Fibroblast growth factor 8 (Fgf8) is a secreted signaling protein expressed in numerous temporospatial domains that are potentially relevant to cardiovascular development. However, the pathogenesis of complex cardiac and outflow tract defects observed in Fgf8-deficient mice, and the specific source(s) of Fgf8 required for outflow tract formation and subsequent remodeling are unknown. A detailed examination of the timing and location of Fgf8 production revealed previously unappreciated expression in a subset of primary heart field cells; Fgf8 is also expressed throughout the anterior heart field (AHF) mesoderm and in pharyngeal endoderm at the crescent and early somite stages. We used conditional mutagenesis to examine the requirements for Fgf8 function in these different expression domains during heart and outflow tract morphogenesis. Formation of the primary heart tube and the addition of right ventricular and outflow tract myocardium depend on autocrine Fgf8 signaling in cardiac crescent mesoderm. Loss of Fgf8 in this domain resulted in decreased expression of the Fgf8 target gene Erm,and aberrant production of Isl1 and its target Mef2c in the anterior heart field, thus linking Fgf8 signaling with transcription factor networks that regulate survival and proliferation of the anterior heart field. We further found that mesodermal- and endodermal-derived Fgf8 perform specific functions during outflow tract remodeling: mesodermal Fgf8 is required for correct alignment of the outflow tract and ventricles, whereas activity of Fgf8 emanating from pharyngeal endoderm regulates outflow tract septation. These findings provide a novel insight into how the formation and remodeling of primary and anterior heart field-derived structures rely on Fgf8 signals from discrete temporospatial domains.

;Gravitropism and, to a lesser extent, phototropism have been characterized in primary roots, but little is known about structural/functional aspects of these tropisms in lateral roots. Therefore, in this study, we report on tropistic responses in lateral roots of Arabidopsis thaliana. Lateral roots initially are plagiogravitropic, but when they reach a length of approximately 10 mm, these roots grow downward and exhibit positive orthogravitropism. Light and electron microscopic studies demonstrate a correlation between positive gravitropism and development of columella cells with large, sedimented amyloplasts in wild-type plants. Lateral roots display negative phototropism in response to white and blue light and positive phototropism in response to red light. As is the case with primary roots, the photoresponse is weak relative to the graviresponse, but phototropism is readily apparent in starchless mutant plants, which are impaired in gravitropism. To our knowledge, this is the first report of phototropism of lateral roots in any plant species.

A single course of antenatal betamethasone (B) is administered to women with threatened preterm delivery to advance fetal lung maturation. Lung maturation is associated with increased breakdown and remodeling of the lung basement membrane and extracellular matrix. These processes are regulated by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) which are present in the fetomaternal membranes of the placenta. We examined the effects of a single course of antenatal B on maternal and placental MMPs and TIMPs. A prospective pilot study of three groups of pregnant patients was conducted. Group I (n=21): women who were antenatally treated with a single course of B, and who delivered ≤36 weeks gestation; Group II (n=7): untreated women who delivered ≤36 weeks (preterm controls); and Group III (n=15): untreated women who delivered ≥38 weeks (term controls). Group I was subdivided into women delivering: a) ≤2 weeks (IA, n=13); and b) ≥2 weeks (IB, n=8) post B treatment. Maternal blood and placental samples were collected at the time of delivery for MMP-2, MMP-9, TIMP-1 and TIMP-2 levels. Placental levels were standardized using total cellular protein levels. Maternal MMP-2 levels (ng/mL) were significantly lower in Group II (14.5Ϯ1.2, p≤0.05) than Groups IA (17.9Ϯ0.67) IB (18.4Ϯ0.6), and III (18.2Ϯ0.83). Maternal MMP-9 and TIMP-1 remained unchanged. Maternal TIMP-2 was suppressed in Group IA (38.6Ϯ10.0, p≤0.05) and Group II (38.5Ϯ4.5, p≤0.05) compared to Group III (83.7Ϯ12.3), however, in Group IB, the levels were increased (65.0Ϯ7.9, p≤0.01) compared to Groups IA and II. As a result, a higher MMP-2 to TIMP-2 ratio was noted in Group IA (0.54Ϯ0.06, p≤0.01) than Groups IB (0.32Ϯ0.04) and III (0.33Ϯ0.04); and a lower ratio was noted in Group IB (0.32Ϯ0.04, p≤0.05) than Group II (0.40Ϯ0.04). In the placenta, MMP-2 levels were increased in Group IA (117.3Ϯ3.9, p≤0.05) compared to Groups II (100.8Ϯ6.0) and III (104.5Ϯ3.7). Interestingly, TIMP-1 levels were 10-fold higher in maternal blood than placenta, whereas TIMP-2 levels were 2-fold greater in placenta than maternal blood. Data are mean±SEM. A single course of antenatal B acutely enhances maternal and placental MMP-2 synthesis and maternal MMP-2/TIMP-2 ratio. Increased MMP-2 activity may result in increased collagen breakdown, and suggests a mechanism for B's effect on fetal lung maturation. Decreased MMP-2 by 2 weeks post B treatment reflects abatement of the effects of antenatal B known to occur by 7-10 days.

In order to understand the molecular and cellular mechanisms whereby mutations in TBX3 cause the complex congenital malformations seen in humans with Ulnar-mammary syndrome, we generated a series of mutant alleles using gene targeting in mice. Since it has previously been shown that Tbx3 null mouse mutants die in mid-gestation, we generated both conditional and hypomorphic alleles to examine the effects of altered gene dosage globally, and in a tissue specific manner using Cre/loxP methodology. We used RT-PCR to determine relative levels of Tbx3 mRNA and were able to correlate gene expression with genotype and with multiple phenotypes, including cardiovascular dysfunction as assessed by in utero echocardiography, limb malformations, and embryonic lethality.

Tbx genes have important roles in mammalian embryogenesis. In particular, mutations in TBX3 and TBX5 cause Ulnar-mammary and Holt-Oram syndromes in humans, respectively. These transcription factors are expressed in the early stages of limb development and disruption of these genes in the aforementioned syndromes results in variable limb defects. Importantly, Tbx3 is the only known Tbx gene expressed in both the early limb mesenchyme and in the Apical Ectodermal Ridge (AER), which is a crucial signaling center directing limb outgrowth. It has previously been shown that Tbx3 null mouse mutants die in mid gestation, which prevents a complete analysis of the roles of this protein in limb development. Therefore, we developed a conditional mutagenesis system using gene targeting in mice. This system employs conditional alleles of Tbx3 and a series of Cre-recombinase expressing drivers which permits us to perform tissue- specific ablation of Tbx3 function in the AER versus the limb mesenchyme, and as compared with ablation in all limb precursors. This approach allows us to interrogate the expression domain-specific roles of Tbx3 in gene regulation during limb morphogenesis. We will present anatomic and molecular phenotypes of these domain-specific mutants and offer hypotheses about the potential regulatory roles played by Tbx3 during normal and abnormal limb morphogenesis.