Late-emigrating trunk neural crest cells in turtle embryos generate an osteogenic ectomesenchyme in the plastron.

BACKGROUND: The turtle plastron is composed of a keratinized epidermis overlying nine dermal bones. Its developmental origin has been controversial; recent evidence suggests that the plastral bones derive from trunk neural crest cells (NCCs). RESULTS: This study extends the observations that there is a turtle-specific, second wave of trunk NCC delamination and migration, after the original NCCs have reached their destination and differentiated. This second wave was confirmed by immunohistochemistry in whole-mounts and serial sections, by injecting DiI (1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate) into the lumen of the neural tube and tracing labeled cells into the plastron, and by isolating neural tubes from older turtle embryos and observing delaminating NCCs. This later migration gives rise to a plastral ectomesenchyme that expresses NCC markers and can be induced to initiate bone formation. CONCLUSIONS: The NCCs of this second migration have properties similar to those of the earlier NCCs, but also express markers characteristic of cranial NCCs. The majority of the cells of the plastron mesenchyme express neural crest markers, and have osteogenic differentiation capabilities that are similar or identical to craniofacial ectomesenchyme. Our evidence supports the contention that turtle plastron bones are derived from a late emigrating population of cells derived from the trunk neural crest.

Circulating KRAS G12D but not G12V is associated with survival in metastatic pancreatic ductal adenocarcinoma.

While high circulating tumor DNA (ctDNA) levels are associated with poor survival for multiple cancers, variant-specific differences in the association of ctDNA levels and survival have not been examined. Here we investigate KRAS ctDNA (ctKRAS) variant-specific associations with overall and progression-free survival (OS/PFS) in first-line metastatic pancreatic ductal adenocarcinoma (mPDAC) for patients receiving chemoimmunotherapy ("PRINCE", NCT03214250), and an independent cohort receiving standard of care (SOC) chemotherapy. For PRINCE, higher baseline plasma levels are associated with worse OS for ctKRAS G12D (log-rank p = 0.0010) but not G12V (p = 0.7101), even with adjustment for clinical covariates. Early, on-therapy clearance of G12D (p = 0.0002), but not G12V (p = 0.4058), strongly associates with OS for PRINCE. Similar results are obtained for the SOC cohort, and for PFS in both cohorts. These results suggest ctKRAS G12D but not G12V as a promising prognostic biomarker for mPDAC and that G12D clearance could also serve as an early biomarker of response.

Differences in physiological and biochemical responses to short-term flooding among the three avocado (Persea americana Mill.) races.

Avocado (P. americana Mill.) trees are classified into three botanical races, Mexican (M), Guatemalan (G), and West Indian (WI), each distinguished by their geographical centers of origin. While avocados are considered highly sensitive to flooding stress, comparative responses of the different races to short-term flooding are not known. This study assessed the differences in physiological and biochemical responses among clonal, non-grafted avocado cultivars of each race to short-term (2-3 days) flooding. In two separate experiments, each with different cultivars of each race, container-grown trees were divided into two treatments: 1) flooded and 2) non-flooded. Net CO2 assimilation (A), stomatal conductance (gs), and transpiration (Tr) were measured periodically over time beginning the day before treatments were imposed, through the flooding period, and during a recovery period (after unflooding). At the end of the experiments, concentrations of sugars in leaves, stems, and roots, and reactive oxygen species (ROS), antioxidants, and osmolytes in leaves and roots were determined. Guatemalan trees were more sensitive to short-term flooding than M or WI trees based on decreased A, gs, and Tr and survival of flooded trees. Guatemalan trees generally had less partitioning of sugars, particularly mannoheptulose, to the roots of flooded compared to non-flooded trees. Principal component analysis showed distinct clustering of flooded trees by race based on ROS and antioxidant profiles. Thus, differential partitioning of sugars and ROS and antioxidant responses to flooding among races may explain the greater flooding sensitivity of G trees compared to M and WI trees.

A Rosaceae Family-Level Approach To Identify Loci Influencing Soluble Solids Content in Blackberry for DNA-Informed Breeding.

A Rosaceae family-level candidate gene approach was used to identify genes associated with sugar content in blackberry (Rubus subgenus Rubus). Three regions conserved among apple (Malus × domestica), peach (Prunus persica), and alpine strawberry (Fragaria vesca) were identified that contained previously detected sweetness-related quantitative trait loci (QTL) in at least two of the crops. Sugar related genes from these conserved regions and 789 sugar-associated apple genes were used to identify 279 Rubus candidate transcripts. A Hyb-Seq approach was used in conjunction with PacBio sequencing to generate haplotype level sequence information of sugar-related genes for 40 cultivars with high and low soluble solids content from the University of Arkansas and USDA blackberry breeding programs. Polymorphisms were identified relative to the 'Hillquist' blackberry (R. argutus) and ORUS 4115-3 black raspberry (R. occidentalis) genomes and tested for their association with soluble solids content (SSC). A total of 173 alleles were identified that were significantly (α = 0.05) associated with SSC. KASP genotyping was conducted for 92 of these alleles on a validation set of blackberries from each breeding program and 48 markers were identified that were significantly associated with SSC. One QTL, qSSC-Ruh-ch1.1, identified in both breeding programs accounted for an increase of 1.5 °Brix and the polymorphisms were detected in the intron space of a sucrose synthase gene. This discovery represents the first environmentally stable sweetness QTL identified in blackberry. The approach demonstrated in this study can be used to develop breeding tools for other crops that have not yet benefited directly from the genomics revolution.

Antiangiogenic treatment delays chondrocyte maturation and bone formation during limb skeletogenesis.

Hypertrophic chondrocytes have important roles in promoting invasion of cartilage by blood vessels and its replacement with bone. However, it is unclear whether blood vessels exert reciprocal positive influences on chondrocyte maturation and function. Therefore, we implanted beads containing the antiangiogenic molecule squalamine around humeral anlagen in chick embryo wing buds and monitored the effects over time. Fluorescence microscopy showed that the drug diffused from the beads and accumulated in humeral perichondrial tissues, indicating that these tissues were the predominant targets of drug action. Diaphyseal chondrocyte maturation was indeed delayed in squalamine-treated humeri, as indicated by reduced cell hypertrophy and expression of type X collagen, transferrin, and Indian hedgehog (Ihh). Although reduced in amount, Ihh maintained a striking distribution in treated and control humeri, being associated with diaphyseal chondrocytes as well as inner perichondrial layer. These decreases were accompanied by lack of cartilage invasion and tartrate-resistant acid phosphatase-positive (TRAP+) cells and a significant longitudinal growth retardation. Recovery occurred at later developmental times, when in fact expression in treated humeri of markers such as matrix metalloproteinase 9 (MMP-9) and connective tissue growth factor (CTGF) appeared to exceed that in controls. Treating primary cultures of hypertrophic chondrocytes and osteoblasts with squalamine revealed no obvious changes in cell phenotype. These data provide evidence that perichondrial tissues and blood vessels in particular influence chondrocyte maturation in a positive manner and may cooperate with hypertrophic chondrocytes in dictating the normal pace and location of the transition from cartilage to bone.

The contribution of neural crest cells to the nuchal bone and plastron of the turtle shell.

The origin of the turtle plastron is not well understood, and these nine bones have been homologized to the exoskeletal components of the clavicles, the interclavicular bone, and gastralia. Earlier data from our laboratory showed that the plastral bone-forming cells stained positively for HNK-1 and PDGFRα, two markers of skeletogenic neural crest cells. We have now shown that the HNK-1(+) cells are also positive for p75 and FoxD3, affirming their neural crest identity. These cells originate from the dorsal neural tube of stage-17 turtle embryos, several days after the original wave of neural crest cells have migrated and differentiated. Moreover, we have demonstrated the existence of a staging area, above the neural tube and vertebrae, where these late-emigrating neural crest cells collect. After residing in the carapacial staging area, these cells migrate to form the plastral bones. We also demonstrate that one bone of the carapace, the nuchal bone, also stains with HNK-1 and with antibodies to PDGFRα. The nuchal bone shares several other properties with the plastral bones, suggesting that it, too, is derived from neural crest cells. Alligator gastralia stain for HNK-1, while their ribs do not, thus suggesting that the gastralial precursor may also be derived from neural crest cells.

Evidence that a late-emerging population of trunk neural crest cells forms the plastron bones in the turtle Trachemys scripta.

The origin of the turtle plastron is not known, but these nine bones have been homologized to the exoskeletal components of the clavicles, the interclavicular bone, and gastralia. Earlier evidence from our laboratory showed that the bone-forming cells of the plastron were positive for HNK-1 and PDGFRalpha, two markers of the skeletogenic neural crest. This study looks at the embryonic origin of these plastron-forming cells. We show that the HNK-1+ cells are also positive for p75 and FoxD3, confirming their neural crest identity, and that they originate from the dorsal neural tube of stage 17 turtle embryos, several days after the original wave of neural crest cells have migrated and differentiated. DiI studies show that these are migratory cells, and they can be observed in the lateral regions of the embryo and can be seen forming intramembranous bone in the ventral (plastron) regions. Before migrating ventrally, these late-emerging neural crest cells reside for over a week in a carapacial staging area above the neural tube and vertebrae. It is speculated that this staging area is where they lose the inability to form skeletal cells.