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.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.     

Misexpression of Gbx2 throughout the mesencephalon by a conditional gain‐of‐function transgene leads to deletion of the midbrain and cerebellum in mice

The mouse homeobox gene, Gbx2, is expressed in discreet domains in the neural tube and plays a key role in forebrain and hindbrain development. Previous studies have demonstrated that mutual inhibition between Gbx2 and Otx2, which are respectively expressed in the anterior and posterior parts of the neural plate, positions the prospective midbrain–hindbrain junction. We describe here a conditional Gbx2 gain‐of‐function transgenic mouse line, Gbx2‐GOF, which expresses Gbx2 and red fluorescence protein, mCherry, upon Cre‐mediated recombination. In the absence of Cre, β‐galactosidase is broadly expressed in mouse embryos and adult brains carrying the transgene. By combining Gbx2‐GOF and En1^Cre knock‐in allele, we activated expression of Gbx2 and mCherry throughout the mesencephalon (mes) and rhombomere 1 (r1). The ectopic expression of Gbx2 causes an anterior shift of the mes/r1 junction at embryonic day 10.5. Interestingly, we found that persistent expression of Gbx2 throughout the mes/r1 region largely abolishes expression of the isthmic organizer gene Fgf8, leading to deletion of the midbrain and cerebellum at later stages. Our data suggest that the juxtaposition of the expression domains of Gbx2 and Otx2 within the mes/r1 area is essential for the maintenance of Fgf8 expression. Furthermore, the Gbx2‐GOF transgenic line is suitable for functional study of Gbx2 during development. genesis 47:667–673, 2009. © 2009 Wiley‐Liss, Inc.     

Region- and cell type-selective expression of the evolutionarily conserved Nolz-1/zfp503 gene in the developing mouse hindbrain

Nolz-1/Zfp503, a zinc finger-containing gene, is a mammalian member of the SP1-related nocA/elb/tlp-1 gene family. Previous studies have shown that Nolz-1 homologs are important for patterning the rhombomeres in zebrafish hindbrain. We therefore studied the expression pattern of Nolz-1 in the developing mouse hindbrain. Nolz-1 mRNA expression was detected in the prospective rhombomere 3, 5 and caudal regions as early as E8.75. After E11.5, Nolz-1-positive cells were organized as distinct cell clusters, and they were largely non-overlapped with either Pax2-positive or Phox2b-positive domains. Most interestingly, we found that Nolz-1 was specifically expressed by Phox2b-negative/Isl1/2-positive somatic motor neurons, but not by Phox2b-positive/Isl1/2-positive branchial and visceral motor neurons, suggesting that Nolz-1 may regulate development of somatic motor neurons in the hindbrain. In addition to be expressed in differentiating post-mitotic neurons, Nolz-1 was also expressed by progenitor cells in the ventricular zone located in the dorsal part of aqueduct and the alar plates of hindbrain, which suggests a regulatory role of Nolz-1 in the germinal zone. Taken together, based on its domain- and cell type-selective pattern, Nolz-1 may involve in regulation of various developmental processes, including regional patterning and cell-type specification and differentiation in the developing mouse hindbrain.     

In situ expression of heat shock proteins,Hsc73, Hsj2 and Hsp86 in the developing tooth germ of mouse lower first molar

This study examined the detailed gene expression pattern of three different heat shock proteins (HSPs), Hsc73, Hsj2, and Hsp86, by means of an in situ hybridization method. Hsc73, Hsj2, and Hsp86 were shown in our previous study to be differentially expressed in the mouse embryonic mandible at day 10.5 (E10.5) gestational age. These HSP genes showed similar expression patterns during development of the mouse lower first molar. HSPs-expressing cells were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5, and then were slightly localized at E12 in an area where the tooth germ of the lower first molar is estimated to be formed. A strong expression of HSPs was observed in the tooth germ at E13.5. At the cap stage, HSPs were expressed in the enamel organ and dental papilla. At the bell stage, HSPs were distinctly expressed in the inner enamel epithelium and dental papilla cells facing the inner enamel epithelial layer, which later differentiate into ameloblasts and odontoblasts, respectively. This study is the first report in which Hsc73, Hsj2, and Hsp86 were distinctly expressed in the developing tooth germ, thus suggesting these HSPs are related to the development and differentiation of odontogenic cells.     

In situ expression of the mitochondrial ATPase6 gene in the developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles on embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to identify genes expressed at its beginning. Adenosine triphosphate synthase subunit a (Atpase6) is one of the highly expressed genes in the E12.0 mandible including tooth germs. In situ hybridization was conducted using the mouse mandibular first molar from E10.5 to E18.0 to determine the precise expression patterns of Atpase6 mRNA in the developing tooth germ. Atpase6 mRNA was strongly expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0 and E13.0. Strong in situ signals were observed in the epithelium at E14.0, and in the enamel organ excluded the area of the primary enamel knot at E15.0. Atpase6 was strongly expressed in the inner enamel epithelium, the adjacent stratum intermedium, and the outer enamel epithelium in the cervical loops from E16.0 to E18.0. In addition, strong Atpase6 signals were coincidently demonstrated in various developing cranio-facial organs. These results suggest that Atpase6 participates in the high energy-utilizing functions of the cells related to the initiation and the development of the tooth germ as well as those of the other cranio-facial organs.     

The expression pattern of Follistatin-like 1 in mouse central nervous system development

Follistatin-like 1 (Fstl1), also named TSC-36 (TGF-β-stimulated clone 36), was first cloned from the mouse osteoblastic MC3T3-E1 cell line and can be up-regulated by TGF-β. To better study the function of Fstl1 during the development of the mouse central nervous system (CNS), we examined Fstl1 expression in the developing mouse CNS, in detail, by in situ hybridization. Our results show that Fstl1 is strongly expressed in the telencephalon, diencephalon, brainstem, limbic system and spinal cord. In the telencephalon, Fstl1 positive cells are mainly located in the ventricular zone (VZ) and the subventricular zone (SVZ); a relatively weak signal was observed in layers II and III of the neocortex at postnatal stages. Fstl1 expression is robust in the developing hippocampus and persists to P20. In the developing diencephalon and hindbrain, abundant Fstl1 signals were also detected in nuclei including the medial habenular nucleus, the medial dorsal nucleus, the cochlear nuclei and so on. In addition, a strong expression of Fstl1 was detected in the thalamencephalic signal center, as well as in the olfactory cortex from E14.5 to P0. Meanwhile, Fstl1 was expressed in the septal area and the cingulate gyrus of the limbic system after birth. A high level of expression was also observed in the ventral horn of the spinal cord. These results indicate that Fstl1 may play an important role during CNS development in the mouse.     

Utility of HoxB2 enhancer‐mediated Cre activity for functional studies in the developing inner ear

The rhombomere 4(r4)‐restricted expression of the mouse Hoxb2 gene is regulated by a 1.4‐kb enhancer‐containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2‐r4‐Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5–E12.5, in the cochleovestibular and facio‐acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2‐r4‐Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected. genesis 47:361–365, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.     

Localization of androgen receptor mRNA-containing cells in avian respiratory-vocal nuclei: An in situ hybridization study

Song development and song pattern formation in oscine songbirds are influenced by steroid hormones such as estrogens and androgens, and the control of vocal pattern generation is mediated via a network of interconnected vocal and respiratory nuclei. The main components of the respiratory part of the network are the expiratory and inspiratory premotor nuclei, known as retroambigualis (RAm) and the rostral ventral respiratory group (rVRG), respectively. These respiratory components play an integral role in song production either by providing the expiratory pulses of air required for each and every song syllable, or by controlling inspiration between syllables in the form of minibreaths, and between phrases for major replenishments of air. Here we analyze the distribution of androgen receptors (AR) and estrogen receptors (ER) in the midbrain and hindbrain of male and female zebra finches, and male canaries and green finches, using in situ hybridization with cRNA probes of the zebra finch AR and ER. ERmRNA was not expressed in any of the respiratory-vocal nuclei of the midbrain or hindbrain, but ARmRNA was expressed in the tracheosyringeal motor nucleus (nXIIts) and in RAm and rVRG. The size of the ARmRNA defined RAm and rVRG was similar in male and female zebra finches, but the size of ARmRNA defined nXIIts was slightly sexual dimorphic. Previously undescribed areas of ARmRNA expression outside the respiratory-vocal network in the brain stem were the nucleus semilunaris and layers 10–12 of the optic tectum. ARmRNA expression in the respiratory-vocal nuclei of adult male songbirds, adult female zebra finches, and juvenile zebra finches suggests that the temporal pattern of learned and unlearned vocalizations is sensitive to androgen-dependent mechanisms mediated by RAm and rVRG. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 865–876, 1997     

High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI, was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut⁺) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His⁺, Mut⁺). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that (10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at a broad pH range of 7.0–10.5 and at a temperature of 30°C or below.     

Expression of Frizzled10 in mouse central nervous system

Frizzled transmembrane proteins (Fzd) are receptors of Wnts, and they play key roles during central nervous system (CNS) development in vertebrates. Here we report the expression pattern of Frizzled10 in mouse CNS from embryonic stages to adulthood. Frizzled10 is expressed strongly at embryonic days E8.5 and E9.5 in the neural tube and tail bud. At E10.5, Frizzled10 is expressed in the forebrain vesicle, the fourth ventricle and the dorsal spinal cord. From E12.5 to E16.5, Frizzled10 expression is mainly observed in the cortical hem/fimbria, the neuroepithelium of the third ventricular zone, midbrain, developing cerebellum, and dorsal spinal cord. At P0, with the exception of expression in the fimbria, Frizzled10 mRNA expression is limited to specific nuclei including the ventral posterior thalamic nucleus (VP) and the dorsal lateral geniculate nucleus (DLG) in the developing thalamus as well as in the proliferative ventricular zone of the developing cerebellum. From P20 to adult, Frizzled10 mRNA is detected only in the internal capsule (ic). Our data show that expression of Frizzled10 is very strong during embryonic development of the CNS and suggest that Frizzled10 may play an essential role in spatial and temporal regulation during neural development.     

Expression of protocadherin-9 and protocadherin-17 in the nervous system of the embryonic zebrafish

In this study we analyzed expression patterns of two δ-protocadherins, protocadherin-9 and protocadherin-17, in the developing zebrafish using in situ hybridization and RT-PCR methods. Both protocadherins were mainly detected in the embryonic central nervous system, but each showed a distinct expression pattern. Protocadherin-9 message (Pcdh9) was expressed after 10 h post fertilization (hpf). It was found mainly in small clusters of cells in the anteroventral forebrain and ventrolateral hindbrain, and scattered cells throughout the spinal cord of young embryos (24 hpf). Pcdh9 expression in the hindbrain was segmental, reflecting a neuromeric organization, which became more evident at 34 hpf. As development proceeded, Pcdh9 expression increased throughout the brain, while its expression in the spinal cord was greatly reduced. Pcdh9 was also found in the developing retina and statoacoustic ganglion. Protocadherin-17 message (Pcdh17) expression began much earlier (1.5–2 hpf) than Pcdh9. Similar to Pcdh9 expression, Pcdh17 expression was found mainly in the anteroventral forebrain at 24 hpf, but its expression in the hindbrain and spinal cord, confined mainly to lateroventral regions of the hindbrain and anterior spinal cord, was more restricted than Pcdh9. As development proceeded, Pcdh17 expression was increased both in the brain and spinal cord: detected throughout the brain of two- and three-day old embryos, strongly expressed in the retina and in lateral regions of spinal cord in two-day old embryos. Its expression in the retina and spinal cord was reduced in three-day old embryos. Our results showed that expression of these two protocadherins was both spatially and temporally regulated.