Impaired primitive streak assembly in the mouse amnionless ( amn ) mutant results in the absence of non-axial trunk mesoderm, a derivative of the middle region of the primitive streak. In addition, the epiblast of amn mutants fails to increase significantly in size after E7.0, indicating that middle primitive streak assembly is mechanistically tied to the growth of the embryo during gastrulation. Amn, a novel transmembrane protein, is expressed exclusively in an extra-embryonic tissue, visceral endoderm (VE), during the early post-implantation stages. We show that Amn is also expressed in kidney proximal tubules (KPT) and intestinal epithelium,which, like the VE, are polarized epithelia specialized for resorption and secretion. To explore whether Amn participates in the development or function of KPT and intestinal epithelia and to gain insight into the function of Amn during gastrulation, we constructed Amn -/- ES cell↔+/+blastocyst chimeras. While chimeras form anatomically normal kidneys and intestine, they exhibit variable, selective proteinuria, a sign of KPT malfunction. In humans, AMN has been genetically connected to Cubilin( CUBN ), a multi-ligand scavenger receptor expressed by KPT, intestine and yolk sac. Loss of CUBN, the intestinal intrinsic factor (IF)-vitamin B12 receptor, results in hereditary megaloblastic anemia (MGA1), owing to vitamin B12 malabsorption. The recent report of MGA1 families with mutations in AMN suggests that AMN functions in the same pathway as CUBN. We demonstrate that Cubn is not properly localized to the cell surface in Amn -/- tissues in the embryo and adult mouse, and that adult chimeras exhibit selective proteinuria of Cubn ligands. This study demonstrates that Amn is an essential component of the Cubn receptor complex in vivo and suggests that Amn/Cubn is required for endocytosis/transcytosis of one or more ligands in the VE during gastrulation to coordinate growth and patterning of the embryo. Furthermore, as AMN is apparently not required for gastrulation in humans, the developmental requirements for Amn/Cubn function may not be evolutionarily conserved, possibly reflecting differences between species in the role and organization of extra-embryonic tissues.

The c- kit receptor tyrosine kinase belongs to the PDGF/CSF-1/c- kit receptor subfamily. The kit -ligand, KL, also called steel factor, is synthesized from two alter natively spliced mRNAs as transmembrane proteins that can either be proteolytically cleaved to produce soluble forms of KL or can function as cell-associated molecules. The c- kit receptor kinase and KL are encoded at the white spotting ( W ) and steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl locus cause deficiencies in gametogenesis, melanogenesis and hematopoiesis. The c- kit receptor is expressed in the cellular targets of W and Sl mutations, while KL is expressed in their microenvironment. In melanogenesis, c- kit is expressed in melanoblasts from the time they leave the neural crest and expression continues during embryonic development and in the melanocytes of postnatal animals. In gametogenesis c- kit is expressed in primordial germ cells, in spermatogonia, and in primordial and growing oocytes, implying a role at three distinct stages of gametogenesis. Many mutant alleles are known at W and Sl loci and their phenotypes vary in the degree of severity in the different cellular targets of the mutations. While many W and Sl alleles severely affect primordial germ cells (PGC), several mild Sl alleles have weak effects on PGCs and exhibit differential male or female sterility. Steel Panda ( Sl pan ) is a KL expression mutation in which KL RNA transcript levels are reduced in most tissues analyzed. In female Sl pan /Sl pan mice, ovarian follicle development is arrested at the one layered cuboidal stage as a result of reduced KL expression in follicle cells, indicating a role for c- kit in oocyte growth. W sh is a c- kit expression mutation, which affects mast cells and melanogenesis. While the mast cell defect results from lack of c- kit expression, the pigmentation deficiency appears to stem from ectopic c- kit receptor expression in the somitic dermatome at the time of migration of melanoblasts from the neural crest to the periphery. It is proposed that the ectopic c- kit expression in W sh mice affects early melanogenesis in a dominant fashion. The “sash” or white belt of W sh/ + animals and some other mutant mice is explained by the varying density of melanoblasts along the body axis of wild-type embryos.