ABSTRACT
Understanding the cellular events that underlie epithelial morphogenesis is a key problem in developmental biology. Here, we describe a new transgenic mouse line that makes it possible to visualize individual cells specifically in the Wolffian duct and ureteric bud, the epithelial structures that give rise to the collecting system of the kidney. myr-Venus, a membrane-associated form of the fluorescent protein Venus, was expressed in the ureteric bud lineage under the control of the Hoxb7 promoter. In Hoxb7/myr-Venus mice, the outlines of all Wolffian duct and ureteric bud epithelial cells are strongly labeled at all stages of urogenital development, allowing the shapes and arrangements of individual cells to be readily observed by confocal microscopy of freshly excised or cultured kidneys. This strain should be extremely useful for studies of cell behavior during ureteric bud branching morphogenesis in wild type and mutant mouse lines.
Subject(s)
Kidney/embryology , Ureter/embryology , Animals , Animals, Newborn , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Cell Shape , DNA Primers/genetics , Gene Expression , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Imaging, Three-Dimensional , Kidney/cytology , Kidney/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Mice, Inbred CBA , Mice, Transgenic , Microscopy, Confocal , Organ Culture Techniques , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ureter/cytology , Ureter/metabolismABSTRACT
The RET receptor tyrosine kinase plays a critical role in the development of the enteric nervous system (ENS) and the kidney. Upon glial-cell-line-derived neurotrophic factor (GDNF) stimulation, RET can activate a variety of intracellular signals, including the Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K)/AKT, and RAC1/JUN NH(2)-terminal kinase (JNK) pathways. We recently demonstrated that the RAC1/JNK pathway is regulated by serine phosphorylation at the juxtamembrane region of RET in a cAMP-dependent manner. To determine the importance of cAMP-dependent modification of the RET signal in vivo, we generated mutant mice in which serine residue 697, a putative protein kinase A (PKA) phosphorylation site, was replaced with alanine (designated S697A mice). Homozygous S697A mutant mice lacked the ENS in the distal colon, resulting from a migration defect of enteric neural crest cells (ENCCs). In vitro organ culture showed an impaired chemoattractant response of the mutant ENCCs to GDNF. JNK activation by GDNF but not ERK, AKT and SRC activation was markedly reduced in neurons derived from the mutant mice. The JNK inhibitor SP600125 and the PKA inhibitor KT5720 suppressed migration of the ENCCs in cultured guts from wild-type mice to comparable degrees. Thus, these findings indicated that cAMP-dependent modification of RET function regulates the JNK signaling responsible for proper migration of the ENCCs in the developing gut.
Subject(s)
Cell Movement/physiology , Enteric Nervous System/embryology , Mutation/genetics , Neural Crest/physiology , Proto-Oncogene Proteins c-ret/genetics , Signal Transduction/physiology , Animals , Anthracenes/pharmacology , Carbazoles/pharmacology , Cell Movement/drug effects , Colon/innervation , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Indoles/pharmacology , Mice , Mice, Mutant Strains , Pyrroles/pharmacologyABSTRACT
Mesenchymal-epithelial interactions are an important source of information for pattern formation during organogenesis. In the developing excretory system, one of the secreted mesenchymal factors thought to play a critical role in patterning the growth and branching of the epithelial ureteric bud is GDNF. We have tested the requirement for GDNF as a paracrine chemoattractive factor by altering its site of expression during excretory system development. Normally, GDNF is secreted by the metanephric mesenchyme and acts via receptors on the Wolffian duct and ureteric bud epithelium. Misexpression of GDNF in the Wolffian duct and ureteric buds resulted in formation of multiple, ectopic buds, which branched independently of the metanephric mesenchyme. This confirmed the ability of GDNF to induce ureter outgrowth and epithelial branching in vivo. However, in mutant mice lacking endogenous GDNF, kidney development was rescued to a substantial degree by GDNF supplied only by the Wolffian duct and ureteric bud. These results indicate that mesenchymal GDNF is not required as a chemoattractive factor to pattern the growth of the ureteric bud within the developing kidney, and that any positional information provided by the mesenchymal expression of GDNF may provide for renal branching morphogenesis is redundant with other signals.
