Lhx6 and Lhx8 are two closely related LIM-homeobox genes that share strong sequence homology and overlapping expression patterns in the developing mouse ventral telencephalon. Our previous published studies have shown that Lhx8 is required for the generation of several major groups of cholinergic neurons in the telencephalon while Lhx6 contributes to the proper positioning and differentiation of GABA-ergic interneurons in the neocortex and hippocampus. Both Lhx6 and Lhx8 are prominently expressed in the medial ganglionic eminence (MGE) of the developing ventral telencephalon. However, the globus pallidus, a major structure of the basal ganglia derived from the MGE, forms normally in either the Lhx6 or the Lhx8 single mutant. This raised the possibility that the two genes share redundant functions in the development of the globus pallidus. To address this issue, we generated and analyzed mutants lacking the function of both Lhx6 and Lhx8. Indeed, the combined functional ablation of these two genes impairs the formation of the globus pallidus. Furthermore, we detected severe defects in the tangential migration of GABAergic interneurons from the MGE to the cortex in Lhx6/Lhx8 double mutant that are more severe than those previously observed in the Lhx6 single mutant. We conclude that Lhx6 and Lhx8 play important, if partially redundant, roles in controlling the development of the telencephalon. Ldb1 encodes a co-regulator known to mediate the function of a number of Lhx genes. In an effort to show that Lhx6 and Lhx8 are also dependent on Ldb1, we generated mice carrying a floxed Ldb1 allele and crossed them with a transgenic line that expresses the Cre recombinase under the control of the regulatory elements of the Nkx2.1 gene. Nkx2.1 acts as an upstream regulator for both Lhx6 and Lhx8 in the ventral telencephalon. The Ldb1/Nkx2.1-Cre conditional mutants show a loss of Ldb1 immunostaining in the MGE. Moreover, they display a defect in the development of cholinergic or GABAergic neurons similar to that observed in either Lhx8 or Lhx6 single mutants. Likewise, the functional ablation of Ldb1 phenocopies the impairment in the development of the globus pallidus observed in Lhx6/Lhx8 double mutants. Another set of LIM-homeobox genes, Lhx 2, Lhx9 and Lmx1b, together with the gene encoding the Ldb1 co-regulator of transcription, play prominent roles in the development of the forelimbs and the hind limbs of the mouse. With the help of loss-of-function analysis of various combinations of these genes we were able to establish that simultaneous loss of Lhx2 and Lhx9 resulted in patterning and growth defects along the proximodistal (PD) and anteroposterior (AP) limb axes. Similar, but more severe, phenotypes were observed when the activities of all three LIM-homeobox genes, Lmx1b, Lhx2 and Lhx9, were significantly reduced by Cre-mediated knockdown of the gene encoding their obligatory cofactor Ldb1. This reveals that the dorsal limb-specific factor Lmx1b can thus partially compensate for the function of Lhx2 and Lhx9 in regulating AP and PD limb patterning and outgrowth. Mechanistically, the action of these three LIM-homeobox genes is tied in with signaling events that precede subsequent limb development. We demonstrated that Lhx2 and Lhx9 can fully substitute for each other, and that Lmx1b is partially redundant in controlling the production of output signals in mesenchymal cells of the early limb bud in response to FGF8 and Shh signaling. IFT172 is a structural component of primary cilia of the cell. We used a knockout approach to study its function in mouse development.Mutants exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. They seldom survive mid-gestation. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Global brain-patterning defects occur along the dorsalventral (DV) and anteriorposterior (AP) axes of the embryo. We were able to show that Ift172 plays multiple roles, such as involvement in early regulation of FGF8 at the midbrainhindbrain boundary, maintenance of the isthmic organizer, and mediating the function of the node (the early embryonic organizer) and the formation of head organizing center (the anterior mesendoderm, or AME). Our findings suggest that forebrain and midhindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. All these important early controls of embryonic development appear to hinge on cilia morphogenesis and cilia-mediated signaling. The reprogramming of somatic cells to an early embryonic state of development is a research goal of utmost biomedical importance. This is because such pluripotent cells potentially can give rise to virtually any cell of the body via specialized multistage differentiation programs. Shinya Yamanakas seminal work, demonstrating that pluripotency can be induced in mammalian somatic cells via viral transfection of four transcription factors (c-Myc, Sox2, Oct4 and Klf4) has revolutionized current research. Work in a rapidly increasing number of laboratories worldwide is focused on the potential of induced pluripotent stem (iPS) cells to serve as diagnostic tools and as a source for treatment of human disorders. Reprogramming experiments did not start with the generation of iPS cells. Efforts have been going on for years to find suitable ways of turning back the natural course of events that lead from a pluripotent cell to a fully differentiated end product. Best known among the results of these earlier efforts are the generation of embryonic stem cells via transfer of somatic nuclei into oocytes and the reprogramming of somatic cells via fusion with embryonic stem (ES) cells. Our own reprogramming studies began with the latter approach. We knew about work by others demonstrating that fusion of somatic cells with ES cells can yield pluripotent cell-cell hybrids, albeit at low fusion and reprogramming efficiencies. In our own experiments, briefly outlined in last years report, we examined the ability of undifferentiated ES cell lines to reprogram the nuclei of mouse embryo fibroblasts (MEF) through cell-cell fusion. Activated baculovirus induced fusion events in 70-85% of the cells, resulting in efficient reprogramming. The resulting MEF/ES cell hybrids, although nearly tetraploid, exclusively expressed ES markers and exhibited characteristics of normal ES cells. When comparing the reprogramming potency of four well known ES cell lines (R1, J1, E14, C57BL/6), we noticed that E14 cells stood out as significantly less potent in their reprogramming ability. Analysis of histone modifications demonstrated that low reprogramming potency was correlated with reduced H3 lysine 9 acetylation (H3K9ac) levels. Treatment of E14 cells with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) significantly increased H3K9 acetylation levels, as well as their reprogramming capacity to a level observed in R1 cells. Furthermore, treatment of E14 cells with HDAC inhibitors can increase the size and differentiation state of teratomas resulting from injection of the cells into immune tolerant mice. This allowed us to conclude that H3K9 acetylation levels are correlated with ES cell pluripotency and reprogramming efficiency.

Project Start
Project End
Budget Start
Budget End
Support Year
37
Fiscal Year
2009
Total Cost
$2,124,183
Indirect Cost
City
State
Country
Zip Code
Narkis, Ginat; Tzchori, Itai; Cohen, Tsadok et al. (2012) Isl1 and Ldb co-regulators of transcription are essential early determinants of mouse limb development. Dev Dyn 241:787-91
Flandin, Pierre; Zhao, Yangu; Vogt, Daniel et al. (2011) Lhx6 and Lhx8 coordinately induce neuronal expression of Shh that controls the generation of interneuron progenitors. Neuron 70:939-50
Hezroni, Hadas; Tzchori, Itai; Davidi, Anna et al. (2011) H3K9 histone acetylation predicts pluripotency and reprogramming capacity of ES cells. Nucleus 2:300-9
Hopper-Borge, Elizabeth A; Churchill, Timothy; Paulose, Chelsy et al. (2011) Contribution of Abcc10 (Mrp7) to in vivo paclitaxel resistance as assessed in Abcc10(-/-) mice. Cancer Res 71:3649-57
Li, LiQi; Jothi, Raja; Cui, Kairong et al. (2011) Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells. Nat Immunol 12:129-36
Phillips, Matthew D; Mukhopadhyay, Mahua; Poscablo, Cristina et al. (2011) Dkk1 and Dkk2 regulate epicardial specification during mouse heart development. Int J Cardiol 150:186-92
Fossat, Nicolas; Jones, Vanessa; Khoo, Poh-Lynn et al. (2011) Stringent requirement of a proper level of canonical WNT signalling activity for head formation in mouse embryo. Development 138:667-76
Zhao, Yangu; Mailloux, Christina M; Hermesz, Edit et al. (2010) A role of the LIM-homeobox gene Lhx2 in the regulation of pituitary development. Dev Biol 337:313-23
Mayers, Chantal M; Wadell, Jennifer; McLean, Kate et al. (2010) The Rho guanine nucleotide exchange factor AKAP13 (BRX) is essential for cardiac development in mice. J Biol Chem 285:12344-54
Miquelajáuregui, Amaya; Varela-Echavarría, Alfredo; Ceci, M Laura et al. (2010) LIM-homeobox gene Lhx5 is required for normal development of Cajal-Retzius cells. J Neurosci 30:10551-62

Showing the most recent 10 out of 19 publications