Deficiency of the placenta- and yolk sac-specific tristetraprolin family member ZFP36L3 identifies likely mRNA targets and an unexpected link to placental iron metabolism.

The ZFP36L3 protein is a rodent-specific, placenta- and yolk sac-specific member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins. These proteins bind to AU-rich elements in target mRNAs, and promote their deadenylation and decay. We addressed the hypotheses that the absence of ZFP36L3 would result in the accumulation of target transcripts in placenta and/or yolk sac, and that some of these would be important for female reproductive physiology and overall fecundity. Mice deficient in ZFP36L3 exhibited decreased neonatal survival rates, but no apparent morphological changes in the placenta or surviving offspring. We found Zfp36l3 to be paternally imprinted, with profound parent-of-origin effects on gene expression. The protein was highly expressed in the syncytiotrophoblast cells of the labyrinth layer of the placenta, and the epithelial cells of the yolk sac. RNA-Seq of placental mRNA from Zfp36l3 knockout (KO) mice revealed many significantly upregulated transcripts, whereas there were few changes in KO yolk sacs. Many of the upregulated placental transcripts exhibited decreased decay rates in differentiated trophoblast stem cells derived from KO blastocysts. Several dozen transcripts were deemed high probability targets of ZFP36L3; these include proteins known to be involved in trophoblast and placenta physiology. Type 1 transferrin receptor mRNA was unexpectedly decreased in KO placentas, despite an increase in its stability in KO stem cells. This receptor is crucial for placental iron uptake, and its decrease was accompanied by decreased iron stores in the KO fetus, suggesting that this intrauterine deficiency might have deleterious consequences in later life.

Efficient division and sampling of cell colonies using microcup arrays.

A microengineered array to sample clonal colonies is described. The cells were cultured on an array of individually releasable elements until the colonies expanded to cover multiple elements. Single elements were released using a laser-based system and collected to sample cells from individual colonies. A greater than an 85% rate in splitting and collecting colonies was achieved using a 3-dimensional cup-like design or "microcup". Surface modification using patterned titanium deposition of the glass substrate improved the stability of microcup adhesion to the glass while enabling minimization of the laser energy for splitting the colonies. Smaller microcup dimensions and slotting the microcup walls reduced the time needed for colonies to expand into multiple microcups. The stem cell colony retained on the array and the collected fraction within released microcups remained undifferentiated and viable. The colony samples were characterized by both reporter gene expression and a destructive assay (PCR) to identify target colonies. The platform is envisioned as a means to rapidly establish cell lines using a destructive assay to identify desired clones.

Switching of melanocyte pigmentation associated with pituitary pars intermedia tumors in Rb+/- and p27-/- female mice with yellow pelage.

As an incidental finding in a study of mammary tumorigenesis, two lines of genetically engineered mice were observed to develop pigmentation changes of the fur. Mice with targeted mutations of the Rb1 (Rb) and Cdkn1b (p27kip1) genes were crossed from C57BL/6 (black coat color; eumelanin) and 129Sv (wild-type agouti coat color) backgrounds, respectively, to one with a dominant yellow coat color (phaeomelanin) carrying a transgene for Agouti under a keratinocyte specific promoter. Both Rb+/- and p27-/- mice developed pituitary tumors of the pars intermedia that were associated with a switch to black (eumelanic) fur but were not observed in sibling Rb+/+ and p27+/+ mice. This phenomenon was observed first in the vibrissae and, subsequently one to two weeks later, as periorbital and dorsal patches, and was associated with pituitary lesions larger than four millimeters in the longest dimension. In Rb+/- mice, pigmentation change preceded a moribund state attributable to the tumors by two to four weeks, whereas in p27-/- mice, the pigmentation alteration was earlier, more gradual, and prolonged. The switch from phaeomelanin to eumelanin in the fur is most likely due to out-competition of the agouti gene product by alpha-melanocyte-stimulating hormone from the pituitary tumors, an effect masked in black or agouti mice.