The vertebrate heart is formed from diverse embryonic territories, including the first and second heart fields. The second heart field (SHF) gives rise to the right ventricle and outflow tract, yet its evolutionary origins are unclear. We found that heart progenitor cells of the simple chordate Ciona intestinalis also generate precursors of the atrial siphon muscles (ASMs). These precursors express Islet and Tbx1/10, evocative of the splanchnic mesoderm that produces the lower jaw muscles and SHF of vertebrates. Evidence is presented that the transcription factor COE is a critical determinant of ASM fate. We propose that the last common ancestor of tunicates and vertebrates possessed multipotent cardiopharyngeal muscle precursors, and that their reallocation might have contributed to the emergence of the SHF.

BACKGROUND AND OBJECTIVES: Academic primary care clinics often care for children from underserved populations affected by food insecurity. Clinical-community collaborations could help mitigate such risk. We sought to design, implement, refine, and evaluate Keeping Infants Nourished and Developing (KIND), a collaborative intervention focused on food-insecure families with infants. METHODS: Pediatricians and community collaborators codeveloped processes to link food-insecure families with infants to supplementary infant formula, educational materials, and clinic and community resources. Intervention evaluation was done prospectively by using time-series analysis and descriptive statistics to characterize and enumerate those served by KIND during its first 2 years. Analyses assessed demographic, clinical, and social risk outcomes, including completion of preventive services and referral to social work or our medical-legal partnership. Comparisons were made between those receiving and not receiving KIND by using χ2 statistics. RESULTS: During the 2-year study period, 1042 families with infants received KIND. Recipients were more likely than nonrecipients to have completed a lead test and developmental screen (both P < .001), and they were more likely to have received a full set of well-infant visits by 14 months (42.0% vs 28.7%; P < .0001). Those receiving KIND also were significantly more likely to have been referred to social work (29.2% vs 17.6%; P < .0001) or the medical-legal partnership (14.8% vs 5.7%; P < .0001). Weight-for-length at 9 months did not statistically differ between groups. CONCLUSIONS: A clinical-community collaborative enabled pediatric providers to address influential social determinants of health. This food insecurity–focused intervention was associated with improved preventive care outcomes for the infants served.

The frog Xenopus , an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus .

Aims-To evaluate the expression of vascular endothelial growth factor (VEGF) in uveal melanomas and correlate its presence with tumour characteristics and systemic metastasis. Methods-47 cases of ciliochoroidal melanoma enucleated between 1983 and 1993 were retrieved from the pathology archives at the University of Western Ontario. ParaYn sections stained with haematoxylin and eosin, periodic acid SchiV, and periodic acid SchiV without haematoxylin after bleaching of melanin were examined. The expression of VEGF protein was examined by an immunoalkaline phosphatase method following antigen retrieval, using an antibody to VEGF and vector red as the chromogen. The intensity of VEGF immunoreactivity was graded on a scale of 0-7 and correlated with tumour cell type, tumour size, presence or absence of necrosis, pigmentation, mitotic activity, microvascular density, and microvascular pattern. Results-VEGF immunoreactivity was present in 44/47 tumours (94%): the intensity was graded as very weak (1-2) in 29/47 (62%) and as weak or greater in 15/47 (32%). VEGF was also found in the ciliary epithelium, smooth muscle of the ciliary body and iris, retinal ganglion cells, inner photoreceptor segments, and the retinal pigment epithelium. Follow up data were available in 43/47 patients (91.5%), with a median follow up time of 10 years. 16/43 (37%) patients developed metastases. VEGF expression in melanoma was linked to the presence of tumour necrosis and the degree of pigmentation but no statistically significant relation with microvascular pattern, tumour size, or microvascular density was found. There was no statistically significant correlation between VEGF expression and metastasis. Conclusions-Most ciliochoroidal melanomas express VEGF and expression is correlated with the presence of necrosis but not with the occurrence of systemic metastasis or tumour angiogenesis. (Br J Ophthalmol 2000;84:750-756)

Powered flight was fundamental to the establishment and radiation of birds. However, flight has been lost multiple times throughout avian evolution. Convergent losses of flight within the ratites (flightless paleognaths, including the emu and ostrich) often coincide with reduced wings. Although there is a wealth of anatomical knowledge for several ratites, the genetic mechanisms causing these changes remain debated. Here, we use a multidisciplinary approach employing embryological, genetic, and genomic techniques to interrogate the mechanisms underlying forelimb heterochrony in emu embryos. We show that the initiation of limb formation, an epithelial to mesenchymal transition (EMT) in the lateral plate mesoderm (LPM) and myoblast migration into the LPM, occur at equivalent stages in the emu and chick. However, the emu forelimb fails to subsequently proliferate. The unique emu forelimb expression of Nkx2.5, previously associated with diminished wing development, initiates after this stage (concomitant with myoblast migration into the LPM) and is therefore unlikely to cause this developmental delay. In contrast, RNA sequencing of limb tissue reveals significantly lower Fgf10 expression in the emu forelimb. Artificially increasing Fgf10 expression in the emu LPM induces ectodermal Fgf8 expression and a limb bud. Analyzing open chromatin reveals differentially active regulatory elements near Fgf10 and Sall-1 in the emu wing, and the Sall-1 enhancer activity is dependent on a likely Fgf-mediated Ets transcription factor-binding site. Taken together, our results suggest that regulatory changes result in lower expression of Fgf10 and a concomitant failure to express genes required for limb proliferation in the early emu wing bud.

We hypothesized that signaling through multiple mitogenactivated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of softtissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions. [Mol Cancer Ther 2008;7(3):648 -58]

SUMMARY Anatomical proportions are robustly maintained in individuals that vary enormously in size, both within a species and between members of related taxa. However, the mechanisms underlying scaling are still poorly understood. We have examined this phenomenon in the context of the patterning of the ventral neural tube in response to a gradient of the morphogen Sonic hedgehog (SHH) in the chick and zebra finch, two species that differ in size during the time of neural tube patterning. We find that scaling is achieved, at least in part, by altering the sensitivity of the target cells to SHH and appears to be achieved by modulating the ratio of the repressive and activating transcriptional regulators, GLI2 and GLI3. This mechanism contrasts with previous experimental and theoretical analyses of morphogenic scaling that have focused on compensatory changes in the morphogen gradient itself.

Amphibian neural development occurs as a two-step process:(1) induction specifies a neural fate in undifferentiated ectoderm; and (2) transformation induces posterior spinal cord and hindbrain. Signaling through the Fgf, retinoic acid (RA) and Wnt/β-catenin pathways is necessary and sufficient to induce posterior fates in the neural plate, yet a mechanistic understanding of the process is lacking. Here, we screened for factors enriched in posterior neural tissue and identify spalt-like 4 (sall4), which is induced by Fgf. Knockdown of Sall4 results in loss of spinal cord marker expression and increased expression of pou5f3.2 (oct25), pou5f3.3 (oct60) and pou5f3.1 (oct91) (collectively, pou5f3 genes), the closest Xenopus homologs of mammalian stem cell factor Pou5f1 (Oct4). Overexpression of the pou5f3 genes results in the loss of spinal cord identity and knockdown of pou5f3 function restores spinal cord marker expression in Sall4 morphants. Finally, knockdown of Sall4 blocks the posteriorizing effects of Fgf and RA signaling in the neurectoderm. These results suggest that Sall4, activated by posteriorizing signals, represses the pou5f3 genes to provide a permissive environment allowing for additional Wnt/Fgf/RA signals to posteriorize the neural plate.