Functional analysis of BMP4 signaling in mouse neural development

During early mouse neural development, bone morphogenetic protein (BMP) signaling patterns the dorsal neural tube and defines distinct neural progenitor cell domains along the dorsoventral axis. Unlike the ventral signaling molecule Sonic hedgehog, which has long-range activity by establishing a concentration gradient in the ventral neural tube, these dorsally expressed BMPs appear to have a limited domain of action. This raises questions as to how BMP activity is restricted locally and how restricted BMP signaling directs dorsal neural patterning and differentiation. I hypothesize that BMPs are restricted in the dorsal neural tube for correct dorsoventral patterning. ^ Previous studies have shown that the positively charged basic amino acids located at the N-terminus of several BMPs are essential for heparin binding and diffusion. This provides a novel tool to address these questions. Here I adapted a UAS/GAL4 bigenic mouse system to control the ectopic expression of BMP4 and a mutant form of BMP4 that lacks a subset of the N-terminal basic amino acids. The target genes, UAS-Bmp4 and UAS-mBmp4 , were introduced into the Hprt locus by gene targeting in mouse embryonic stem cells. The expression of the GAL4 transactivator was driven by a roof plate specific Wnt1 promoter. ^ The bigenic mouse embryos exhibit phenotype variations, ranging from mid/hindbrain defects, hemorrhage, and eye abnormalities to vasculture formation. Embryonic death starts around E11.5 because of severe hemorrhage. The different expression levels of the activated transgene may account for the phenotype variation. Further marker analysis reveals that mutant BMP4 induces ectopic expression of the dorsal markers MSX1/2 and PAX7 in the ventral neural tube. In addition, the expression of the ventral neural marker NKX2.2 is affected by the expanded BMP4 activity, indicating that ectopic BMP signaling can antagonize ventral signaling. Comparison of the phenotypes of the Wnt1/ Bmp4 and Wnt1/mBmp4 bigenic embryos that express transgenes at the same level, respectively, shows that mutant BMP4 causes the expansion of dorsal neural fates ventrally while wild type BMP4 does not, suggesting that mutant BMP4 acts farther than wild type BMP4. Together, these data suggest that the N-terminus basic amino acid core controls BMP4 long-range activity in neural development, and that BMP signaling patterns the dorsal neural tube through a secondary signaling pathway that involves homeodomain transcription factors MSX1/2 and PAX7. ^

Ser88 phosphorylation regulates dynein light chain 1 (DLC1) function in the mouse mammary gland

Dynein light chain 1 (DLC1) is a highly conserved and ubiquitously expressed protein which might have critical cellular function as total loss of DLC1 caused Drosophila embryonic death. Despite many proteins and RNAs interaction with it identified, DLC1's function(s) and regulation are largely unknown. Recently, DLC1 was identified as a physiological substrate of P21-activate kinase 1(Pak1) kinase from a human mammary cDNA library in a yeast-2-hybridization screening assay. Studies in primary human tumors and cell culture implicated that DLC1 could promote mammary cancerous phenotypes, and more importantly, Ser88 phosphorylation of DLC1by Pak1 kinase was found to be essential for DLC1's tumorigenic activities. Based on the above tissue culture studies, we hypothesized that Ser88 phosphorylation regulates DLC1. ^ To test this hypothesis, we generated two transgenic mouse models: MMTV-DLC1 and MMTV-DLC1-S88A mice with mammary specific expression of the DLC1 and DLC1-S88A cDNAs. Both of the transgenic mice mammary glands showed rare tumor incidence which indicated DLC1 alone may not be sufficient for tumorigenesis in vivo. However, these mice showed a significant alteration of mammary development. Mammary glands from the MMTV-DLC1 mice had hyperbranching and alveolar hyperplasia, with elevated cell proliferation. Intriguingly, these phenotypes were not seen in the mammary glands from the MMTV-S88A mice. Furthermore, while MMTV-DLC1 glands were normal during involution, MMTV-S88A mice showed accelerated mammary involution with increase apoptosis and altered expression of involution-associated genes. Further analysis of the MMTV-S88A glands showed they had increased steady state level of Bim protein which might be responsible for the early involution. Finally, our in vitro data showed that Ser88 phosphorylation abolished DLC1 dimer and consequently might disturb its interaction with Bim and destabilize Bim. ^ Collectively, our findings provided in vivo evidence that Ser88 phosphorylation of DLC1 can regulate DLC1's function. In addition, Ser88 phosphorylation might be critical for DLC1 dimer-monomer transition. ^

Life and death of the embryonic female reproductive tract

Regardless of genetic sex, amniotes develop two sets of genital ducts, the Wolffian and Müllerian ducts. Normal sexual development requires the differentiation of one duct and the regression of the other. I show that cells in the rostral most region of the coelomic epithelium (CE) are specified to a Müllerian duct fate beginning at Tail Somite Stage 19 (TS19). The Müllerian duct (MD) invaginates from the CE where it extends caudally to and reaches the Wolffian duct (WD) by TS22. Upon contact, the MD elongates to the urogenital sinus separating the WD from the CE and its formation is complete by TS34. During its elongation, the MD is associated with and dependent upon the WD and I have identified the mechanism for MD elongation. Using the Rosa26 reporter to fate map the WD, I show that the WD does not contribute cells to the MD. Using an in vitro recombinant explant culture assay I show that the entire length of the MD is derived from the CE. Furthermore, I analyzed cell proliferation and developed an in vitro assay to show that a small population of cells at the caudal tip proliferates, laying the foundation for the formation of the MD. I also show that during its formation, the MD has a distinctive mesoepithelial character. The MD in males regresses under the influence of Anti-Müllerian Hormone (AMH). Through tissue-specific gene inactivation I have identified that Acvr1 and Bmpr1a and Smad1, Smad5 and Smad8 function redundantly in transducing the AMH signal. In females the MD differentiates into an epithelial tube and eventually the female reproductive tract. However, the exact tissue into which the MD differentiates has not been determined. I therefore generated a MD specific Cre allele that will allow for the fate mapping of the MD in both females males. The MD utilizes a unique form of tubulogenesis during development and to my knowledge is the only tubule that relies upon a signal from and the presence of another distinct epithelial tube for its formation.^

GENOME-WIDE PROFILING UNVEILS CRITICIAL FUNCTIONS OF p53 IN HUMAN EMBRYONIC STEM CELLS

Embryonic stem cells (ESCs) possess two unique characteristics: infinite self-renewal and the potential to differentiate into almost every cell type (pluripotency). Recently, global expression analyses of metastatic breast and lung cancers revealed an ESC-like expression program or signature, specifically for cancers that are mutant for p53 function. Surprisingly, although p53 is widely recognized as the guardian of the genome, due to its roles in cell cycle checkpoints, programmed cell death or senescence, relatively little is known about p53 functions in normal cells, especially in ESCs. My hypothesis is that p53 has specific transcription regulatory functions in human ESCs (hESCs) that a) oppose pluripotency and b) protect the stem cell genome in response to DNA damage and stress signaling. In mouse ESCs, these roles are believed to coincide, as p53 promotes differentiation in response to DNA damage, but this is unexplored in hESCs. To determine the biological roles of p53, specifically in hESCs, we mapped genome-wide chromatin interactions of p53 by chromatin immunoprecipitation and massively parallel tag sequencing (ChIP-Seq), and did so under three VIdifferent conditions of hESC status: pluripotency, differentiation-initiated and DNA-damage-induced. ChIP-Seq showed that p53 is enriched at distinct, induction-specific gene loci during each of these different conditions. Microarray gene expression analysis and functional annotation of the distinct p53-target genes revealed that p53 regulates specific genes encoding developmental regulators, which are expressed in differentiation-initiated but not DNA- damaged hESCs. We further discovered that, in response to differentiation signaling, p53 binds regions of chromatin that are repressed but also poised for rapid activation by core pluripotency factors OCT4 and NANOG in pluripotent hESCs. In response to DNA damage, genes associated with migration and motility are targeted by p53; whereas, the prime targets of p53 in control of cell death are conserved for p53 regulation in both differentiation and DNA damage. Our genome-wide profiling and bioinformatics analyses show that p53 occupies a special set of developmental regulatory genes during early differentiation of hESCs and functions in an induction-specific manner. In conclusion, our research unveiled previously unknown functions of p53 in ESC biology, which augments our understanding of one of the most deregulated proteins in human cancers.