High sensitivity analysis of gene expression in single embryonic somites using coupled reverse transcription-polymerase chain reaction

Sanzo, James F.; Tuan, Rocky S.

doi:10.1007/bf02752693

Cited by 4 publications

(3 citation statements)

References 18 publications

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“…Hcy treatment per se induced spinal NTD at ages E4 and E6, and in some cases, embryos of the Hcy group showed two or more categories of spinal NTD. FA pretreatment did not always prevent this event, Sanzo and Tuan (1998). because embryos of the FA 1 Hcy group also displayed NTD at both embryonic ages. However, the incidence of NTD was lower in embryos of the FA 1 Hcy group than in the Hcy group.…”

Section: Spinal Cord Morphologymentioning

confidence: 90%

“…RT‐qPCR was performed using HT 7900 Fast Real‐Time PCR System (Applied Biosystems). Specific primer sequences were the same used previously by Sanzo and Tuan () for Pax 1, Pax 9, β‐actin and glyceraldehyde‐3‐phosphate dehydrogenase ( GAPDH ) (Table ). β‐actin and GAPDH were used as housekeeping genes, and the results are provided as level of expression relative to the control.…”

Section: Methodsmentioning

confidence: 99%

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Homocysteine causes disruptions in spinal cord morphology and changes the expression of Pax 1/9 and Sox 9 gene products in the axial mesenchyme

Kobus

Ammar

Nazari

et al. 2013

Birth Defects Research

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Section: Spinal Cord Morphologymentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Homocysteine causes disruptions in spinal cord morphology and changes the expression of Pax 1/9 and Sox 9 gene products in the axial mesenchyme

Kobus

Ammar

Nazari

et al. 2013

Birth Defects Research

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show abstract

“…For PCR, an aliquot of premixed reagents (10 × PCR buffer II, 25 mM MgCl 2 , 10 mM dNTPs, 0.5 μl AmpliTaq AS; Perkin‐Elmer) and gene‐specific primers ( Pax‐1, Pax‐9, or GAPDH; 25 pM, IDT Technologies) was added to the cDNA (total volume: 50 μl). Primer sequences were the same as used previously (Sanzo and Tuan, 1998). Reactions were performed in a Perkin‐Elmer DNA Thermal Cycler under the following conditions: 94°C for 5 minutes (denaturation), 72°C for 2 minutes (extension), and 54°C for 2 minutes (annealing).…”

Section: Methodsmentioning

confidence: 99%

Expression of the paired-box genesPax-1 andPax-9 in limb skeleton development

1999

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Vertebrate Pax genes encode a family of transcription factors that play important roles in embryonic patterning and morphogenesis. Two closely related Pax genes, Pax‐1 and Pax‐9, are associated with early axial and limb skeleton development. To investigate the role of these genes in cartilage formation we have examined the expression profiles of Pax‐1 and Pax‐9 in developing chick limb mesenchyme in vivo and in vitro. Both transcripts are detected by reverse transcription polymerase chain reaction and Northern blotting throughout chick limb development, from the early bud stages (Hamburger‐Hamilton 20–23) to fully patterned appendages (stage 30). Whole‐mount in situ hybridization reveals complex, nonoverlapping expression domains of these two genes. Pax‐1 transcripts first appear at the anterior proximal margin of the limb buds, while Pax‐9 is expressed more distally at what will be the junction of the autopod and the zeugopod. In situ hybridization to serial sections of the girdles reveals that in the pectoral region Pax‐1 is expressed proximally in condensed mesenchyme surrounding the junction of the developing scapula, humerus, and coracoid. In the pelvis, Pax‐1 is expressed between the femur and the developing acetabulum and along the ventral edge of the ischium; this transcript was also found in the distal hindlimb along the posterior edge of the fibula. Pax‐9 transcripts were not detected in the pectoral girdle at any stage, and only weakly in the pelvis along the ventral ischial margin. In the distal parts of both wings and legs, however, Pax‐9 is strongly expressed between the anterior embryonic cartilages (e.g., distal radius or tibia) and the anterior ectodermal ridge. The expression of both genes was strongest in undifferentiated cells of precartilage condensations or at the margins of differentiated cartilages, and was absent from cartilage itself. In micromass cultures of chondrifying limb bud mesenchyme expression of Pax‐1 and Pax‐9 is maintained for up to 3 days in vitro, most strongly at the end of the culture period during chondrogenic differentiation. As seen in vivo, transcripts are found in loose mesenchyme cells at the outer margins of developing cartilage nodules, and are absent from differentiated chondrocytes at the nodule center. Taken together, these investigations extend previous studies of Pax‐1 and Pax‐9 expression in embryonic limb development while validating limb bud mesenchyme culture as an accessible experimental system for the study of Pax gene function and regulation. Our in vivo and in vitro observations are discussed with reference to 1) the relationship between somitic and limb expression of these two Pax genes, 2) what regulates this expression in different regions of the embryo, and 3) the putative cellular functions of Pax‐1 and Pax‐9 in embryonic skeletogenesis. Dev Dyn 1999;214:101–115. © 1999 Wiley‐Liss, Inc.

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Exposure to homocysteine leads to cell cycle damage and reactive gliosis in the developing brain

Cecchini

Bourckhardt

Jaramillo

et al. 2019

Reproductive Toxicology

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High sensitivity analysis of gene expression in single embryonic somites using coupled reverse transcription-polymerase chain reaction

Cited by 4 publications

References 18 publications

Homocysteine causes disruptions in spinal cord morphology and changes the expression of Pax 1/9 and Sox 9 gene products in the axial mesenchyme

Homocysteine causes disruptions in spinal cord morphology and changes the expression of Pax 1/9 and Sox 9 gene products in the axial mesenchyme

Expression of the paired-box genesPax-1 andPax-9 in limb skeleton development

Exposure to homocysteine leads to cell cycle damage and reactive gliosis in the developing brain

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