Transitory expression of Dlx5 and Dlx6 in maxillary arch precursors is essential for upper jaw morphogenesis

Mouse strains and breeding

All animal experimentation was performed in accordance to French national regulations and approved by the MNHN ethical committee (approval n° 68–028r1). For this study we used about 35 dams (including 10 WT, 5 Dlx5^lacZ/+; 3 Dlx5/6+/-; 12 B6.129S4-Gt(ROSA)26Sor^tm1Sor/J; 5 B6(Cg)-Dlx5^{tm1(cre/ERT2)Zjh}/J) and analyzed about 120 embryos, the exact record of animals used, litters obtained, embryos genotyped and number of embryos per litter is on record in our animal house. WT animals were form Charles River France and were maintained in the MNHN mouse facility which is officially certified by the French National Animal well being committee.

Dlx5^lacZ/+ knock-in mice were maintained on a mixed B6/D2 genetic background¹⁸. Double Dlx5 and Dlx6 (Dlx5/6) mutant mice were maintained and genotyped as reported¹⁹. The inducible Cre driver strain B6(Cg)-Dlx5^{tm1(cre/ERT2)Zjh}/J (designed by Z. J. Huang²⁰), and the lacZ Cre reporter strain B6.129S4-Gt(ROSA)26Sor^tm1Sor/J (R26R-lacZ) were purchased from Jackson Laboratory (#10705 and #003309 respectively; Maine, USA) through Charles River Laboratories (L’Arbresle, France) and maintained on a C57BL/6J genetic background through heterozygous mating. Double heterozygous embryos were obtained through bi-directional crosses. Induction of Cre recombinase activity was obtained upon single intraperitoneal injection of 5mg of tamoxifen (Sigma-Aldrich), in corn oil. Tamoxifen preparation and administration in pregnant dams followed the Jackson Laboratory’s Guidelines and CNRS/MNHN Animal Handling Guidelines. Dams were 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^18,21. Vehicle (corn oil) injection in double heterozygous mice did not yield leaking ß-galactosidase activity.

Histology and 3D reconstruction

Heads from 18.5 dpc (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 in²² 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.

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^15,16. To better visualize the jaw phenotype of Dlx5/6 mutants, we performed 3D reconstruction of craniofacial elements of 18.5 dpc (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 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.

Figure 1. Three-dimensional reconstruction of the dentary and maxillary bones of 18.5 dpc 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 purple. Middle row: Dorsal view of the dentary bone of WT and Dlx5/6^-/- 18.5 dpc mice. Lower row: Ventral view of the maxillary components of WT and Dlx5/6^-/- 18.5 dpc 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 arch-like structure deriving from upper jaw transformation.

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 (6.25 dpc). We monitored ß-gal reporter activity from ts15 (10 dpc) to ts20 (12.5 dpc). At ts15 we observed a stream of ß-gal-positive cells extending from the lambdoidal junction, which joins the olfactory pit with the distal maxillary arch^17,23, 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’).

Figure 2. Lineage of Dlx5-expressing cells in the maxillary arch.

ß-Galactosidase activity in the cephalic region of Dlx5-lacZ (A,A’) and Dlx5-creERT2; R26R-lacZ mouse embryos (B–D’). In all cases pregnant dams were treated with tamoxifen at 6.25 dpc/Theiler stage 9 (ts9) and embryos were collected at the indicated Theiler stage. (A,A’) As expected, even after tamoxifen treatment, Dlx5 is expressed in the mandibular arch (md), in the olfactory pit (olf), in the otic vesicle (ov), in the basal telencephalon (bt) and in the hind limb (hl), but not in the maxillary arch. (B,B’) Permanent activation of lacZ reporter expression in derivatives of Dlx5-expressing early progenitors (ts9) reveals the presence of a positive cellular contingent in the ts15 lambdoidal junction (λ) between the olfactory pit and the maxillary process. (C,C’; D,D’) At later developmental stages (ts19, ts20) a contingent of lacZ positive cells populates the distal domain of the maxillary arch. hl, hind limb; md, mandibular arch; mx, maxillary arch; olf, olfactory pit; ov, otic vesicle; bt, basal telencephalon; λ, lambdoidal junction; red asterisk/black arrowheads, territories of the maxillary arch colonized by derivatives of Dlx5-expressing progenitors. Bar: A–D 1mm; A’–D’ 250µm.