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The LIM homeobox containing genes of the LIM-3 group, Lhx3 and Lhx4, are critical for normal development. Both genes are involved in the formation of the pituitary and the motoneuron system and loss of either gene causes perinatal lethality. Previous studies had shown that Lhx3 is overexpressed in hyperplastic placentas of mouse interspecies hybrids. To determine the role of LHX3 in the mouse placenta, we performed expression and function analyses. Our results show that Lhx3 exhibits specific spatial and temporal expression in the mouse placenta. However, deletion of Lhx3 does not produce a placental phenotype. To test whether this is due to functional substitution by Lhx4, we performed a phenotype analysis of Lhx3-/-; Lhx4-/- double-mutant placentas. A subset of Lhx3-/-; Lhx4-/- placentas exhibited abnormal structure of the labyrinth. However, absence of both LIM-3 genes did not interfere with placental transport nor consistently with expression of target genes such as Gnrhr. Thus, LHX3 and LHX4 appear to be dispensable for placental development and function.

Different causes, such as maternal diabetes, cloning by nuclear transfer, interspecific hybridization, and deletion of some genes such as Esx1, Ipl, or Cdkn1c, may underlie placental overgrowth. In a previous study, we carried out comparative gene expression analysis in three models of placental hyperplasias, cloning, interspecies hybridization (IHPD), and Esx1 deletion. This study identified a large number of genes that exhibited differential expression between normal and enlarged placentas; however, it remained unclear how altered expression of any specific gene was related to any specific placental phenotype. In the present study, we focused on two genes, Car2 and Ncam1, which both exhibited increased expression in interspecies and cloned hyperplastic placentas. Apart from a detailed expression analysis of both genes during normal murine placentation, we also assessed morphology of placentas that were null for Car2 or Ncam1. Finally, we attempted to rescue placental hyperplasia in a congenic model of IHPD by decreasing transcript levels of Car2 or Ncam1. In situ analysis showed that both genes are expressed mainly in the spongiotrophoblast, however, expression patterns exhibited significant variability during development. Contrary to expectations, homozygous deletion of either Car2 or Ncam1 did not result in placental phenotypes. However, expression analysis of Car3 and Ncam2, which can take over the function of Car2 and Ncam1, respectively, indicated a possible rescue mechanism, as Car3 and Ncam2 were expressed in spongiotrophoblast of Car2 and Ncam1 mutant placentas. On the other hand, downregulation of either Car2 or Ncam1 did not rescue any of the placental phenotypes of AT24 placentas, a congenic model for interspecies hybrid placentas. This strongly suggested that altered expression of Car2 and Ncam1 is a downstream event in placental hyperplasia., (Copyright 2005 Wiley-Liss, Inc.)

The role of cranial neural crest cells in the formation of visceral arch musculature was investigated in the Mexican axolotl, Ambystoma mexicanum. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, perchlorate) labeling and green fluorescent protein (GFP) mRNA injections combined with unilateral transplantations of neural folds showed that neural crest cells contribute to the connective tissues but not the myofibers of developing visceral arch muscles in the mandibular, hyoid, and branchial arches. Extirpations of individual cranial neural crest streams demonstrated that neural crest cells are necessary for correct morphogenesis of visceral arch muscles. These do, however, initially develop in their proper positions also in the absence of cranial neural crest. Visceral arch muscles forming in the absence of neural crest cells start to differentiate at their origins but fail to extend toward their insertions and may have a frayed appearance. Our data indicate that visceral arch muscle positioning is controlled by factors that do not have a neural crest origin. We suggest that the cranial neural crest-derived connective tissues provide directional guidance important for the proper extension of the cranial muscles and the subsequent attachment to the insertion on the correct cartilage. In a comparative context, our data from the Mexican axolotl support the view that the cranial neural crest plays a fundamental role in the development of not only the skeleton of the vertebrate head but also in the morphogenesis of the cranial muscles and that this might be a primitive feature of cranial development in vertebrates., (2004 Wiley-Liss, Inc.)