Defining Atoh1 function and regulation in avian supporting cells during auditory hair cell regeneration

Thesis (Ph.D.)--University of Washington, 2016-06

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E-17) in the rat such that embryonic day 19 (E-19) wounds do not re-epithelialize. Moreover, wounds created in E-17 fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E-17 and E-19 skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E-17 and E-19 skin. c-fos and c-jun induction was transient in E-17 skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E-19 skin, AP-11 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E-17 skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

Methylglyoxal, glyoxalses and cell proliferation

The metabolic function of the glyoxalase system was investigated in (a) the differentiation and proliferation of human tumour cells in vitro, (b) the cell-free assembly of microtubules and (c) in the red blood cells during hyperglycaemia associated with Diabetes Mellitus. Chemically-induced differentiation of human promyelocytic HL60 leukaemia cells to neutrophils, and K562 erythroleukaemia cells, was accompanied by a decrease and an increase in the activity of glyoxalase I, respectively. Growth-arrest of Burkitt's lymphoma Raji cells and GM892 lymphoblastoid cells was accompanied by an increase and a decrease in the activity of glyoxalase I respectively. However, differentiation and growth arrest generally proceeded with an increase in the activity of glyoxalase II. Glyoxalase I activity did not consistently correlate with cell differentiation or proliferation status; hence, it is unlikely that glyoxalase I activity is either an indicator or a regulator of cell differentiation or proliferation. Conversely, glyoxalase II activity consistently increased during cell differentiation and growth-arrest and may be both an indicator and regulator of cell differentiation or proliferation. This may be related to the control of cellular microtubule assembly. S-D-Lactoylglutathione potentiated the cell-free, GTP-promoted assembly of microtubules. The effect was dose-related and was inhibited by glyoxalase II. During assembly, S-D-lactoylglutathione was consumed. This suggests that the glyoxalase system, through the influence of S-D-lactoylglutathione, may regulate the assembly of microtubules in cellular systems The whole blood concentrations of methylglyoxal and S-D-lactoylglutathione were increased in Diabetes Mellitus. There was no significant difference between red blood cell glyoxalase activities in diabetics, compared to healthy controls. However, insulin-dependent diabetic patients with retinopathy had a significantly higher glyoxalase I activity and a lower glyoxalase II activity, than patients without retinopathy. Diabetic retinopathy correlated with high glyoxalase I activity and low glyoxalase II activity and suggests the glyoxalase system may be involved in the development of diabetic complications.

Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivo

Background Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type I diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. Methods ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. Results In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. in contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation. Copyright (c) 2009 John Wiley & Sons, Ltd.

Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss- and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1(BAC):yfp) × Tg(kdrl:ras-cherry)(s916) embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.

Changes of 5-hydroxymethylcytosine distribution during myeloid and lymphoid differentiation of CD34+ cells

BACKGROUND: Hematopoietic stem cell renewal and differentiation are regulated through epigenetic processes. The conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) by ten-eleven-translocation enzymes provides new insights into the epigenetic regulation of gene expression during development. Here, we studied the potential gene regulatory role of 5hmC during human hematopoiesis.

RESULTS: We used reduced representation of 5-hydroxymethylcytosine profiling (RRHP) to characterize 5hmC distribution in CD34+ cells, CD4+ T cells, CD19+ B cells, CD14+ monocytes and granulocytes. In all analyzed blood cell types, the presence of 5hmC at gene bodies correlates positively with gene expression, and highest 5hmC levels are found around transcription start sites of highly expressed genes. In CD34+ cells, 5hmC primes for the expression of genes regulating myeloid and lymphoid lineage commitment. Throughout blood cell differentiation, intragenic 5hmC is maintained at genes that are highly expressed and required for acquisition of the mature blood cell phenotype. Moreover, in CD34+ cells, the presence of 5hmC at enhancers associates with increased binding of RUNX1 and FLI1, transcription factors essential for hematopoiesis.

CONCLUSIONS: Our study provides a comprehensive genome-wide overview of 5hmC distribution in human hematopoietic cells and new insights into the epigenetic regulation of gene expression during human hematopoiesis.

The Role of Morphogens in B Cell Development

Morphogens are signalling molecules that play a significant role in modulation of cell fate and development. Hedgehog proteins (Hh) are morphogens that have been shown to be involved in the development of immune cells. In this study, it is demonstrated that treatment of B cells with rShh, can increase B cell activation and also promote survival of B cells at 18hours post-stimulus. Also, at this time point, there was found to be an increase in secretion of antibody isotypes and IL-6. By 40hours post-stimulus, it was observed that the level of B cell activation was apparently arrested in treated B cells, whereas the level of activation continued to rise in untreated B cells. Interestingly, it was observed that there was an increase in the percentage of; CD23-CD25+ B cells when B cells were treated with rShh and this was accompanied by an increase in apoptosis. Consistent with this finding in relation to apoptosis, there was an increased expression of the pro-apoptotic protein Bnip3 in B cells treated with rShh by 40hours post-stimulus. It was observed that there were three subsets of B cells arising in our culture at 40hours, which were all found to possess different characteristics. It was demonstrated that treatment with rShh can increase B cell differentiation towards FO-I at 18hours post-stimulus. By 40hours post-stimulus, Hh signalling can divert differentiation away from the FO-I B cell towards the T2-MZP, which was accompanied by an increase in IL-10 secretion. Gene expression analysis revealed that Hh signalling could modulate a number of molecules involved in delivering the BCR signal into the cells such as Btk, Nfatc1 and Traf2. Additionally, deletion of Dhh, showed that there was a skewed peripheral B cell development in the Dhh-/- mice. Overall, our data demonstrate that Hh signalling can regulate the development of B cells in response to an activation stimulus by strengthening the BCR signalling pathway.

The Role of HBD-2 and HBD-3 in Human T Cell Development

Human β-defensins (hBDs) are a family of cationic peptides able to directly kill a wide range of microorganisms including bacteria, fungi and viruses. In addition to their antimicrobial activities, defensins also contribute to the modulation of both the host innate and adaptive immunity. In this project, we demonstrate that the αCD3/28 co-stimulation of human CD4+ T cells in the presence of 10μg/ml hBD-2 or hBD-3 together causes an up-regulation in numbers of CD4+CD69+CD25+ and CD4+CD69-CD25+ T cell subsets, indicating that the treatment of hBD-2 and 3 enhances CD4+ T cell activation. Consistent with this finding, proliferation assay using CFSE suggests that hBD-2 and hBD-3 treatment in vitro induces the proliferation of CD4+ T cells following by 96hrs culture. Analysis of expression of the regulatory T cells (Tregs) specific marker, FoxP3, reveals a shift in the CD4+CD127-CD25+ Treg subset at 18hrs. However, at the later time point, we found that the percentage of FoxP3+cells decreased in the CD4+CD127-CD25+ Treg population, whereas the presence of the FoxP3+CTLA-4+ Treg subset increased. These data indicate that Treg suppressive function may be potentially defective following the co-incubation of purified T cells with either hBD-2 or hBD-3 for 42hrs in vitro due to the apparent loss of FoxP3 expression. We further characterise the role of hBD-2 and hBD-3 in driving human CD4+ T cells polarisation. Our in vitro data suggests that treatment with hBD-2 and hBD-3 can not only induces effector T cell (Teff) differentiation into RORγt+T-bet+ (Th17/Th1) cells, but can also trigger the differentiation of Treg expressing RORγt and T-bet rather than the master controller of Treg function, FoxP3. This apparent plasticity of T cell phenotype allows them to convert from Treg to Th1/17-like effector T cell phenotype following 18hrs in culture. By 42hrs in culture, treatment with hBD-2 and hBD-3 induced both Teff cell and Treg cell differentiation towards the Th17-like phenotype. Compared with the treatment with hBD-2, treatment with hBD-3 induced a more pronounced effect to increase levels of RORγt in CD4+ T cells. This elevated expression may, in turn, be responsible for the induction of higher IL-17A secretion. Consistent with this idea, it was found that treatment with hBD-3 but not hBD-2 was capable of inducing the higher level of secretion of IL-17A. Additionally, treatment with hBD-3 induced an increased expression of IL-6, which is capable of driving the differentiation of naïve T cells towards IL-17-producing Th17 cells. Functionally, using the Treg suppression assay, the data suggested that hBD-2 may dampen down Treg cell ability to induce suppression of Teff cell activity. Interestingly, co-culture with hBD-2 would also appear to increase Teff cell resistance to Treg immunoregulation in vitro. Further investigation using microarray gene analysis revealed chemokine C-C motif ligand 1 (CCL1) as potential genes responding to hBD-2 treatment. The blockade of CCL1 has been reported to inhibit Treg suppressive function. Thus, this study explored the function of these antimicrobial candidates in regulating CD4+ T cell plasticity which could result in hBD-2 and hBD-3 being able to regulate its own production, but also may regulate Treg and Teff cell development and function, thus strengthening the link between innate and adaptive immunity

Role for cell-to-cell communication in stem cell specification toward pancreatic progenitors: relevance to the design of novel therapies for diabetes.

Thesis (Ph.D.)--University of Washington, 2016-08

Sampling strategies for characterization of neuropeptides using mass spectrometry and functional implications into cell-cell signaling

In this dissertation, there are developed different analytical strategies to discover and characterize mammalian brain peptides using small amount of tissues. The magnocellular neurons of rat supraoptic nucleus in tissue and cell culture served as the main model to study neuropeptides, in addition to hippocampal neurons and mouse embryonic pituitaries. The neuropeptidomcis studies described here use different extraction methods on tissue or cell culture combined with mass spectrometry (MS) techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). These strategies lead to the identification of multiple peptides from the rat/mouse brain in tissue and cell cultures, including novel compounds One of the goals in this dissertation was to optimize sample preparations on samples isolated from well-defined brain regions for mass spectrometric analysis. Here, the neuropeptidomics study of the SON resulted in the identification of 85 peptides, including 20 unique peptides from known prohormones. This study includes mass spectrometric analysis even from individually isolated magnocellular neuroendocrine cells, where vasopressin and several other peptides are detected. At the same time, it was shown that the same approach could be applied to analyze peptides isolated from a similar hypothalamic region, the suprachiasmatic nucleus (SCN). Although there were some overlaps regarding the detection of the peptides in the two brain nuclei, different peptides were detected specific to each nucleus. Among other peptides, provasopressin fragments were specifically detected in the SON while angiotensin I, somatostatin-14, neurokinin B, galanin, and vasoactive-intestinal peptide (VIP) were detected in the SCN only. Lists of peptides were generated from both brain regions for comparison of the peptidome of SON and SCN nuclei. Moving from analysis of magnocellular neurons in tissue to cell culture, the direct peptidomics of the magnocellular and hippocampal neurons led to the detection of 10 peaks that were assigned to previously characterized peptides and 17 peaks that remain unassigned. Peptides from the vasopressin prohormone and secretogranin-2 are attributed to magnocellular neurons, whereas neurokinin A, peptide J, and neurokinin B are attributed to cultured hippocampal neurons. This approach enabled the elucidation of cell-specific prohormone processing and the discovery of cell-cell signaling peptides. The peptides with roles in the development of the pituitary were analyzed using transgenic mice. Hes1 KO is a genetically modified mouse that lives only e18.5 (embryonic days). Anterior pituitaries of Hes1 null mice exhibit hypoplasia due to increased cell death and reduced proliferation and in the intermediate lobe, the cells differentiate abnormally into somatotropes instead of melanotropes. These previous findings demonstrate that Hes1 has multiple roles in pituitary development, cell differentiation, and cell fate. AVP was detected in all samples. Interestingly, somatostatin [92-100] and provasopressin [151-168] were detected in the mutant but not in the wild type or heterozygous pituitaries while somatostatin-14 was detected only in the heterozygous pituitary. In addition, the putative peptide corresponding to m/z 1330.2 and POMC [205-222] are detected in the mutant and heterozygous pituitaries, but not in the wild type. These results indicate that Hes1 influences the processing of different prohormones having possible roles during development and opens new directions for further developmental studies. This research demonstrates the robust capabilities of MS, which ensures the unbiased direct analysis of peptides extracted from complex biological systems and allows addressing important questions to understand cell-cell signaling in the brain.

Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Ceruloplasmin is regulated by copper and lactational hormones in PMC42-LA mammary epithelial cell culture models

Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 μM). However, there was a significant (P < 0.05) increase (∼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.

Physiological investigation of periosteum structure and its application in periosteum tissue engineering

Although many different materials, techniques and methods, including artificial or engineered bone substitutes, have been used to repair various bone defects, the restoration of critical-sized bone defects caused by trauma, surgery or congenital malformation is still a great challenge to orthopedic surgeons. One important fact that has been neglected in the pursuit of resolutions for large bone defect healing is that most physiological bone defect healing needs the periosteum and stripping off the periosteum may result in non-union or non-healed bone defects. Periosteum plays very important roles not only in bone development but also in bone defect healing. The purpose of this project was to construct a functional periosteum in vitro using a single stem cell source and then test its ability to aid the repair of critical-sized bone defect in animal models. This project was designed with three separate but closely-linked parts which in the end led to four independent papers. The first part of this study investigated the structural and cellular features in periostea from diaphyseal and metaphyseal bone surfaces in rats of different ages or with osteoporosis. Histological and immunohistological methods were used in this part of the study. Results revealed that the structure and cell populations in periosteum are both age-related and site-specific. The diaphyseal periosteum showed age-related degeneration, whereas the metaphyseal periosteum is more destructive in older aged rats. The periosteum from osteoporotic bones differs from normal bones both in terms of structure and cell populations. This is especially evident in the cambial layer of the metaphyseal area. Bone resorption appears to be more active in the periosteum from osteoporotic bones, whereas bone formation activity is comparable between the osteoporotic and normal bone. The dysregulation of bone resorption and formation in the periosteum may also be the effect of the interaction between various neural pathways and the cell populations residing within it. One of the most important aspects in periosteum engineering is how to introduce new blood vessels into the engineered periosteum to help form vascularized bone tissues in bone defect areas. The second part of this study was designed to investigate the possibility of differentiating bone marrow stromal cells (BMSCs) into the endothelial cells and using them to construct vascularized periosteum. The endothelial cell differentiation of BMSCs was induced in pro-angiogenic media under both normoxia and CoCl2 (hypoxia-mimicking agent)-induced hypoxia conditions. The VEGF/PEDF expression pattern, endothelial cell specific marker expression, in vitro and in vivo vascularization ability of BMSCs cultured in different situations were assessed. Results revealed that BMSCs most likely cannot be differentiated into endothelial cells through the application of pro-angiogenic growth factors or by culturing under CoCl2-induced hypoxic conditions. However, they may be involved in angiogenesis as regulators under both normoxia and hypoxia conditions. Two major angiogenesis-related growth factors, VEGF (pro-angiogenic) and PEDF (anti-angiogenic) were found to have altered their expressions in accordance with the extracellular environment. BMSCs treated with the hypoxia-mimicking agent CoCl2 expressed more VEGF and less PEDF and enhanced the vascularization of subcutaneous implants in vivo. Based on the findings of the second part, the CoCl2 pre-treated BMSCs were used to construct periosteum, and the in vivo vascularization and osteogenesis of the constructed periosteum were assessed in the third part of this project. The findings of the third part revealed that BMSCs pre-treated with CoCl2 could enhance both ectopic and orthotopic osteogenesis of BMSCs-derived osteoblasts and vascularization at the early osteogenic stage, and the endothelial cells (HUVECs), which were used as positive control, were only capable of promoting osteogenesis after four-weeks. The subcutaneous area of the mouse is most likely inappropriate for assessing new bone formation on collagen scaffolds. This study demonstrated the potential application of CoCl2 pre-treated BMSCs in the tissue engineering not only for periosteum but also bone or other vascularized tissues. In summary, the structure and cell populations in periosteum are age-related, site-specific and closely linked with bone health status. BMSCs as a stem cell source for periosteum engineering are not endothelial cell progenitors but regulators, and CoCl2-treated BMSCs expressed more VEGF and less PEDF. These CoCl2-treated BMSCs enhanced both vascularization and osteogenesis in constructed periosteum transplanted in vivo.

Engineering an Ecosystem : Taking Cues from Nature’s Paradigm to Build Tissue in the Lab and the Body

This manuscript took a 'top down' approach to understanding survival of inhabitant cells in the ecosystem bone, working from higher to lower length and time scales through the hierarchical ecosystem of bone. Our working hypothesis is that nature “engineered” the skeleton using a 'bottom up' approach,where mechanical properties of cells emerge from their adaptation to their local me-chanical milieu. Cell aggregation and formation of higher order anisotropic struc- ture results in emergent architectures through cell differentiation and extracellular matrix secretion. These emergent properties, including mechanical properties and architecture, result in mechanical adaptation at length scales and longer time scales which are most relevant for the survival of the vertebrate organism [Knothe Tate and von Recum 2009]. We are currently using insights from this approach to har-ness nature’s regeneration potential and to engineer novel mechanoactive materials [Knothe Tate et al. 2007, Knothe Tate et al. 2009]. In addition to potential applications of these exciting insights, these studies may provide important clues to evolution and development of vertebrate animals. For instance, one might ask why mesenchymal stem cells condense at all? There is a putative advantage to self-assembly and cooperation, but this advantage is somewhat outweighed by the need for infrastructural complexity (e.g., circulatory systems comprised of specific differentiated cell types which in turn form conduits and pumps to overcome limitations of mass transport via diffusion, for example; dif-fusion is untenable for multicellular organisms larger than 250 microns in diameter. A better question might be: Why do cells build skeletal tissue? Once cooperatingcells in tissues begin to deplete local sources of food in their aquatic environment, those that have evolved a means to locomote likely have an evolutionary advantage. Once the environment becomes less aquarian and more terrestrial, self-assembled organisms with the ability to move on land might have conferred evolutionary ad-vantages as well. So did the cytoskeleton evolve several length scales, enabling the emergence of skeletal architecture for vertebrate animals? Did the evolutionary advantage of motility over noncompliant terrestrial substrates (walking on land) favor adaptations including emergence of intracellular architecture (changes in the cytoskeleton and upregulation of structural protein manufacture), inter-cellular con- densation, mineralization of tissues, and emergence of higher order architectures?How far does evolutionary Darwinism extend and how can we exploit this knowl- edge to engineer smart materials and architectures on Earth and new, exploratory environments?[Knothe Tate et al. 2008]. We are limited only by our ability to imagine. Ultimately, we aim to understand nature, mimic nature, guide nature and/or exploit nature’s engineering paradigms without engineer-ing ourselves out of existence.