Previously titled : Transitory expression of and in maxillary arch precursors is essential for upper jaw Dlx 5 Dlx 6 morphogenesis

Asymmetric, articulated jaws are characteristic of most vertebrate species; they derive from the first pharyngeal arch (PA1) which generates both maxillary and mandibular components. PA1 is colonized by cranial neural crest cells (CNCCs) which give rise to most bones and tendons of the jaws. The elements formed by different CNCCs contingents are specified by the combinatorial expression of genes. and are predominantly expressed by Dlx Dlx5  Dlx6 mandibular CNCCs. Analysis of the phenotype of and double mutant Dlx5 Dlx6 mice has suggested that they are necessary and sufficient to specify mandibular identity. Here, using 3D reconstruction, we show that inactivation of and does not only affect the mandibular arch, but results in the Dlx5 Dlx6 simultaneous transformation of mandibular and maxillary skeletal elements which assume a similar morphology with gain of symmetry. As and Dlx5Dlx6 -expressing cells are not found in the maxillary bud, we have examined the lineage of -expressing progenitors using an genetic approach. We Dlx5 in vivo find that a contingent of cells deriving from epithelial precursors transiently expressing participate in the formation of the maxillary arch. These cells Dlx5 are mostly located in the distal part of the maxillary arch and might derive from its lambdoidal junction with the olfactory pit. Our observations provide the first genetic demonstration of the ‘Hinge and Caps’ model[1]. We support the notion that ‘cap’ signals could originate from epithelial derivatives of expressing Dlx5progenitors which migrate and colonize the maxillary arch epithelium. Our results imply that Dlx5 and Dlx6 control upper and lower jaw morphogenesis through different coordinated mechanisms to generate functional, articulated jaws. Referee Status:


Abstract
Asymmetric, articulated jaws are characteristic of most vertebrate species; they derive from the first pharyngeal arch (PA1) which generates both maxillary and mandibular components. PA1 is colonized by cranial neural crest cells (CNCCs) which give rise to most bones and tendons of the jaws. The elements formed by different CNCCs contingents are specified by the combinatorial expression of genes. and are predominantly expressed by Dlx Dlx5 Dlx6 mandibular CNCCs. Analysis of the phenotype of and double mutant Dlx5 Dlx6 mice has suggested that they are necessary and sufficient to specify mandibular identity. Here, using 3D reconstruction, we show that inactivation of and does not only affect the mandibular arch, but results in the Dlx5 Dlx6 simultaneous transformation of mandibular and maxillary skeletal elements which assume a similar morphology with gain of symmetry. As and Dlx5-Dlx6 -expressing cells are not found in the maxillary bud, we have examined the lineage of -expressing progenitors using an genetic approach. We Dlx5 in vivo find that a contingent of cells deriving from epithelial precursors transiently expressing participate in the formation of the maxillary arch. These cells Dlx5 are mostly located in the distal part of the maxillary arch and might derive from its lambdoidal junction with the olfactory pit. Our observations provide the first genetic demonstration of the 'Hinge and Caps' model [1]. We support the notion that 'cap' signals could originate from epithelial derivatives of expressing Dlx5progenitors which migrate and colonize the maxillary arch epithelium. Our results imply that Dlx5 and Dlx6 control upper and lower jaw morphogenesis through different coordinated mechanisms to generate functional, articulated jaws. The skull of most vertebrates is characterized by the presence of articulated, asymmetric jaws which support the function of a muscularized oral cavity 2,3 . During embryonic development, the upper and lower jaws derive from the maxillary and mandibular processes of the first pharyngeal arch (PA1). Most cartilaginous and dermatocranial derivatives of PA1 are formed by Cranial Neural Crest Cells (CNCCs) 4-9 . During migration, signals emanating from the endoderm and possibly other PA1 components instruct the CNCCs to unfold the morphogenetic process of the jaws 8,10,11 . The nested expression of Dlx homeobox genes, vertebrate homologues of Drosophila Distal-less, has a fundamental role in the specification of the dorsoventral patterning of PA1 derivatives 2,12 . While Dlx1 and Dlx2 are expressed by CNCCs of the maxillary and mandibular components of PA1, Dlx5 and Dlx6 transcripts are present only in mandibular CNCCs. Targeted simultaneous inactivation of Dlx5 and Dlx6 13,14 results in the transformation of lower jaw into upper jawlike structures, underlining the importance of these genes for lower jaw identity. This phenotype is more severe than those observed in both single mutants (for instance, see 2,13,15,16 ). The activation of Dlx5 and Dlx6 by endothelin-1 signalling is necessary and sufficient to define lower jaw identity 17-21 . Interestingly it has been observed 13,14 that, after inactivation of Dlx5 and Dlx6, maxillary components are also affected despite the fact that these genes are not expressed by maxillary CNCCs. This observation could be accounted for by the presence of shared Dlx5/6-dependent signalling centres in proximity to the extremities of both the mandibular and maxillary arches; this notion gave rise to the so-called "Hinge and Caps" model of jaw organization 1,3,22 . In its original formulation this model predicts the presence of two opposing morphogen gradients, one emanating from the region of the upper/lower jaw articulation (hinge) and one from the distal extremities of PA1 (caps); the origin and nature of these signals remain elusive. Here we revisit the effects of Dlx5 and Dlx6 double inactivation on jaw development and, using a transgenic lineage tracing approach, we reveal that the maxillary arch epithelium harbours a cellular contingent derived from frontonasal Dlx5-expressing progenitors. Our findings suggest that transient Dlx5/6 expression could program these epithelial cells to provide the cues needed for maxillary arch morphogenesis. Dams were anesthetized in a chamber containing 2.5% isoflurane in oxygen, euthanized by cervical dislocation at indicated stages and embryos were collected in phosphate-buffered saline (PBS), then staged and fixed by immersion in ice-cold fixative (2% paraformaldehyde/0.2% glutaraldehyde) for 5 to 15 minutes (depending upon their developmental stage).

β-galactosidase detection
For lacZ expression, embryos were fixed for 15-30 min in 4% paraformaldehyde; X-gal staining was performed as described previously 15,25 . Vehicle (corn oil) injection in double heterozygous mice did not yield leaking β-galactosidase activity.
Histology and 3D reconstruction Heads from 18.5dpc (days post coitum) Dlx5/6 -/and wild type mouse embryos were fixed in Bouin's solution (Sigma, France), embedded in paraffin and complete sets of frontal or parasagittal serial sections (12µm) were prepared. All sections were stained by Mallory's trichrome as in 21 and photographed (Nikon Digital Site DS-FI1). Pictures were aligned, piled and registered using the Fiji plug-in of NIH ImageJ "Register Virtual Stack Slices" (http://fiji. sc/wiki/index.php/Register_Virtual_Stack_Slices). 3D segmentation was performed with Mimics (Materialise, Belgium: http://biomedical. materialise.com/mimics) and visualized using Adobe Acrobat 9 pro.

Results
Dlx5/6 inactivation results in upper and lower jaw transformation with gain of symmetry Previous reports suggest that double inactivation of Dlx5 and Dlx6 results in lower-to-upper jaw transformation; these reports also indicated that the upper jaw of these mice is not normal 13,14 . To better visualize the jaw phenotype of Dlx5/6 mutants, we performed 3D reconstructions of craniofacial elements of 18.5dpc (days post coitum) embryos. Frontal view of the mutant jaws ( Figure 1, upper panel) shows an obvious gain of symmetry compared to a WT animal. Examining the defects of the lower and upper jaws separately ( Figure 1, middle and lower panels), it is evident that both are transformed. In the absence of Dlx5 and Dlx6 the dentary and the upper

Amendments from Version 2
We would like to thank Dr. Knight for his rapid review of our report. The text of the manuscript has been modified to include more references on Dlx5 and Dlx6 gene expression and single mutant reports during craniofacial morphogenesis.

Figure 1. Three-dimensional reconstruction of the dentary and maxillary bones of 18.5dpc wild type and Dlx5/6 -/mouse embryos.
Upper row: Frontal view of WT and Dlx5/6 -/oral apparatus. Skeletal elements are grey, the tongue is red and incisors are violet. Middle row: Dorsal view of the dentary bone of WT and Dlx5/6 -/-18.5dpc mice. Lower row: Ventral view of the maxillary components of WT and Dlx5/6 -/-18.5dpc mice. Note that the inactivation of Dlx5/6 results in the transformation of both lower and upper jaw skeletal elements into new structures which appear more similar to each other than to their WT counterpart. cp, coronoid processes; dt, dentary bone; li, lower incisor; t, tongue; ui, upper incisor; za, zygomatic arch; za*, zygomatic arch-like structure deriving from lower jaw transformation; za', zygomatic archlike structure deriving from upper jaw transformation.
jaw bones do not form correctly and are replaced by remarkably similar skeletal structures. In the mutant embryos, both the upper and lower jaw skeletal elements are reduced in size, are not fused in the midline, and display a lateral process positionally homologous to the wild type zygomatic arch. Thus the upper and lower jaw mutant bones resemble each other more closely than usually found in their normal counterparts.

Transient Dlx5 expression in maxillary arch progenitors
In Dlx5-lacZ heterozygous Theiler stage (ts) 19 (12 dpc) embryos the reporter is active in the olfactory pit and mandibular arch, but not in the maxillary arch; this pattern of expression does not change upon tamoxifen treatment of the pregnant dam ( Figure 2A, A').
To understand the origin of the Dlx5/6-dependent defect of the upper jaw we used a genetic approach to follow the lineage of Dlx5-precursors in the head. To this end we brought the R26R-lacZ reporter into the Dlx5-creERT2 driver background and we activated cre-recombinase activity by tamoxifen treatment of the pregnant dam at ts9 (7 dpc). We monitored β-Gal reporter activity from ts15 (10 dpc) to ts20 (12.5 dpc) (n=10 embryos per stage). At ts15 we observed a stream of β-Gal-positive cells extending from the lambdoidal junction, which joins the olfactory pit with the distal maxillary arch 1,26 , towards the body of the maxillary arch ( Figure 2B, B'). At ts19 and ts20 ( Figure 2C, C'; D, D') reporter-expressing cells are found in the upper epithelial lining of the maxillary arch (arrowheads in Figure 2C', 2D') and in two distinct proximal and distal territories of the arch body (red asterisk in Figure 2C').
To determine more precisely the tissue distribution of craniofacial derivatives of Dlx5-positive cells, we set apart two illustrative among the ten embryos per stage, and performed serial paraffin sections of Dlx5-creERT2; R26R-lacZ β-Gal-stained mouse embryos (12.5 dpc) after tamoxifen treatment of pregnant dams at 7dpc/ Theiler stage 9 (ts9) (Figure 3). While in the mandibular arch β-Gal staining is limited, as expected, to CNCCs derivatives, the analysis of the complete set of serial sections shows that only epithelial cells lining the maxillary arch are positive. As no Dlx5-positive epithelial cells are present in the maxillary arch of normal embryos, we conclude that a population of epithelial cells derived from the Dlx5-positive frontonasal process participates to the formation of the maxillary arch.

Discussion
In this study we have re-examined the skeletal jaw phenotype of Dlx5/6 mutant mice. We confirm that both the mandibular and maxillary arches are transformed. The profound change in the shape of the maxillary arch is difficult to explain, as this region does not derive from a Dlx5/6-expressing territory. Indeed, in normal embryos maxillary CNCCs and the overlying epithelium do not express Dlx5 and Dlx6. Lineage analysis to identify derivatives of We have previously shown that early Dlx5 and Dlx6 expression in the anterior neural fold is essential for nasal capsule patterning 29 ; our present findings suggest that the same population of cells could also contribute to maxillary patterning.

Competing interests
No competing interests were disclosed.

Grant information
This research was partially supported by the EU Consortium IDEAL (HEALTH-F2-2011-259679) to GL.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This is an interesting story and it is intriguing that genes function in different cellular compartments to Dlx pattern the upper and lower jaws, especially considering our assumptions about the homology of upper and lower skeletal elements in the pharyngeal arches. I look forward to reading about the conditional mouse alleles.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed.

5.
that expressing cells are restricted to ectoderm, although I could not determine how many animals Dlx5 were examined to determine this. The authors further state that these cells no longer express when Dlx5 they arrive at this point, but again this is not shown, but rather referenced by other work. The authors argue that this ectodermal positioning of the -lineage cells in the maxillary arch, in conjunction with Dlx5 the phenotype of the double mutant, argues that the 'caps' signal is an ectoderm signal. The Dlx5/6 authors then cite unpublished data for a CNCC-specific knockout of and state this has no Dlx5/6 phenotype in the maxillary arch, but fail to show this. This paper is interesting and posits an attractive idea -that a discrete population of cells located at a point in the ectoderm can act as a positional signal to pattern forming skeletal elements. The lineage tracing experiments are elegant and could be elaborated on to show if the restriction of the lineage cells are Dlx5 restricted to the ectoderm throughout the maxillary arch, as only one plane is shown in Figure 3.
One thing that I was not able to glean from this work, is whether there is any expression or Dlx6 Dlx6 -derived cells in the maxillary arch and if so, is this in ectoderm-derived or CNCC-derived cells? This is critical to ascertain as the premise of the work is that a expressing cell lineage in the ectoderm is The authors should show that the expression of is absent from the maxillary arch at Dlx5 comparable stages to the staining (this is referenced, but not shown at comparable stages to lacZ the stained animals in ) lacZ I would like to see what expression of is in the maxillary arch at comparable stages to those Dlx6 examined by the transgene.

Dlx5-lacZ
I would like to see the data for the mice in which is knocked out in CNCC (unpublished data Dlx5 cited in the Discussion). The central premise of the paper is that the lineage cells in the Dlx5 epithelial of the maxillary arch are acting as the caps, in which case it is crucial to show that a knockout of in the CNCC does not affect the morphology of the maxillary skeleton.

Dlx5/6
Please reference descriptions of maxillary arches in and single mutants, describe the Dlx5 Dlx6 expression of both genes in the maxilla and highlight the redundancy between these genes in arch patterning. These should be incorporated into the Discussion.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.

Competing Interests:
No competing interests were disclosed.

Competing Interests:
Author Response 10 Sep 2014 , CNRS/MNHN, France Giovanni Levi "The authors display 3D reconstructions emphasising the symmetry of the transformed skeletal elements, but do not elaborate beyond the characterisations of these mutants that haven previously been performed." As discussed in our first revision in response to Referee 1, in this manuscript we have focused our attention on the simultaneous alteration of both upper and lower jaws of double mutant Dlx5/6 embryos. In previous reports, the skeletal analysis was interpreted suggesting that the lower jaw was transformed into an upper jaw with gain of symmetry. Now we stress the fact that the acquisition of new symmetry derives from morphological changes occurring both in upper and lower jaws. Figure 3."

"The lineage tracing experiments are elegant and could be elaborated on to show if the restriction of the Dlx5 lineage cells are restricted to the ectoderm throughout the maxillary arch, as only one plane is shown in
As stated in the revised version of the paper we have analysed complete sets of serially sectioned embryos and we conclude that lineage derivatives are restricted to the ectoderm in each Dlx5 section analysed. We have now added the sentence: "the analysis of the complete set of serial sections" to stress this point. The specific section shown in Fig. 3 was chosen as it shows simultaneously the maxillary and the mandibular arches.

"One thing that I was not able to glean from this work, is whether there is any Dlx6 expression or Dlx6-derived cells in the maxillary arch and if so, is this in ectoderm-derived or CNCC-derived cells?" and … "The authors further state that these cells no longer express Dlx5 when they arrive at this point, but again this is not shown, but rather referenced by other work."
In situ hybridization and transcriptomic analyses by our group as well as others has, to our opinion, settled the issue ; Dlx6 expression in maxillary-fated cells in the nasal prominence. We have added a paragraph at the end of the discussion to emphasize this issue, including the sentence: 'As the compound mutant Dlx5/6 displays an upper jaw malformation which is not observed in either single mutant (Jeong , et al. 2008), it appears that and exert redundant functions not only on lower, but also on upper Dlx5 Dlx6 jaw morphogenesis. Whether there is a specific and transiently-expressing cell population Dlx6 during upper jaw morphogenesis remains, ideally, to be formally determined using an inducible targeted strain…' Cre-Dlx6 "The authors then describe the expression of an inducible Dlx5 transgene in the maxillary arch and show that Dlx5 expressing cells are restricted to ectoderm, although I could not determine how many animals were examined to determine this." We have now modified the text to include this data (n=10 embryos per stage).
"The authors then cite unpublished data for a CNCC-specific knockout of Dlx5/6 and state this has no phenotype in the maxillary arch, but fail to show this" no phenotype in the maxillary arch, but fail to show this" The extensive analysis of this new series of conditional mutant is the subject of another Dlx5/6 paper in preparation in which we include also the use of other tissue-specific cre-recombinase mice. Deletion of and in CNCCs, from E7 on, results in a mandibular phenotype without Dlx5 Dlx6 any detectable alteration of maxillary skeletal elements.
"How many animals were examined for lacZ reporter gene expression in the maxillary arch by paraffin wax sectioning? Another example and an indication of the number of animals examined is required." As stated above (and now included in the text) we have analysed 10 embryos per stage. Illustrative results are provided in Figures 2 and 3. The results section has now been modified to precise this point. As stated above, the analysis of this new series of conditional mutant is the subject of Dlx5/6 another paper in preparation in which we include also the use of other tissue-specific cre-recombinase mice. A remarkable morphological feature of these mutant mice is that after deletion only in CNCCs the lower jaw is transformed, but the nasal capsule and the upper Dlx5/6 jaw are normal reinforcing the notion that act on different cellular populations. If the reviewer Dlx5/6 is interested in seeing some of these images we are glad to provide them personally to him, however, in order to maintain the novelty of our results we cannot attach these pictures to this reply as on this will be published online. F1000Research "Please reference descriptions of maxillary arches in Dlx5 and Dlx6 single mutants, describe the expression of both genes in the maxilla and highlight the redundancy between these genes in arch patterning. These should be incorporated into the Discussion." We added a sentence stating that the morphological defects observed in the jaws of single mutants were previously described to be less severe than in the double mutant (Jeong , 2008 et al. ), and we pointed to relevant references. The changes made by Gitton and colleagues in this revised manuscript have improved and clarified their new finding regarding Dlx5/6 contributions to upper jaw morphogenesis. In particular, the addition of Figure 3 and the changes to the text more clearly describe the role of epithelial expression of to Dlx5 upper jaw patterning. However, I do not necessarily agree with the statement that these "observations provide the first genetic demonstration of the 'Hinge and Caps' model" (p. In this manuscript, Gitton and colleagues explore the role of Dlx5/6 in upper jaw morphogenesis. Dlx5/6 have largely been recognized for their role in lower jaw identity, based on the fact that loss of these genes in mice results in a loss of lower jaw identity. Previous reports have further suggested that loss of Dlx5/6 in mice causes a transformation of identity from that of lower jaw to upper jaw. In this manuscript, Gitton and colleagues present 3D reconstructions of WT and Dlx5/6 mutant mouse jaws, which allow for a more detailed analysis of the jaw phenotype. They note that the Dlx5/6 jaws not only exhibit dysmorphic lower jaw structures, but the upper jaw elements are also abnormal. They propose two hypotheses that could explain this data: 1) That loss of Dlx5 in the epithelia overlying the developing upper jaw primorida disrupts signaling to the underlying CNC (as previously hypothesized by the Hinge and Caps model of jaw development), or 2) that Dlx5 is transiently expressed in cells that will later populate the maxillary arch, and that this transient expression is essential for subsequent upper jaw morphogenesis. Using lineage tracing experiments, the authors conclude that Dlx5 is indeed transiently expressed in precursors that will populate the maxillary arch, and also provide support for the Hinge and Caps model.
The question that Gitton and colleagues proposed is an important one, as the role of Dlx5/6 in jaw mutants, we think that what she calls the "two hypotheses" of this paper needs further Dlx5/6 consideration. This paper is based on experimental evidence. We are not formulating any hypothesis, but we provide experimental evidence supporting an existing hypothesis: the "Hinge and Caps hypothesis" (for instance ). We show that indeed cells derived from the Fish JL , 2011 et al. frontonasal epithelium after losing the expression of migrate to the epithelium overlaying the Dlx5/6 maxillary arch. This is what we meant saying " "; in no way did cells populating the maxillary arch. we hint to the possibility that mesenchymal cells populating the maxillary arch did express at any time . The whole text of the manuscript has been reformulated to clarify this point. We have Dlx5/6 now added a new figure (Figure 3) demonstrating experimentally that derivatives of positive Dlx5/6 cells in the upper jaw are epithelial and not mesenchymal. To make this point even clearer we have changed the title and several sentences of the paper referring now to " epithelial precursors". Dlx5/6 Regarding the first hypothesis that the reviewer claims that we have formulated: "That loss of Dlx5 in the epithelia overlying the developing upper jaw primorida disrupts signaling to the underlying it is CNC (as previously hypothesized by the Hinge and Caps model of jaw development)" important to note that is NEVER expressed by the epithelia overlying the developing upper Dlx5 jaw primordia. What we show is that derivatives of cells from the frontonasal primordial (FNP) migrate, after having downregulated , to the upper jaw and then play an important role in Dlx5/6 defining upper jaw identity. These cells carry therefore a "memory" of having expressed Dlx5/6 before migrating to the epithelia overlying the upper jaw primordia.
As the reviewer asks : ", to clarify this point, it would be nice to see sections of the embryos shown ." in Figure 2 that would clearly show where Lac-Z is expressed-in the epithelia or the mesenchyme we have added Figure 3. :

Other minor points
The authors state that CNCCs populating PA1 come from the prosencephalic and anterior mesenchepalic neural folds. In fact, neural crest populating PA1 derives from the posterior mesencephalon and the first and second rhombomeres of the hindbrain. We removed the sentence as the origin of CNCCs is not particularly relevant to the paper.
It would be more appropriate to say that the evolution of asymmetric jaws has been important for the diversification of vertebrates, as the symmetric jaws of sharks are quite sufficient for predation. We agree with the reviewer and the discussion has been modified accordingly including the cited reference.
Thanking you again for the time and energy you give to the reviewing process, Sincerely yours, YG, NNN, GL No competing interests were disclosed. Competing Interests: