DEV028621 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Paralogue Hox gene expression patterns in mouse pharyngeal arches. (A-H) Whole-mount in situ hybridization on E10.0 wild-type (WT) embryos using Hoxa2 (A), Hoxb2 (B), Hoxa3 (C), Hoxb3 (D), Hoxd3 (E), Hoxa4 (F), Hoxb4 (G) and Hoxd4 (H) antisense probes. CP, dorsolateral circumpharyngeal ridge. (I-K) In situ hybridization on frontal cryosections of E10.0 embryos using antisense Hoxb3 (I), Hoxb4 (J) and Hoxd4 (K) probes. PA3, third pharyngeal arch; PA4, fourth pharyngeal arch.

• Supplemental Figure S2 -

  Fig. S2. Selective deletion of Hoxa3 expression in NCCs of Wnt1::Cre/Hoxa^flox/del mutant embryos. (A-D) Whole-mount in situ hybridization on wild-type (WT) (A,B) and Wnt1::Cre/Hoxa^flox/del (C,D) E10.0 embryos using an antisense Hoxa3 probe. Lateral (A,C) and dorsal (B,D) views are shown. The arrows in C show the absence of Hoxa3 expression in the NCCs of third (PA3) and fourth (PA4) pharyngeal arches, as well as in the dorsal Wnt1⁺ rhombic lip, when compared with wild type in A. In D, note the persistence of Hoxa3 expression in the neural tube (NT).

• Supplemental Figure S3 -

  Fig. S3. Characterisation of the R4::Cre driver. (A,B) Alkaline phosphatase (AP) staining on whole-mount (A) and frontals vibratome sections (B) of E10.5 R4::Cre;Z/AP mouse embryo. Note that AP staining is selectively restricted to the second arch (PA2), whereas the other PAs are devoid of AP expression. Within PA2, expression is confined to the NCC-derived mesenchyme and excluded from the mesodermal core (Mes). (C-F) Skeletal preparations from Hoxa2^-/- (C,E) and R4::Cre/Hoxa2^flox/flox (D,F) mutant newborns. Note that the conditional Hoxa2 inactivation using the R4::Cre driver recapitulates the full Hoxa2 knockout second arch skeletal phenotype. The analysis strongly suggests that the R4::Cre driver is expressed in the vast majority of PA2 NCC progenitors. Asterisk represents the absence of the lesser horn of the hyoid bone. H, hyoid bone; I, incus; I2, ectopic incus; G*, transformed gonial bone; M, malleus; M2, ectopic malleus; MC, Meckel’s cartilage; MC2, ectopic Meckel’s cartilage; PA1 to PA4, first to fourth pharyngeal arches; R4, rhombomere 4; T, tympanic bone; T2, ectopic tympanic bone; TC, thyroid cartilage.

• Supplemental Figure S4 -

  Fig. S4. Normal Hoxb2 expression in Hoxa-deleted mutant embryos (A,B) Whole-mount in situ hybridization on wild-type (WT) (A) and Wnt1::Cre/Hoxa^flox/del (B) E10.0 embryos using an antisense Hoxb2 probe. The blacks arrows in B show the persistence of normal Hoxb2 expression in the third (PA3) and fourth (PA4) pharyngeal arches. A normal Hoxb2 expression pattern is also detected in the neural tube, as expected. PA1 and PA2, first and second pharyngeal arches, respectively; R, rhombomere.