Year: 2009
Type: article
Abstract: KLINE genes in microarray analyses performed on MEFs (mouse embryonic fibroblasts; >30 transcripts) and embryonic brain (>700 transcripts). Importantly, although changes in transcript levels are significant statistically and reproducible over large num- bers of individual samples, most changes are relatively small in magnitude, usually less than twofold. Included in this category is the Nipbl transcript itself, which is ubiquitously expressed and which was found to be downregulated by 30% in both adult and embryonic tissues derived from Nipbl 564/þ mice. In contrast, we found no evidence for elevated PSCS (precocious sister chromatid separation) in any cells derived from Nipbl 564/þ mice (fibroblasts, embryonic stem cells, and splenocytes), suggesting that cohesion defects do not occur, or if they do, are very subtle. Altogether, our data support the idea that the phenotypes observed in the Nipbl heterozygous mouse, and by extension in CdLS, are the consequence of subtle dysregulation of the expression of numerous genes, which results from a decrease in Nipbl function. Acknowledgment This work was supported by NIH grant P01HD052860. Modeling Early Developmental Defects in Cornelia de Lange Syndrome Using the Zebrafish Akihiko Muto 1,2 , Trevor Hoffman 1,2 , Shimako Kawauchi 2,3,4 , Thomas F. Schilling 1,2,5 , Arthur D. Lander 1,2,5 and Anne L. Calof 2,3,4,5 Department of Developmental and Cell Biology, 2 Developmental Biology Center, 3 Department of Anatomy and Neurobiology, 4 Center for Hearing Research, and 5 Center for Complex Biological Systems, University of California, Irvine, CA 92697 Nipbl (the ortholog of Drosophila Nipped-B) has been identified as a gene in which heterozygous mutations are associated with at least 50% of cases of Cornelia de Lange Syndrome (CdLS). Nipbl and its relatives encode cohesin regulatory proteins, which are thought to be involved in sister chromatid cohesion and DNA repair. Studies in Drosophila have revealed that Nipped-B also plays roles in developmental gene expression, possibly by influencing the strength of long-range promoter–enhancer interactions. Our re- cent studies in Nipblþ/ mutant mice strongly support the notion that widespread transcriptional dysregulation underlies the devel- opmental abnormalities in CdLS. However, the precise targets of Nipbl and the mechanism(s) by which it regulates gene expression remain unclear. To address these questions, we have chosen the zebrafish (Danio rerio) as a model system in which gene expression levels are easily manipulated by injecting morpholino antisense oligonucleotides (MO) and/or in vitro synthesized mRNA. Zebra- fish have two Nipbl orthologs (designated zNipbl-1 and zNipbl-2), and both genes are expressed ubiquitously during embryogenesis. Predicted amino acid (a.a.) sequences reveal that the N-terminal 200 a.a. and the C-terminal 1,800 a.a—the latter of which contains multiple HEAT domains—are highly conserved (66–80% identical) among zebrafish and mammalian proteins. MO knock- down in fish embryos, using MOs directed against both zNipbl1 and zNipbl2, causes a variety of developmental defects, including ab- normalities in heart and gut development, which overlap with abnormalities seen in CdLS. In Nipbl-MO-injected embryos (zNipbl-morphants), migration of cardiac progenitors is delayed, leading to cardia bifida (split heart) in about 15% of morphants. In addition, morphants exhibit reduced expression of an endodermal marker, foxA3, which correlates with later mis-localization of visceral organs (liver and pancreas) as well as abnormal gut looping. Such abnormalities of visceral organs are also common findings in CdLS. Consistent with the observations of aberrant morphology of heart and visceral organs in zNipbl-morphants, microarray and in situ hybridization analyses of gene expression reveals reduced expression of other endodermal genes (gata5, sox32, sox17, and foxA2). Although these endodermal genes are all known to be regulated by Nodal signaling, expression of Nodal genes, as well as the Nodal mesodermal target genes no tail and goosecoid, are not affected by zNipbl-MO, suggesting that at least some endodermal genes are directly regulated by Nipbl. These results suggest a mechanism by which Nipbl regulates endodermal development and a hypothesis for the etiology of heart and gut abnormalities observed in individuals with CdLS. We are further testing this hypothesis by examining endodermal gene expression in Nipbl- heterozygous mutant mice. Moreover, we are also examining functional relationships between Nipbl and cohesin on gene ex- pression by simultaneously manipulating cohesin expression in zebrafish embryos. Acknowledgment This work was supported by NIH P01-HD052860. Cohesin-Dependent Regulation of Gene Expression in Zebrafish Julia A. Horsfield 1 , Sasha H. Anagnostou 2 , Jimmy Kuang-Hsien Hu 2 *, Kitty Hsiao Yu Cho 2 , Robert Geisler 3 , Graham Lieschke 4 , Kathryn E. Crosier 2 , Philip S. Crosier 2 Department of Pathology, Dunedin School of Medicine, University of Otago, New Zealand; 2 Department of Molecular Medicine and Pathol- ogy, School of Medical Sciences, The University of Auckland, Auckland, New Zealand; 3 Max Planck Institute fur Entwicklungsbiologie, Tubin- gen, Germany; 4 Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia *Present address: Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. Cohesion or ‘‘pairing’’ of sister chromatids is essential to ensure each daughter cell receives the correct number of chromosomes during cell division. Sister chromatid cohesion is mediated by cohesin, a multimeric protein complex comprising at least four subunits: Rad21/Scc1, SA/Scc3, Smc1, and Smc3. We identified the cohesin subunit rad21 in a zebrafish forward genetic screen for positive regulators of runx1. Runx transcription factors are critical for determining cell fate in many cell types, and maintaining balanced levels of Runx protein is essential for normal development. Deregulated expression of runx genes leads to cancers and developmental disorders. 12-somite zebrafish embryos mutant for rad21 or morphant for Smc3 lack hematopoietic expression of runx1, and fail to express runx3. Partial failure of blood development in rad21 mutants is rescued by microinjection of runx1 mRNA. Significantly, loss of just one copy of the rad21 gene caused a reduction in runx1 transcription and reduced expression of neuro- nal genes ascl1a and ascl1b. rad21 mutant embryos also have neurodevelopmental abnormalities, consistent with the idea that neuronal development is particularly sensitive to loss of cohesin function.
Authors: Arthur D. Lander, Akihiko Muto, Trevor L. Hoffman, Shimako Kawauchi, Thomas F. Schilling, Anne L. Calof
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2401434408