MicroRNAs in Palatogenesis and Cleft Palate

Abstract

Palatogenesis requires a precise spatiotemporal regulation of gene expression, which is controlled by an intricate network of transcription factors and their corresponding DNA motifs. Even minor perturbations of this network may cause cleft palate, the most common congenital craniofacial defect in humans. MicroRNAs (miRNAs), a class of small regulatory non-coding RNAs, have elicited strong interest as key regulators of embryological development, and as etiological factors in disease. MiRNAs function as post-transcriptional repressors of gene expression and are therefore able to fine-tune gene regulatory networks. Several miRNAs are already identified to be involved in congenital diseases. Recent evidence from research in zebrafish and mice indicates that miRNAs are key factors in both normal palatogenesis and cleft palate formation. Here, we provide an overview of recently identified molecular mechanisms underlying palatogenesis involving specific miRNAs, and discuss how dysregulation of these miRNAs may result in cleft palate.

Keywords: cleft palate; genetics; miRNA; palatogenesis; post-transcriptional regulation.

Figure 1

Palatogenesis. Illustration of the developing secondary palate (highlighted in green) through frontal sections of a mouse embryo with the timing of the relevant stages below. MxP, maxillary process; MdP, mandibular process; NS, nasal septum; T, tongue; highlighted in green: palatal shelves.

Figure 2

MiRNA biogenesis. Mature miRNAs are encoded in the genome and form after a series of enzymatic cleavages from two possible precursor molecules; primary miRNAs (pri-miRNAs) or Mirtrons. Pri-miRNAs, following the canonical pathway, are transcribed as long hairpin RNAs that are recognized by the RNA-binding DiGeorge syndrome critical region 8 protein (DGCR8). Many pri-miRNAs are often transcribed simultaneously due to clustering of several miRNA genes (Ambros et al., 2003). DGCR8 then directs the RNase III endonuclease DROSHA to cleave the base of the hairpin to produce ~70 nucleotide hairpins known as pre-miRNA. Mirtrons, following the non-canonical pathway, bypass the microprocessor as they are transcribed as part of the introns of protein coding genes and are as such spliced by the spliceosome (Berezikov et al., 2007). Splicing also produces ~70 nucleotide hairpins known as pre-miRNA. The pre-miRNA is transported to the cytoplasm by exportin 5 where it is cleaved by another RNase III endonuclease known as DICER to ~20 nucleotide miRNA duplexes with protruding 2 nucleotide 3′ ends. The resulting mature miRNA is released and a guiding strand is incorporated into the RNA-induced silencing complex (RISC).

Figure 3

MiRNA involvement in congenital disease. Alterations affecting miRNA activity by changing target recognition or modulating their expression. ^*Single nucleotide polymorphisms (SNPs) within miRNAs are likely involved in complex disease (common disease, common variant hypothesis). ¹Environmental factors can directly or indirectly regulate miRNA expression independent of any germline miRNA alteration (Zhao et al., 2008). ²A SNP/mutation within the miRNA seed sequence can alter both its processing and target recognition, while a change outside the seed sequence only alters miRNA processing (Duan et al., 2007). ³ Large copy number variants lead to syndromes while subtle ones (those only detectable via molecular methods) are predicted to be involved in complex diseases (Shelling and Ferguson, 2007). ⁴Germline alterations of regulators belonging to one of the two miRNA biogenesis/ processing pathways (i.e., the pathways involving mature miRNA generation from pri-miRNAs or Mirtrons, see Figure 2) will only change the expression level of mature miRNAs being generated through this pathway (Finnegan and Pasquinelli, 2013). Epigenetic changes in this context refer to functional changes without a change in the DNA sequence, such as methylation and histone modification.

Figure 4

Homozygous conditional deletion of Dicer in neural crest derived mesenchyme and oral ectoderm. Coronal sections of E18.5 wild-type and Wnt1-Cre; Dicer^f/f (a,b) and E16.5 wild type and Pitx2-Cre; Dicer^f/f (c,d). Black arrow: (left) palate. (a,b) Adapted from Nie et al. (2011). (c,d) Adapted from Cao et al. (2010). pa, palate; tb, tooth bud; Mc, Meckel's cartilage; To, tongue.