Esther Arman

Distinct GATA6- and laminin-dependent mechanisms regulate endodermal and ectodermal embryonic stem cell fates

This study investigates the establishment of alternative cell fates during embryoid body differentiation when ES cells diverge into two epithelia simulating the pre-gastrulation endoderm and ectoderm. We report that endoderm differentiation and endoderm-specific gene expression, such as expression of laminin 1 subunits, is controlled by GATA6 induced by FGF. Subsequently, differentiation of the non-polar primitive ectoderm into columnar epithelium of the epiblast is induced by laminin 1. Using GATA6 transformed Lamc1-null endoderm-like cells, we demonstrate that laminin 1 exhibited by the basement membrane induces epiblast differentiation and cavitation by cell-to-matrix/matrix-to-cell interactions that are similar to the in vivo crosstalk in the early embryo. Pharmacological and dominant-negative inhibitors reveal that the cell shape change of epiblast differentiation requires ROCK, the Rho kinase. We also show that pluripotent ES cells display laminin receptors; hence, these stem cells may serve as target for columnar ectoderm differentiation. Laminin is not bound by endoderm derivatives; therefore, the sub-endodermal basement membrane is anchored selectively to the ectoderm, conveying polarity to its assembly and to the differentiation induced by it. Unique to these interactions is their flow through two cell layers connected by laminin 1 and their involvement in the differentiation of two epithelia from the same stem cell pool: one into endoderm controlled by FGF and GATA6; and the other into epiblast regulated by laminin 1 and Rho kinase.

Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane

Basement membranes are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Our previous analysis of a fibroblast growth factor (FGF) receptor mutation raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. Here we test this hypothesis and demonstrate that constitutively active Akt/PKB, an important downstream element of phosphoinositide 3′-kinase signaling, induces the synthesis of laminin-1 and collagen IV isotypes and causes their translocation to the basement membrane. By using promoter–reporter constructs, we show that constitutively active phosphoinositide 3′-kinase-p110 or Akt/PKB activates, whereas dominant negative Akt/PKB inhibits, transcription of laminin β1 and collagen IV α1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary–T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of basement membrane formation. The possible role of Akt/PKB-induced laminin and collagen IV synthesis in cell survival and differentiation will be discussed.

Protein tyrosine phosphatases ε and α perform nonredundant roles in osteoclasts

The closely related tyrosine phosphatases PTPa and PTPe fulfill distinct roles in osteoclasts. The various effects of each PTP on podosome organization in osteoclasts are caused by their distinct N-termini. The function of PTPe in these cells requires the presence of its 12 N-terminal residues, in particular serine 2.

Massive osteopetrosis caused by giant, non-functional osteoclasts in R51Q SNX10 mutant mice

In this study we report on the establishment and characterization of a novel knock-in mouse model that is homozygous for the R51Q mutation in the sorting nexin 10 (SNX10) protein. This mutation leads to massive, early-onset, and widespread osteopetrosis in the mutant mice, similar to that observed in humans who are homozygous for this mutation. The diseased mice exhibit multiple additional characteristics of the corresponding human osteopetrosis, including missing and impacted teeth, occasional osteomyelitis, stunted growth, failure to thrive, and a significantly-reduced lifespan. The phenotype of homozygous R51Q SNX10 osteoclasts is unique and defines a novel form of ARO that combines both lack of bone-resorbing activity and reduced cell numbers in vivo. Furthermore, mutant osteoclasts grown on bone develop a giant cell morphology, reaching sizes that are up to three orders of magnitude larger than osteoclasts from wild-type or heterozygous mice. These large osteoclasts display poor survival in vitro, which may account for their fewer numbers in vivo. Electron microscopy studies indicate that homozygous mutant osteoclasts exhibit severely impaired ruffled borders and are incapable of resorbing bone, providing a clear cellular basis for the osteopetrotic phenotype. We propose that the R51Q SNX10 mutation directly causes osteoporosis by affecting both osteoclast formation and function. We further conclude that the maximal size of osteoclasts is determined by an active and genetically-regulated mechanism in which SNX10 participates, and that it is disrupted by the R51Q SNX10 mutation.Summary The molecular mechanisms that regulate fusion of monocytes into functional osteoclasts are virtually unknown. We describe a knock-in mouse model for the R51Q mutation in sorting nexin 10 (SNX10) that exhibits osteopetrosis and related symptoms of patients of autosomal recessive osteopetrosis linked to this mutation. Osteopetrosis arises in homozygous R51Q SNX10 mice due to a unique combination of reduced numbers of osteoclasts that are non-functional. Fusion of mutant monocytes is deregulated and occurs rapidly and continuously to form giant, non-functional osteoclasts. Mutant osteoclasts mature quickly and survive poorly in vitro, possibly accounting for their scarcity in vivo. These cells also exhibit impaired ruffled borders, which are required for bone resorption, providing an additional basis for the osteopetrotic phenotype. More broadly, we propose that the maximal size of osteoclasts is actively determined by a genetically-regulated, cell-autonomous mechanism that limits precursor cell fusion, and for which SNX10 is required.