Cortical morphology at birth reflects spatiotemporal patterns of gene expression in the fetal human brain.

Interruption to gestation through preterm birth can significantly impact cortical development and have long-lasting adverse effects on neurodevelopmental outcome. We compared cortical morphology captured by high-resolution, multimodal magnetic resonance imaging (MRI) in n = 292 healthy newborn infants (mean age at birth = 39.9 weeks) with regional patterns of gene expression in the fetal cortex across gestation (n = 156 samples from 16 brains, aged 12 to 37 postconceptional weeks [pcw]). We tested the hypothesis that noninvasive measures of cortical structure at birth mirror areal differences in cortical gene expression across gestation, and in a cohort of n = 64 preterm infants (mean age at birth = 32.0 weeks), we tested whether cortical alterations observed after preterm birth were associated with altered gene expression in specific developmental cell populations. Neonatal cortical structure was aligned to differential patterns of cell-specific gene expression in the fetal cortex. Principal component analysis (PCA) of 6 measures of cortical morphology and microstructure showed that cortical regions were ordered along a principal axis, with primary cortex clearly separated from heteromodal cortex. This axis was correlated with estimated tissue maturity, indexed by differential expression of genes expressed by progenitor cells and neurons, and engaged in stem cell differentiation, neuron migration, and forebrain development. Preterm birth was associated with altered regional MRI metrics and patterns of differential gene expression in glial cell populations. The spatial patterning of gene expression in the developing cortex was thus mirrored by regional variation in cortical morphology and microstructure at term, and this was disrupted by preterm birth. This work provides a framework to link molecular mechanisms to noninvasive measures of cortical development in early life and highlights novel pathways to injury in neonatal populations at increased risk of neurodevelopmental disorder.

Variability in the efficacy of a standardized antenatal steroid treatment was independent of maternal or fetal plasma drug levels: evidence from a sheep model of pregnancy.

BACKGROUND: Administration of antenatal steroids is standard of care for women assessed to be at imminent risk of preterm delivery. There is a marked variation in antenatal steroid dosing strategy, selection for treatment criteria, and agent choice worldwide. This, combined with very limited optimization of antenatal steroid use per se, means that treatment efficacy is highly variable, and the rate of respiratory distress syndrome is decreased to perhaps as low as 40%. In some cases, antenatal steroid use is associated with limited benefit and potential harm. OBJECTIVE: We hypothesized that individual differences in maternofetal steroid exposure would contribute to observed variability in antenatal steroid treatment efficacy. Using a chronically catheterized sheep model of pregnancy, we aimed to explore the relationship between maternofetal steroid exposure and antenatal steroid treatment efficacy as determined by functional lung maturation in preterm lambs undergoing ventilation. STUDY DESIGN: Ewes carrying a single fetus underwent surgery to catheterize a fetal and maternal jugular vein at 119 days' gestation. Animals recovered for 24 hours before being randomized to either (1) a single maternal intramuscular injection of 2 mL saline (negative control group, n=10) or (2) a single maternal intramuscular injection of 0.25 mg/kg betamethasone phosphate plus acetate (antenatal steroid group, n=20). Serial maternal and fetal plasma samples were collected from each animal after 48 hours before fetuses were delivered and ventilated for 30 minutes. Total and free plasma betamethasone concentration was measured by mass spectrometry. Fetal lung tissue was collected for analysis using quantitative polymerase chain reaction. RESULTS: One animal from the control group and one animal from the antenatal steroid group did not complete their treatment protocol and were removed from analyses. Animals in the antenatal steroid group were divided into a responder subgroup (n=12/19) and a nonresponder subgroup (n=7/19) using a cutoff of partial pressure of arterial CO2 at 30-minute ventilation within 2 standard deviations of the mean value from saline-treated negative control group animals. Although antenatal steroid improved fetal lung maturation in the undivided antenatal steroid group and in the responder subgroup both physiologically (blood gas- and ventilation-related data) and biochemically (messenger ribonucleic acid expression related to fetal lung maturation), these values did not improve relative to saline-treated control group animals in the antenatal steroid nonresponder subgroup. No differences in betamethasone distribution, clearance, or protein binding were identified between the antenatal steroid responder and nonresponder subgroups. CONCLUSION: This study correlated individual maternofetal steroid exposures with preterm lung maturation as determined by pulmonary ventilation. Herein, approximately 40% of preterm lambs exposed to antenatal steroids had lung maturation that was not significantly different to saline-treated control group animals. These nonresponsive animals received maternal and fetal betamethasone exposures identical to animals that had a significant improvement in functional lung maturation. These data suggest that the efficacy of antenatal steroid therapy is not solely determined by maternofetal drug levels and that individual fetal or maternal factors may play a role in determining treatment outcomes in response to glucocorticoid signaling.

Oral dosing for antenatal corticosteroids in the Rhesus macaque.

Antenatal corticosteroids (ACS) are standard of care for women at risk of preterm delivery, although choice of drug, dose or route have not been systematically evaluated. Further, ACS are infrequently used in low resource environments where most of the mortality from prematurity occurs. We report proof of principle experiments to test betamethasone-phosphate (Beta-P) or dexamethasone-phosphate (Dex-P) given orally in comparison to the clinical treatment with the intramuscular combination drug beta-phosphate plus beta-acetate in a Rhesus Macaque model. First, we performed pharmacokinetic studies in non-pregnant monkeys to compare blood levels of the steroids using oral dosing with Beta-P, Dex-P and an effective maternal intramuscular dose of the beta-acetate component of the clinical treatment. We then evaluated maternal and fetal blood steroid levels with limited fetal sampling under ultrasound guidance in pregnant macaques. We found that oral Beta is more slowly cleared from plasma than oral Dex. The blood levels of both drugs were lower in maternal plasma of pregnant than in non-pregnant macaques. Using the pharmacokinetic data, we treated groups of 6-8 pregnant monkeys with oral Beta-P, oral Dex-P, or the maternal intramuscular clinical treatment and saline controls and measured pressure-volume curves to assess corticosteroid effects on lung maturation at 5d. Oral Beta-P improved the pressure-volume curves similarly to the clinical treatment. Oral Dex-P gave more variable and nonsignificant responses. We then compared gene expression in the fetal lung, liver and hippocampus between oral Beta-P and the clinical treatment by RNA-sequencing. The transcriptomes were largely similar with small gene expression differences in the lung and liver, and no differences in the hippocampus between the groups. As proof of principle, ACS therapy can be effective using inexpensive and widely available oral drugs. Clinical dosing strategies must carefully consider the pharmacokinetics of oral Beta-P or Dex-P to minimize fetal exposure while achieving the desired treatment responses.

Amniotic fluid LPCAT1 mRNA correlates with the lamellar body count.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is required in the biosynthesis of pulmonary surfactant. This short communication describes our assessment of LPCAT1 mRNA levels in human amniotic fluid. We found a direct correlation between LPCAT1 mRNA copies and the amniotic fluid lamellar body count (LBC). This finding corroborates an association between LPCAT1 and surfactant phospholipid biosynthesis in humans. It may provide a model for future research in perinatal medicine.

Systems biology evaluation of cell-free amniotic fluid transcriptome of term and preterm infants to detect fetal maturity.

BACKGROUND: Amniotic fluid (AF) is a proximal fluid to the fetus containing higher amounts of cell-free fetal RNA/DNA than maternal serum, thereby making it a promising source for identifying novel biomarkers that predict fetal development and organ maturation. Our aim was to compare AF transcriptomic profiles at different time points in pregnancy to demonstrate unique genetic signatures that would serve as potential biomarkers indicative of fetal maturation. METHODS: We isolated AF RNA from 16 women at different time points in pregnancy: 4 from 18 to 24 weeks, 6 from 34 to 36 weeks, and 6 from 39 to 40 weeks. RNA-sequencing was performed on cell-free RNA. Gene expression and splicing analyses were performed in conjunction with cell-type and pathway predictions. RESULTS: Sample-level analysis at different time points in pregnancy demonstrated a strong correlation with cell types found in the intrauterine environment and fetal respiratory, digestive and external barrier tissues of the fetus, using high-confidence cellular molecular markers. While some RNAs and splice variants were present throughout pregnancy, many transcripts were uniquely expressed at different time points in pregnancy and associated with distinct neonatal co-morbidities (respiratory distress and gavage feeding), indicating fetal immaturity. CONCLUSION: The AF transcriptome exhibits unique cell/organ-selective expression patterns at different time points in pregnancy that can potentially identify fetal organ maturity and predict neonatal morbidity. Developing novel biomarkers indicative of the maturation of multiple organ systems can improve upon our current methods of fetal maturity testing which focus solely on the lung, and will better inform obstetrical decisions regarding delivery timing.

An exon 5-less splice variant of the extracellular calcium-sensing receptor rescues absence of the full-length receptor in the developing mouse lung.

The authors have recently demonstrated that, in the developing mouse lung, fetal plasma Ca(2+) suppresses branching morphogenesis and cell proliferation while promoting fluid secretion via activation of the extracellular Ca(2+)-sensing receptor (CaSR). The aim of the current study was to further elucidate the role of Ca(2+) in lung development by studying the effects of extracellular Ca(2+) on fetal lung development in mice lacking the CaSR. These mice were produced by exon 5 deletion in the CaSR gene. Since such a maneuver has been known to induce the expression of an exon 5-less splice variant of the CaSR in some tissues, the molecular and functional expression of this splice variant in the developing mouse lung was also investigated. Whereas there was a mild in vivo phenotype observed in these mice, in vitro sensitivity of Casr(-/-) lung explants to specific activators of the CaSR was unaffected. These results imply that compensatory expression of an exon 5-less splice variant rescues CaSR function in this mouse model and therefore a lung-specific, complete CaSR knockout model must be developed to fully appreciate the role for this receptor in lung development and the contribution of its ablation to postnatal respiratory disease.

Dexamethasone and betamethasone for prenatal lung maturation: differences in vascular endothelial growth factor expression and alveolarization in rats.

BACKGROUND: Fetal and postnatal lung development is regulated by glucocorticoids. The use of antenatal corticosteroids is reported to produce effects on vascular endothelial growth factor (VEGF), which plays a crucial role in pulmonary development. OBJECTIVES: The purpose of this study was to compare pulmonary VEGF expression in newborn rats that were exposed to antenatal betamethasone versus dexamethasone and to evaluate its impact on the alveolarization period of rats (0-14 days of life). METHODS: Betamethasone, dexamethasone or equivalent saline solution (control group) was administered to pregnant rats on 20th and 21st days of gestation. Pulmonary VEGF mRNA, VEGF protein expression, and alveolarization changes were evaluated at birth and at 14 days of life. RESULTS: Betamethasone and dexamethasone were observed to have different actions on VEGF expression with a correlation with alveolarization on both days of study. Antenatal dexamethasone decreased VEGF expression, betamethasone tended to produce the induction of the expression of VEGF, and moreover, betamethasone did not produce a decrease in alveolarization as seen in the animals that received dexamethasone. CONCLUSIONS: Our results support the notion that betamethasone could be a better choice than dexamethasone for antenatal lung maturation.

Expression of Iroquois genes is up-regulated during early lung development in the nitrofen-induced pulmonary hypoplasia.

BACKGROUND/PURPOSE: Iroquois homeobox (Irx) genes have been implicated in the early lung morphogenesis of vertebrates. Irx1-3 and Irx5 gene expression is seen in fetal lung in rodents up to day (D) 18.5 of gestation. Fetal lung in Irx knockdown mice shows loss of mesenchyme and dilated airspaces, whereas nitrofen-induced hypoplastic lung displays thickened mesenchyme and diminished airspaces. We hypothesized that the Irx genes are up-regulated during early lung morphogenesis in the nitrofen-induced hypoplastic lung. METHODS: Pregnant rats were exposed either to olive oil or nitrofen on D9. Fetal lungs harvested on D15 were divided into control and nitrofen groups; and the lungs harvested on D18 were divided into control, nitrofen without congenital diaphragmatic hernia (CDH[-]), and nitrofen with CDH (CDH[+]). Irx gene expression levels were analyzed by reverse transcriptase polymerase chain reaction. Immunohistochemistry was performed to evaluate protein expression of Irx family. RESULTS: Pulmonary Irx1-3 and Irx5 messenger RNA expression levels were significantly up-regulated in nitrofen group compared with controls at D15. On D15, Irx immunoreactivity was increased in nitrofen-induced hypoplastic lung compared with controls. CONCLUSION: Overexpression of Irx genes in the early lung development may cause pulmonary hypoplasia in the nitrofen CDH model by inducing lung dysmorphogenesis with thickened mesenchyme and diminished airspaces.

Perinatal exposure to di-(2-ethylhexyl) phthalate leads to restricted growth and delayed lung maturation in newborn rats.

BACKGROUND: Pregnant women and infants have significant exposures to the most commonly used plasticizer di-(2-ethylhexyl) phthalate (DEHP). OBJECTIVES: This study was designed to evaluate the effects of DEHP exposure on growth and lung maturation in rats and determine if DEHP regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene (Hsd11b1) expression in the lung tissue plays a role in its effects on lung maturation. METHOD: Pregnant Sprague-Dawley rats were treated from gestational day 12 to postnatal day (PND) 21 with DEHP orally at dosages of 0, 10, 100 or 750 mg/kg/day, respectively (n=8 for each group). Two rat pups (one male and one female) from each litter were sacrificed at PND 1 and 21. Body weight was measured and the lung was processed for histology and calculation of lung interstitial tissue proportion as well as real-time PCR determination of the expressions of Hsd11b1, surfactant associated protein-A1 gene (Sftpa1) and B gene (Sftpb). RESULTS: The perinatal DEHP exposure led to a dose dependent intrauterine and postnatal growth restriction (P<0.001). High dose DEHP (750 mg/kg/day) exposure led to decreased gas-exchange space as evidenced by increased lung interstitial tissue proportion (P<0.001), but did not cause significant changes in Hsd11b1, Sftpa1 or Sftpb gene expression in the rat lung at PND 1. The DEHP-induced change in lung histology remained significant at PND 21 with improvement despite continual exposure to DEHP. CONCLUSION: Perinatal DEHP exposure leads to growth restriction and delayed lung maturation in newborn rats.

Spatiotemporal alteration in phosphatidylinositide 3-kinase-serine/threonine protein kinase B signaling in the nitrofen-induced hypoplastic lung.

PURPOSE: The pathogenesis of pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) is not fully understood. The serine/threonine protein kinase B (AKT) plays important roles for lung morphogenesis through epithelial-mesenchymal interaction in phosphatidylinositide 3-kinase (PI3K)-dependent manner. It has been reported that the lung explant morphogenesis in mice is interfered by inhibitors of the PI3K-AKT pathway. We hypothesized that PI3K and AKT gene and protein expression/distribution are altered during epithelial morphogenesis in the nitrofen-induced hypoplastic lung. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, D18, and D21 and divided into 3 groups as follows: control, nitrofen with CDH (CDH[-]), and nitrofen without CDH (CDH[+]) (n = 8 at each time-point, respectively). Reverse transcription polymerase chain reaction and immunohistochemistry were performed. RESULTS: Messenger RNA expression levels of PI3K at D21 was significantly decreased in CDH(-) and CDH(+) group (5.71 +/- 0.85 and 6.80 +/- 0.88, respectively) compared to controls (8.95 +/- 3.22; P < .05). Messenger RNA levels of AKT were also significantly decreased at D18 in CDH(-) and CDH(+) lungs (1.21 +/- 0.16 and 1.20 +/- 0.32, respectively) compared to controls (1.62 +/- 0.14; P < .01). The PI3K immunoreactivity was diminished in the distal epithelium at D18 and decreased in the overall intensity at D21 in hypoplastic lungs compared to controls. The AKT immunoreactivity was decreased in mesenchyme at D18 and decreased overall intensity at D21 in CDH lungs compared to controls. CONCLUSION: Spatiotemporal alteration of pulmonary PI3K and AKT gene and protein expression during epithelial morphogenesis may interfere with epithelial-mesenchymal interaction, causing pulmonary hypoplasia in CDH by disrupting PI3K-AKT signaling pathway.

Loss of betaarrestin1 and betaarrestin2 contributes to pulmonary hypoplasia and neonatal lethality in mice.

Less is known about the connection between the malfunction of betaarrestins and developmental defects as the mice with either of two betaarrestin isoforms knockout appear normal. In order to address the biological function of betaarrestins during developmental process, we generate betaarrestin1/2 double knockout mice. We found that betaarrestin1/2 dual-null mice developed respiratory distress and atelectasis that subsequently caused neonatal death. Morphological examination revealed type II pneumocyte immaturity. Our results indicate that not only betaarrestin1/2 double knockout lung tissue show disturbances in cell proliferation but betaarrestin1 and betaarrestin2 contribute to pulmonary surfactant complex generation during pulmonary maturation. Intra-amniotic delivery of recombinant adenovirus expressing betaarrestin1 or betaarrestin2 enhances surfactant-associated proteins synthesis in vivo. Our mRNA microarray data further reveal that betaarrestin1/2 double knockout results in downregulation of a significant proportion of genes involved in several lung morphogenesis processes. Together, our study demonstrates that betaarrestin1 and betaarrestin2 collaborate in embryonic development processes for epithelial pneumocyte differentiation and lung maturation.

The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Pulmonary neuroendocrine cells (PNECs) are the first cell type to differentiate within the primitive airway epithelium, suggesting a possible role in lung development. The differentiation of PNECs in fetal lung is governed by proneural genes such as the mammalian homolog of the achaete-scute complex (Mash-1) and a related transcription factor, hairy and enhancer of split1 (Hes-1). We examined the expression of Mash-1 and a downstream transcription factor Prox-1 in the developing mouse lung of wild-type and respective knockout mouse models. During early stages (embryonic day 12, E12) of development, only some PNECs expressed Mash-1 and Prox-1, but by E15, all PNECs coexpressed both transcription factors. PNECs failed to develop in Mash-1 but not in Prox-1-null mice, indicating that Mash-1 is essential for the initiation of the PNEC phenotype, whereas Prox-1 is associated with the development of this phenotype. As lung develops within a low O(2) environment (fetal euoxia, pO(2) approximately 20 to 30 mm Hg), we examined the effects of hypoxia on PNEC differentiation. Organ cultures of fetal mouse lungs at E12 and E16 were maintained under either 20% O(2) (normoxia, Nox) or 5% O(2) (hypoxia, Hox) and were examined every 24 h for up to 6 days in culture. In E12 explants, Hox enhanced branching morphogenesis and increased cell proliferation, but PNEC numbers and Mash-1 expression were significantly reduced. This effect could be reversed by switching the explants back to Nox. In contrast, Hox had no apparent effect on Hes-1 expression. Similarly, Hox had no effect on airway branching, PNEC numbers, or Mash-1 expression in E16 explants, indicating locked-in developmental programming. We suggest that during early stages of lung development, pO(2) concentration in concert with neurogenic gene expression modulates PNEC phenotype. Thus, disturbances in intrauterine pO(2) homeostasis could alter the functional maturation of the PNEC system and hence be involved in the pathogenesis of various perinatal pulmonary disorders.

Upregulation of Slit-2 and Slit-3 gene expressions in the nitrofen-induced hypoplastic lung.

PURPOSE: The pathogenesis of pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) is not clearly understood. Slit-2 and Slit-3 are expressed in fetal lung and play a key role in directing the functional organization and differentiation of lung mesenchyme during branching morphogenesis. We hypothesized that the pulmonary gene expression levels of Slit genes are altered in the nitrofen-induced CDH. MATERIALS AND METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15 and D21 and divided into 2 groups as follows: CDH (n = 9 at each time-point) and control (n = 9 at each time-point). The pulmonary gene expression levels of Slit-2, Slit-3, Robo1, and Robo2 were analyzed by real time reverse transcription polymerase chain reaction. Student's t test or Mann-Whitney U test was used for statistical analysis. RESULTS: Relative messenger RNA expression levels of Slit-2 and Slit-3 were significantly increased in CDH lungs compared to control at both D15 and D21 (P < .05). However, there were no significant differences between CDH and controls in the pulmonary gene expression levels of Robo1 and Robo2 at each time-point. CONCLUSION: Our results provide evidence, for the first time, that Slit genes are upregulated in nitrofen-induced hypoplastic lungs in both early and late stages of lung development. Altered pulmonary Slit gene expression may disrupt branching lung morphogenesis resulting in pulmonary hypoplasia.

Gene expression analysis after prenatal tracheal ligation in fetal rat as a model of stimulated lung growth.

PURPOSE: Prenatal tracheal occlusion or ligation (TL) has been proven to accelerate lung growth, but the mechanism of this is poorly understood. To increase understanding of the biological mechanisms involved in growth stimulation after TL in the fetal lung, we performed Global gene expression analysis using microarray technology. MATERIAL AND METHODS: Sprague-Dawley rats underwent surgery on gestational day 19. After a small hysterotomy, the trachea was mobilized and tied. As controls, we used littermates to manipulated fetuses. On day 21, fetuses were removed and lungs harvested. Global gene expression analysis was performed using Affymetrix Platform and the RAE 230 set arrays (Affymetrix Inc, Santa Clara, Calif). For validation of microarray data, we performed real time polymerase chain reaction (PCR) of the most significant upregulated or downregulated genes, combined with immunohistochemical (IHC) analysis of lung sections. RESULTS: In the group that underwent TL, several growth factors had an increased expression including connective tissue growth factor (CTGF), insulin-like growth factor 1 (IGF-1), and fibroblast growth factor 18 (FGF-18). Some of the genes that were downregulated in the group that underwent TL compared with controls were surfactant protein A (SP-A), apolipoprotein E (Apo-E), and phospholipase group II A2 (plg2a2). These results could be confirmed with real time PCR and IHC studies. DISCUSSION: Tracheal occlusion or ligation is a well-documented stimulator of fetal lung growth, and the present study provides novel insights into the underlying molecular mechanisms, with increased expression of genes and proteins with growth factor activity. One of these growth factors, CTGF, has never been previously described in this model. Also, decreased levels of genes involved in surfactant metabolism were observed, providing molecular insights into the decreased surfactant production that is known to occur in TL. Increased understanding of the molecular mechanisms that control lung growth may be the key to develop novel therapeutic techniques to stimulate prenatal and/or postnatal lung growth.

Repeated ethanol exposure during late gestation alters the maturation and innate immune status of the ovine fetal lung.

Little is known about the effects of fetal ethanol exposure on lung development. Our aim was to determine the effects of repeated ethanol exposure during late gestation on fetal lung growth, maturation, and inflammatory status. Pregnant ewes were chronically catheterized at 91 days of gestational age (DGA; term approximately 147 days). From 95-133 DGA, ewes were given a 1-h daily infusion of either 0.75 g ethanol/kg (n = 9) or saline (n = 8), with tissue collection at 134 DGA. Fetal lungs were examined for changes in tissue growth, structure, maturation, inflammation, and oxidative stress. Between treatment groups, there were no differences in lung weight, DNA and protein contents, percent proliferating and apoptotic cells, tissue and air-space fractions, alveolar number and mean linear intercept, septal thickness, type-II cell number and elastin content. Ethanol exposure caused a 75% increase in pulmonary collagen I alpha1 mRNA levels (P < 0.05) and a significant increase in collagen deposition. Surfactant protein (SP)-A and SP-B mRNA levels were approximately one third of control levels following ethanol exposure (P < 0.05). The mRNA levels of the proinflammatory cytokines interleukin (IL)-1beta and IL-8 were also lower (P < 0.05) in ethanol-exposed fetuses compared with controls. Pulmonary malondialdehyde levels tended to be increased (P = 0.07) in ethanol-exposed fetuses. Daily exposure of the fetus to ethanol during the last third of gestation alters extracellular matrix deposition and surfactant protein gene expression, which could increase the risk of respiratory distress syndrome after birth. Changes to the innate immune status of the fetus could increase the susceptibility of the neonatal lungs to infection.

Role of secretoglobin 3A2 in lung development.

RATIONALE: Secretoglobin 3A2 (SCGB3A2) was originally identified as a downstream target in lung for the homeodomain transcription factor NKX2-1, whose null mutation resulted in severely hypoplastic lungs. A very low level of SCGB3A2 is expressed in lungs at Embryonic Day (E) 11.5 during mouse development, which markedly increases by E16.5, the time when lung undergoes dramatic morphologic changes, suggesting that SCGB3A2 may be involved in lung development in addition to a known role in lung inflammation. OBJECTIVES: To determine whether SCGB3A2 plays a role in lung development. METHODS: To assess a potential role for SCGB3A2 during early lung development, wild-type and Nkx2-1-null fetal lungs of early developmental stages were subjected to ex vivo organ culture in the presence of SCGB3A2. Nkx2-1-null fetuses were exposed to SCGB3A2 during early organogenesis period through intravenous administration of this protein to Nkx2-1-heterozygous pregnant females carrying these null fetuses. Cultured lungs and fetal lungs were subjected to histologic and immunohistochemical analyses. To assess a role for SCGB3A2 in late lung development, SCGB3A2 was administered to pregnant wild-type females during mid- to late organogenesis stages, and the preterm pups and/or their lungs were evaluated for extent of maturity using breathing motion, gross morphology and histology of lungs, expression of gestational stage-specific genes, and phospholipid profiles. MEASUREMENTS AND MAIN RESULTS: SCGB3A2 significantly promoted both early and late stages of lung development. CONCLUSIONS: SCGB3A2 is a novel growth factor in lung.

Sexual dimorphism of gonadal development.

Sexual dimorphism is a term describing morphological differences between the sexes, but is often extended to include all differences observed between females and males. Sex differentiation in vertebrates is by definition sexually dimorphic and starts at the level of the sex chromosomes. In this review the sexual dimorphism of gonadal differentiation is discussed, with a focus on human development. In the embryo, the indifferent gonadal anlagen harbours four different cell lineages with bipotential fates dependent on the sex of the individual. The different paths taken by these cell lineages in male and female development are reviewed, along with other sexually dimorphic features of gonadal development. These include sex-determining genes, timing of events, dependence on germ cells, spatial organization of stromal cells, steroidogenic cells types, and other aspects.

Effects of acute alcohol intoxication in the second trimester of pregnancy on development of the murine fetal lung.

OBJECTIVE: We hypothesized that administration of alcohol during the second trimester of gestation at the pseudoglandular phase of lung development might lead to aberrant differentiation and growth, similar to that seen in congenital cystic adenomatoid malformation in human. We further hypothesized that these effects would be apparent morphologically and by altered Hoxb5 expression. STUDY DESIGN: C57BL/6J mice, exposed to ethanol at embryonic day (E) 11.5 to E13.5, which corresponds to the pseudoglandular stage of lung development, were examined at E18.5. The lungs were analyzed histologically by immunostaining. RESULTS: The average body and lung weight of alcohol-exposed (AE) fetuses were lower than those of control fetuses, the reduction in lung mass being more than the body weight. Histology showed that AE lungs were less developed and exhibited higher expression of Hoxb5 in AE lungs than controls. CONCLUSION: Alcohol exposure at E13.5 affected fetal lung development, with delayed differentiation and increased Hoxb5.

Perinatal ablation of the mouse lens causes multiple anterior chamber defects.

PURPOSE: The purpose of this study was to reassess the role of the lens as an "embryonic organizer" of ocular tissues. METHODS: We ablated the lens in mice by lens-specific expression of an attenuated version of diphtheria toxin A subunit(Tox176) driven by a modified crystallin promoter. Alterations in the differentiation programs of ocular tissues were examined by hematoxylin and eosin staining, in situ hybridization, and immunohistochemistry. RESULTS: Transgenic mice in the family OVE1757 exhibited severe microphakia. Apoptotic lens fibers were seen by embryonic day 15 (E15) and the lenses were completely ablated by post natal day 8. Multiple defects were seen in the anterior chamber. Corneal endothelial cells did not differentiate properly. The mesenchymal cells that would normally give rise to the endothelial layer were found to express N-cadherin, but they failed to form tight junctions and undergo a mesenchymal-to-epithelial transition. Although early specification of the presumptive ciliary body and iris was detected, subsequent differentiation of the iris was blocked. No dramatic changes were seen in the development of the retina. CONCLUSIONS: These results support the hypothesis that an intact lens is essential for proper differentiation of both the corneal endothelium and the iris and that the lens "organizes" the development of tissues in the anterior chamber.