Henry M. Sucov

An Essential Role for Retinoid Receptors RARβ and RXRγ In Long-Term Potentiation and Depression

Hippocampal long-term potentiation (LTP) and long-term depression (LTD) are the most widely studied forms of synaptic plasticity thought to underlie spatial learning and memory. We report here that RARbeta deficiency in mice virtually eliminates hippocampal CA1 LTP and LTD. It also results in substantial performance deficits in spatial learning and memory tasks. Surprisingly, RXRgamma null mice exhibit a distinct phenotype in which LTD is lost whereas LTP is normal. Thus, while retinoid receptors contribute to both LTP and LTD, they do so in different ways. These findings not only genetically uncouple LTP and LTD but also reveal a novel and unexpected role for vitamin A in higher cognitive functions.

Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation

To facilitate the elucidation of the genetic events that may play an important role in the development or tumorigenesis of the prostate gland, we have generated a transgenic mouse line with prostate-specific expression of Cre recombinase. This line, named PB-Cre4, carries the Cre gene under the control of a composite promoter, ARR2PB which is a derivative of the rat prostate-specific probasin (PB) promoter. Based on RT-PCR detection of Cre mRNA in PB-Cre4 mice or Cre-mediated activation of LacZ activity in PB-Cre4/R26R double transgenic mice, it is conclusively demonstrated that Cre expression is post-natal and prostatic epithelium-specific. Although the Cre recombination is detected in all lobes of the mouse prostate, there is a significant difference in expression levels between the lobes, being highest in the lateral lobe, followed by the ventral, and then the dorsal and anterior lobes. Besides the prostate gland, no other tissues of the adult PB-Cre4 mice demonstrate significant Cre expression, except for a few scattered areas in the gonads and the stroma of the seminal vesicle. By crossing the PB-Cre4 animals with floxed RXRalpha allelic mice, we demonstrate that mice, whose conventional knockout of this gene is lethal in embryogenesis, could be propagated with selective inactivation of RXRalpha in the prostate. Taken together, the results show that the PB-Cre4 mice have high levels of Cre expression and a high penetrance in the prostatic epithelium. The PB-Cre4 mice will be a useful resource for genetic-based studies on prostate development and prostatic disease.

Hepatocyte-Specific Mutation Establishes Retinoid X Receptor α as a Heterodimeric Integrator of Multiple Physiological Processes in the Liver

ABSTRACT A large number of physiological processes in the adult liver are regulated by nuclear receptors that require heterodimerization with retinoid X receptors (RXRs). In this study, we have usedcre-mediated recombination to disrupt the mouse RXRα gene specifically in hepatocytes. Although such mice are viable, molecular and biochemical parameters indicate that every one of the examined metabolic pathways in the liver (mediated by RXR heterodimerization with PPARα, CARβ, PXR, LXR, and FXR) is compromised in the absence of RXRα. These data demonstrate the presence of a complex circuitry in which RXRα is integrated into a number of diverse physiological pathways as a common regulatory component of cholesterol, fatty acid, bile acid, steroid, and xenobiotic metabolism and homeostasis.

RXR alpha deficiency confers genetic susceptibility for aortic sac, conotruncal, atrioventricular cushion, and ventricular muscle defects in mice.

Retinoid-dependent pathways play a central role in regulating cardiac morphogenesis. Recently, we characterized gene-targeted RXR alpha -/- embryos, which display an atrial-like ventricular phenotype with the development of heart failure and lethality at embryonic day 14.5. To quantitate the frequency and complexity of cardiac morphogenic defects, we now use microdissection and scanning electron microscopy to examine 107 wild-type, heterozygous, and homozygous embryos at embryonic day 13.5, 14.5, and 15.5. RXR alpha -/- embryos display complex defects, including ventricular septal, atrioventricular cushion, and conotruncal ridge defects, with double outlet right ventricle, aorticopulmonary window, and persistent truncus arteriosus. In addition, heterozygous RXR alpha embryos display a predisposition for trabecular and papillary muscle defects, ventricular septal defects, conotruncal ridge defects, atrioventricular cushion defects, and pulmonic stenosis. Lastly, we show that the intermediate anatomic phenotype displayed by heterozygous embryos is mirrored in the molecular marker MLC-2a. The intermediate phenotype of RXR alpha heterozygous embryos documents a gene dosage effect for RXR alpha in maintaining normal cardiac morphogenesis. In addition, some defects in RXR alpha mutant mice are phenocopies of human congenital heart defects, thereby suggesting that a relative deficiency in RXR alpha or molecules downstream in its signaling pathway may represent congenital heart disease-susceptibility genes.

Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development†

The knowledge concerning fetal hepatic stellate cells (HSCs) is scarce, and their cell lineage and functions are largely unknown. The current study isolated fetal liver mesenchymal cells from a mouse expressing β‐galactosidase under the control of Msx2 promoter by fluorescence‐activated cell sorting (FACS) and surveyed marker genes by microarray analysis. Based on the location and immunostaining with conventional and newly disclosed markers, we have identified three distinct populations of fetal liver mesenchymal cells expressing both desmin and p75 neurotrophin receptor (p75NTR): HSCs in the liver parenchyma; perivascular mesenchymal cells expressing α‐smooth muscle actin (α‐SMA); and submesothelial cells associated with the basal lamina beneath mesothelial cells and expressing activated leukocyte cell adhesion molecule (ALCAM) and platelet‐derived growth factor receptor α. A transitional cell type from the submesothelial cell phenotype to fetal HSCs was also identified near the liver surface. Mesothelial cells expressed podoplanin and ALCAM. Ki‐67 staining showed that proliferative activity of the submesothelial cells is higher than that of mesothelial cells and transitional cells. Using anti‐ALCAM antibodies, submesothelial and mesothelial cells were isolated by FACS. The ALCAM+ cells expressed hepatocyte growth factor and pleiotrophin. In culture, the ALCAM+ cells rapidly acquired myofibroblastic morphology and α‐SMA expression. The ALCAM+ cells formed intracellular lipid droplets when embedded in collagen gel and treated with retinol, suggesting the potential for ALCAM+ cells to differentiate to HSCs. Finally, we demonstrated that fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are all derived from mesoderm by using MesP1‐Cre and ROSA26 reporter mice. Conclusion: Fetal HSCs, submesothelial cells, and perivascular mesenchymal cells are mesodermal in origin, and ALCAM+ submesothelial cells may be a precursor for HSCs in developing liver. (HEPATOLOGY 2009.)

Msx2 and Twist cooperatively control the development of the neural crest-derived skeletogenic mesenchyme of the murine skull vault.

The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2 mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.

Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest.

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2α, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.

A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. I. Authentication of the cloned gene and its developmental expression.

The developing sea urchin embryo forms endoskeletal CaCO3 containing spicules which are elaborated by the primary mesenchyme cells, descendants of the micromeres, beginning at gastrulation. In this and the accompanying paper [H. M. Sucov, S. Benson, J. J. Robinson, R. J. Britten, F. Wilt, and E. H. Davidson (1987) Dev. Biol. 120, 507-519] the isolation and characterization of a gene that encodes a 50-kDa spicule matrix glycoprotein that we call SM50 are described. A cloned cDNA isolated from a lambda gt11 library was used in hybrid-selected translation and hybrid arrest of translation experiments to verify that the cDNA encodes a spicule matrix protein. The cognate RNA transcript encodes a 50-kDa protein which is precipitated by polyclonal antisera against spicule matrix proteins and is present only in polyadenylated RNA at stages known to be making a spicule. The cloned cDNA sequence described in the accompanying paper was used to follow the time of expression of the cognate gene by RNA blotting analysis. The 2.2-kb mRNA is first detected at late cleavage stages and rapidly accumulates as the primary mesenchyme forms, reaching an apparent maximum concentration in the late gastrula and pluteus stages. The cDNA was also used to identify the cells that contain the transcripts by hybridization in situ. Hybridization to cellular transcripts is first detected in primary mesenchyme cells as they enter the blastocoel, and transcripts are confined to these cells during spicule formation and subsequent development.

Retinoic acid and retinoic acid receptors in development

The vitamin A derivative retinoic acid (RA) and related compounds (retinoids) are utilized as signaling molecules in a diverse array of developmental and physiological regulatory processes, including many important in the developing and mature nervous system. Retinoids function by interaction with high affinity receptors of the nuclear receptor family, which also mediate the effects of steroid and thyroid hormones and which act in the nucleus as transcription factors. This review summarizes current knowledge of the molecular mechanisms of retinoid action, the complex role of retinoid receptors in a variety of hormonal signaling processes, and illustrates current efforts to more fully understand the biological functions of retinoid receptors through analysis of downstream gene regulatory networks and studies of mouse gene knockout systems.

Endothelins are vascular-derived axonal guidance cues for developing sympathetic neurons

During development, sympathetic neurons extend axons along a myriad of distinct trajectories, often consisting of arteries, to innervate one of a large variety of distinct final target tissues. Whether or not subsets of neurons within complex sympathetic ganglia are predetermined to innervate select end-organs is unknown. Here we demonstrate in mouse embryos that the endothelin family member Edn3 (ref. 1), acting through the endothelin receptor EdnrA (refs 2, 3), directs extension of axons of a subset of sympathetic neurons from the superior cervical ganglion to a preferred intermediate target, the external carotid artery, which serves as the gateway to select targets, including the salivary glands. These findings establish a previously unknown mechanism of axonal pathfinding involving vascular-derived endothelins, and have broad implications for endothelins as general mediators of axonal growth and guidance in the developing nervous system. Moreover, they suggest a model in which newborn sympathetic neurons distinguish and choose between distinct vascular trajectories to innervate their appropriate end organs.