Facile methods for reusing laboratory plastic in developmental biology experiments.

Plastic pollution negatively affects ecosystems and human health globally, with single-use plastic representing the majority of marine litter in some areas. Life science laboratories prefer pristine conditions for experimental reliability and therefore make use of factory standardized single-use plastic products. This contributes to overall plastic waste in the United States and globally. Here, we investigate the potential of reusing plastic culture dishes and subsequently propose methods to mitigate single-use plastic waste in developmental biology research laboratories. We tested the efficacy of bleach and ethyl alcohol in sterilizing used dishes. We then tested the feasibility of washing and reusing plastic to culture Xenopus laevis embryos subjected to various manipulations. Cleaning and reusing laboratory plastic did not affect the development or survival of X. laevis, indicating that these cleaning methods do not adversely affect experimental outcome and can be used to sterilize plastic before reuse or recycling. Lastly, we performed a survey of various life science laboratories to estimate both waste reduction and savings associated with recycling single-use plastics. Standardization of these procedures would allow research laboratories to benefit economically while practicing environmentally conscious consumption.

Developmental biology: A 5'Hoxd-Gli3 balance in tetrapod axial polarity.

Almost all living tetrapods exhibit postaxial dominance in digit formation, apart from urodele amphibians, which show preaxial dominance. Recent work shines light on the genetic differences between the two modes of limb development, suggesting that differences in 5'Hoxd expression, mediated by Gli3, may explain the switch in axial polarity.

Limb positioning and initiation: An evolutionary context of pattern and formation.

Before limbs or fins, can be patterned and grow they must be initiated. Initiation of the limb first involves designating a portion of lateral plate mesoderm along the flank as the site of the future limb. Following specification, a myriad of cellular and molecular events interact to generate a bud that will grow and form the limb. The past three decades has provided a wealth of understanding on how those events generate the limb bud and how variations in them result in different limb forms. Comparatively, much less attention has been given to the earliest steps of limb formation and what impacts altering the position and initiation of the limb have had on evolution. Here, we first review the processes and pathways involved in these two phases of limb initiation, as determined from amniote model systems. We then broaden our scope to examine how variation in the limb initiation module has contributed to biological diversity in amniotes. Finally, we review what is known about limb initiation in fish and amphibians, and consider what mechanisms are conserved across vertebrates.

Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression.

The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.

Attenuated Fgf Signaling Underlies the Forelimb Heterochrony in the Emu Dromaius novaehollandiae.

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.

Developmental Biology: Hox Timing Determines Limb Placement.

Hox genes are known to determine vertebral identity along with being required for normal limb patterning. A new study now finds that differential expression timing of Hox genes in the lateral plate mesoderm determines limb placement as well.

Noggin is required for first pharyngeal arch differentiation in the frog Xenopus tropicalis.

The dorsal ventral axis of vertebrates requires high BMP activity for ventral development and inhibition of BMP activity for dorsal development. Presumptive dorsal regions of the embryo are protected from the ventralizing activity of BMPs by the secretion of BMP antagonists from the mesoderm. Noggin, one such antagonist, binds BMP ligands and prevents them from binding their receptors, however, a unique role for Noggin in amphibian development has remained unclear. Previously, we used zinc-finger nucleases to mutagenize the noggin locus in Xenopus tropicalis. Here, we report on the phenotype of noggin mutant frogs as a result of breeding null mutations to homozygosity. Early homozygous noggin mutant embryos are indistinguishable from wildtype siblings, with normal neural induction and neural tube closure. However, in late tadpole stages mutants present severe ventral craniofacial defects, notably a fusion of Meckel's cartilage to the palatoquadrate cartilage. Consistent with a noggin loss-of-function, mutants show expansions of BMP target gene expression and the mutant phenotype can be rescued with transient BMP inhibition. These results demonstrate that in amphibians, Noggin is dispensable for early embryonic patterning but is critical for cranial skeletogenesis.

Saunders's framework for understanding limb development as a platform for investigating limb evolution.

John W. Saunders, Jr. made seminal discoveries unveiling how chick embryos develop their limbs. He discovered the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the domains of interdigital cell death within the developing limb and determined their function through experimental analysis. These discoveries provided the basis for subsequent molecular understanding of how vertebrate limbs are induced, patterned, and differentiated. These mechanisms are strongly conserved among the vast diversity of tetrapod limbs suggesting that relatively minor changes and tweaks to the molecular cascades are responsible for the diversity observed in nature. Analysis of the pathway systems first identified by Saunders in the context of animals displaying limb reduction show how alterations in these pathways have resulted in multiple mechanisms of limb and digit loss. Other classes of modification to these same patterning systems are seen at the root of other, novel limb morphological alterations and elaborations.

Forging a pediatric primary care-community partnership to support food-insecure families.

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.

Spalt-like 4 promotes posterior neural fates via repression of pou5f3 family members in Xenopus.

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/ß-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.

Targeted gene disruption with engineered zinc-finger nucleases (ZFNs).

Reverse genetics in Xenopus has been limited to knockdown strategies using antisense morpholino oligonucleotides (MOs). Recently, engineered zinc-finger nucleases have been used to induce targeted mutations resulting in null alleles. Zinc-finger nuclease (ZFN) technology has been adapted to induce null mutations in many systems previously refractory to targeted gene inactivation. Here we provide a general protocol for inducing targeted mutations in Xenopus tropicalis using ZFNs, a method to detect resulting mutations, and the steps to generate homozygous mutant embryos.

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.

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.

Early chordate origins of the vertebrate second heart field.

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.

Abciximab: a review and update for clinicians.

The glycoprotein (GP) IIb/IIIa receptor serves as the final common pathway of platelet-thrombus formation. Thus, the GP IIb/IIIa receptor has been identified as a target for the prevention of thrombus formation during acute coronary syndromes and/or percutaneous coronary intervention. While there are several intravenous GP IIb/IIIa inhibitors available, abciximab has proven to be a dependable agent with unique properties. Based on American College of Cardiology, American Heart Association and Society for Angiography and Interventions guidelines, compared with the other available intravenous GP IIb/IIIa inhibitors, abciximab receives the highest recommendation for use in patients with ST-elevation myocardial infarction undergoing primary percutaneous coronary intervention. Abciximab is also unique in that there is no need for renal dosing and its effects are mostly reversible with platelet transfusions.

Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma.

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue 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.

Vulnerable plaque intervention: State of the art.

Progressive atherosclerotic disease is responsible for many of the late adverse clinical events that detract from the high procedural and clinical success of percutaneous coronary intervention. Despite recent advances in catheter based technology for the treatment of obstructive coronary artery disease, the greater risk to the patient over time may in fact come from the significant rate of acute coronary events triggered by nonculprit and/or nonobstructive coronary artery lesions. These areas of vulnerability within the epicardial coronary tree have generated a great deal of interest surrounding the concepts of vulnerable plaque (VP), vulnerable blood and the vulnerable patient. This 'state of the art' review discusses the limitations of coronary angiography alone in providing risk assessment; reviews the underlying biological concepts of VP; discusses evolving noninvasive and invasive imaging technologies for the detection of VP; and finally provides a futuristic look at how the field of interventional cardiology may transcend the traditional angiogram and move toward a more comprehensive treatment approach that benefits the patients' overall coronary health.

Anthrax lethal toxin inhibits growth of and vascular endothelial growth factor release from endothelial cells expressing the human herpes virus 8 viral G protein coupled receptor.

PURPOSE: In this study, we tested the hypothesis that inhibition of mitogen-activated protein kinase kinases (MKK) inhibits tumor growth by acting on angiogenic signaling and by extension may form the basis of an effective strategy for treatment of Kaposi's sarcoma. EXPERIMENTAL DESIGN: Murine endothelial cells expressing the human herpes virus 8 G protein-coupled receptor (vGPCR-SVEC) were treated with anthrax lethal toxin (LeTx). LeTx is a binary toxin ordinarily secreted by Bacillus anthracis and is composed of two proteins: protective antigen (the binding moiety) and lethal factor (the active moiety). Lethal factor is a protease that cleaves and inactivates MKKs. RESULTS: In vitro, treatment of vGPCR-SVEC with LeTx inhibited MKK signaling, moderately inhibited cell proliferation, and blocked the ability of these cells to form colonies in soft agar. Treatment with LeTx also blocked the ability of these cells to release several angioproliferative cytokines, notably vascular endothelial growth factor (VEGF). In contrast, inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 with U0126 caused a substantial inhibition of proliferation but only modestly inhibited VEGF release. In xenograft models, i.v. injection of LeTx caused reduced tumor growth characterized immunohistochemically by inhibition of MKK signaling, decreased rates of proliferation, and reduced levels of VEGF and VEGF receptor 2, with a corresponding decrease in vascular density. CONCLUSIONS: These data support a role for MKK signaling in tumor growth and vascularization and are consistent with the hypothesis that inhibition of MKK signaling by LeTx or a similar agent may be an effective strategy for the treatment of Kaposi's sarcoma as well as other vascular tumors.