Conserved expression of the PRELI domain containing 2 gene (<Emphasis Type="Italic">Prelid2</Emphasis>) during mid-later-gestation mouse embryogenesis

Prelid2, which belongs to the PRELI domain containing family, is identified as a conserved evolution gene. The expression and regulation during embryonic development of the prelid2 gene is unknown. In this study, we investigated the prelid2 gene expression and regulation using mouse embryos model, by in situ hybridization analysis, RT-PCR and bisulfite sequencing. In situ hybridization analysis showed that prelid2 gene expression were found in midbrain, spinal cord, optic eminence, otic vesicle and tail at E9.5 and E10.5 embryos, in forebrain, hindbrain, heart, lung, liver and kidney at E13.5 and E15.5 embryos. Real-time quantitative RT-PCR results verified the expression pattern in the four major mouse organs, brain, heart, lung, and liver during organs differentiation and formation. Bisulfite sequencing illustrated the consistent result of expression and its unmethylation status in the genomic promoter region at E12.5, E18.5, and new born. Thus, the prelid2 gene is a widely-spread, persistently expressed and unmethylated gene in mouse embryonic development. Our results suggest that the PRELI domain containing 2 gene is involved in mouse embryonic development.     

Evolutionarily Conserved, “Acatalytic” Carbonic Anhydrase-Related Protein XI Contains a Sequence Motif Present in the Neuropeptide Sauvagine: The Human CA-RP XI Gene (CA11) Is Embedded between theSecretorGene Cluster and theDBPGene at 19q13.3

Conserved amino acid motifs are found in numerous expressed genes. Proteins and peptides with functional relationships may be identified using probes designed to hybridize with these motifs. An oligonucleotide probe was prepared to match the sequence of the expected active region of a frog corticotropin-releasing factor-like peptide sauvagine and used to screen a sheep brain cDNA library. A novel 1331-bp cDNA encoding a putative 328-residue protein with a theoretical mass of 36 kDa was identified. The presence of a strong signal sequence indicates that it is a secreted protein. The amino- and carboxy-terminal regions are characterized by several potential phosphorylation sites and binding motifs, suggesting a role in intracellular signal transduction. Although the protein possesses a 7-residue sequence identical to that found in sauvagine, its overall primary structure most closely resembles those of the α-carbonic anhydrases (α-CAs). Moreover, the detection of the human and mouse orthologues in the EST databases, together with an evolutionary analysis, indicates that the protein represents a new member of the α-CA gene family, which we designate carbonic anhydrase-related protein XI (CA-RP XI), encoded byCA11(human) andCar11(mouse, rat). The humanCA11gene appears to be located between the secretor type α(1,2)-fucosyltransferase gene cluster (FUT1–FUT2–FUT2P) and the D-site binding protein gene (DBP) on chromosome 19q13.3. Despite potentially inactivating changes in the active-site residues, CA-RP XI is evolving very slowly in mammals, a property indicative of an important function, which has also been observed in the two other “acatalytic” CA isoforms, CA-RP VIII and CA-RP X, whose functions are unknown.     

Carbonic anhydrases in the mouse harderian gland

The harderian gland is located within the orbit of the eye of most terrestrial vertebrates. It is especially noticeable in rodents, in which it synthesises lipids, porphyrins, and indoles. Various functions have been ascribed to the harderian gland, such as lubrication of the eyes, a site of immune response, and a source of growth factors. Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyse the reaction \( {\text{CO}}_{2} + {\text{H}}_{2} {\text{O}} \Leftrightarrow {\text{H}}^{ + } + {\text{HCO}}_{3}^{ - } \). They are involved in the adjustment of pH in the secretions of different glands. Thirteen enzymatically active isozymes have been described in the mammalian α-CA family. Here, we first investigated the mRNA expression of all 13 active CAs in the mouse harderian gland by quantitative real-time PCR. Nine CA mRNAs were detectable in the gland. Car5b and Car13 showed the highest signals. Car4, Car6, and Car12 showed moderate expression levels, whereas Car2, Car3, Car7, and Car15 mRNAs were barely within the detection limits. Immunohistochemical staining was performed to study the expression of Car2, Car4, Car5b, Car12, and Car13 at the protein level. The epithelial cells were intensively stained for CAVB, whereas only weak signal was detected for CAXIII. Positive signals for CAIV and CAXII were observed in the capillary endothelial cells and the basolateral plasma membrane of the epithelial cells, respectively. This study provides an expression profile of all CAs in the mouse harderian gland. These results should improve our understanding of the distribution of CA isozymes and their potential roles in the function of harderian gland. The high expression of mitochondrial CAVB at both mRNA and protein levels suggests a role in lipid synthesis, a key physiological process of the harderian gland.     

Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures

Brain carbonic anhydrases (CAs) are known to modulate neuronal signalling. Using a novel CA VII (Car7) knockout (KO) mouse as well as a CA II (Car2) KO and a CA II/VII double KO, we show that mature hippocampal pyramidal neurons are endowed with two cytosolic isoforms. CA VII is predominantly expressed by neurons starting around postnatal day 10 (P10). The ubiquitous isoform II is expressed in neurons at P20. Both isoforms enhance bicarbonate‐driven GABAergic excitation during intense GABA_A‐receptor activation. P13–14 CA VII KO mice show behavioural manifestations atypical of experimental febrile seizures (eFS) and a complete absence of electrographic seizures. A low dose of diazepam promotes eFS in P13–P14 rat pups, whereas seizures are blocked at higher concentrations that suppress breathing. Thus, the respiratory alkalosis‐dependent eFS are exacerbated by GABAergic excitation. We found that CA VII mRNA is expressed in the human cerebral cortex before the age when febrile seizures (FS) occur in children. Our data indicate that CA VII is a key molecule in age‐dependent neuronal pH regulation with consequent effects on generation of FS.     

Molecular Cloning,Expression and Chromosomal Localization of Mouse <Emphasis Type="Italic">MM-1</Emphasis>

The protooncogene product Myc associates with many proteins. The isolation of the mouse MM-1; c-Myc binding protein (Myc-Modulator 1) cDNA is described. The cDNA contains a 462 bp open reading frame that encodes a polypeptide of 154 amino acid residues. The deduced amino acid sequence indicates that mouse MM-1 has a 99% identity with the sequence of human MM-1. The expression of mouse MM-1 mRNA was detected in the fetal liver, but its level was 3-fold higher than that in the normal adult liver, and was slightly increased after a partial hepatectomy. It is expressed widely in a variety of adult mouse tissues. Thus, MM-1 may play a role in liver development and growth. A bioinformatics analysis indicates that mouse MM-1 gene consists of 6 exons. Furthermore, the chromosomal location of the mouse MM-1 gene was on the F2-F3 band of chromosome 15, as determined by fluorescence in situ hybridization. The nucleotide sequence data reported in this paper appear in DDBJ, EMBL, and the GenBank nucloetide sequence databases with the following accession number, AF108357.     

Ash2l-1调控小鼠卵黄囊早期造血

作为甲基转移酶MLL/SET1复合体的核心成分之一,ASH2L能够促进组蛋白H3K4me3修饰的形成,并在小鼠早期胚胎发育过程中行使重要功能.在小鼠中,由于启动子的选择性使用,Ash2l会转录成两种不同长度的转录本并形成两种蛋白质亚型:ASH2L-1和ASH2L-2.目前有关该基因在小鼠胚胎发育中的作用机制及不同亚型的功能还不清楚.本文利用CRISPR/Cas9技术特异敲除Ash2l-1并研究该亚型的生理学功能.研究结果发现,当Ash2l-1缺失时,小鼠胚胎在E9.5~E10.5时发生致死.特别是Ash2l-1-/-E9.5胚胎的卵黄囊血管和早期造血发育存在明显缺陷.转录组测序结果显示,Ash2l-1的缺失影响红细胞发育和成熟、血管发生和形成相关基因的表达.H3K4me3的CUT&RUN结果显示,在一些表达下调关键基因的启动子区,H3K4me3修饰水平出现下降.以上结果表明,Ash2l-1在小鼠卵黄囊的早期造血和血管形成过程中是必不可少的,它可能是通过调控关键基因启动子区的H3K4me3修饰水平而控制这些基因的表达,从而在相关过程中行使功能.     

Cat-2 gene expression

Summary Poly(A)⁴ RNA was isolated from maize scutella of different stages of post-germinative development and translated in vitro in a rabbit reticulocyte translation system. Immunoprecipitation of the translation products with CAT-2-specific antibody was used to quantitate the relative levels of translatable CAT-2 mRNA at each stage. The results show a close correlation between the developmental profile of Cat2 gene expression and the profile of CAT-2 mRNA levels. Evidence that the levels of CAT-2 mRNA are regulated by a temporal regulatory gene (Car1) is presented and the possible mechanism(s) of this regulation discussed.This work was supported by Research Grants No. GM22733 and No. GM33817 from the U.S. National Institutes of Health, Public Health Service to J.G.S. This is paper No. 9933 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, USA     

Expression of smoothened in mouse embryonic maxillofacial development

Abstract

Hedgehog (Hh) signaling plays many key roles in the development of Drosophila and vertebrate embryos including regulation of craniofacial development. The seven-transmembrane protein, smoothened (Smo) transduces the Hh signal across the plasma membrane as an essential receptor of PTCHED1/2. There are few studies that evaluate the detailed expression of Smo in mouse embryonic craniofacial development. We investigated the expression patterns of Smo during murine embryonic craniofacial development using in situ hybridization (ISH), studies of whole-mounts and sections, immunohistochemistry, quantitative real time PCR, and Western blot analysis. We found that Smo mRNA was expressed in the face of mouse embryos at 11 and 12.5 days post coitum (dpc). After 13.5 dpc, the expression decreased to a low level and was faintly detected after birth. Smo protein could be detected also in embryos at 11, 12.5, and 14.5 dpc. After 15.5 dpc, the expression was very faint and paralleled the gene expression studies. No expression was detected in whisker follicle during facial development and faint signal was detected in Meckel's cartilage. These findings concerning Smo expression should guide further investigation of sonic Hh signaling pathway gene function during maxillofacial development.     

Genomic organization and expression analysis of mouse kynurenine aminotransferase II, a possible factor in the pathophysiology of Huntington's disease

Decreased levels of the endogenous neuroprotectant kynurenic acid (KYNA) have been observed in the brain of Huntington's Disease (HD) patients and may be related to neuronal loss in this disorder. This reduction may be caused by a dysfunction of kynurenine aminotransferase II (KAT II), the major enzyme responsible for the synthesis of KYNA in the brain. Towards understanding the role of KAT II in HD, we isolated and characterized the cDNA sequence and determined the genomic organization of mouse KAT II (mKat-2). The full length mKat-2 cDNA is 1812 bp, encoding 425 amino acids, and shares 89.9% amino acid similarity with the rat Kat-2 sequence. The gene for mKat-2 is composed of 13 exons divided by 12 intronic sequences. Northern blot analysis demonstrated that mKat-2 mRNA is mainly expressed in kidney and liver. RT-PCR showed mKat-2 expression in the brain starting from at least d11 of embryonic development. An alternative isoform mKat-2β, derived from the usage of novel exons, shows a different expression pattern from mKat-2. Western blot analysis of various mouse tissues shows a 40-kDa protein in brain, heart, kidney, and liver. In the kidney and liver an additional 45-kDa isoform was detected. Use of the BSS chromosomal mapping panel from The Jackson Laboratory indicates that the mKat-2 gene co-segregates with polymorphic markers D8Mit129 and D8Mit128 on mouse Chr 8. Knowledge of the genomic organization, the isoform tissue-specific expression patterns, the chromosomal localization of mKat-2, and the reagents generated here, will provide the tools for further studies and allow generation and characterization of mice that are nullizygous for mKat-2. Received: 5 March 1999 / Accepted: 18 May 1999     

Partial Structure, Chromosome Localization, and Expression of the MouseGirk4Gene

The G protein-gated potassium channel I_KAChconstitutes part of a signaling pathway that mediates the negative chronotropic and inotropic effects of acetylcholine on cardiac physiology. Similar or identical ion channels regulate the excitability of many neurons in response to neurotransmitters. I_KAChis composed of two homologous subunits, GIRK1 and GIRK4. Here we describe a partial genomic structure of the mouseGirk4gene. Two exons containing the complete protein-coding sequence were identified.Girk4was mapped to mouse chromosome 9 (13 cM), consistent with the mapping of humanGIRK4to chromosome 11q23–ter. GIRK4 mRNA was found mainly in mouse heart, with trace levels detected in brain, kidney, lung, and spleen. No detectable levels were observed in skeletal muscle, liver, and testis. The onset of GIRK4 mRNA expression in the developing mouse occurs between Embryonic Days 7 and 11, consistent with the appearance and function of the mouse heart.     

Expression of carbonic anhydrases II, IV, VII, VIII and XII in rat brain after kainic acid induced status epilepticus

Carbonic anhydrases (CAs) are important enzymes in the central nervous system (CNS), where they participate in regulating cerebrospinal fluid (CSF) secretion, blood-brain barrier and glial cell function. Using RT-PCR we found CA XII mRNA in rat and mouse brain. Cloning of rat CA XII revealed 94% homology with the mouse CA XII. To map the putative functional roles of different CAs, we studied the expression and localization of CA II, CA IV, CA VII, CA-related protein (CA-RP) VIII and CA XII mRNAs in rat brain after kainic acid induced epileptic seizures using Northern blot analysis and in situ hybridization. The expression of CA IV, CA VII and CA-RP VIII was somewhat similar: they were expressed in the cortex, hippocampus and midbrain structures and their expression did not change after the kainic acid treatment. The expression of CA II was concentrated in the white matter structures, which is in line with the preferential expression of CA II in the oligodendrocytes. High levels of CA II mRNA were also detected in the choroid plexus. Surprisingly, CA II was induced 3-12 h after seizures in the vulnerable CA1 region. CA XII was expressed in dentate granule cells, cortex and choroid plexus. Kainic acid stimulated CA XII expression throughout the cortical layer I. The observed hippocampal induction of CA II may indicate a pro-apoptotic and/or epileptogenic role of CA II after prolonged seizures. The physiological significance of the observed cortical induction of CA XII remains obscure. Cytosolic CA II is known to participate in CSF secretion, and the high expression of CA XII in the choroid plexus suggests an analogous role for this membrane-bound isozyme.     

Characterization,tissue distribution of resistin gene and the effect of fasting and refeeding on resistin mRNA expression in Siberian sturgeon (Acipenser baerii)

Resistin as an adipokine identified from rodents in 2001 is involved in many biological processes. However, little is known about this gene in fish. We cloned Siberian sturgeon (Acipenser baerii) resistin cDNA of 795 base pairs, encoding 107 amino acids, which showed 38–40% identity to human and rodents. Real-time quantitative PCR showed that the resistin gene was widely distributed in tissues of Siberian sturgeon, with the highest expression in liver. After fasting for 1, 3, 6 and 10 days, the expression of the resistin gene in the liver of Siberian sturgeon decreased significantly, and after refeeding on the 10 days of fasting the resistin mRNA expression increased rapidly, suggesting that resistin may play an important role in liver in response to starvation. Taken together, these results suggest that resistin may be involved in the regulation of energy homeostasis in liver.     

Capn8 promoter directs the expression of Cre recombinase in gastric pit cells of transgenic mice

Gastric pit cells are high‐turnover epithelial cells of the gastric mucosa. They secrete mucus to protect the gastric epithelium from acid and pepsin. To investigate the genetic mechanisms underlying the physiological functions of gastric pit cells, we generated a transgenic mouse line, namely, Capn8‐Cre, in which the expression of Cre recombinase was controlled by the promoter of the intracellular Ca²⁺‐regulated cysteine protease calpain‐8. To test the tissue distribution and excision activity of Cre recombinase, the Capn8‐Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4^Co/Co). Multiple‐tissue PCR and LacZ staining demonstrated that Capn8‐Cre transgenic mouse expressed Cre recombinase in the gastric pit cells. Cre recombinase activity was also detected in the liver and skin tissues. These data suggest that the Capn8‐Cre mouse line described here could be used to dissect gene function in gastric pit cells. genesis 47:674–679, 2009. © 2009 Wiley‐Liss, Inc.     

Embryonic Expression of Human Keratin 18 and K18-β-Galactosidase Fusion Genes in Transgenic Mice

During embryogenesis, EndoB, the mouse form of human keratin 18 (K18), is expressed in a complex spatial and temporal pattern in various embryonic epithelia. We have compared the expression of transgenic human K18 to the endogenous mouse homolog and to the coexpressed, complementary keratin 8 homolog, EndoA, during postimplantation mouse embryogenesis and fetal development in order to determine the developmental expression pattern of the human gene in a mouse environment. The tissue distribution of K18 protein was identical to that of endogenous EndoB in both 7.5- and 13.5-day-old embryos, except for certain heart, eye, and extraembryonic mesodermal tissues in which K18 was not detected. These results indicate that the 10-kb K18 gene specifies appropriate developmental expression in the mouse and support previously reported differences in K18 expression in human and mouse fetal heart. We have also compared the expression patterns of K18 to a series of constructions that utilize the Escherichia coli gene for β-galactosidase (lacZ) as a reporter gene. Some of these constructions were regulated correctly in embryos during development of the germ layers. However, none was expressed consistently in extraembryonic or in adult tissues. Analysis with methylation-sensitive restriction enzymes revealed that hypermethylation of the CpG-rich prokaryotic reporter gene was not the cause of its silence in adult transgenic liver. However, the repressed state of K18-LacZ transgenes in adult liver was correlated with a different chromatin state that lacked diagnostic DNase hypersensitive sites found in K18 transgenic liver. Expression of the lacZ reporter gene did not accurately reflect the developmental pattern of K18 even in constructions that used all available K18 sequences. We conclude that in these contexts, the lacZ gene was not a developmentally neutral reporter gene.