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1.  Disruption of SoxB1 dependent Sonic hedgehog expression in the hypothalamus causes Septo-Optic Dysplasia 
Developmental Cell  2012;22(3):585-596.
Septo-optic dysplasia (SOD) is a congenital brain anomaly that results in pituitary, optic nerve, and midline forebrain defects. The etiology of SOD is poorly understood, with the majority of cases being sporadic. In rare instances, SOD is caused by mutations in Sox2, Sox3 or Hesx1, but how this manifests in disease is not entirely certain. We demonstrate here that mouse embryos lacking Sonic hedgehog (Shh) in the prospective hypothalamus exhibit key features of SOD, including pituitary hypoplasia and absence of the optic disc. The hypothalamic source of Shh is required to maintain gene expression boundaries along the anteroposterior and mediolateral neural axes that are important for proper pituitary and eye development, respectively. We further reveal that Sox2 and Sox3 are dose dependent regulators of Shh transcription, which directly bind and activate a long-range Shh forebrain enhancer. These data indicate that reduced levels of Shh expression in the hypothalamus cause SOD.
PMCID: PMC3306593  PMID: 22421044
Septo-optic dysplasia; Sonic hedgehog; SoxB1; hypothalamus; pituitary; optic disc
2.  Direct transcriptional regulation of Six6 is controlled by SoxB1 binding to a remote forebrain enhancer 
Developmental biology  2012;366(2):393-403.
Six6, a sine oculis homeobox protein, plays a crucial and conserved role in the development of the forebrain and eye. To understand how the expression of Six6 is regulated during embryogenesis, we screened ~250 kb of genomic DNA encompassing the Six6 locus for cis-regulatory elements capable of directing reporter gene expression to sites of Six6 transcription in transgenic mouse embryos. Here, we describe two novel enhancer elements, that are highly conserved in vertebrate species and whose activities recapitulate Six6 expression in the ventral forebrain and eye, respectively. Cross-species comparisons of the Six6 forebrain enhancer sequences revealed highly conserved binding sites matching the consensus for homeodomain and SoxB1 transcription factors. Deletion of either of the binding sites resulted in loss of the forebrain enhancer activity in the ventral forebrain. Moreover, our studies show that members of the SoxB1 family, including Sox2 and Sox3, are expressed in the overlapping region of the ventral forebrain with Six6 and can bind to the Six6 forebrain enhancer. Loss of function of SoxB1 genes in vivo further emphasizes their role in regulating Six6 forebrain enhancer activity. Thus, our data strongly suggest that SoxB1 transcription factors are direct activators of Six6 expression in the ventral forebrain.
PMCID: PMC4009495  PMID: 22561201
Six6; SoxB1; forebrain; Gene expression
3.  Regulation of Thalamic Development by Sonic Hedgehog 
The thalamus is strategically positioned within the caudal diencephalic area of the forebrain, between the mesencephalon and telencephalon. This location is important for unique aspects of thalamic function, to process and relay sensory and motor information to and from the cerebral cortex. How the thalamus comes to reside within this region of the central nervous system has been the subject of much investigation. Extracellular signals secreted from key locations both extrinsic and intrinsic to the thalamic primordium have recently been identified and shown to play important roles in the growth, regionalization, and specification of thalamic progenitors. One factor in particular, the secreted morphogen Sonic hedgehog (Shh), has been implicated in spatiotemporal and threshold models of thalamic development that differ from other areas of the CNS due, in large part, to its expression within two signaling centers, the basal plate and the zona limitans intrathalamica, a dorsally projecting spike that separates the thalamus from the subthalamic region. Shh signaling from these dual sources exhibit unique and overlapping functions in the control of thalamic progenitor identity and nuclei specification. This review will highlight recent advances in our understanding of Shh function during thalamic development, revealing similarities, and differences that exist between species.
PMCID: PMC3328779  PMID: 22529771
thalamus; diencephalon; forebrain; zli; Shh; morphogen
4.  Cis-regulatory mutations in human disease 
Cis-acting regulatory sequences are required for the proper temporal and spatial control of gene expression. Variation in gene expression is highly heritable and a significant determinant of human disease susceptibility. The diversity of human genetic diseases attributed, in whole or in part, to mutations in non-coding regulatory sequences is on the rise. Improvements in genome-wide methods of associating genetic variation with human disease and predicting DNA with cis-regulatory potential are two of the major reasons for these recent advances. This review will highlight select examples from the literature that have successfully integrated genetic and genomic approaches to uncover the molecular basis by which cis-regulatory mutations alter gene expression and contribute to human disease. The fine mapping of disease-causing variants has led to the discovery of novel cis-acting regulatory elements that, in some instances, are located as far away as 1.5 Mb from the target gene. In other cases, the prior knowledge of the regulatory landscape surrounding the gene of interest aided in the selection of enhancers for mutation screening. The success of these studies should provide a framework for following up on the large number of genome-wide association studies that have identified common variants in non-coding regions of the genome that associate with increased risk of human diseases including, diabetes, autism, Crohn's, colorectal cancer, and asthma, to name a few.
PMCID: PMC2742803  PMID: 19641089
cis regulation; transcription; gene expression; human disease
5.  Regulation of a remote Sonic hedgehog forebrain enhancer by the Six3 homeoprotein 
Nature genetics  2008;40(11):1348-1353.
The secreted morphogen, Sonic hedgehog (Shh) is a significant determinant of brain size and craniofacial morphology1–4. In humans, SHH haploinsufficiency results in holoprosencephaly (HPE)5, a defect in anterior midline formation. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a combination of genetic and biochemical experiments to uncover a critical pair of cis and trans acting determinants of Shh forebrain expression. A rare nucleotide variant located 460kb upstream of SHH was discovered in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity capture assay we screened SBE2 sequence for DNA binding proteins and identified members of the Six3/Six6 homeodomain family as candidate regulators of Shh transcription. Six3 and Six6 showed reduced binding affinity for the mutant compared to wild type SBE2 sequence. Moreover, HPE causing mutations in Six3 failed to bind and activate SBE2, whereas, Shh forebrain expression was unaltered in Six6−/− embryos. These data provide a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE.
PMCID: PMC2648611  PMID: 18836447
Shh; gene expression; forebrain; holoprosencephaly
6.  Activation of Class I transcription factors by low level Sonic hedgehog signaling is mediated by Gli2-dependent and independent mechanisms 
Developmental biology  2007;305(1):52-62.
The partitioning of the ventral neural tube into five distinct neuronal progenitor domains is dependent on the morphogenic action of the secreted protein Sonic hedgehog (Shh). The prevailing model stipulates that Class I genes are repressed and Class II genes are activated by high levels of Shh signaling and that sharp progenitor domain boundaries are established by the mutual repression of complementary pairs of Class I and Class II transcription factors. While core elements of this model are supported by experimental evidence, a number of issues remain unresolved. Foremost of these is a more thorough understanding of the mechanism by which Class I genes are regulated. In this study, we describe the consequences of Shh misexpression on Class I and Class II gene expression in the hindbrain of ShhP1 embryos. We observed that an ectopic source of Shh in the otic vesicle of ShhP1 embryos ventralized the adjacent hindbrain by inducing, rather than repressing, the expression of several Class I genes (Pax6, Dbx1, Dbx2). The Shh dependent activation of Class I genes was mediated, in part, by Gli2. These results bear significance on the model of ventral neural tube patterning as they suggest a dual role for Shh in the regulation of Class I genes, whereby low levels of Shh signaling initiate Class I gene transcription, while higher levels restrict the domains of Class I gene expression to intermediate positions of the neural tube through the activation of Class II transcriptional regulators.
PMCID: PMC1914214  PMID: 17321515
Sonic hedgehog; Class I (Pax6,Dbx1,Dbx2); Class II (Nkx2.2,Nkx6.1,Nkx6.2) Gli2; ventral neural tube; morphogen; central nervous system; gene expression
7.  Wnt/β-catenin signaling promotes expansion of Isl-1–positive cardiac progenitor cells through regulation of FGF signaling 
Journal of Clinical Investigation  2007;117(7):1794-1804.
The anterior heart field (AHF), which contributes to the outflow tract and right ventricle of the heart, is defined in part by expression of the LIM homeobox transcription factor Isl-1. The importance of Isl-1–positive cells in cardiac development and homeostasis is underscored by the finding that these cells are required for cardiac development and act as cardiac stem/progenitor cells within the postnatal heart. However, the molecular pathways regulating these cells’ expansion and differentiation are poorly understood. We show that Isl-1–positive AHF progenitor cells in mice were responsive to Wnt/β-catenin signaling, and these responsive cells contributed to the outflow tract and right ventricle of the heart. Loss of Wnt/β-catenin signaling in the AHF caused defective outflow tract and right ventricular development with a decrease in Isl-1–positive progenitors and loss of FGF signaling. Conversely, Wnt gain of function in these cells led to expansion of Isl-1–positive progenitors with a concomitant increase in FGF signaling through activation of a specific set of FGF ligands including FGF3, FGF10, FGF16, and FGF20. These data reveal what we believe to be a novel Wnt-FGF signaling axis required for expansion of Isl-1–positive AHF progenitors and suggest future therapies to increase the number and function of these cells for cardiac regeneration.
PMCID: PMC1891000  PMID: 17607356
8.  Neural crest stem cell maintenance by combinatorial Wnt and BMP signaling 
The Journal of Cell Biology  2005;169(2):309-320.
Canonical Wnt signaling instructively promotes sensory neurogenesis in early neural crest stem cells (eNCSCs) (Lee, H.Y., M. Kléber, L. Hari, V. Brault, U. Suter, M.M. Taketo, R. Kemler, and L. Sommer. 2004. Science. 303:1020–1023). However, during normal development Wnt signaling induces a sensory fate only in a subpopulation of eNCSCs while other cells maintain their stem cell features, despite the presence of Wnt activity. Hence, factors counteracting Wnt signaling must exist. Here, we show that bone morphogenic protein (BMP) signaling antagonizes the sensory fate-inducing activity of Wnt/β-catenin. Intriguingly, Wnt and BMP act synergistically to suppress differentiation and to maintain NCSC marker expression and multipotency. Similar to NCSCs in vivo, NCSCs maintained in culture alter their responsiveness to instructive growth factors with time. Thus, stem cell development is regulated by combinatorial growth factor activities that interact with changing cell-intrinsic cues.
PMCID: PMC2171862  PMID: 15837799

Results 1-8 (8)