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. 2023 Jan 6:13:1039850.
doi: 10.3389/fgene.2022.1039850. eCollection 2022.

Transcriptomic analysis of the upper lip and primary palate development in mice

Sini Cai  1 , Nuo Si  2 , Yanyang Wang  1 , Ningbei Yin  1
Affiliations

Transcriptomic analysis of the upper lip and primary palate development in mice

Sini Cai et al. Front Genet. .

Abstract

Background: Normal fusion of the upper lip and primary palate is a complex process involving a series of characteristic and orderly regulated cellular events. Cleft lip with or without palate (CL/P), one of the most common congenital malformations, may be induced by abnormalities in any of these events. However, less is known about the precise regulatory process in the fusion of the upper lip and primary palate. Methods: Lambdoidal junction tissues of mice from embryonic days 10.5, 11.5, and 12.5- three key fusion stages-were acquired for RNA sequencing. Results: Gene expression profiles in distinct fusion stages of mice were identified. Some of the differentially expressed genes (DEGs) have been reported to affect upper lip and primary palate development. However, other DEGs, such as Krt5, Pax1, Ambn, Hey2, and Tnmd, have not previously been investigated. Gene set enrichment analysis (GSEA) of these DEGs revealed the sequential intensification of Wnt, PI3K-Akt, MAPK, Hippo, and TGF-beta signaling pathways and identified relatively highly expressed genes including Tnn, Wnt3a, and Wnt16. We also observed substantial alternative splicing events during the fusion of the upper lip and primary palate and identified potentially important genes including Gtpbp8, Armcx1, Tle3, and Numa1. Protein-protein interaction (PPI) network analysis identified a series of hub genes, including Col1a2, Fos, Bmp2, Shh, Col1a1, Wnt3a, Anxa1, Gem, etc. Conclusion: Overall, the results of this study provided a comprehensive analysis of the development of the upper lip and primary palate. Our work provides insight into future studies of normal upper lip and primary palate development and the etiology of CL/P.

Keywords: RNA sequencing; cleft lip with or without cleft palate; development; gene expression profile; upper lip and primary palate.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Transcriptomic changes during upper lip and primary palate development. (A) Lambdoidal junction tissues of mice at embryonic days 10.5, 11.5, and 12.5. (B) Histogram showing upregulated and downregulated DEGs for comparison groups E11.5 vs. E10.5, E12.5 vs. E10.5, and E12.5 vs. E11.5, respectively. (C) Volcano plots showing upregulated and downregulated DEGs in comparison group E11.5 vs. E10.5 (upper), E12.5 vs. E11.5 (middle), and E12.5 vs. E10.5 (lower), respectively. (D) Gene ontology analysis of upregulated DEGs in comparison group E11.5 vs. E10.5 (upper), E12.5 vs. E10.5 (middle), and E12.5 vs. E11.5 (lower), respectively. Circle size: gene number; bar length: range of p values. (E) Quantitative PCR validation of representative genes. *p < .05.
FIGURE 2
FIGURE 2
RNAScope validation of development-associated upregulated or downregulated DEGs. (A) Venn diagram showing the amount of shared upregulated or downregulated DEGs among comparison group E11.5 vs. E10.5, E12.5 vs. E11.5, and E12.5 vs. E10.5. (B) Heatmap showing the top ten shared upregulated or downregulated DEGs among E10.5, E11.5, and E12.5. Color key: expression levels, (C) GO terms for shared upregulated (left) and downregulated (right) DEGs from E10.5 to E12.5. (D) RNAScope analysis of the location and expression levels of candidate genes among E10.5, E11.5, and E12.5. MxP: maxillary prominence; LNP: lateral nasal; MNP: medial nasal.
FIGURE 3
FIGURE 3
GSEA analysis of representative significant signal pathways. Gene set enrichment plots of the (A) Wnt, (B) PI3K-AKT, (C) MAPK, (D) Hippo, and (E) Tgf-beta signaling pathways among comparison groups E11.5 vs. E10.5 (left), E12.5 vs. E11.5 (middle), and E12.5 vs. E10.5 (right), respectively. Green curve: enrichment score; vertical red or blue bars: gene positions.
FIGURE 4
FIGURE 4
Signal-gene interaction network. DEGs and significant signal pathways playing essential roles during upper lip and primary palate development among comparison groups (A) E11.5 vs. E10.5, (B) E12.5 vs. E11.5, and (C) E12.5 vs. E10.5 were chosen, and profiled by constructing signal-gene networks, respectively. Inner nodes: signal pathways; surrounding nodes: genes; lines between inner and surrounding nodes: interactions between signaling pathways and genes.
FIGURE 5
FIGURE 5
xpression levels of representative genes from the signal-gene network detected by immunofluorescence in lambdoidal junction tissues of mice at embryonic days 10.5, 11.5, and 12.5. (A) Expression levels of wnt3a, col6a1, and inhba between E10.5 and E11.5. (B) Expression levels of wnt16, fgf18, and chrm1 between E12.5 and E11.5. (C) Expression levels of gdf7, inhba, and ngf between E12.5 and E10.5. MxP: maxillary prominence; LNP: lateral nasal; MNP, medial nasal.
FIGURE 6
FIGURE 6
Changes in alternative splicing events in lambdoidal junction tissues of mice at embryonic days 10.5, 11.5, and 12.5. Development-associated changes in alternative splicing events among comparison groups (A) E11.5 vs. E10.5, (B) E12.5 vs. E11.5, and (C) E12.5 vs. E10.5. Red: changes of △Inc level that first increased and then decreased; blue: △Inc level first decreased and then increased; orange: △Inc level of Numa1 persistently upregulated from E10.5 to E12.5. (D) RT-PCR validation to demonstrate the expression levels of Numa1 from E10.5 to E12.5. IncLevel Difference (△IncLevel) = IncLevel1 − IncLevel2; IncLevel1 = the expression of exon inclusion isoform/the expression of two isoforms (in experimental group 1), IncLevel2 = expression of exon inclusion isoform/expression of two isoforms (in experimental group 2).
FIGURE 7
FIGURE 7
ene ontology analysis showing the top five ranked terms for alternatively spliced genes among comparison groups (A) E11.5 vs. E10.5, (B) E12.5 vs. E11.5, and (C) E12.5 vs. E10.5.
FIGURE 8
FIGURE 8
PPI network of developmentally related DEGs. PPI network of DEGs among comparison groups (A) E11.5 vs. E10.5, (B) E12.5 vs. E11.5, and (C) E12.5 vs. E10.5 constructed in Cytoscape. The top 10 hub nodes (genes) are marked. The numbers in parentheses next to the genes indicate the node degrees.
FIGURE 9
FIGURE 9
xpression levels of representative genes from the PPI network detected by immunofluorescence in lambdoidal junction tissues of mice at embryonic days 10.5, 11.5, and 12.5. (A) Expression levels of col1a2 at E10.5, E11.5, and E12.5. (B) Expression levels of Fos between E11.5 and E12.5. MxP: maxillary prominence; LNP: lateral nasal; MNP: medial nasal.
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