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.