Histone deacetylase 1 and 2 are essential for murine neural crest proliferation, pharyngeal arch development, and craniofacial morphogenesis

Abstract

Background: Craniofacial anomalies involve defective pharyngeal arch development and neural crest function. Copy number variation at 1p35, containing histone deacetylase 1 (Hdac1), or 6q21-22, containing Hdac2, are implicated in patients with craniofacial defects, suggesting an important role in guiding neural crest development. However, the roles of Hdac1 and Hdac2 within neural crest cells remain unknown.

Results: The neural crest and its derivatives express both Hdac1 and Hdac2 during early murine development. Ablation of Hdac1 and Hdac2 within murine neural crest progenitor cells cause severe hemorrhage, atrophic pharyngeal arches, defective head morphogenesis, and complete embryonic lethality. Embryos lacking Hdac1 and Hdac2 in the neural crest exhibit decreased proliferation and increased apoptosis in both the neural tube and the first pharyngeal arch. Mechanistically, loss of Hdac1 and Hdac2 upregulates cyclin-dependent kinase inhibitors Cdkn1a, Cdkn1b, Cdkn1c, Cdkn2b, Cdkn2c, and Tp53 within the first pharyngeal arch.

Conclusions: Our results show that Hdac1 and Hdac2 function redundantly within the neural crest to regulate proliferation and the development of the pharyngeal arches by means of repression of cyclin-dependent kinase inhibitors. Developmental Dynamics 246:1015-1026, 2017. © 2017 Wiley Periodicals, Inc.

Keywords: craniofacial morphogenesis; histone deacetylase; neural crest; pharyngeal arch.

Figure 1

(A) Hdac1^LacZ knock-in allele targeting map and generation scheme. (B–G) Whole-mount X-Gal staining for β-galactosidase (blue, LacZ) of Hdac1^LacZ/+ and Hdac1^+/+ at various developmental stages. B, ventral view of Hdac1^+/+ at E7.75. C, ventral view of Hdac1^LacZ/+ at E7.75. D, right lateral view of Hdac1^+/+ embryo at E8.75. E, right lateral view of Hdac1^LacZ/+ embryo at E8.75. F, right lateral view of Hdac1^+/+ embryo at E9.5. G, right lateral view of Hdac1^LacZ/+ embryo at E9.5.. (H–M) C-terminal Hdac1 (Hdac1) immunostaining (brown) on whole-mount X-Gal stained (LacZ, blue) E9.5 Hdac1^Lacz/+ sagittal sections including the outflow tract (OFT, H), primitive ventricle (PrV, I), atrioventricular canal (AVC, J), midbrain and telencephalic vesicle (MB & TV, K), otic pit (OtP, L), and caudal somites (So, M) (N–S) Hdac2 immunostaining (brown) on whole-mount X-Gal stained (LacZ, blue) E9.5 Hdac1^LacZ/+ sagittal sections including the OFT (N), PrV (O), AVC (P), MB & TV (Q), OtP (R), and So (S). Scale bars 50μm.

Figure 2

(A) Hdac1^Flox conditional allele targeting vector map and generation scheme. (B) Predicted aligned amino acid sequence for Hdac1^WT and recombined Hdac1^Flox (Hdac1^KO) alleles consensus-determining sequences (CDS). (Blue, WT; Green, unchanged; Purple, frameshift mutation; Red, stop codon) (C–F) Western blot analysis of control (Mesp1;1^Het, Hdac2^+/+) and knockout (Mesp1;1^KO, Mesp1;2^KO) P0 heart lysate for Hdac1^C (C), Hdac1^N (E), and Hdac2 (F). Hdac1^C was quantified and normalized to total input α-Tubulin and is presented as mean (SD) (**, p=0.007) (G–J) Hdac1^C and Hdac1^N immunostaining of Mesp1;1^KO and Mesp1;1^Het frontal heart sections at P0 (Black – present; White – absent). (K–N) Hdac1^C (K,M) and Hdac2 (L,N) immunostaining of Wnt1;1^Het2^Het;R26R-LacZ^−/+ and Wnt1;1^KO2^KO;R26R-LacZ^−/+ neural tubes (NT) at E9.5 (Black, present; White, absent). (O–Q) Left lateral (main) and right lateral (inset) images of Hdac1^F/F;Hdac2^F/F (O), Wnt1;1^Het2^KO (P), and Wnt1;1^KO2^Het (Q) pups at birth (White, milk spot; Black, absent milk spot; Red, distended abdomen; Orange, neonatal distress). (R–T) Dissected/fixed P0 Hdac1^F/F;Hdac2^F/F (R), Wnt1;1^Het2^KO (S), and Wnt1;1^KO2^Het (T) palates (PAL). (U–Z) Dissected/fixed P0 Hdac1^F/F;Hdac2^F/F (U–V), Wnt1;1^Het2^KO (W–X), and Wnt1;1^KO2^Het (Y–Z) thymus (TY; U,W,Y) and outflow tract (OFT; V,X,Z). (AA–AF) Hematoxylin and Eosin P0 frontal sections of Hdac1^F/F;Hdac2^F/F (AA–AB), Wnt1;1^Het2^KO (AC–AD), and Wnt1;1^KO2^Het (AE–AF) aortic valves (AA,AC,AE) and pulmonic valves (AB,AD,AF). (AG–AI) Hematoxylin and Eosin staining of Hdac1^F/F;Hdac2^F/F (AG), Wnt1;1^Het2^KO (AH), and Wnt1;1^KO2^Het (AI) P0 hearts shows peri-arterial morphology in the aorta/pulmonary artery. Nuclear hematoxylin counterstain is present. Scale bars 100μM. AO, aorta; PA, pulmonary artery; PAL, palate; TY, thymus; BA, brachiocephalic artery; LCC, left common carotid artery; LS, left subclavian artery; RA, right atria; LA, left atria; RV, right ventricle; LV, left ventricle; PV, pulmonic valve; AV, aortic valve.

Figure 3

(A–D) Wnt1;1^KO2^KO (C–D) and Hdac1^F/F;Hdac2^F/F (A–B) embryos at E11.5 (Orange – cranial blood; Blue – gut blood). (E–H) LacZ-stained Wnt1;1^KO2^KO;R26R-LacZ^−/+ (G–H) and Wnt1;1^Het2^Het;R26R-LacZ^−/+ (E–F) embryos at E10.5 (Red –nuchal angle; Blue, first pharyngeal arch; Orange, differential LacZ staining). (I–L) LacZ-stained Wnt1;1^KO2^KO;R26R-LacZ^−/+ (K–L) and Wnt1;1^Het2^Het;R26R-LacZ^−/+ (I–J) embryos at E9.5 (Red, posterior nuchal angle; Black, frontal swelling; Blue, first pharyngeal arch; Orange, differential LacZ staining). (M–O) First pharyngeal arch area (normalized to PHT length; M) and cranial length (normalized to PHT length; N) at E9.5 in Wnt1;1^Het2^Het;R26R-LacZ^−/+ and Wnt1;1^KO2^KO;R26R-LacZ^−/+. Presented as mean (SD) (***, p=0.0001; **, p=0.0076)(Schematic diagram, O). (P–S) Eosin and LacZ stained E9.5 Frontal sections of Wnt1;1^Het2^Het;R26R-LacZ^−/+ (P,R) and Wnt1;1^KO2^KO;R26R-LacZ^−/+ (Q,S) craniofacial morphology at two levels. (T–W) Eosin/LacZ stained (T–U) or α-smooth muscle actin (V–W, SMA) immunofluorescence of Wnt1;1^Het2^Het;R26R-LacZ^−/+ (T,V) and Wnt1;1^KO2^KO;R26R-LacZ^−/+ (U,W) E9.5 posterior pharyngeal arch arteries (2/3/4/6, Second/Third/Fourth/Sixth arch arteries; White arrows, SMA). Scale bars 100μM. PhA, pharyngeal arch; PHT, primitive heart tube; NT, neural tube; SMA, α-smooth muscle actin.

Figure 4

(A–B) Phospho-histone H3 (PHH3) immunostaining within the first pharyngeal arch in Wnt1;1^Het2^Het;R26R-LacZ^−/+ (Ai, Ai′) and Wnt1;1^KO2^KO;R26R-LacZ^−/+ (B, B′) at E9.5 (Black, PHH3⁺). (C) Quantification of PHH3 positive nuclei per 100 nuclei (proliferative index; presented as mean (SD); *, p=0.022). (D–E) PHH3 immunostaining within the neural tube (NT) in Wnt1;1^Het2^Het;R26R-LacZ^−/+ (D, D′) and Wnt1;1^KO2^KO;R26R-LacZ^−/+ (E, E′) embryos at E9.5 (Black, PHH3⁺). (F) Quantification of PHH3 positive nuclei per 100 nuclei (proliferative index; presented as mean(SD); *, p=0.043). (G–H) Cleaved caspase 3 (CC3) immunostaining within the first pharyngeal arch in Wnt1;1^Het2^Het;R26R-LacZ^−/+ (G, G′) and Wnt1;1^KO2^KO;R26R-LacZ^−/+ (H, H′) at E9.5 (Black, CC3⁺). (I) Quantification of CC3 positive nuclei per 100 nuclei (apoptotic index; presented as mean (SD); **, p=0.0038). (J–K) CC3 immunostaining within the NT in Wnt1;1^Het2^Het;R26R-LacZ^−/+ (J, J′) and Wnt1;1^KO2^KO;R26R-LacZ^−/+ (K, K′) at E9.5 (Black, CC3⁺). (L) Quantification of CC3 positive nuclei per 100 nuclei (apoptotic index; presented as mean (SD); *, p=0.028). (M–R) Relative mRNA levels of in E9.5 Wnt1;1^Het2^Het and Wnt1;1^KO2^KO microdissected first pharyngeal arches of Cdkn2b (M, *, p=0.042), Cdkn2c (N, *, p=0.032), Cdkn1a (O, ***, p=0.0008), Cdkn1b (P, *, p=0.035), Tp53 (Q, ***, p=0.0001), and Cdkn1c (R, **, p=0.0073). NT, neural tube.