Faculty Bibliography

The genetic pathways that partition the developing nervous system into functional systems are largely unknown. The engrailed (En) homeobox transcription factors are candidate regulators of this process in the dorsal midbrain (tectum) and anterior hindbrain (cerebellum). En1 mutants lack most of the tectum and cerebellum and die at birth, whereas En2 mutants are viable with a smaller cerebellum and foliation defects. Our previous studies indicated that the difference in phenotypes is due to the earlier expression of En1 as compared with En2, rather than differences in protein function, since knock-in mice expressing En2 in place of En1 have a normal brain. Here, we uncovered a wider spectrum of functions for the En genes by generating a series of En mutant mice. First, using a conditional allele we demonstrate that En1 is required for cerebellum development only before embryonic day 9, but plays a sustained role in forming the tectum. Second, by removing the endogenous En2 gene in the background of En1 knock-in alleles, we show that Drosophila en is not sufficient to sustain midbrain and cerebellum development in the absence of En2, whereas En2 is more potent than En1 in cerebellum development. Third, based on a differential sensitivity to the dose of En1/2, our studies reveal a genetic subdivision of the tectum into its two functional systems and the medial cerebellum into four regions that have distinct circuitry and molecular coding. Our study suggests that an ;engrailed code' is integral to partitioning the tectum and cerebellum into functional domains

BACKGROUND: B-type natriuretic peptide (BNP) released from cardiac myocytes plays an important role in cardiac homeostasis through cyclic guanosine monophosphate (cGMP) activation. BNP also reduces cardiac remodeling and fibrosis. The antifibrotic effects of BNP are mediated in part by blocking the effects of transforming growth factor beta, a profibrotic cytokine that plays a significant role in cutaneous wound healing. It is unclear if BNP plays any role in cutaneous wound healing. OBJECTIVES: To investigate if BNP levels would be elevated in thermally injured human skin and if human-derived fibroblasts would respond to BNP exposure by increasing levels of cGMP. METHODS: This was an in vitro analysis of human skin. Skin samples and cells were collected from patients with and without thermal injury. The authors stained three skin samples from normal skin (taken at the time of elective cosmetic surgery) with antibodies to BNP and compared these with three tissue samples obtained from burned human skin taken during tangential excision of deep burns. Normal human-derived fibroblasts and keratinocytes were exposed in triplicate to BNP in vitro, and cGMP accumulation was evaluated. Levels of cGMP were quantified and compared with analysis of variance. RESULTS: BNP was present in all specimens of thermally injured skin (especially around collagen, epithelial cells, and endothelial cells) but not in any uninjured skin samples (p = 0.05, single-tailed Fisher's exact test). In vitro grown fibroblasts showed significant increases of cGMP levels with increasing levels of BNP exposure (mean [+/-SD]: 0.6 [+/-0.3], 1.2 [+/-0.2], 4.6 [+/-0.1], and 5.0 [+/-0.9] pmol/mL with BNP concentrations of 0, 10, 500, and 1,000 nmol/L, respectively; p < 0.001). The effect of BNP on keratinocytes was minimal and below the level of quantification. CONCLUSIONS: These findings demonstrate proof of principle that human fibroblasts are responsive to the effects of BNP in vitro and that BNP is present in injured skin, suggesting that BNP may play a role in cutaneous wound healing.

Recent studies have demonstrated that the LIM homeodomain transcription factor Islet1 (Isl1) marks pluripotent cardiovascular progenitor cells and is required for proliferation, survival, and migration of recently defined second heart field progenitors. Factors that are upstream of Isl1 in cardiovascular progenitors have not yet been defined. Here we demonstrate that beta-catenin is required for Isl1 expression in cardiac progenitors, directly regulating the Isl1 promoter. Ablation of beta-catenin in Isl1-expressing progenitors disrupts multiple aspects of cardiogenesis, resulting in embryonic lethality at E13. beta-Catenin is also required upstream of a number of genes required for pharyngeal arch, outflow tract, and/or atrial septal morphogenesis, including Tbx2, Tbx3, Wnt11, Shh, and Pitx2. Our findings demonstrate that beta-catenin signaling regulates proliferation and survival of cardiac progenitors.

For more than 50 years, investigators have unsuccessfully tried to recreate in experimental animals the cardiovascular complications of diabetes seen in humans. In particular, accelerated atherosclerosis and dilated cardiomyopathy, the major causes of mortality in patients with diabetes, have been conspicuously absent in many mouse models of the disease. Under the auspices of the NIH, the Animal Models of Diabetic Complications Consortium has worked to address this issue. This effort has focused on the development of mouse models because of the high level of genomic information available and the many well-developed genetic manipulations that may be performed in mice. Importantly, the consortium has also worked to standardize many methods to assess metabolic and cardiovascular end points for measurement of the diabetic state and its macrovascular complications. Finally, for maximum benefits from these animal models in the study of atherosclerosis and of other diabetic complications, the consortium has created a system for sharing both the animal models and the accumulated phenotypic data with the greater scientific community.

The integrated stress response (ISR) is defined as a highly conserved response to several stresses that converge to the induction of the activating transcription factor 4 (ATF4). Because an uncontrolled response may have deleterious effects, cells have elaborated several negative feedback loops that attenuate the ISR. In the present study, we describe how induction of the human homolog of Drosophila tribbles (TRB3) attenuates the ISR by a negative feedback mechanism. To investigate the role of TRB3 in the control of the ISR, we used the regulation of gene expression by amino acid limitation as a model. The enhanced production of ATF4 upon amino acid starvation results in the induction of a large number of target genes like CHOP (CAAT/enhancer-binding protein-homologous protein), asparagine synthetase (ASNS), or TRB3. We demonstrate that TRB3 overexpression inhibits the transcriptional induction of CHOP and ASNS whereas TRB3 silencing induces the expression of these genes both under normal and stressed conditions. In addition, transcriptional profiling experiments show that TRB3 affects the expression of many ISR-regulated genes. Our results also suggest that TRB3 and ATF4 belong to the same protein complex bound to the sequence involved in the ATF4-dependent regulation of gene expression by amino acid limitation. Collectively, our data identify TRB3 as a negative feedback regulator of the ATF4-dependent transcription and participates to the fine regulation of the ISR.

STUDY DESIGN: In vitro assessment of the effects of recombinant human osteogenic protein-1 (rhOP-1) on the proteoglycan metabolism of human intervertebral disc cells. OBJECTIVES: To determine whether rhOP-1 is effective in stimulating the cell proliferation and proteoglycan metabolism of human intervertebral disc cells cultured in alginate beads. SUMMARY OF THE BACKGROUND DATA: OP-1 has been shown to stimulate the proteoglycan and collagen synthesis of rabbit intervertebral disc cells in vitro. In vivo, a single injection of rhOP-1 restored the disc height of a degenerated disc in the rabbit anular-puncture model. The effect of rhOP-1 on human intervertebral disc cells remains unknown. METHODS: Human nucleus pulposus and anulus fibrosus cells were isolated from the discs of 4 cadaveric spines and one surgical specimen. After preculture for 7 days, alginate beads containing nucleus pulposus and anulus fibrosus cells were cultured for 21 days in media containing 10% fetal bovine serum with 0, 100, or 200 ng/mL rhOP-1 and supplements. The synthesis and accumulation of proteoglycans and the DNA content were biochemically assessed. RESULTS: The addition of rhOP-1 to the media resulted in the prevention of a decreased cell number during culture. Treatment with rhOP-1, compared with the control condition (10% fetal bovine serum), significantly upregulated proteoglycan synthesis and accumulation in alginate beads in all cases tested. A longer exposure over 14 days to rhOP-1 resulted in a pronounced response. The retention of newly-synthesized proteoglycan was higher in the rhOP-1-treated cells than in the control. CONCLUSIONS: rhOP-1 was effective in stimulating the cell proliferation and proteoglycan metabolism of human intervertebral disc cells in vitro. The results supported the hypothesis that an in vivo injection of rhOP-1 may increase the metabolic activity of disc cells or prevent apoptosis of disc cells in a degenerated disc. However, the requirement for a long exposure to rhOP-1 for human cells may suggest the need for a prolonged supply of rhOP-1 by a drug delivery system or by repeated injections.

The mammalian hair follicle is a complex 'mini-organ' thought to form only during development; loss of an adult follicle is considered permanent. However, the possibility that hair follicles develop de novo following wounding was raised in studies on rabbits, mice and even humans fifty years ago. Subsequently, these observations were generally discounted because definitive evidence for follicular neogenesis was not presented. Here we show that, after wounding, hair follicles form de novo in genetically normal adult mice. The regenerated hair follicles establish a stem cell population, express known molecular markers of follicle differentiation, produce a hair shaft and progress through all stages of the hair follicle cycle. Lineage analysis demonstrated that the nascent follicles arise from epithelial cells outside of the hair follicle stem cell niche, suggesting that epidermal cells in the wound assume a hair follicle stem cell phenotype. Inhibition of Wnt signalling after re-epithelialization completely abrogates this wounding-induced folliculogenesis, whereas overexpression of Wnt ligand in the epidermis increases the number of regenerated hair follicles. These remarkable regenerative capabilities of the adult support the notion that wounding induces an embryonic phenotype in skin, and that this provides a window for manipulation of hair follicle neogenesis by Wnt proteins. These findings suggest treatments for wounds, hair loss and other degenerative skin disorders

How specific sensory stimuli evoke specific behaviors is a fundamental problem in neurobiology. In Drosophila, most odorants elicit attraction or avoidance depending on their concentration, as well as their identity [1]. Such odorants, moreover, typically activate combinations of glomeruli in the antennal lobe of the brain [2-4], complicating the dissection of the circuits translating odor recognition into behavior. Carbon dioxide (CO2), in contrast, elicits avoidance over a wide range of concentrations [5, 6] and activates only a single glomerulus, V [5]. The V glomerulus receives projections from olfactory receptor neurons (ORNs) that coexpress two GPCRs, Gr21a and Gr63a, that together comprise a CO2 receptor [7-9]. These CO2-sensitive ORNs, located in the ab1 sensilla of the antenna, are called ab1c neurons [10]. Genetic silencing of ab1c neurons indicates that they are necessary for CO2-avoidance behavior [5]. Whether activation of these neurons alone is sufficient to elicit this behavior, or whether CO2 avoidance requires additional inputs (e.g., from the respiratory system), remains unclear. Here, we show that artificial stimulation of ab1c neurons with light (normally attractive to flies) elicits the avoidance behavior typical of CO2. Thus, avoidance behavior appears hardwired into the olfactory circuitry that detects CO2 in Drosophila

STUDY DESIGN: In vivo study of the effect of an injection of recombinant human osteogenic protein-1 into degenerated discs induced by chondroitinase ABC. OBJECTIVE: To investigate the efficacy of an injection of recombinant human osteogenic protein-1 to induce the recovery of disc height, and biochemical and histologic repair, in discs degenerated through enzymatic digestion by chondroitinase ABC. SUMMARY OF THE BACKGROUND DATA: Chondroitinase ABC is currently proposed as a chemonucleolysis agent; however, postchemonucleolysis degeneration is currently unavoidable. Recombinant human OP-1 has been shown to promote extracellular matrix repair in vitro and in vivo. METHODS: Fifty-four adolescent New Zealand white rabbits were used. Four weeks after an initial injection of chondroitinase ABC (10 mU/disc), 5% lactose (10 microL/disc) or recombinant human osteogenic protein-1 (100 microg in 10 microL lactose/disc) was injected. Disc heights were monitored radiographically at 2-week intervals, and rabbits were killed at 6, 8, 12, and 16 weeks after the initial chondroitinase ABC injections. The intervertebral discs were subjected to histologic and biochemical analyses. RESULTS: Significant disc space narrowing was observed in both groups 2 weeks after the injection of chondroitinase ABC. In the chondroitinase ABC/lactose group, this narrowing progressed after the vehicle injection and was sustained for up to 16 weeks. In the chondroitinase ABC/recombinant human osteogenic protein-1 group, the disc height index showed a significant increase at 6 weeks (lactose vs. recombinant human osteogenic protein-1; P < 0.01); this recovery was sustained for up to 16 weeks. The proteoglycan content was higher in the chondroitinase ABC/recombinant human osteogenic protein-1 group than in the chondroitinase ABC/lactose group. However, histologic changes, after the recombinant human osteogenic protein-1 injection, were not observed. CONCLUSIONS: A single injection of recombinant human osteogenic protein-1 into a rabbit disc dramatically reversed the decrease in disc height induced by chondroitinase ABC chemonucleolysis. The recovery was significant and sustained over the next 12 weeks. The therapeutic effects of both chondroitinase ABC chemonucleolysis and recombinant human osteogenic protein-1 injections should be further explored in higher animals before it is applied to humans.