chondrocytes of the retroarticular process), indicating these joint-adjacent chondrocytes have an intermediate identity distinct from more ventral chondrocytes, which are minimally labeled. The boundary between these two types of chondrocytes in wild type is located at the site of the ectopic Meckel’s joint inbarx1 mutants (Fig. 4E, arrowhead).

The reducedsox9a andsox10 expression at the ectopic Meckel’s joint inbarx1 mutants (Fig. 4D,F) strongly suggests that the chondrogenic program has failed to initiate. To test whether chondrocyte cellular function is also absent, we immunostained for collagen type II, a hallmark of cartilage matrix (Kronenberg, 2003), in conjunction with transgenic labeling with the chondrocyte-specific transgenesox9a:EGFP (Fig. 4G, jj). At 72 hpf, transgenically labeled chondrocytes are present in wild-type Meckel’s and palatoquadrate cartilages, which are divided by the weaklysox9a:EGFP-expressing joint region. Chondrocytes actively secrete collagen type II at this time, indicated by immunoreactivity surrounding the cartilage cells (Fig. 4G, mc, pq), whereas collagen type II is not detected in the jaw joint (Fig. 4G, jj). Inbarx1 mutants, transgenically labeled chondrocytes of the palatoquadrate as well as both the independent elements of the divided Meckel’s cartilage (Fig. 4H, mi, mv) secrete collagen type II, whereas collagen is undetectable in both the endogenous and ectopic joints (Fig. 4H, jj, mj).

These analyses of chondrocyte and joint cell identity suggest that joint cells are gained at the expense of chondrocytes inbarx1 mutants. This conclusion motivates the hypothesis that undifferentiated precursor cells in the subintermediate domain have the potential to differentiate into either chondrocytes (as they do in wild type), or joint cells (as inbarx1 mutants). To test this hypothesis, we monitored ectomesenchyme cells as they differentiated into joint cells by monitoringtrps1:EGFP expression (supplementary material Movie 2, green). Analysis of wild-type animals revealed that thetrps1:EGFP reporter is initially broadly expressed at low levels in precursor cells (50-53 hpf). By 60 hpf in wild types, differentiating joint cells begin to adopt their characteristic morphology and upregulatetrps1:EGFP. By 70 hpf, a stripe of very brighttrps1:EGFP-positive joint cells can be seen adjacent to the more weakly expressing chondrocytes of Meckel’s cartilage intermediate region, as well as the palatoquadrate. Inbarx1 mutants, the broad weaktrps1 reporter expression from 50-53 hpf in undifferentiated cells looks similar to that in wild types. However, by 60 hpf,barx1 mutants begin to display an excess of brightlytrps1:EGFP-expressing cells compared with wild types, and by 70 hpf a bulbous accumulation of brighttrps1:EGFP cells, which we interpret to be ectopic joint cells, is present inbarx1 mutants.

To see whether tissue drift or cell migration contributes to the ectopic joint cells, we enlarged single confocal sections from the recordings and tracked small groups of isolatedhand2:Crimson-positive undifferentiated cells and saw no such movement (supplementary material Movie 3, arrow). Specifically, as tracked cells differentiated they remained distal to the developing jaw joint at positions where cartilage or joints develop in wild types or mutants, respectively (Fig. 4A-H). Hence, these findings suggest that cell rearrangement does not contribute to the phenotypic changes between wild types and mutants, and that subintermediate domainbarx1 is necessary for ectomesenchyme to differentiate into cartilage versus joints.

Joint loss infurina mutants requiresbarx1

The hypothesis thatbarx1 represses joint formation, predicts that upregulation ofbarx1 in the intermediate domain should lead to the loss of the endogenous jaw joint. We can examine this prediction genetically: both expandedbarx1 and joint loss are present when Edn1 signaling is partially lost, as infurina mutants (Walker et al., 2006), and our hypothesis predicts that the joint loss in these mutants depends onbarx1 being functional in thefurina mutant background. It follows thatbarx1;furina double mutants should not show joint loss. Consistent with our previous studies, we found thatfurina single mutants have a joint-loss phenotype (Fig. 5C, asterisk), as opposed to the joint-gain phenotype inbarx1 single mutants (Fig. 5B, mj). Strikingly, we discovered that mutations inbarx1 partially rescue the joint-loss phenotype seen infurina single mutants (Fig. 5D, jj;supplementary material Table S2). These findings suggest that the loss of joints infurina mutants is, at least in part, due to ectopicbarx1. Moreover, the discovery thatbarx1 is epistatic tofurina in compound mutants is consistent with our understanding of the order of gene activity:barx1 acts downstream offurina.

To further test the hypothesis thatbarx1 is sufficient to repress joint formation, we designed an experiment to directly overexpressbarx1. Injection of a control construct encoding nuclear mCherry protein under the control of thefli1a promoter (Das and Crump, 2012) results in mosaics in which clones of transgenic cells populate the pharyngeal arches, including occasionally the intermediate domain (data not shown). Animals injected with a construct encodingbarx1 driven by thefli1a promoter, display variable joint loss, including clones of ectopic chondrocytes in the jaw joint (Fig. 5F, asterisk), as well as complete jaw joint fusions (Fig. 5G, asterisk). We concludebarx1 is both necessary (Fig. 4) and sufficient (Fig. 5) for joint repression.

hand2 is required for endogenous and ectopic joints, and is negatively regulated bybarx1

The above analyses showbarx1 functions in the Edn1 signaling pathway to repress joints. By contrast, the Edn1 target genehand2, in addition to its being required for ventral cartilage development, is required for jaw joint formation (Miller et al., 2003), suggesting the two transcription factors might negatively interact. Epistasis analysis supports such interaction: inhand2;barx1 double mutants, the ectopic Meckel’s joint characteristic ofbarx1 single mutants is rescued (Fig. 6D). Furthermore, the endogenous jaw joint is lost as in thehand2 single mutants (Fig. 6C, asterisk). Hence, the two genes interact genetically, withhand2 epistatic tobarx1 for both phenotypes (supplementary material Table S3). These data placehand2 downstream tobarx1 in a negative regulatory pathway (Avery and Wasserman, 1992).

Negative regulation ofhand2 bybarx1 is also indicated byin situ hybridization. We observed no consistent difference inhand2 expression between wild types andbarx1 mutants during the pharyngula period (Fig. 7A,B). By contrast, during the hatching period,hand2 expression is dorsally expanded inbarx1 mutants (Fig. 7C-H). Specifically, in wild-type animals,hand2 expression is restricted to ventral undifferentiated midline cells of the developing first arch skeleton (Fig. 7C,E,G, arrows), whereas inbarx1 mutantshand2 is upregulated and dorsally expanded towards the joint (Fig. 7D,F,H, arrowheads). Interestingly,hand2 expands to fill the domain normally occupied bybarx1 (Fig. 3E, arrowhead), resulting in an ectopic boundary betweenhand2 and the intermediate domain genenkx3.2 (Fig. 7D,F, arrowhead). These results strongly suggest thatbarx1 represseshand2.

Thathand2 expression is unchanged inbarx1 mutants in the pharyngula period, but is expanded during differentiation in the hatching period, motivates the hypothesis thatbarx1 downregulateshand2 only as cells differentiate, as we could examine by time-lapse analysis with transgenic reporters. In wild types before chondrocyte differentiation begins, ourhand2 reporterhand2:Crimson is strongly expressed in the ventral undifferentiated ectomesenchyme (supplementary material Movie 4, note lack of the green chondrocyte reportersox9a:EGFP in early frames). As chondrocyte differentiation initiates and thesox9a reporter is upregulated (in addition to the expressing cells organizing and enlarging into their characteristic chondrocyte morphology), thehand2 reporter downregulates in the differentiating chondrocytes of Meckel’s cartilage, compared with the strong expression in the surrounding perichondrium (Fig. 7I, arrow;supplementary material Movie 4). Inbarx1 mutants, by contrast,hand2 fails to downregulate as efficiently, resulting in an expandedhand2 domain (Fig. 7J, arrowhead), consistent with the results of our staticin situ experiments.

hand2 regulatesbarx1 expression

Expression ofhand2 andbarx1 partially overlap during the pharyngula period, but expression resolves into exclusive domains during the hatching period, as might be explained by reciprocal negative interaction, and as we examined byin situ hybridization forbarx1 inhand2 mutants. During the pharyngula period, we found decreased ventralbarx1 expression inhand2 mutants (Fig. 8B, arrow). By contrast, we observed a large expansion in ventralbarx1 expression during the hatching period (Fig. 8D, arrowhead). Hence, repression is dynamic and complex:hand2 activates ventralbarx1 expression during the pharyngula stage, yet repressesbarx1 expression during the hatching period.

barx1 repression prevents skeletal boundaries from developing into joints

We discovered that two separate populations of chondrocytes exist in Meckel’s cartilage. A population located adjacent to the jaw joint, and distinct from the joint cells themselves, consists of cells that aretrps1 andnkx3.2 positive, and have a rounded morphology. A second population distal to the joint istrps1 andnkx3.2 negative, with stacked, columnar cell morphology. We propose that a cryptic boundary with the potential to develop into a joint separates these two cartilage modules in wild-type animals. Moreover, that organ attachment sites correlate with ectopic joint sites suggest some unknown singular characteristic is present in wild types that marks the location where ectopic joints form in mutants. Through removal ofbarx1, the singularity of this location is clearly revealed, as joint repression is lifted and the cryptic joint is unveiled. A simple body plan (hidden boundaries/joints) seemingly is layered on top of a more complex one.

The data presented here demonstrate that during patterning, Edn1 signaling precisely positionsbarx1 such that the joint-repressive properties ofbarx1 function to prevent the ectopic Meckel’s joint, but not the jaw joint from developing (Fig. 9A). Other Edn1 pathway mutants, such asedn1, plcb3 andmef2ca, also have ectopicbarx1 in the intermediate domain and lack joints (Miller et al., 2000;Miller et al., 2007;Walker et al., 2006;Walker et al., 2007). Our model predicts that removingbarx1 function from these mutants (by generating compound mutants) would rescue the joint loss phenotype similar to the results obtained inbarx1;furina double mutants. The model inFig. 9 is incomplete however, for in addition to the endothelin 1 pathway, a number of other signaling systems such as BMP and Notch signaling are also crucial for zebrafish joint development. Specifically, misexpression of the BMP ligandbmp4 (Zuniga et al., 2011), a dominant-negative BMP receptordnBmpr1a (Alexander et al., 2011) or the Notch ligandJAG1 (Zuniga et al., 2010), all result in joint loss. It would be interesting to learn whether these joint loss phenotypes also involve ectopicbarx1 in the intermediate domain. In fact, evidence already exists thatbarx1 expression can be regulated by BMP (Barlow et al., 1999;Sperber and Dawid, 2008;Tucker et al., 1998) or Notch (Mitsiadis et al., 2010) signaling. In this scenario,barx1 would serve as a node for crosstalk between several signaling pathways that sculpt the joint.

We have previously demonstrated thattrps1:EGFP expands inhand2 mutants (Talbot et al., 2010), and here we showtrps1:EGFP also expands inbarx1 mutants. Yet, we conclude these genes,barx1 andhand2, have opposite effects on cell fate:hand2 is required for joints, whereasbarx1 represses joints. This apparent paradox can be reconciled by the fact that in addition to joint cells,trps1:EGFP also marks cartilage cells near the joint (e.g. cartilage of the retroarticular process istrps1:EGFP positive). Therefore, it seems likely that the expandedtrps1:EGFP inhand2 mutants represents an expansion of intermediate domain cartilage, not of joint cells. Further supporting this model, cells positive for the intermediate markernkx3.2 and the cartilage markersox9a are ventrally expanded inhand2 mutants (Talbot et al., 2010). Yet another line of evidence that joint-adjacent cartilage is expanded inhand2 mutants comes from examining the morphology of ventral cells inhand2 mutants. Morphological joints are invariably lost inhand2 mutants, and rounded interlocking chondrocytes constitute what remains of Meckel’s cartilage inhand2 mutant animals, reminiscent of the Meckel’s cartilage intermediate domain morphology (Fig. 6D).

Support for the hinge and caps model

During the hatching period, the mandibular region of the developing jaw can be subdivided into three nonoverlapping domains (Fig. 9B). The distalmosthand2-positive domain and the proximalnkx3.2 domain flank the subintermediatebarx1 expression domain. This configuration fits nicely with the recently proposed hinge and caps model of jaw development (Depew and Compagnucci, 2008;Medeiros and Crump, 2012). Briefly, this model posits a hinge region connects the mandibular and maxillary aspects of arch 1. The maxillary and mandibular regions of the arch have an inherent polarity with a cap region at their distalmost end. In such a model, thebarx1 domain between thenkx3.2 hinge andhand2 cap is a module that can be altered during the course of evolution.barx1 (this work) as well as several other genes (Miller et al., 2003;Talbot et al., 2010) are dramatically expanded when thehand2 cap is disrupted, with disastrous consequences for jaw development. Our work supports a model in which ventral-mosthand2 expression maintains a cap of undifferentiated cells that serves to keep the cartilage modules dorsally contained during chondrocyte differentiation. Thatbarx1 reciprocally represseshand2 suggests that the differentiating cartilage modules also feed back to restrict the cap. In fact, inbarx1 mutants thehand2 cap is dorsally expanded such that it directly juxtaposes thenkx3.2 hinge, suggesting the subintermediate domain module is completely absent in these mutant conditions (Fig. 9B).

barx1 function in the evolution of development

The ability to develop a jointed jaw fostered a revolution in feeding strategies among early vertebrates (Janvier, 1996;Mallatt, 1996). In this study, we propose a model in whichbarx1 functions as a joint repressor such that increasedbarx1 leads to joint loss, and decreasedbarx1 results in joint gain. Therefore, it is tempting to hypothesize that changes inBarx1 function may be relevant to the advent of jaw hinges in evolution. The only available developmental model for the assemblage of jawless stem vertebrates from which gnathostomes likely evolved is the lamprey. Modern approaches complement classical studies to reveal that larval lamprey develop cartilaginous mouth support structures, but do not develop dorsal-ventral articulations, i.e. jaw joints (Cattell et al., 2011;Johnels and Sundström-Schött, 1948). Interestingly,Barx is expressed in intermediate domain cells in lamprey, which do not develop into joint cells (Cerny et al., 2010). One might speculate, then, that exclusion ofBarx from the intermediate domain of early gnathostomes, perhaps resulting from an increase in Edn1 signaling, was a crucial step in the evolution of the jaw joint. By this scenario, the absence of joints and expandedbarx1 in zebrafishfurina mutants would be atavistic, resembling the primitive jawless condition seen in lamprey.

The peculiar arrangement of cartilage and joints inbarx1 mutant zebrafish is reminiscent of that seen in anuran tadpoles: two joints form in the first arch larval skeleton (the jaw joint and intramandibular joint) carving out three independent elements (the palatoquadrate, Meckel’s and infrarostral cartilages) (de Beer, 1938;Svensson and Haas, 2005). It would be interesting to test the hypothesis that development of the second (intramandibular) joint in anurans involves changes inbarx1 function.

One might predict that theBarx1 mutant mouse skeleton will exhibit disruptions similar to those we report here for zebrafish. Indeed,Barx1 mutant mice display a gap in the stapes (Paul Sharpe, personal communication) reminiscent of the gap we report for the zebrafish equivalent, the hyosymplectic (supplementary material Fig. S1J). These phenotypes speak to the conservation of at least some aspects ofBarx1 function across vertebrates. However, arch 1Barx1 expression was reported to be unaffected by losses in Edn1 signaling in mice (Clouthier et al., 2000), highlighting potential differences across vertebrates in the genetic network we propose.

Our findings extend several studies describing remarkable roles forBarx1 in cell fate specification. First,Barx1 was reported to promote the development of molars versus incisors (Miletich et al., 2005;Tucker et al., 1998). More recently,Barx1 was shown to promote stomach versus intestine development (Kim et al., 2005). In keeping with this theme, we now provide evidence thatBarx1 functions to promote cartilage versus joint development. A tantalizing idea has been put forth proposing that evolutionary changes inBarx1 could have had profound changes in feeding strategy in mammals by simultaneously altering two organs involved in feeding: the dentition and digestive system (Miletich et al., 2005). Our findings broaden this idea to include vertebrates beyond mammals, and add the origin and subsequent modification of the hinged jaw skeleton to the list ofBarx1-controlled feeding anatomy. It is intriguing to consider thatBarx was instrumental in the success of early vertebrates.

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