Analysis of Uncharacterized mKiaa1211 Expression during Mouse Development and Cardiovascular Morphogenesis

Abstract

Mammalian Kiaa1211 and Kiaa1211-like are a homologous pair of uncharacterized, highly conserved genes cloned from fetal and adult brain cDNA libraries. Herein we map the in utero spatiotemporal expression of mKiaa1211 and mKiaa1211L mRNA and their expression patterns in postnatal testis, skin, gastrointestinal, and adipose progenitor tissues. Significantly, mKiaa1211 is present throughout the early stages of mouse heart development, particularly in the second heart field (SHF) lineage as it differentiates from mesenchymal cells into cardiomyocytes. We also show that mKiaa1211 is expressed within several early neuronal tissues destined to give rise to central, peripheral, and sympathetic nervous system structures. Expression profiling revealed that the paralog mKiaa1211L is not expressed during the normal developmental process and that mKiaa1211 expression was noticeably absent from most adult terminally differentiated tissues. Finally, we confirm that a previously uncharacterized CRISPR/CAS-generated mKiaa1211 mouse mutant allele is hypomorphic.

Keywords: hypomorph; mKiaa1211; mKiaa1211-like; mRNA expression; mouse heart morphogenesis; neural development; postnatal progenitor tissues; testis.

Figure 1

Phylogenetic, genomic structure and comparative qPCR analysis of uncharacterized mKiaa1211 and mKiaa1211L genes. (A) Based on NCBI Reference Sequence similarity, mKiaa1211 (originally called C530008M17Rik) and Kiaa1211L mouse paralogs, along with human and rat orthologs were aligned and a phylogenetic tree was constructed without distance corrections. Chromosomal locations, NCBI Reference Sequence numbers, and the percent identity matrix (created by Clustal2.1) are shown. (B) Ensemble schematic of mouse mKiaa1211 6835 bp transcript, with 9 exons (boxes) of 15 total shown. Lines connecting the boxes are introns. Filled boxes are coding sequence, and empty, unfilled boxes are the 3′ untranslated region. The site of the mKiaa1211 in situ hybridization probe used in Figure 2, Figure 3, Figure 4 and Figure 5 is indicated. (C–E) Quantitative PCR analysis of mKiaa1211 mRNA levels during development (C), in developmentally staged isolated hearts (D), and in separate atrial and ventricular heart chambers at E12.5, newborn, and adult stages (E). (F) Comparison of mKiaa1211 and mKiaa1211L (indicated via *) mRNA expression levels in isolated E11.5 hearts (25.8 vs. 32.4 median cycles) and adult testis (23.6 vs. 31.1 median cycles) but roughly equivalent low levels in adult lungs. qPCR data are presented as a logarithmic plot of relative expression, where a value of 1 indicates no difference between E13.5 whole embryo in (C), E12.5 isolated heart in (D), and newborn atria in (E); and values <1 indicate reduced and >1 indicate increased expression. Error bars represent SD.

Figure 2

Developmental analysis of mKiaa1211 expression. (A) Non-radioactive in situ hybridization detection using digoxigenin (DIG)-labeled mKiaa1211 antisense cDNA probe on E8 transverse sections revealed mRNA expression (purple/blue stain) in neural folds (nf) and second heart field (SHF)/first branchial arch/outflow tract region (arrows), where the primitive tubular heart attaches to the pharyngeal foregut (fore). (B) At E9, mKiaa1211 is present in sagittal sections in the neural tube (nt) and fore- and hindbrain, as well as in the branchial arch/second heart field region (arrow). (C) Similarly, mKiaa1211 is present in the sagittal E14 section neural tube, hindbrain, and brain, as well as the dorsal root ganglia (drg). (D–K) mKiaa1211 is expressed throughout the transverse E10 (D), E11 (E), and E12.5 neural tubes (F), except in the floor plate (arrowhead (D,F)); but is lost in the fetal and newborn (Nb) marginal layers (*H,I) and roof plate (arrowhead (I)) of the central nervous system spinal cord. In newborn sagittal brain, mKiaa1211 is present in cortex (cx), olfactory bulb (ob), and hippocampus (arrowhead (K)). mKiaa1211 is also expressed as the dorsal root ganglia are formed (E11–birth), and during morphogenesis of tyrosine hydroxylase (TH)-positive ganglia (G) of the sympathetic nervous system ((F–J) arrows). mKiaa1211 is detectable in the embryonic sinus venosus (* F) and fetal vagal (X) trunks (arrowhead (H)), along with some punctate expression in E14.5 lung (H). Scale bars: (A,B,F–H) = 100 µm; (C–E) = 50 µm; (K) = 200 µm. Abbreviations: avc—atrioventricular cushion; 4th—fourth ventricle; fb—forebrain; h—heart; oft—outflow tract; v—heart ventricle.

Figure 3

Analysis of mKiaa1211 cardiovascular expression. (A) Whole mount mKiaa1211 expression is present in E9.5 neural tube (nt), as well as in the SHF (arrow). Additionally, mKiaa1211 is expressed in glossopharyngeal ganglia (arrowhead). Note staining in otic vesicle (*) is artefactual trapping. (B,C) Non-radioactive in situ hybridization (B) and αSMA myocyte marker antibody (C) in adjacent transversely sectioned E10.5 embryo. Note mKiaa1211 and αSMA overlap in the outflow tract/SHF region and right ventricle (rv), specifically in the cardiomyocyte outer layer (arrowheads (B,C)). mKiaa1211 is also expressed in the second branchial arch (2^nd) and in mesenchyme (arrows B) between cardinal veins and αSMA-positive dorsal aorta (arrows (C)). (C) Whole mount mKiaa1211 expression is present in neural tube (nt), as well as in the SHF (arrow). Additionally, mKiaa1211 is expressed in glossopharyngeal ganglia (arrowhead). Note staining in otic vesicle (*) is artefactual trapping. (D) mKiaa1211 is in the E10.5 inflow region, in the walls of the atria (arrowheads) and foregut (fore) endoderm. (E,F) mKiaa1211 is in both left and right horns of the E12.5 sinus venosus (* in (E)), neural tube, sympathetic ganglia (arrows), and dorsal root ganglia (drg) in transverse sections. Similarly, mKiaa1211 is in the sagittal E14 wall of the vena cava (vc) just before it enters the right atrium (arrow (F)). (G,H) Adult atrial mKiaa1211 expression is in a punctate pattern amongst myocytes (arrowheads, (H)) but is absent from overlying endothelial lineage (arrow). (I) Non-radioactive in situ hybridization detection using DIG-labeled (negative control) mKiaa1211 sense cDNA probe did not reveal any specific expression in E10.5 embryos. Scale bars: (B,C) = 50 µm; (D) = 20 µm; (E–H) = 100 µm. Abbreviations: 1st—first branchial arch; a—atria; bw—thoracic body wall; lb—limb bud; lj—lower jaw; ra—right atria; rv—right ventricle; vent—ventricle.

Figure 4

Spatiotemporal analysis of fetal and adult mKiaa1211 expression. (A–C) Non-radioactive in situ hybridization in adult testis revealed very robust expression in both mitotic spermatogonia and meiotic primary spermatocytes Type A and B (that contain dispersed chromatin, arrow (C)). Proliferating cells were identified using phosphohistone H3 (pHH3) immunohistochemistry on adjacent section (C). Spermatids and mature sperm (s) do not express mKiaa1211. (D–H) mKiaa1211 is expressed throughout gastrointestinal tract development, with punctate expression present in transverse E14 mesenchymal cells located in close apposition to the endoderm (arrow (D), which is the intestinal stem cell niche). In newborn (E–G) and adult (H) transverse sections, mKiaa1211 is continually expressed in the crypt stem cell niche (arrow (E,F,H)) prior to their undergoing pHH3+ proliferation (arrow (G)). (I) mKiaa1211 is present in fetal, newborn, and adult skin, with limited expression present in abdominal newborn skin epidermis (epi) but robust expression in the root sheath and connective tissue/bulge (stem cell region, arrows (I)) surrounding the hair follicles but is absent from the dermis and dermal papilla/hair bulb itself. (J,K) mKiaa1211 is also expressed punctately in brown adipose (* in (J)) and fibrous septae (arrow (J)) and in white adipose tissue nuclei (arrowheads (K)). Scale bars: (A,H) = 100 µm; (B–D,J) = 20 µm; (E–G) = 50 µm, (I) = 10 µm. Abbreviations: a—gastrointestinal (GI) adventitia layer; dp—dermal papilla; epi—epithelium; hf—hair follicle; hypo—hypodermis; l—large lipid droplet; m—GI muscularis layer; v—villi.

Figure 5

Analysis of mKiaa1211 mouse mutant phenotype. (A,B) Using CRISPR-generated mKiaa1211 mouse mutant (C530008M17Rik^em1(IMPC)J/J) mice, histology showed that mKiaa1211/mKiaa1211 homozygous adult testis and epididymis were grossly normal (B) when compared to age-matched littermate controls (A). (C,D) Non-radioactive in situ hybridization detection of mKiaa1211 revealed homozygous mutant testis exhibits significantly reduced mRNA levels (arrows (D)) when compared to controls (C), despite similar patterns of expression. (E) Quantitative PCR analysis confirmed that duplicate mKiaa1211/mKiaa1211 homozygous adult testis express ~60% less mKiaa1211 mRNA levels (indicated by *) but that mKiaa1211L levels are unaffected, when compared to age-matched littermate controls. (F,G) Homozygous proliferating spermatogonia (identified using pHH3 antibody) were detected in roughly equal numbers. (H–K) Immunohistochemistry verified that homozygous mutant (I,K) and control (arrows (H,J)) testis express equivalent cytoplasmic Hook1 protein in spermatids, as well as Dazl protein in spermatogonia nucleus. Scale bars: (A,B) = 200 µm; (C,D) = 100 µm; (F–K) = 20 µm. Abbreviations: e—epididymis.