Conditional deletion of the human ortholog gene Dicer1 in Pax2-Cre expression domain impairs orofacial development

Abstract

Background: Orofacial clefts are common worldwide and result from insufficient growth and/or fusion during the genesis of the derivatives of the first pharyngeal arch and the frontonasal prominence. Recent studies in mice carrying conditional and tissue-specific deletions of the human ortholog Dicer1, an RNAse III family member, have highlighted its importance in cell survival, differentiation, proliferation, and morphogenesis. Nevertheless, information regarding Dicer1 and its dependent microRNAs (miRNAs) in mammalian palatogenesis and orofacial development is limited.

Aims: To describe the craniofacial phenotype, gain insight into potential mechanisms underlying the orofacial defects in the Pax2-Cre/Dicer1 CKO mouse, and shed light on the role of Dicer1 in mammalian palatogenesis.

Materials and methods: Histological and molecular assays of wild type (WT) and Pax2-Cre/Dicer1(loxP/loxP) (Dicer1 CKO) mice dissected tissues have been performed to characterize and analyze the orofacial dysmorphism in Pax2-Cre/Dicer1(loxP/loxP) mouse.

Results: Dicer1 CKO mice exhibit late embryonic lethality and severe craniofacial dysmorphism, including a secondary palatal cleft. Further analysis suggest that Dicer1 deletion neither impacts primary palatal development nor the initial stages of secondary palatal formation. Instead, Dicer1 is implicated in growth, differentiation, mineralization, and survival of cells in the lateral palatal shelves. Histological and molecular analysis demonstrates that secondary palatal development becomes morphologically arrested prior to mineralization around E13.5 with a significant increase in the expression levels of apoptotic markers (P < 0.01).

Conclusions: Pax2-Cre-mediated Dicer1 deletion disrupts lateral palatal outgrowth and bone mineralization during palatal shelf development, therefore providing a mammalian model for investigating the role of miRNA-mediated signaling pathways during palatogenesis.

Keywords: Cleft palate; Dicer1; cranial neural crest cells; microRNAs; palatogenesis.

Figure 1

Pax2-Cre-mediated deletion of Dicer1 results in craniofacial abnormalities and secondary palatal cleft. a, c. Frontal and lateral views of WT mice. b, d. Frontal and lateral views of Dicer CKO mouse. Bar = 2mm

Figure 2

X - gal staining in Pax2-Cre/DicerloxP/WT/Rosa26R (a-c) and Dicer1 CKO (d) mice. MHB: mid-hindbrain; PS: palatal shelf; NC: nasal cavity; NS: nasal septum; PP: primary palate; SP: secondary palate. Bar = 50 μm (a, b); 1mm (c, d). Palatal rim delineated by dotted line

Figure 3

In situ hybridization with Dicer1 probe at E12.5. a-b. Head. c-d. Palate. e-f. Brain. HB: hindbrain; MB: midbrain; FB: forebrain; PP: primary palate; PS: palatal shelves; NC: nasal cavity. Bar = 50 μm (a–d); 2 mm (e–f). Palatal rim delineated

Figure 4

Comparative palatal development. a, c, e. WT. b, d, f. Dicer1 CKO. NT: neural tube; MP: mandibular process; PS: palatal shelves; OC: oral cavity; P: secondary palate; NS: nasal septum; T: tongue. Bar = 1mm (a, b); 500 μm (c-f). H and E staining

Figure 5

Dicer1 CKO cranioskeletal staining. NB: nasal bone; PX: premaxilla; MX: maxilla; F: frontal; PA: parietal; Z: zygoma; T: temporal; TR: tympanic ring; M: mandible; IP: intraparietal; EO: exoccipital; V: vomer; P: palatine; PS: presphenoid; BS: basiphenoid; HB: hyoid. Bar = 2 mm

Figure 6

Von Kossa staining showing absence of mineralization in coronal sections of Dicer1 CKO palatal region at E17.5. a. WT. b. Dicer1 CKO. Boxed region is magnified in (c, d). T: tongue; PS: lateral palatal shelves; SP: secondary palate. Bar = 500 μm

Figure 7

EdU (green) and ApopTag (red) staining in coronal sections of E11.5 Dicer1 CKO. a, c. WT. b, d. Dicer1 CKO. High magnification of dotted boxes shown in white inset box. NT: neural tube. Bar = 500 μm (a, b); 20μm (C, D)

Figure 8

EdU (green) and ApopTag (red) staining at later time points. Right. WT. Left. Dicer1 CKO. Inset (d) highlights apoptosis. Boxes in (a, b), (e, f) shown in (c, d), (g, h), respectively. DAPI (blue). T: tongue; PS: palatal shelves; SP: secondary palate. Bar = 500μm

Figure 9

Comparative RT Q-PCR analysis of apoptotic (Caspase 3 and p53) and quiescence (p21) markers in palatal tissue during development. The SDs were within 1% of the mean. ^*P < 0.01; ^**P < 0.001. Exact P values are provided within the results section

Figure 10

RT Q - PCR demonstrates differential, but significant depletion of miR-101b, -140, and -145 in Dicer1 CKO palatal tissue during palate development. The SDs were within 1% of the mean. ^*P < 0.001. Exact P values are provided within the results section