The method of mouse embryoid body establishment affects structure and developmental gene expression.

To investigate formation of the three primary germ layers in mouse embryoid bodies (EBs), we observed changes in structure and gene expression over a 7-day culture period. We compared these changes using two methods for EB formation: hanging drop (HD) and static suspension culture (SSC). Light microscopy showed that a stratified columnar epithelial layer developed on the surface of EBs formed using the HD method. From Day 3 in culture, ultrastructural changes occurred in the aligned cellular membranes. Condensation of actin filaments was followed by formation of complicated adherent junctions and dilatation of intercellular canaliculi containing well-developed microvilli. These changes were more marked in EBs formed by the HD method than the SSC method. On Day 5 of culture, Brachyury gene expression, a marker for mesoderm formation, was detected only with the HD method. Nestin, an ectoderm marker, and Foxa2, an endoderm marker, were expressed with both methods. These results suggest that in EBs formed with the HD method, actin formation and Brachyury gene expression mark the transition from two to three primary germ layers. Additionally, the HD method promotes more rapid and complete development of mouse EBs than does the SSC method. While the SSC method is simple and easy to use, it needs improvement to form more complete EBs.

Increased expression of proapoptotic BMCC1, a novel gene with the BNIP2 and Cdc42GAP homology (BCH) domain, is associated with favorable prognosis in human neuroblastomas.

Differential screening of the genes obtained from cDNA libraries of primary neuroblastomas (NBLs) between the favorable and unfavorable subsets has identified a novel gene BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1). Its 350 kDa protein product possessed a Bcl2-/adenovirus E1B nineteen kDa-interacting protein 2 (BNIP2) and Cdc42GAP homology domain in the COOH-terminus in addition to P-loop and a coiled-coil region near the NH2-terminus. High levels of BMCC1 expression were detected in the human nervous system as well as spinal cord, brain and dorsal root ganglion in mouse embryo. The immunohistochemical study revealed that BMCC1 was positively stained in the cytoplasm of favorable NBL cells but not in unfavorable ones with MYCN amplification. The quantitative real-time reverse transcription-PCR using 98 primary NBLs showed that high expression of BMCC1 was a significant indicator of favorable NBL. In primary culture of newborn mice superior cervical ganglion (SCG) neurons, mBMCC1 expression was downregulated after nerve growth factor (NGF)-induced differentiation, and upregulated during the NGF-depletion-induced apoptosis. Furthermore, the proapoptotic function of BMCC1 was also suggested by increased expression in CHP134 NBL cells undergoing apoptosis after treatment with retinoic acid, and by an enhanced apoptosis after depletion of NGF in the SCG neurons obtained from newborn mice transgenic with BMCC1 in primary culture. Thus, BMCC1 is a new member of prognostic factors for NBL and may play an important role in regulating differentiation, survival and aggressiveness of the tumor cells.

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes.

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro.

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.

Targeted disruption of the mouse homologue of the Drosophila polyhomeotic gene leads to altered anteroposterior patterning and neural crest defects.

The rae28 gene is a mouse homologue of the Drosophila polyhomeotic gene (Nomura, M., Takihara, Y. and Shimada, K. (1994) Differentiation 57, 39-50), which is a member of the Polycomb group (Pc-G) of genes (DeCamillis, M., Cheng, N., Pierre, D. and Brock, H.W. (1992) Genes Dev. 6, 223-232). The Pc-G genes are required for the correct expression of the Homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. To study the role of the rae28 gene in mouse development, we generated rae28-deficient mice by gene targeting in embryonic stem cells. The rae28-/- homozygous mice exhibited perinatal lethality, posterior skeletal transformations and defects in neural crest-related tissues, including ocular abnormalities, cleft palate, parathyroid and thymic hypoplasia and cardiac anomalies. The anterior boundaries of Hoxa-3, a-4, a-5, b-3, b-4 and d-4 expression were shifted rostrally in the paraxial mesoderm of the rae28-/- homozygous embryos, and those of Hoxb-3 and b-4 expression were also similarly altered in the rhombomeres and/or pharyngeal arches. These altered Hox codes were presumed to be correlated with the posterior skeletal transformations and neural crest defects observed in the rae28-/- homozygous mice. These results indicate that the rae28 gene is involved in the regulation of Hox gene expression and segment specification during paraxial mesoderm and neural crest development.

The human homologue of the murine Llglh gene (LLGL) maps within the Smith-Magenis syndrome region in 17p11.2.

We have isolated and characterized the human homologue of the murine Llglh gene, which was originally isolated as a homologue of a Drosophila tumor suppressor gene 1(2)gl (lethal(2) giant larvae). In the mouse, Llglh is thought to play an important role during brain development as a regulatory target of Hoxc8. The human homologue of Llglh (LLGL) encodes a protein consisting of 1,033 amino acids. This gene was mapped by fluorescence in situ hybridization (FISH) to human chromosome 17p11.2, a region that is typically deleted in patients with Smith-Magenis syndrome (SMS). In our FISH analysis of metaphase chromosomes of four SMS patients, a probe representing LLGL failed in each case to hybridize to one of the two chromosome 17 homologues, indicating that this gene may play a role in the pathogenesis of SMS.

A mouse homologue of the Drosophila tumour-suppressor gene l(2)gl controlled by Hox-C8 in vivo.

The homeobox is a 183-base-pair DNA sequence originally found in Drosophila segmentation and homeotic genes. In Drosophila, homeotic genes are clustered in the Antennapedia and Bithorax complexes, collectively called the homeotic gene complex (HOM-C). In the mouse genome, about 40 homeobox genes (Hox) are clustered in four chromosomal regions (Hox A to D). The Hox genes are arranged in the same order and have the same anteroposterior pattern of expression as their structural homologue in the HOM-C, suggesting that they control mouse pattern formation in the same way that HOM-C members do in Drosophila. Homeobox gene products are believed to be transcription factors that regulate expression of target genes. A few candidate target genes have been identified in Drosophila by various approaches but the Hox gene targets are poorly understood, mostly because of limitations in the available approaches. Here we identify several candidate Hox gene targets, including a mouse homologue of the Drosophila tumour-suppressor gene l(2)gl, by immunopurification of DNA sequences bound to a Hox protein in native chromatin.