Physicochemical and Inflammatory Analysis of Unconjugated and Conjugated Bone-Binding Carbon Dots.

Carbon nanodots (CDs) have drawn significant attention for their potential uses in diagnostic and therapeutic applications due to their small size, tissue biocompatibility, stable photoluminescence, and modifiable surface groups. However, the effect of cargo molecules on CD photoluminescence and their ability to interact with tissues are not fully understood. Our previous work has shown that CDs produced from the acidic oxidation of carbon nanopowder can bind to mineralized bone with high affinity and specificity in a zebrafish animal model system. Using this model, we investigated the impact of loading Cy5 and biotin cargo on CDs' photoluminescence and bone-binding properties. We report that CD cargo loading alters CD photoluminescence in a pH- and cargo-dependent manner without interfering with the CDs' bone binding properties. In a reciprocal analysis, we show that cargo loading of CDs does not affect the cargo's fluorescence. Significantly, CDs do not trigger nitric oxide production in a mouse macrophage assay, suggesting that they are noninflammatory. Together, these results further support the development of carbon nanopowder-derived CDs for the precise delivery of therapeutic agents to bone tissue.

In vivo characterization of carbon dots-bone interactions: toward the development of bone-specific nanocarriers for drug delivery.

Current treatments for osteoporosis and other bone degenerative diseases predominately rely on preventing further bone erosion rather than restoring bone mass, as the latter treatments can unintentionally trigger cancer development by undiscriminatingly promoting cell proliferation. One approach to circumvent this problem is through the development of novel chemical carriers to deliver drug agents specifically to bones. We have recently shown that carbon nanodots (C-dots) synthesized from carbon nanopowder can bind with high affinity and specificity to developing bones in the larval zebrafish. Larval bones, however, are physiologically different from adult bones in their growth, repair, and regeneration properties. Here we report that C-dots can bind to adult zebrafish bones and that this binding is highly specific to areas of appositional growth. C-dots deposition occurred within 30 minutes after delivery and was highly selective, with bones undergoing regeneration and repair showing higher levels of C-dots deposition than bones undergoing normal homeostatic turnover. Importantly, C-dots deposition did not interfere with bone regeneration or the animal's health. Together, our results establish C-dots as a potential novel vehicle for the targeted delivery of drugs to treat adult bone disease.

A theoretical model of neural maturation in the developing chick spinal cord.

Cellular differentiation is a tightly regulated process under the control of intricate signaling and transcription factors interaction network working in coordination. These interactions make the systems dynamic, robust and stable but also difficult to dissect. In the spinal cord, recent work has shown that a network of FGF, WNT and Retinoic Acid (RA) signaling factors regulate neural maturation by directing the activity of a transcription factor network that contains CDX at its core. Here we have used partial and ordinary (Hill) differential equation based models to understand the spatiotemporal dynamics of the FGF/WNT/RA and the CDX/transcription factor networks, alone and in combination. We show that in both networks, the strength of interaction among network partners impacts the dynamics, behavior and output of the system. In the signaling network, interaction strength determine the position and size of discrete regions of cell differentiation and small changes in the strength of the interactions among networking partners can result in a signal overriding, balancing or oscillating with another signal. We also show that the spatiotemporal information generated by the signaling network can be conveyed to the CDX/transcription network to produces a transition zone that separates regions of high cell potency from regions of cell differentiation, in agreement with most in vivo observations. Importantly, one emerging property of the networks is their robustness to extrinsic disturbances, which allows the system to retain or canalize NP cells in developmental trajectories. This analysis provides a model for the interaction conditions underlying spinal cord cell maturation during embryonic axial elongation.

CDX4 regulates the progression of neural maturation in the spinal cord.

The progression of cells down different lineage pathways is a collaborative effort between networks of extracellular signals and intracellular transcription factors. In the vertebrate spinal cord, FGF, Wnt and Retinoic Acid signaling pathways regulate the progressive caudal-to-rostral maturation of neural progenitors by regulating a poorly understood gene regulatory network of transcription factors. We have mapped out this gene regulatory network in the chicken pre-neural tube, identifying CDX4 as a dual-function core component that simultaneously regulates gradual loss of cell potency and acquisition of differentiation states: in a caudal-to-rostral direction, CDX4 represses the early neural differentiation marker Nkx1.2 and promotes the late neural differentiation marker Pax6. Significantly, CDX4 prevents premature PAX6-dependent neural differentiation by blocking Ngn2 activation. This regulation of CDX4 over Pax6 is restricted to the rostral pre-neural tube by Retinoic Acid signaling. Together, our results show that in the spinal cord, CDX4 is part of the gene regulatory network controlling the sequential and progressive transition of states from high to low potency during neural progenitor maturation. Given CDX well-known involvement in Hox gene regulation, we propose that CDX factors coordinate the maturation and axial specification of neural progenitor cells during spinal cord development.

Carbon dots: promising biomaterials for bone-specific imaging and drug delivery.

Bone-related diseases and dysfunctions are heavy burdens on our increasingly aged society. One important strategy to relieve this problem is through early detection and treatment of bone-related diseases. Towards this goal, there has been constant interest in developing novel bone-specific materials for imaging and drug delivery. Currently, however, materials that have high affinity and specificity towards bone are very limited. Carbon dots (C-dots) synthesized from carbon nanopowder bind to calcified bones in vivo with high affinity and specificity. In this study we show that bone binding is highly unique to a specific type of C-dot, and that this binding is non-toxic. Significantly, C-dots derived from other raw materials did not show any bone binding properties. These differences are attributed to the differences in surface chemistry of C-dot preparations, highlighting the heterogeneous nature of C-dots. Importantly, bone-binding by carbon nanopowder derived C-dots is not significantly altered by chemical functionalization of their surface. These unique properties indicate the potential applications of carbon nanopowder-derived C-dots as highly bone-specific bioimaging agents and drug carriers.

CDX4 and retinoic acid interact to position the hindbrain-spinal cord transition.

The sub-division of the posterior-most territory of the neural plate results in the formation of two distinct neural structures, the hindbrain and the spinal cord. Although many of the molecular signals regulating the development of these individual structures have been elucidated, the mechanisms involved in delineating the boundary between the hindbrain and spinal cord remain elusive. Two molecules, retinoic acid (RA) and the Cdx4 transcription factor have been previously implicated as important regulators of hindbrain and spinal cord development, respectively. Here, we provide evidence that suggests multiple regulatory interactions occur between RA signaling and the Cdx4 transcription factor to establish the anterior-posterior (AP) position of the transition between the hindbrain and spinal cord. Using chemical inhibitors to alter RA concentrations and morpholinos to knock-down Cdx4 function in zebrafish, we show that Cdx4 acts to prevent RA degradation in the presumptive spinal cord domain by suppressing expression of the RA degradation enzyme, Cyp26a1. In the hindbrain, RA signaling modulates its own concentration by activating the expression of cyp26a1 and inhibiting the expansion of cdx4. Therefore, interactions between Cyp26a1 and Cdx4 modulate RA levels along the AP axis to segregate the posterior neural plate into the hindbrain and spinal cord territories.

"Dark" carbon dots specifically "light-up" calcified zebrafish bones.

Because accidents, disease and aging compromise the structural and physiological functions of bones, the development of an in vivo bone imaging test is critical to identify, detect and diagnose bone related development and dysfunctions. Recent advances in fluorescence instrumentation offer a new alternative for traditional bone imaging methods. However, the development of new in vivo bone imaging fluorescence materials has significantly lagged behind. Here we show that carbon dot nanoparticles (C-dots) with low quantum yield ("dark") bind to calcified bone structures of live zebrafish larvae with high affinity and selectivity. Binding resulted in a strong enhancement of luminescence that was not observed in other tissues, including non-calcified endochondral elements. Retention of C-dots by bones was very stable, long lasting, and with no detectable toxicity. Furthermore, we found C-dots to be a suitable carrier to deliver fluorescein to bones. These observations support a novel and revolutionary use of C-dots as highly specific bioagents for bone imaging and diagnosis, and as bone-specific drug delivery vehicles.

Spatiotemporal analysis of zebrafish hox gene regulation by Cdx4.

BACKGROUND: Cdx factors expressed in caudal regions of vertebrate embryos regulate hox patterning gene transcription. While loss of Cdx function is known to shift hox spatial expression domains posteriorly, the mechanism underlying the shift is not understood. We addressed this problem by analyzing the spatiotemporal expression profile of all 49 zebrafish hox genes in wild-type and Cdx4-deficient embryos. RESULTS: Loss of Cdx4 had distinct effects on hox spatial expression in a paralogous group-dependent manner: in the head, group 4 expression was expanded posteriorly; in the trunk, group 5-10 expression was shifted posteriorly; and in the tail, group 11-13 genes were expressed in the tail bud but not in more differentiated tissues. In the trunk neural tissue, loss of Cdx4 severely delayed both transcriptional activation of hox genes during the initiation phase, and the anterior-ward expansion of hox expression domains during the establishment phase. In contrast, in the trunk mesoderm, loss of Cdx4 only delayed the hox initiation phase. CONCLUSIONS: These results indicate that Cdx4 differentially regulates the transcription of head, trunk and tail hox genes. In the trunk, Cdx4 conveys spatial positional information to axial tissues primarily by regulating the time of hox gene transcriptional activation during the initiation phase.

Retinoic acid regulates size, pattern and alignment of tissues at the head-trunk transition.

At the head-trunk transition, hindbrain and spinal cord alignment to occipital and vertebral bones is crucial for coherent neural and skeletal system organization. Changes in neural or mesodermal tissue configuration arising from defects in the specification, patterning or relative axial placement of territories can severely compromise their integration and function. Here, we show that coordination of neural and mesodermal tissue at the zebrafish head-trunk transition crucially depends on two novel activities of the signaling factor retinoic acid (RA): one specifying the size and the other specifying the axial position relative to mesodermal structures of the hindbrain territory. These activities are each independent but coordinated with the well-established function of RA in hindbrain patterning. Using neural and mesodermal landmarks we demonstrate that the functions of RA in aligning neural and mesodermal tissues temporally precede the specification of hindbrain and spinal cord territories and the activation of hox transcription. Using cell transplantation assays we show that RA activity in the neuroepithelium regulates hindbrain patterning directly and territory size specification indirectly. This indirect function is partially dependent on Wnts but independent of FGFs. Importantly, RA specifies and patterns the hindbrain territory by antagonizing the activity of the spinal cord specification gene cdx4; loss of Cdx4 rescues the defects associated with the loss of RA, including the reduction in hindbrain size and the loss of posterior rhombomeres. We propose that at the head-trunk transition, RA coordinates specification, patterning and alignment of neural and mesodermal tissues that are essential for the organization and function of the neural and skeletal systems.

Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss.

Hereditary hearing loss is characterized by a high degree of genetic heterogeneity. Here we present OTOGL mutations, a homozygous one base pair deletion (c.1430 delT) causing a frameshift (p.Val477Glufs(∗)25) in a large consanguineous family and two compound heterozygous mutations, c.547C>T (p.Arg183(∗)) and c.5238+5G>A, in a nonconsanguineous family with moderate nonsyndromic sensorineural hearing loss. OTOGL maps to the DFNB84 locus at 12q21.31 and encodes otogelin-like, which has structural similarities to the epithelial-secreted mucin protein family. We demonstrate that Otogl is expressed in the inner ear of vertebrates with a transcription level that is high in embryonic, lower in neonatal, and much lower in adult stages. Otogelin-like is localized to the acellular membranes of the cochlea and the vestibular system and to a variety of inner ear cells located underneath these membranes. Knocking down of otogl with morpholinos in zebrafish leads to sensorineural hearing loss and anatomical changes in the inner ear, supporting that otogelin-like is essential for normal inner ear function. We propose that OTOGL mutations affect the production and/or function of acellular structures of the inner ear, which ultimately leads to sensorineural hearing loss.

Current perspectives in zebrafish reverse genetics: moving forward.

Use of the zebrafish as a model of vertebrate development and disease has expanded dramatically over the past decade. While many articles have discussed the strengths of zebrafish forward genetics (the phenotype-driven approach), there has been less emphasis on equally important and frequently used reverse genetics (the candidate gene-driven approach). Here we review both current and prospective reverse genetic techniques that are applicable to the zebrafish model. We include discussion of pharmacological approaches, popular gain-of-function and knockdown approaches, and gene targeting strategies. We consider the need for temporal and spatial control over gain/loss of gene function, and discuss available and developing techniques to achieve this end. Our goal is both to reveal the current technical advantages of the zebrafish and to highlight those areas where work is still required to allow this system to be exploited to full advantage.

Repression of the hindbrain developmental program by Cdx factors is required for the specification of the vertebrate spinal cord.

The spinal cord is a unique vertebrate feature that originates, together with the hindbrain, from the caudal neural plate. Whereas the hindbrain subdivides into rhombomeres, the spinal cord remains unsegmented. We have identified Cdx transcription factors as key determinants of the spinal cord region in zebrafish. Loss of Cdx1a and Cdx4 functions causes posterior expansion of the hindbrain at the expense of the unsegmented spinal cord. By contrast, cdx4 overexpression in the hindbrain impairs rhombomere segmentation and patterning and induces the expression of spinal cord-specific genes. Using cell transplantation, we demonstrate that Cdx factors function directly within the neural ectoderm to specify spinal cord. Overexpression of 5' Hox genes fails to rescue hindbrain and spinal cord defects associated with cdx1a/cdx4 loss-of-function, suggesting a Hox-independent mechanism of spinal cord specification. In the absence of Cdx function, the caudal neural plate retains hindbrain characteristics and remains responsive to surrounding signals, particularly retinoic acid, in a manner similar to the native hindbrain. We propose that by preventing the posterior-most region of the neural plate from following a hindbrain developmental program, Cdx factors help determine the size of the prospective hindbrain and spinal cord territories.

Determination of embryonic polarity in a regulative system: evidence for endogenous inhibitors acting sequentially during primitive streak formation in the chick embryo.

Avian embryos have a remarkable capacity to regulate: when a pre-primitive streak stage embryo is cut into fragments, each fragment can spontaneously initiate formation of a complete embryonic axis. We investigate the signalling pathways that initiate primitive streak formation and the mechanisms that ensure that only a single axis normally forms. As reported previously, an ectopic primitive streak can be induced by misexpression of Vg1 in the marginal zone. We now show that Vg1 induces an inhibitor that travels across the embryo (3 mm distance) in less than 6 hours. We provide evidence that this inhibitor acts early in the cascade of events downstream of Vg1. We also show that FGF signalling is required for primitive streak formation, in cooperation with Nodal and Chordin. We suggest that three sequential inhibitory steps ensure that a single axis develops in the normal embryo: an early inhibitor that spreads throughout the embryo (which can be induced by Vg1), a second inhibition by Cerberus from the underlying hypoblast, and finally a late inhibition from Lefty emitted by the primitive streak itself.

A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo.

In the chick embryo, two secreted factors have recently be shown to cooperate in inducing the first axial structure, the primitive streak: cWnt8C (normally expressed around the circumference of the embryo, in the marginal zone) and the TGF beta superfamily member cVg1 (expressed in the posterior part of the marginal zone) (Development 128 (2001) 2915). Misexpression of Vg1 in the anterior marginal zone induces an ectopic primitive streak and recapitulates the morphological changes associated with normal primitive streak formation. Here, we analyse the time-course of appearance and disappearance of expression of 12 genes (cVg1, Lef1, Nodal, FGF8, cWnt8C, cBra, cNot1, goosecoid, HNF3 beta, Chordin, Otx2 and Sox3, whose normal expression is also polarized at early stages of development) in response to cVg1 misexpression in the anterior marginal zone. We show that a hierarchy of gene expression accompanies induction of the ectopic axis, reminiscent of the order in which the same genes begin to be expressed in the normal embryo.