Reconstitution of Mammary Epithelial Morphogenesis by Murine Embryonic Stem Cells Undergoing Hematopoietic Stem Cell Differentiation

Background

Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.

Methodology/Principal Findings

To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES) cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs) under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.

Conclusions/Significance

Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.     

Atrophic thyroid follicles and inner ear defects reminiscent of cochlear hypothyroidism in Slc26a4-related deafness

Thyroid hormone is essential for inner ear development and is required for auditory system maturation. Human mutations in SLC26A4 lead to a syndromic form of deafness with enlargement of the thyroid gland (Pendred syndrome) and non-syndromic deafness (DFNB4). We describe mice with an Slc26a4 mutation, Slc26a4 ^loop/loop , which are profoundly deaf but show a normal sized thyroid gland, mimicking non-syndromic clinical signs. Histological analysis of the thyroid gland revealed defective morphology, with a majority of atrophic microfollicles, while measurable thyroid hormone in blood serum was within the normal range. Characterization of the inner ear showed a spectrum of morphological and molecular defects consistent with inner ear pathology, as seen in hypothyroidism or disrupted thyroid hormone action. The pathological inner ear hallmarks included thicker tectorial membrane with reduced β-tectorin protein expression, the absence of BK channel expression of inner hair cells, and reduced inner ear bone calcification. Our study demonstrates that deafness in Slc26a4 ^loop/loop mice correlates with thyroid pathology, postulating that sub-clinical thyroid morphological defects may be present in some DFNB4 individuals with a normal sized thyroid gland. We propose that insufficient availability of thyroid hormone during inner ear development plays an important role in the mechanism underlying deafness as a result of SLC26A4 mutations.     

Predetermined Flake Production at the Lower/Middle Paleolithic Boundary: Yabrudian Scraper-Blank Technology

While predetermined débitage technologies are recognized beginning with the middle Acheulian, the Middle Paleolithic is usually associated with a sharp increase in their use. A study of scraper-blank technology from three Yabrudian assemblages retrieved from the early part of the Acheulo-Yabrudian complex of Tabun Cave (ca. 415–320 kyr) demonstrates a calculated and preplanned production, even if it does not show the same complexity and elaboration as in the Levallois technology. These scraper dominated assemblages show an organization of production based on an intensive use of predetermination blank technology already in place at the end of the Lower Paleolithic of the Levant. These results provide a novel perspective on the differences and similarities between the Lower and Middle Paleolithic industries. We suggest that there was a change in the paradigm in the way hominins exploited stone tools: in many Middle Paleolithic assemblages the potential of the stone tools for hafting was a central feature, in the Lower Paleolithic ergonometric considerations of manual prehension were central to the design of blanks and tools.     

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.     

The enzymatic activity of phosphoglucose isomerase is not required for its cytokine function

PGI is a housekeeping gene encoding phosphoglucose isomerase (PGI) a glycolytic enzyme that also functions as a cytokine (autocrine motility factor (AMF)/neuroleukin/maturation factor) upon secretion from the cell and binding to its 78 kDa seven-transmembrane domain receptor (gp78/AMF-R). PGI contains a CXXC motif, characteristic of redox proteins and possibly evolutionarily related to the CC and CXC motif of the chemokine gene family. Using site-directed mutagenesis, single- and double-deletion (CXC, CC) mutants were created by deleting amino acids 331 and 332 of human PGI, respectively. The mutant proteins lost their enzymatic activity; however, neither of the deletions augmented the proteins' binding affinity to the receptor and all maintained cytokine function. The results demonstrate that the enzymatic activity of PGI is not essential for either receptor binding or cytokine function of human PGI.     

Plasma Factor in Red Blood Cells Adhesion to Endothelial Cells: Humans and Rats

Erythrocyte adhesion to the vascular endothelium is one of the key determinants of microcirculatory blood flow. Adhesion is a complex process determined by the intricate interaction among red blood cells (RBC), plasma factors, and the vascular endothelium. Rats are commonly used as disease models to investigate the pathophysiology of various hematological disease processes occurring in humans and their response to prospective treatments. The aim of our study was to characterize the adhesion of RBC in adult blood from rat and human subjects, in order to test the validity of rat models for adhesion-related disease processes. We demonstrated that adhesion of RBC from rats (rRBC), to endothelial cells (EC) in plasma-free buffer, is stronger than from human subjects (hRBC). In addition, plasma proteins induced elevation of hRBC (eightfold) but depression of rRBC (threefold) adhesion to EC. It is thus suggested to be aware of the difference in RBC/EC interaction for human and rat subjects, when studying models of blood flow.     

What's hot about otoferlin

Mutations in the otoferlin (OTOF) gene lead to profound hearing loss in humans. Interestingly, a number of missense otoferlin mutations cause hearing defects but only at higher body temperature, and the reasons for this have been elusive until now. A study published in this issue of The EMBO Journal (Strenzke et al, 2016 ) adds insight into the underlying mechanisms for this heat‐dependent hearing loss.     

The GPSM2/LGN GoLoco motifs are essential for hearing

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn ^ΔC mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn ^ΔC allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.