Abstract
Concurrent Session
8:30 AM
Friday, January 29, 1999
Characterization Of Trunk Neural Tube, A Novel Cdna Fragment, Isolated Via Differential Display.
Neural crest cells give rise to a diverse range of cell types, including neurons,glia, melanocytes, cartilage, and bone. While many studies have focused on the mechanisms underlying migration and differentiation of neural crest cells, few have investigated how differences arise in neural crest populations along the rostral caudal axis. For example, cranial neural crest cells give rise to cartilage and bone, while trunk neural crest cells do not. Using differential display, we isolated a number of differentially expressed genes that may influence cell fate decisions in subpopulations of neural crest cells. We focused on TNT, a trunk neural tube gene which was selectively expressed in the trunk, but not in the cranial neural tissue. Sequence and whole mount in situ hybridization analysis revealed that the TNT cDNA fragment is truly differentially expressed in the trunk neural tube. For 9–11 somite stage chick embryos, TNT is expressed in the trunk neural plate and neural folds. TNT is highly expressed in the tail bud and diffusely expressed in the trunk neural tube for 18 and 30 somite stage chick embryos. Later in development, Hamburger and Hamilton stages 20 and 24, TNT is expressed in the dorsolateral aspect of the trunk neural tube. We are currently screening a cDNA library to obtain the full-length clone of the TNT gene. We have cloned and are sequencing a candidate full-length clone of TNT, 2.5 kilobases in length. Once obtained we hope to gain more insight into the role of TNT in trunk neural crest development using tissue transplantation and retrovirally-mediated gene transfer. This research will help elucidate some of the key events and gene products responsible for differences in neural crest populations along the rostral caudal axis of chick embryos. In addition, these findings may be useful for future studies of birth defects involving malformation of the neural crest.
* The first two authors contributed equally and are listed in alphabetical order.
Vegf Mrna Expression Is Not Decreased In Pulmonary Hypoplasia Due To Oligohydramnios In The Fetal Rat At The End Of Gestation.
Oligohydramnios (O) during pregnancy often results in significant pulmonary hypoplasia and is also associated with diminution of the pulmonary circulation. Angiogenesis of the circulation is stimulated by vascular endothelial growth factor (VEGF). However, little is known about the role of VEGF during abnormal fetal lung growth. We investigated whether VEGF expression is altered during diminished growth of the lung and decreased pulmonary vascular development due to O. We induced O on day 16 in 10 pregnant rat mothers. For half of the fetuses in each litter (O), the amniotic sacs were punctured and the fluid allowed to drain for the remainder of the pregnancy, sham-operated littermates were used as paired controls (C). We harvested the fetal lungs on day 19 (n=5) or day 21 (n=5) and performed RT-PCR using primers for VEGF, normalized to actin and converted to densitometric units. In addition, 7 unoperated pregnant rat mothers had their fetuses harvested at day 19 or 21 to use as unoperated control comparisons (U). The data are mean ± sem and were compared by paired and unpaired t-tests among groups.
Although the O lungs at both 19 and 21 days were smaller compared to C and U controls, unexpectedly the lung VEGF mRNA was not decreased in O, and in fact it was increased compared to the unoperated control state. This would suggest pulmonary hypoplasia is not the result of decreased VEGF. We speculate perhaps the VEGF expression increases in this condition in a compensatory response, similar to the increased VEGF seen in other injury.
Autoregulation Of Nkx2.1: Identification Of A Novel Binding Site.
Phenotypic diversity & cellular specialization are accomplished by sequential gene activation. Many of the initial steps in the activation of regulatory genes are dependent on mesoderm-endoderm interactions. Subsequent expression of these genes must be maintained during morphogenesis to yield a functionally appropriate organ. A parsimonious way of accomplishing this task is by autoregulation. NKX2.1 is a homeodomain transcriptional regulator whose activity is critical in lung, thyroid & brain morphogenesis. Nkx2.1(-/-) neonates exhibit lung abnormalities & die of respiratory insufficiency. Also, absence of NKX2.1 is implicated in pathogenesis of BPD. Structural & functional analysis of the Nkx2.1 indicates presence of a highly complex array of cis-active regulatory DNA elements located 5′ of exon I & within intron 1. DNA sequence of Nkx2.1 includes putative NKX2.1 binding sites, indicating the possibility for autoregulation. In this study, we demonstrate that: 1) The NKX2.1 binding sites are functionally capable of binding NKX2.1 & directing transcription, & 2) An element 5′ of exon I, also confers significant autoregulation. DNA sequence of this element forms a palindromic structure which contains a novel binding site for NKX2.1. Point mutations in this sequence abrogate in vitro binding to, & reduce transactivation of reporter genes by NKX2.1. Thus, we have identified a novel DNA sequence on the Nkx2.1 gene which serves as a target, binding site for NKX2.1 mediating autoregulation. HL56221, HL56590, HL60231 & the Hastings Foundation
Developmental Expression Of D7, A Novel Gene Identified In The Embryonic Mouse Heart.
SP Pak, HL Bartlett, and SE Klewer (intr. by MH Witte), Department of Pediatrics, University of Arizona College of Medicine, Tucson, AZ.
Little is currently known regarding the molecular regulation of heart development. D7 is a novel gene isolated from the embryonic mouse heart. The purpose of this current study is to determine the normal spatial and temporal expression patterns of D7 transcripts during mouse development. cDNA probes were generated from both the 5′ and 3′ regions of a 2.6 kb D7 clone by RT-PCR. Nucleotide sequences of the cloned cDNA fragments were confirmed prior to use in Northern blot analyses and in situ hybridization studies. Northern blot analysis of whole mouse embryo RNA (E7-E17) using a 32P-labeled probe from the 5′ region of D7 detects 3 distinct mRNA transcripts (1.35, 3.5, and 4 kb) in all stages examined. The 1.35 kb transcript is more abundant than the larger transcripts at each of the developmental stages. Preliminary in situ hybridization studies in E12.5-13 mouse embryos with antisense probes from the 5′ region of D7 demonstrate a ubiquitous expression pattern while antisense probe from the 3′ region of the D7 gene demonstrate a more restricted expression pattern within the developing heart endothelium, the limb buds and the somite regions. These results indicate that D7 has multiple mRNA transcripts expressed during development. The restricted expression pattern of the larger D7 transcripts suggest a possible role in endothelial-mediated remodeling events during organogenesis. Additional studies are being conducted to more fully characterize the complete spatial and temporal expression of D7 mRNA transcripts during mouse development.
Vegf Mrna Expression Increases And Vegfr-2/Flk Receptor Mrna Expression Decreases In The Fetal Rat As Gestation Advances.
Angiogenesis and development of the fetal pulmonary circulation parallels the growth and development of the fetal lung. Vascular endothelial growth factor (VEGF) stimulates angiogenesis and is believed to have a vital role in the development of blood vessels, but little is known about its role in the fetal development of the lung, a highly vascular organ. VEGF binds to tyrosine kinase receptors (VEGFR-1/Flt and VEGFR-2/Flk) to stimulate angiogenesis, and these receptors have been found in fetal lung tissue. We investigated whether VEGF expression and mRNA expression of one of its receptors, VEGFR-2/Flk increases during a period of rapid growth of the fetal lung and increased pulmonary vascular development at the end of gestation. We harvested lungs from fetal rat pups on day 19 (n=10) or day 21 (n=7) and performed RT-PCR using primers for VEGF and its receptor VEGFR-2/Flk, normalized to β actin and converted to densitometric units. The data are mean ± sem and were compared by unpaired t-tests between time periods.
During this two day period, the fetal lungs almost doubled in size. As anticipated, we found a significant 5-fold increase in VEGF expression during this period of increased vascularity of the lung. In contrast, we found a decrease in the VEGFR-2/Flk receptor expression during this same period. We speculate that increased VEGF expression stimulates development of the fetal pulmonary circulation and is important for development of the fetal lung during this greatly increased growth. It appears that this fetal vascular development is less dependent on increased receptor expression. It may be that receptor turnover is altered at the end of gestation or that other receptors for VEGF, namely VEGFR-1/Flt are more important at the end of gestation in the fetal rat lung.
Assessment Of Fetal Spinal Cord Cell Death Due To Amniotic Fluid Urea Concentrations.
S. Vasudevan, Emory University School of Medicine, Atlanta, GA.;
In the myelomeningocele lesion, failed closure of the neural tube during gestation results in the exposure of the developing spinal cord to the intrauterine environment. Thus far, experiments from this lab and others have proposed that the damage to the exposed spinal cord in this malformation is twofold: mechanical and chemical. The chemical factors refer to the contents of the intrauterine environment, namely the amniotic fluid. Other laboratories have been able to show that exposure of rat fetal spinal cord cells to second trimester human amniotic fluid causes a significant amount of cell death which may be responsible for the neural deficits associated with myelomeningocele. Preliminary experiments in this lab have suggested that the same is true when applied to the human fetal spinal cord cells. In an attempt to analyze the chemical aspect of fetal spinal cell damage, the present study hypothesizes that urea is the primary toxic component of the amniotic fluid causing the damage and cell death typical to the myelomeningocele lesion. Explant cultures were prepared from 1st trimester human fetal spinal cords. They were grown in pure media for 72 hours to establish a foundation and to initiate growth. The cultures were then treated with 28, 34, 44 week gestational concentrations of urea, values of which were obtained from Benzie et al. An LDH/ELISA assay (Cytotox96-Promega) was performed on supernatant collected after 72 hours of urea treatment to ascertain cell death. A few cultures were treated with lysis solution to have a maximum LDH reference. Pictures were taken before and after urea treatment. LDH levels were 10.99 u/L for controls, 13.94 u/L for 28 weeks, 13.78 u/L for 34 weeks, 11.59 u/L for 44 weeks and 37.6 u/L for the maximum lysis. No significant cell death was present in the experimentals whereas the maximum lysis is significantly higher. This data shows that late gestational amniotic fluid concentrations of urea are not toxic enough to cause a significant amount of cell death and therefore is not the primary toxic agent of amniotic fluid. Other substances in amniotic fluid may work in concordance with the urea to achieve a significant amount of damage.
Understanding The Role Of Homeobox Gene Msx-2 In The Development And Proliferation Of Epidermal And Hair Keratinocytes Using Msx-2 Transgenic Mice.
In the field of dermatopathology, there is the continual search for molecules involved in the development and cycling of skin and its appendages. This understanding is imperative when trying to develop new treatment options for wound healing, regeneration, and alopecia. Recently, several signaling molecules were identified to be involved in skin development. Of particular interest is homeobox gene Msx-2 which is hypothesized to be involved in the control of the growth of keratinocytes.
Appearance Of Receptor Active Insulin-Like Growth Factor-I (Igf-I) In Blood After Feeding In The Suckling Rat.
Insulin-Like Growth Factor-I (IGF-I) is a potent mitogenic peptide believed to be involved in postnatal growth regulation. In addition to endogenous production, sucklings of many species, including the rat, ingest significant quantities of IGF-I in breast milk. In recent work from our laboratory, we have demonstrated that sucklings fed an artificial diet + IGF-I had serum IGF-I levels similar to dam fed controls and 2X that of animals fed the artificial diet alone. We examined the hypothesis that IGF's may be absorbed in bioactive form from the gastrointestinal tract to the portal circulation and beyond. The experimental design consisted of using fasted suckling rats of 10 days of age (n=22). Radiolabelled rhIGF-I (4x106 cpm, 100 µl volume) was given in a 1:1 mixture of appropriate aged rat milk via orogastric tube at time “0.” At 5, 10, 20, and 30 minutes post administration, portal blood was obtained by venipuncture while under anesthesia. Within 30-60 seconds after portal venipuncture, cardiac (“peripheral”) blood was also removed. Blood specimens were weighed to determine volume and counted in a gamma counter to determine radioactivity (cpm). Results are expressed as per 100 µl of blood to account for differences in volume of blood obtained during venipuncture. Results show that IGF-I radioactivity in portal blood was higher than in peripheral blood and peaked at 20 min post ingestion. Size exclusion gel chromatography was performed on all samples and confirmed that 30-40% of cpm eluted in the position of “native” IGF-I. HPLC performed on selected peaks from portal blood was identical to the elution position of rhIGF-I. In addition, selected peak fractions from portal blood isolates were exposed to competitive binding assays using a crude placental membrane preparation bearing IGF-I receptors. This assay confirmed the presence of receptor active IGF-I in portal blood samples from animals fed the radiolabelled IGF-I preparation. We conclude that IGF-I is absorbed in a receptor active form from the gastrointestinal tract of the suckling rat. Furthermore, since we have previously demonstrated that intravenously injected IGF-I passes into bile in a receptor active form in the infant rat, this latter series of studies would also lend credence to the concept of an enterohepatic circulation for IGF in the suckling.