Developmental microRNA expression profiling of murine embryonic orofacial tissue

Abstract

Background: Orofacial development is a multifaceted process involving precise, spatio-temporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs involved in gene silencing, represent critical regulators of cell and tissue differentiation. MicroRNA gene expression profiling is an effective means of acquiring novel and valuable information regarding the expression and regulation of genes, under the control of miRNA, involved in mammalian orofacial development.

Methods: To identify differentially expressed miRNAs during mammalian orofacial ontogenesis, miRNA expression profiles from gestation day (GD) -12, -13 and -14 murine orofacial tissue were compared utilizing miRXplore microarrays from Miltenyi Biotech. Quantitative real-time PCR was utilized for validation of gene expression changes. Cluster analysis of the microarray data was conducted with the clValid R package and the UPGMA clustering method. Functional relationships between selected miRNAs were investigated using Ingenuity Pathway Analysis.

Results: Expression of over 26% of the 588 murine miRNA genes examined was detected in murine orofacial tissues from GD-12-GD-14. Among these expressed genes, several clusters were seen to be developmentally regulated. Differential expression of miRNAs within such clusters wereshown to target genes encoding proteins involved in cell proliferation, cell adhesion, differentiation, apoptosis and epithelial-mesenchymal transformation, all processes critical for normal orofacial development.

Conclusions: Using miRNA microarray technology, unique gene expression signatures of hundreds of miRNAs in embryonic orofacial tissue were defined. Gene targeting and functional analysis revealed that the expression of numerous protein-encoding genes, crucial to normal orofacial ontogeny, may be regulated by specific miRNAs.

Figure 1

Photomicrographs of ventral views of the developing orofacial region of a GD-13 mouse embryo. (A) Upper and lower lips and jaws (maxilla and mandible). (B) The embryonic oral cavity. The lower half of the photo contains the roof of the oral cavity with the maxillary processes, primary palate, and secondary palatal processes; the upper half contains the base/floor of the oral cavity showing the tongue and the mandible. (C) A magnified view of the roof of the oral cavity: note that the upper lip and the primary palate are completely formed, and the developing secondary palatal shelves are derived from the medial aspect of each maxillary process. The region demarcated by the black line was excised from GD-13 embryos for extraction of total RNA. Corresponding regions were dissected from the developing orofacial region of GD-12 and GD-14 embryos. UL = upper lip; LL = lower lip; Mx = maxilla; Md = mandible; P1 = primary palate; P2 = secondary palate; T = tongue. (Reprinted from Mukhopadhyay et al., 2006).

Figure 2

Heat maps (hierarchical clusters) of differentially expressed miRNAs in developing murine orofacial tissue. Heat maps (hierarchical clusters) of all 90 miRNAs found to be significantly differentially expressed (adjusted p value < 0.05) for at least one of the three comparisons between gestational days (GD-13 vs. GD-12, GD-14 vs. GD-12, and GD-14 vs. GD-13; see Tables 2, 3, and 4, respectively) in developing murine orofacial tissue. Each row of the heat map represents a gene, and each column represents a time point in development (as labeled at the bottom; GD-12-1 = Gestation Day 12-sample replicate 1, etc.). Pink indicates an increase in miRNA gene expression (relative to the other expression measurements in the same row), whereas blue indicates a decrease.

Figure 3

Hierarchical cluster analysis illustrating six patterns of expression of microRNAs undergoing significant alteration in expression during murine orofacial development. Gene expression data sets from orofacial tissue from each of the critical days of orofacial development (GD-12, GD-13, and GD-14) were filtered and clustered as detailed in the Methods section. The solid red line on each graph represents the average miRNA expression pattern for the miRNA cluster, and the lighter gray lines of the graph represent the individual expression patterns for each miRNA. The expression pattern for each gene was normalized to mean 0 and SD 1 to better reflect the similarities between patterns based on the correlation measure (the measure used for clustering). The number above each graph indicates the number of miRNAs in the respective cluster. The lists of miRNA genes (90 genes) making up each cluster can be found in tabular form as supplementary material (Supplementary Table 4).

Figure 4

Computational gene interaction predictions: selected microRNA gene network in developing orofacial tissue. A network with selected genes encoding microRNAs was constructed with IPA software. Several differentially regulated microRNA genes from the study were used to construct a gene association map as shown in this figure. Solid lines specify direct relationships, whereas dotted lines indicate indirect interactions.

Figure 5

Computational gene interaction predictions: selected microRNA gene network in developing orofacial tissue. A network with selected genes encoding microRNAs was constructed with IPA software. Several differentially regulated microRNA genes from the study were used to construct a gene association map (Fig. 4) for predicting various cellular and molecular events (as shown in this figure) operative within the developing mouse orofacial tissue. Solid lines specify direct relationships, whereas dotted lines indicate indirect interactions.

Figure 6

Computational gene interaction predictions: selected microRNA gene network in the developing orofacial tissue. A network with selected genes encoding microRNAs was constructed with IPA software. Several differentially regulated microRNA genes from the study were used to construct a gene association map (Fig. 4) for highlighting diverse signaling pathways (as shown in this figure) operative within the developing mouse orofacial tissue. Solid lines specify direct relationships, whereas dotted lines indicate indirect interactions.

Figure 7

Computational gene interaction predictions: gene network with microRNAs demonstrating enhanced expression in developing orofacial tissue (GD-13 vs. GD-12) and their target genes. A network with selected genes encoding microRNAs (orange) with increased expression on GD-13 versus GD-12 developing orofacial tissue and their known or predicted target genes (yellow) critical for orofacial ontogenesis was constructed with IPA software and the miRDB (http://mirdb.org/miRDB/) database. Solid lines specify direct relationships, whereas dotted lines indicate indirect interactions.

Figure 8

Computational gene interaction predictions: gene network with microRNAs demonstrating diminished expression in developing orofacial tissue (GD-13 vs. GD-12) and their target genes. A network with selected genes encoding microRNAs (green) with decreased expression on GD-13 versus GD-12 developing orofacial tissue and their known or predicted target genes (yellow) critical for orofacial ontogenesis was constructed with IPA software and the miRDB (http://mirdb.org/miRDB/) database. Solid lines specify direct relationships, whereas dotted lines indicate indirect interactions.