Stromal cells mediate retinoid-dependent functions essential for renal development.
The essential role of vitamin A and its metabolites, retinoids, in kidney development has been demonstrated in vitamin A deficiency and gene targeting studies. Retinoids signal via nuclear transcription factors belonging to the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families. Inactivation of RARaplpha and RARbeta2 receptors together, but not singly, resulted in renal malformations, suggesting that within a given renal cell type, their concerted function is required for renal morphogenesis. At birth, RARalpha beta2(-) mutants displayed small kidneys, containing few ureteric bud branches, reduced numbers of nephrons and lacking the nephrogenic zone where new nephrons are continuously added. These observations have prompted us to investigate the role of RARalpha and RARbeta2 in renal development in detail. We have found that within the embryonic kidney, RARalpha and RARbeta2 are colocalized in stromal cells, but not in other renal cell types, suggesting that stromal cells mediate retinoid-dependent functions essential for renal development. Analysis of RARalpha beta2(-) mutant kidneys at embryonic stages revealed that nephrons were formed and revealed no changes in the intensity or distribution of molecular markers specific for different metanephric mesenchymal cell types. In contrast the development of the collecting duct system was greatly impaired in RARalpha beta2(-) mutant kidneys. Fewer ureteric bud branches were present, and ureteric bud ends were positioned abnormally, at a distance from the renal capsule. Analysis of genes important for ureteric bud morphogenesis revealed that the proto-oncogene c-ret was downregulated. Our results suggest that RARalpha and RARbeta2 are required for generating stromal cell signals that maintain c-ret expression in the embryonic kidney. Since c-ret signaling is required for ureteric bud morphogenesis, loss of c-ret expression is a likely cause of impaired ureteric bud branching in RARalpha beta2(-) mutants. (+info
FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression.
Beads containing recombinant FGF8 (FGF8-beads) were implanted in the prospective caudal diencephalon or midbrain of chick embryos at stages 9-12. This induced the neuroepithelium rostral and caudal to the FGF8-bead to form two ectopic, mirror-image midbrains. Furthermore, cells in direct contact with the bead formed an outgrowth that protruded laterally from the neural tube. Tissue within such lateral outgrowths developed proximally into isthmic nuclei and distally into a cerebellum-like structure. These morphogenetic effects were apparently due to FGF8-mediated changes in gene expression in the vicinity of the bead, including a repressive effect on Otx2 and an inductive effect on En1, Fgf8 and Wnt1 expression. The ectopic Fgf8 and Wnt1 expression domains formed nearly complete concentric rings around the FGF8-bead, with the Wnt1 ring outermost. These observations suggest that FGF8 induces the formation of a ring-like ectopic signaling center (organizer) in the lateral wall of the brain, similar to the one that normally encircles the neural tube at the isthmic constriction, which is located at the boundary between the prospective midbrain and hindbrain. This ectopic isthmic organizer apparently sends long-range patterning signals both rostrally and caudally, resulting in the development of the two ectopic midbrains. Interestingly, our data suggest that these inductive signals spread readily in a caudal direction, but are inhibited from spreading rostrally across diencephalic neuromere boundaries. These results provide insights into the mechanism by which FGF8 induces an ectopic organizer and suggest that a negative feedback loop between Fgf8 and Otx2 plays a key role in patterning the midbrain and anterior hindbrain. (+info
A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo.
Morphogenesis depends on the precise control of basic cellular processes such as cell proliferation and differentiation. Wnt5a may regulate these processes since it is expressed in a gradient at the caudal end of the growing embryo during gastrulation, and later in the distal-most aspect of several structures that extend from the body. A loss-of-function mutation of Wnt5a leads to an inability to extend the A-P axis due to a progressive reduction in the size of caudal structures. In the limbs, truncation of the proximal skeleton and absence of distal digits correlates with reduced proliferation of putative progenitor cells within the progress zone. However, expression of progress zone markers, and several genes implicated in distal outgrowth and patterning including Distalless, Hoxd and Fgf family members was not altered. Taken together with the outgrowth defects observed in the developing face, ears and genitals, our data indicates that Wnt5a regulates a pathway common to many structures whose development requires extension from the primary body axis. The reduced number of proliferating cells in both the progress zone and the primitive streak mesoderm suggests that one function of Wnt5a is to regulate the proliferation of progenitor cells. (+info
The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping.
Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut. (+info
The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development.
Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. Recently, heterozygous mutations of this gene were found to cause human congenital heart disease (Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and Seidman, J. G. (1998) Science 281, 108-111). To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, we have generated and analyzed mutant mice completely null for Csx/Nkx2.5. Homozygous null embryos showed arrest of cardiac development after looping and poor development of blood vessels. Moreover, there were severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 was disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there were almost no ES cell-derived cardiac myocytes, while there were substantial contributions of Csx /Nkx2.5-deficient cells in other organs. Whole-mount &bgr;-gal staining of chimeric embryos showed that more than 20% contribution of Csx/Nkx2. 5-deficient cells in the heart arrested cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 did not abolish initial heart looping, (2) there was no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes, (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart, (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes, (5) Csx/Nkx2. 5 null cells exert dominant interfering effects on cardiac development, and (6) there were severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos. (+info
Requirement of a novel gene, Xin, in cardiac morphogenesis.
A novel gene, Xin, from chick (cXin) and mouse (mXin) embryonic hearts, may be required for cardiac morphogenesis and looping. Both cloned cDNAs have a single open reading frame, encoding proteins with 2,562 and 1,677 amino acids for cXin and mXin, respectively. The derived amino acid sequences share 46% similarity. The overall domain structures of the predicted cXin and mXin proteins, including proline-rich regions, 16 amino acid repeats, DNA-binding domains, SH3-binding motifs and nuclear localization signals, are highly conserved. Northern blot analyses detect a single message of 8.9 and 5.8 kilo base (kb) from both cardiac and skeletal muscle of chick and mouse, respectively. In situ hybridization reveals that the cXin gene is specifically expressed in cardiac progenitor cells of chick embryos as early as stage 8, prior to heart tube formation. cXin continues to be expressed in the myocardium of developing hearts. By stage 15, cXin expression is also detected in the myotomes of developing somites. Immunofluorescence microscopy reveals that the mXin protein is colocalized with N-cadherin and connexin-43 in the intercalated discs of adult mouse hearts. Incubation of stage 6 chick embryos with cXin antisense oligonucleotides results in abnormal cardiac morphogenesis and an alteration of cardiac looping. The myocardium of the affected hearts becomes thickened and tends to form multiple invaginations into the heart cavity. This abnormal cellular process may account in part for the abnormal looping. cXin expression can be induced by bone morphogenetic protein (BMP) in explants of anterior medial mesoendoderm from stage 6 chick embryos, a tissue that is normally non-cardiogenic. This induction occurs following the BMP-mediated induction of two cardiac-restricted transcription factors, Nkx2.5 and MEF2C. Furthermore, either MEF2C or Nkx2.5 can transactivate a luciferase reporter driven by the mXin promoter in mouse fibroblasts. These results suggest that Xin may participate in a BMP-Nkx2.5-MEF2C pathway to control cardiac morphogenesis and looping. (+info
Insect evolution: Redesigning the fruitfly.
Homeotic mutations in Drosophila can result in dramatic phenotypes that suggest the possibility for rapid morphological evolution, but dissection of the genetic pathway downstream of Ultrabithorax is beginning to reveal how wing morphology may have evolved by more gradual transformations. (+info
Ultrabithorax function in butterfly wings and the evolution of insect wing patterns.
BACKGROUND: . The morphological and functional evolution of appendages has played a critical role in animal evolution, but the developmental genetic mechanisms underlying appendage diversity are not understood. Given that homologous appendage development is controlled by the same Hox gene in different organisms, and that Hox genes are transcription factors, diversity may evolve from changes in the regulation of Hox target genes. Two impediments to understanding the role of Hox genes in morphological evolution have been the limited number of organisms in which Hox gene function can be studied and the paucity of known Hox-regulated target genes. We have therefore analyzed a butterfly homeotic mutant 'Hindsight', in which portions of the ventral hindwing pattern are transformed to ventral forewing identity, and we have compared the regulation of target genes by the Ultrabithorax (Ubx) gene product in Lepidopteran and Dipteran hindwings. RESULTS: . We show that Ubx gene expression is lost from patches of cells in developing Hindsight hindwings, correlating with changes in wing pigmentation, color pattern elements, and scale morphology. We use this mutant to study how regulation of target genes by Ubx protein differs between species. We find that several Ubx-regulated genes in the Drosophila haltere are not repressed by Ubx in butterfly hindwings, but that Distal-less (Dll) expression is regulated by Ubx in a unique manner in butterflies. CONCLUSIONS: . The morphological diversification of insect hindwings has involved the acquisition of different sets of target genes by Ubx in different lineages. Changes in Hox-regulated target gene sets are, in general, likely to underlie the morphological divergence of homologous structures between animals. (+info