TheamnGene Product Is Required in Extraembryonic Tissues for the Generation of Middle Primitive Streak Derivatives

Abstract

The primitive streak is the defining feature of the gastrulating mouse embryo. Currently, little is known in the mouse about the mechanisms that mediate the assembly of the primitive streak or about the signaling pathways that specify the different types of mesoderm and endoderm generated from the streak. To gain insight into primitive streak assembly and function, we have conducted a detailed phenotypic characterization of amnionless, a transgene-induced insertional mouse mutation that arrests embryonic development during gastrulation. Our histological and molecular analyses, when examined in the context of the mouse gastrula fate map, lead to the model that middle streak formation is specifically impaired in the amnionless mutant. Significantly, these observations argue that the formation of the middle streak is mediated by a pathway that is genetically separable from those that direct the specification of the distal and proximal streak regions. Intriguingly, our findings from wt ES cell left and right arrow amnionless-/- blastocyst chimeras indicate that this proposed separate pathway for middle streak formation is dependent on amnionless gene functions in the visceral endoderm.

Delivery of endosomes to lysosomes via microautophagy in the visceral endoderm of mouse embryos

“…As suggested by the name, the embryos lacking amnionless exhibit defective gastrulation, with severe disorganisation of the embryonic ectoderm and embryonic mesoderm. The cubilin mutant exhibits a developmental defect similar to that of the amnionless mutant 47,49 . Loss of function of p18, a lipid raft adaptor essential for the MEK-ERK pathway and mammalian target of rapamycin complex 1 signalling, results in a similar phenotype to that of amnionless 26 .…”

Mouse amnionless, which is required for primitive streak assembly,mediates cell-surface localization and endocytic function of cubilin on visceral endoderm and kidney proximal tubules

“…In situ hybridization studies were carried out as previously described (Kalantry et al, 2001;Tomihara-Newberger et al, 1998). Immunohistochemistry was performed with the Discovery staining module at the MSKCC Molecular Cytology Core Facility (Ventana Medical Systems).…”

“…Our earlier studies on the amnionless (amn) mutant highlighted a previously undefined role for the VE in patterning the primitive streak during gastrulation (Kalantry et al, 2001;Tomihara-Newberger et al, 1998). The primitive streak, a transient structure in amniotes, orchestrates epiblast cell behaviors required to generate definitive ectoderm, mesoderm and endoderm.…”

“…Proximal streak produces extraembryonic mesoderm and germ cells; middle streak produces lateral plate, intermediate and paraxial components of trunk mesoderm; and distal streak produces cardiac mesoderm and node-derived axial mesendoderm. amn mutants do not assemble a functional middle streak and specifically fail to form non-midline trunk mesoderm (Tomihara-Newberger et al, 1998). In addition, amn mutants are growth impaired after E7.0, indicating that streak assembly is mechanistically tied to the growth of the embryo.…”

ADAMTS13 is expressed in hepatic stellate cells

“…The liver tissue blocks were sectioned at 4 mm in thickness with a microtome and used in in situ hybridization using the procedures as previously described. 14 To generate probes for hybridization, an ADAMTS13 cDNA from human fetal liver cDNA library spanning nucleotide 541-1680 and inserted in pBSII-SK þ , kindly provided by David Ginsburg (Howard Hughes Medical Institutes, Ann Arbor, MI, USA) was used as the template. The plasmid was used to prepare labeled antisense or sense RNA by in vitro transcription with bacteriophage T7 or T3 RNA polymerase in the presence of 12 mmol/l UTP and 4 mmol/l [ 33 P]UTP.…”