Key Points Calreticulin mutants responsible for myeloproliferative neoplasms specifically activate the thrombopoietin receptor and in turn JAK2. Activation of the thrombopoietin receptor requires the glycan binding site and a novel C-terminal tail of the mutant calreticulin.

Cilia are organelles that protrude from the apical surface of most eukaryotic cells. According to their structure and motility, they are classified into three groups 1 . Primary monocilia, present in most cells, lack a central pair of microtubules (9+0 structure), and play several roles in mechanosensation and cell signaling. Nodal cilia have a 9+0 structure but, unlike primary cilia, they move and generate an asymmetric distribution of morphogenetic cues in the node, thereby contributing to laterality 2 . The third group is composed of motile 9+2 cilia that cover epithelial cells lining airways, reproductive tracts, and cerebral ventricles. Motile cilia play crucial functions in clearing mucus and debris in the airways and may assist the transit of sperm and eggs in genital tracts [3][4] . In the early postnatal mammalian brain, neuroepithelial cells that line the cerebral ventricles leave the cell cycle and differentiate into a monolayer of ependymal cells. At the end of maturation, the apical surface of ependymal cells bears dozens of cilia that beat in coordinate manner to facilitate the circulation of the cerebrospinal fluid (CSF), from sites of production in choroid plexuses to sites of absorption in subarachnoid spaces. In mice, mutations in genes involved in the assembly or structure of ependymal cilia, such as Mdnah5 5 , Ift88 (also known as Tg737 or Polaris) 6 , and Hy3 7-8 affect cilia genesis, CSF dynamics, and result in hydrocephalus. Thus far, however, little is known about the genetic factors that govern ependymal cilia polarization and the relationship between the polarity and the development and function of these organelles.Planar cell polarity (PCP), also known as tissue polarity, controls the polarization of epithelial cells in a plane perpendicular to their apicobasal axis. It was initially described in Drosophila, where it affects the stereotypic arrangement of cuticular hairs, sensory bristles, and Supplementary Fig. 1a, b). RT-PCR and (Supplementary Fig. 1c).Using the knocked-in beta-galactosidase reporter, we monitored the expression of Celsr2 in heterozygous mice. Consistent with published data [24][25][26] , Celsr2 expression was detected in all brain areas, from E11.5 to P5 (Fig. 1a-h). Celsr2 mutant mice develop progressive hydrocephalusCelsr2 mutant mice were viable and fertile, except for some females that had vaginal atresia. At birth, their brain did not display any flagrant morphological abnormality, suggesting that Celsr2 is not critical for cerebral embryonic development. However, a progressive ventricular dilation appeared between P5 and P10 with variable severity between animals, and became evident at P21 (Fig. 2a,b).The lateral ventricles were enlarged, and the septum had an abnormal triangular shape, due to 6 6 reduction of the dorsal part of the lateral septum. We did not observe any stenosis or constriction at the level of the foramen of Monro or of the aqueduct. The subcommissural organ (SCO), a structure thought to play a role in non-communicating hydrocephalus, was...

The extracellular compartment where bone resorption occurs, between the osteoclast and bone matrix, is shown in this report to be actively acidified. The weak base acridine orange accumulates within this compartment but dissipates after incubation with ammonium chloride. Upon removal of ammonium chloride, the cells are able to rapidly reacidify this compartment. The highly convoluted plasma membrane of the osteoclast facing this acidic compartment (ruffled border) is shown to contain a 100-kD integral membrane protein otherwise present in limiting membranes of lysosomes and other related acidified organelles

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During liver development, hepatocytes and biliary cells differentiate from common progenitors called hepatoblasts. The factors that control hepatoblast fate decision are unknown. Here we report that a gradient of activin/ TGF␤ signaling controls hepatoblast differentiation. High activin/TGF␤ signaling is required near the portal vein for differentiation of biliary cells. The Onecut transcription factors HNF-6 and OC-2 inhibit activin/TGF␤ signaling in the parenchyma, and this allows normal hepatocyte differentiation. In the absence of Onecut factors, the shape of the activin/TGF␤ gradient is perturbed and the hepatoblasts differentiate into hybrid cells that display characteristics of both hepatocytes and biliary cells. Thus, a gradient of activin/TGF␤ signaling modulated by Onecut factors is required to segregate the hepatocytic and the biliary lineages.Supplemental material is available at http://www.genesdev.org.

Nephrolithiasis (kidney stones) affects 5-10% of adults and is most commonly associated with hypercalciuria, which may be due to monogenic renal tubular disorders. One such hypercalciuric disorder is Dent's disease, which is characterized by renal proximal tubular defects that include low molecular weight proteinuria, aminoaciduria and glycosuria, together with rickets in some patients. Dent's disease is due to inactivating mutations of the renal-specific voltage-gated chloride channel, CLC-5, which is expressed in the proximal tubule, thick ascending limb and collecting duct. The subcellular localization of CLC-5 to the proximal tubular endosomes has suggested a role in endocytosis, and to facilitate in vivo investigations of CLC-5 in Dent's disease we generated mice lacking CLC-5 by targeted gene disruption. CLC-5-deficient mice developed renal tubular defects which included low molecular weight (<70 kDa) proteinuria, generalized aminoaciduria that was more pronounced for neutral and polar amino acids, and glycosuria. They also developed hypercalciuria and renal calcium deposits and some had deformities of the spine. Furthermore, endocytosis as assessed by horseradish peroxidase uptake in the proximal tubule was severely impaired in CLC-5-deficient mice, thereby demonstrating a role for CLC-5 in endosomal uptake of low molecular weight proteins. Thus, CLC-5-deficient mice provide a model for Dent's disease and this will help in elucidating the function of this chloride channel in endocytosis and renal calcium homeostasis.

Dent's disease, which is a renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is associated with inactivating mutations of the X-linked chloride channel, CLC-5. However, the manner in which a functional loss of CLC-5 leads to such diverse renal abnormalities remains to be defined. In order to elucidate this, we performed studies to determine the segmental expression of CLC-5 in the human kidney and to define its intracellular distribution. We raised and characterized antisera against human CLC-5, and identified by immunoblotting an 83 kDa band corresponding to CLC-5 in human kidney cortex and medulla. Immunohistochemistry revealed CLC-5 expression in the epithelial cells lining the proximal tubules and the thick ascending limbs of Henle's loop, and in intercalated cells of the collecting ducts. Studies of subcellular human kidney fractions established that CLC-5 distribution was associated best with that of Rab4, which is a marker of recycling early endosomes. In addition, confocal microscopy studies using the proximal tubular cell model of opossum kidney cells, which endogenously expressed CLC-5, revealed that CLC-5 co-localized with the albumin-containing endocytic vesicles that form part of the receptor-mediated endocytic pathway. Thus, CLC-5 is expressed at multiple sites in the human nephron and is likely to have a role in the receptor-mediated endocytic pathway. Furthermore, the functional loss of CLC-5 in the proximal tubules and the thick ascending limbs provides an explanation for the occurrences of low molecular weight proteinuria and hypercalciuria, respectively. These results help to elucidate further the patho-physiological basis of the renal tubular defects of Dent's disease.

The ␤-amyloid peptide (A␤) is the major constituent of the amyloid core of senile plaques found in the brain of patients with Alzheimer disease. A␤ is produced by the sequential cleavage of the amyloid precursor protein (APP) by ␤-and ␥-secretases. Cleavage of APP by ␥-secretase also generates the APP intracellular C-terminal domain (AICD) peptide, which might be involved in regulation of gene transcription. APP contains three Gly-XXX-Gly (GXXXG) motifs in its juxtamembrane and transmembrane (TM) regions. Such motifs are known to promote dimerization via close apposition of TM sequences. We demonstrate that pairwise replacement of glycines by leucines or isoleucines, but not alanines, in a GXXXG motif led to a drastic reduction of A␤40 and A␤42 secretion. ␤-Cleavage of mutant APP was not inhibited, and reduction of A␤ secretion resulted from inhibition of ␥-cleavage. It was anticipated that decreased ␥-cleavage of mutant APP would result from inhibition of its dimerization. Surprisingly, mutations of the GXXXG motif actually enhanced dimerization of the APP C-terminal fragments, possibly via a different TM ␣-helical interface. Increased dimerization of the TM APP C-terminal domain did not affect AICD production.The progressive deposition of ␤-amyloid peptide (A␤) 3 leading to the formation of senile plaques is an invariant feature of Alzheimer disease. A␤ is a 39 -43-amino acid peptide, with two major isoforms of 40 and 42 amino acids (1, 2). A␤ is produced by the amyloidogenic cleavage of its precursor, the amyloid precursor protein or APP (3).The amyloidogenic processing of APP is initiated by ␤-cleavage within the lumenal/extracellular domain of the protein.The ␤-cleavage of APP is performed by the BACE proteins (BACE1 and -2) that are integral membrane proteins belonging to the aspartyl protease family (4 -8). ␤-Cleavage produces a 99-amino acid, membrane-anchored APP C-terminal fragment (␤CTF), which is further cleaved by the ␥-secretase activity to generate A␤. The ␥-secretase activity is contained in a high molecular weight multiprotein complex formed at least by the following proteins: a presenilin (PS1 or PS2), nicastrin (Nct), Pen-2, and Aph-1 (9). The activity of the ␥-secretase complex is also required for the generation of the intracellular fragment named (APP intracellular C-terminal domain (AICD). AICD was shown to translocate to the nucleus (10, 11), and there is growing experimental evidence suggesting a role for AICD in the regulation of gene transcription (12-17) even if the identity of APP target genes remains a matter of debate (18). The ␥-secretase complex, therefore, plays a central role in the onset and progression of Alzheimer disease not only because proteolysis of ␤CTF controls the production of A␤, but it also controls the intracellular signaling associated with APP, which in turn might regulate the expression of genes involved in the disease. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "ad...