Molecular and cellular mechanisms underlying the evolution of form and function in the amniote jaw.

The amniote jaw complex is a remarkable amalgamation of derivatives from distinct embryonic cell lineages. During development, the cells in these lineages experience concerted movements, migrations, and signaling interactions that take them from their initial origins to their final destinations and imbue their derivatives with aspects of form including their axial orientation, anatomical identity, size, and shape. Perturbations along the way can produce defects and disease, but also generate the variation necessary for jaw evolution and adaptation. We focus on molecular and cellular mechanisms that regulate form in the amniote jaw complex, and that enable structural and functional integration. Special emphasis is placed on the role of cranial neural crest mesenchyme (NCM) during the species-specific patterning of bone, cartilage, tendon, muscle, and other jaw tissues. We also address the effects of biomechanical forces during jaw development and discuss ways in which certain molecular and cellular responses add adaptive and evolutionary plasticity to jaw morphology. Overall, we highlight how variation in molecular and cellular programs can promote the phenomenal diversity and functional morphology achieved during amniote jaw evolution or lead to the range of jaw defects and disease that affect the human condition

A Possible Contribution of Altered Cathepsin B Expression to the Development of Skin Sclerosis and Vasculopathy in Systemic Sclerosis

Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1+/− and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1+/− mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc

Modeling Reveals Bistability and Low-Pass Filtering in the Network Module Determining Blood Stem Cell Fate

Combinatorial regulation of gene expression is ubiquitous in eukaryotes with multiple inputs converging on regulatory control elements. The dynamic properties of these elements determine the functionality of genetic networks regulating differentiation and development. Here we propose a method to quantitatively characterize the regulatory output of distant enhancers with a biophysical approach that recursively determines free energies of protein-protein and protein-DNA interactions from experimental analysis of transcriptional reporter libraries. We apply this method to model the Scl-Gata2-Fli1 triad—a network module important for cell fate specification of hematopoietic stem cells. We show that this triad module is inherently bistable with irreversible transitions in response to physiologically relevant signals such as Notch, Bmp4 and Gata1 and we use the model to predict the sensitivity of the network to mutations. We also show that the triad acts as a low-pass filter by switching between steady states only in response to signals that persist for longer than a minimum duration threshold. We have found that the auto-regulation loops connecting the slow-degrading Scl to Gata2 and Fli1 are crucial for this low-pass filtering property. Taken together our analysis not only reveals new insights into hematopoietic stem cell regulatory network functionality but also provides a novel and widely applicable strategy to incorporate experimental measurements into dynamical network models

Induced Pluripotent Stem Cells Produced From Cryopreserved Pygmy Sperm Whale (Kogia Breviceps) Lung Tissue

Concern over marine mammal health has risen in the last decade due to increases in mortality, unusual mortality events and previously undetected pathologies. Developing environmental trends, such as global warming and acute environmental insults, such as large-scale oil spills have been implicated. Unfortunately, the limited availability of biological samples hinders basic biological/toxicological studies. To compensate for the limited availability of samples and to make the most of rare samples that do become available, we have pursued methods to establish and expand cultures of primary cell types and reconstituted tissues from marine mammals for long-term use as surrogates for freshly isolated samples. To this end, we first developed a method to cryopreserve tissues from deceased/stranded individuals and thereby initiate establishment of a tissue bank biorepository. We were able to establish conditions and perform this successfully on lung tissue from a Pygmy Sperm Whale (PSW; Kogia breviceps). Using these conditions, we were able to establish cultures of viable primary lung cell types from tissue fragments that had been cryopreserved several months earlier (immediately after the stranding event). We then applied genetic or chemical means for generating induced pluripotent stem (iPS) cells to one of these primary cultures (lung fibroblasts). We observed that the genetic means, involving the forced expression of Klf4, Oct3/4, Sox2 and Myc, did produce PSW cells with long-term expansion and differentiative capability, while the chemical means using valproic acid (driving histone deacetylase inhibition and chromatin decondensation) did not. Finally, we employed specialized culture conditions to differentiate in bulk PSW cells with vascular endothelial and airway epithelial-like properties. The cryopreservation of viable tissue samples and the generation of iPS-like cells with unlimited expansion & pluripotent differentiative capacities from marine mammals is anticipated to have far-reaching impacts on levels ranging from basic biology to environmental policy related to stressors of marine and land mammalian health

HoxC6 regulation of early mammary epithelial growth and differentiation: implications for breast cancer potential.

Abstract Abstract #2010 Background: Early menarche and breast development are increasingly common risk factors for breast cancer. Epigenetic mechanisms implicated include natural and anthropogenic steroids, such as estrogens, that disrupt endocrine signaling and dysregulate stem cell homeostasis. Animal models show that, although a proliferative response to estrogens in mammary epithelium is not observed until near puberty, in utero or neonatal exposure can recapitulate aberrant breast development. The molecular basis for the delay in proliferative response remains poorly understood and assessments of agent activity, potency and risk remain inaccurate. Here, we provide a candidate molecular mechanism involving the homeobox transcription factor HoxC6, through which more accurate assessments may be facilitated.&amp;#x2028; Materials &amp; Methods: Genetically and hormonally manipulated in vivo mouse models (HoxC6 targeted disruption, C6KO; and mammary epithelial-specific inducible HoxC6, MMTV-rtTA TRE-HoxC6-IRES-EGFP) and in vitro human breast cancer cell lines (MCF10A, MCF7 and MDA-MB-231; "231") were used to assess impacts on mammary epithelial growth, differentiation and expression of HoxC6 and its direct target genes IGFBP3, CD44 and FGFR2. Cell lines were manipulated using HoxC6 shRNA and cDNA lentivirus and activated Akt isoform retroviral expression vectors. Gene expression and signaling were measured by quantitative RT-qPCR, IHC, Western blot and using the BioPlex200 Multiplex System.&amp;#x2028; Results: RT-qPCR analysis shows HoxC6 expression elevated in well-differentiated cell lines (MCF10A, 5.06X; MCF7, 5.16X) and decreased in more aggressive tumor cell lines (-1.95X, 231 cells; relative to A549 cells). HoxC6 knockdown in MCF10A cells results in an inhibition of cellular growth and decreased branching in 3D cultures and dysregulation of HoxC6 target genes. These results are consistent with in vivo results in which C6KO causes defective postnatal mammary epithelial growth/branching. Rescue of growth defects in knockdown MCF10A cells is achieved to varying extents with myristoylated Akt isoforms, however morphological defects persist and are associated with EMT. HoxC6 induction in stem cells is associated with altered self-renewal and differentiation. HoxC6 repression by estrogens was assessed in vivo under conditions of neonatal estrogen exposure and estrogen-free diets, showing alterations in HoxC6 and target gene expressions and other phenotypic changes.&amp;#x2028; Discussion: Based on our results, we will present a model in which HoxC6 is estrogen-responsive and coordinates mammary stem/progenitor cell homeostatic pathways, including FGF and GH-IGF1 signaling in the control of pool size and branching morphogenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2010.</jats:p