Cell Sheet Morphogenesis: Dorsal Closure in Drosophila melanogaster as a Model System.

Dorsal closure is a key process during Drosophila morphogenesis that models cell sheet movements in chordates, including neural tube closure, palate formation, and wound healing. Closure occurs midway through embryogenesis and entails circumferential elongation of lateral epidermal cell sheets that close a dorsal hole filled with amnioserosa cells. Signaling pathways regulate the function of cellular structures and processes, including Actomyosin and microtubule cytoskeletons, cell-cell/cell-matrix adhesion complexes, and endocytosis/vesicle trafficking. These orchestrate complex shape changes and movements that entail interactions between five distinct cell types. Genetic and laser perturbation studies establish that closure is robust, resilient, and the consequence of redundancy that contributes to four distinct biophysical processes: contraction of the amnioserosa, contraction of supracellular Actomyosin cables, elongation (stretching?) of the lateral epidermis, and zipping together of two converging cell sheets. What triggers closure and what the emergent properties are that give rise to its extraordinary resilience and fidelity remain key, extant questions.

Remodeling Tissue Interfaces and the Thermodynamics of Zipping during Dorsal Closure in Drosophila.

Dorsal closure during Drosophila embryogenesis is an important model system for investigating the biomechanics of morphogenesis. During closure, two flanks of lateral epidermis (with actomyosin-rich purse strings near each leading edge) close an eye-shaped opening that is filled with amnioserosa. At each canthus (corner of the eye) a zipping process remodels the tissue interfaces between the leading edges of the lateral epidermis and the amnioserosa. We investigated zipping dynamics and found that apposing leading edge cells come together at their apical ends and then square off basally to form a lateral junction. Meanwhile, the purse strings act as contractile elastic rods bent toward the embryo interior near each canthus. We propose that a canthus-localized force contributes to both bending the ends of the purse strings and the formation of lateral junctions. We developed a thermodynamic model for zipping based on three-dimensional remodeling of the tissue interfaces and the reaction dynamics of adhesion molecules in junctions and elsewhere, which we applied to zipping during unperturbed wild-type closure and to laser or genetically perturbed closure. We identified two processes that can contribute to the zipping mechanism, consistent with experiments, distinguished by whether amnioserosa dynamics do or do not augment canthus adhesion dynamics.

Cell ingression and apical shape oscillations during dorsal closure in Drosophila.

Programmed patterns of gene expression, cell-cell signaling, and cellular forces cause morphogenic movements during dorsal closure. We investigated the apical cell-shape changes that characterize amnioserosa cells during dorsal closure in Drosophila embryos with in vivo imaging of green-fluorescent-protein-labeled DE-cadherin. Time-lapsed, confocal images were assessed with a novel segmentation algorithm, Fourier analysis, and kinematic and dynamical modeling. We found two generic processes, reversible oscillations in apical cross-sectional area and cell ingression characterized by persistent loss of apical area. We quantified a time-dependent, spatially-averaged sum of intracellular and intercellular forces acting on each cell's apical belt of DE-cadherin. We observed that a substantial fraction of amnioserosa cells ingress near the leading edges of lateral epidermis, consistent with the view that ingression can be regulated by leading-edge cells. This is in addition to previously observed ingression processes associated with zipping and apoptosis. Although there is cell-to-cell variability in the maximum rate for decreasing apical area (0.3-9.5 µm(2)/min), the rate for completing ingression is remarkably constant (0.83 cells/min, r(2) > 0.99). We propose that this constant ingression rate contributes to the spatiotemporal regularity of mechanical stress exerted by the amnioserosa on each leading edge during closure.

Neuromelanins isolated from different regions of the human brain exhibit a common surface photoionization threshold.

Neuromelanin isolated from the premotor cortex, cerebellum, putamen, globus pallidus and corpus callosum of the human brain is studied by scanning probe and photoelectron emission microscopies and the results are compared with previously published work on neuromelanin from the substantia nigra. Scanning electron microscopy reveals common structure for all neuromelanins. All exhibit spherical entities of diameters between 200 and 400 nm, composed of smaller spherical substructures, approximately 30 nm in diameter. These features are similar to that observed for many melanin systems including Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy images were collected for all neuromelanins at specific wavelengths of ultraviolet light between 248 and 413 nm, using the spontaneous emission output from the Duke free electron laser. Analysis of the data establishes a common threshold photoionization potential for neuromelanins of 4.7 +/- 0.2 eV, corresponding to an oxidation potential of -0.3 +/- 0.2 V vs the normal hydrogen electrode (NHE). These results are consistent with previously reported potentials for neuromelanin from the substantia nigra of 4.5 +/- 0.2 eV (-0.1 +/- 0.2 V vs NHE). All neuromelanins exhibit a common low surface oxidation potential, reflecting their eumelanic component and their inability to trigger redox processes with neurotoxic effect.

Unified biophysical mechanism for cell-shape oscillations and cell ingression.

We describe a mechanochemical and percolation cascade that augments myosin's regulatory network to tune cytoskeletal forces. Actomyosin forces collectively generate cytoskeletal forces during cell oscillations and ingression, which we quantify by elastic percolation of the internally driven, cross-linked actin network. Contractile units can produce relatively large, oscillatory forces that disrupt crosslinks to reduce cytoskeletal forces. A (reverse) Hopf bifurcation switches contractile units to produce smaller, steady forces that enhance crosslinking and consequently boost cytoskeletal forces to promote ingression. We describe cell-shape changes and cell ingression in terms of intercellular force imbalances along common cell junctions.

The surface oxidation potential of melanosomes measured by free electron laser-photoelectron emission microscopy.

A technique for measuring the photoionization spectrum and the photoelectron emission threshold of a microscopic structured material is presented. The theoretical underpinning of the experiment and the accuracy of the measurements are discussed. The technique is applied to titanium silicide nanostructures and melanosomes isolated from human hair, human and bovine retinal pigment epithelium cells, and the ink sac of Sepia officinalis. A common photothreshold of 4.5 +/- 0.2 eV is found for this set of melanosomes and is attributed to the photoionization of the eumelanin pigment. The relationship between the photoionization threshold and the electrochemical potential referenced to the normal hydrogen electrode is used to quantify the surface oxidation potential of the melanosome. The developed technique is used to examine the effect of iron chelation on the surface oxidation potential of Sepia melanosomes. The surface oxidation potential is insensitive to bound Fe(III) up to saturation, suggesting that the metal is bound to the interior of the granule. This result is discussed in relation to the age-dependent accumulation of iron in human melanosomes in both the eye and brain.

Age-dependent photoionization thresholds of melanosomes and lipofuscin isolated from human retinal pigment epithelium cells.

Melanosomes and lipofuscin were isolated from 14-, 59-, and 76-year-old, human retinal pigment epithelium specimens and examined. The morphological features of these samples were studied by scanning electron microscopy and atomic force microscopy, and the photoionization properties were examined by photoelectron emission microscopy. Ovoid- and rod-shaped melanosomes were observed. The size of the granules and the distribution between the two shapes show no significant age-dependent change. However, there is a higher occurrence of irregularly shaped aggregates of small round granules in older samples which suggests degradation or damage to melanosomes occurs with age. The melanosomes from the 14-year-old donor eye are well characterized by a single photoionization threshold, 4.1 eV, while the two older melanosomes exhibit two thresholds around 4.4 and 3.6 eV. Lipofuscin from both young and old cells show two thresholds, 4.4 and 3.4 eV. The similarity of the potentials observed for aged melanosomes and lipofuscin suggest that the lower threshold in the melanosome sample reflects lipofuscin deposited the surface of the melanosome. The amount, however, is not sufficient to alter the density of the melanosome, and therefore these granules do not separate in a sucrose gradient at densities characteristic of the typical melanolipofuscin granule. These data suggest that thin deposits of lipofuscin on the surface of retinal pigment epithelium melanosomes are common in the aged eye and that this renders the melanosomes more pro-oxidant.

Photoionization thresholds of melanins obtained from free electron laser-photoelectron emission microscopy, femtosecond transient absorption spectroscopy and electron paramagnetic resonance measurements of oxygen photoconsumption.

Free electron laser-photoelectron emission microscopy (FEL-PEEM), femtosecond absorption spectroscopy and electron paramagnetic resonance (EPR) measurements of oxygen photoconsumption were used to probe the threshold potential for ionization of eumelanosomes and pheomelanosomes isolated from human hair. FEL-PEEM data show that both pigments are characterized by an ionization threshold at 282 nm. However, pheomelanosomes exhibit a second ionization threshold at 326 nm, which is interpreted to be reflective of the benzothiazine structural motif present in pheomelanin and absent in eumelanin. The lower ionization threshold for pheomelanin is supported by femtosecond transient absorption spectroscopy. Unlike photolysis at 350 nm, following excitation of solubalized synthetic pheomelanin at 303 nm, the transient spectrum observed between 500 and 700 nm matches that for the solvated electron, indicating the photoionization threshold for the solubalized pigment is between 350 and 303 nm. For the same synthetic pheomelanin, EPR oximetry experiments reveal an increased rate of oxygen uptake between 338 nm and 323 nm, narrowing the threshold for photoionization to sit between these two wavelengths. These results on the solubalized synthetic pigment are consistent with the FEL-PEEM results on the human melanosomes. The lower ionization potential observed for pheomelanin could be an important part of the explanation for the greater incidence rate of UV-induced skin cancers in red-haired individuals.

Quantifying dorsal closure in three dimensions.

Dorsal closure is an essential stage of Drosophila embryogenesis and is a powerful model system for morphogenesis, wound healing, and tissue biomechanics. During closure, two flanks of lateral epidermis close an eye-shaped dorsal opening that is filled with amnioserosa. The two flanks of lateral epidermis are zipped together at each canthus ("corner" of the eye). Actomyosin-rich purse strings are localized at each of the two leading edges of lateral epidermis ("lids" of the eye). Here we report that each purse string indents the dorsal surface at each leading edge. The amnioserosa tissue bulges outward during the early-to-mid stages of closure to form a remarkably smooth, asymmetric dome indicative of an isotropic and uniform surface tension. Internal pressure of the embryo and tissue elastic properties help to shape the dorsal surface.

Oxidation potentials of human eumelanosomes and pheomelanosomes.

Eumelanosomes and pheomelanosomes isolated from black and red human hair, respectively, were studied by photoelectron emission microscopy (PEEM). PEEM images were collected at various wavelengths between 207 and 344 nm, using the spontaneous emission output of the Duke OK-4 free electron laser (FEL). Analysis of the FEL-PEEM data revealed ionization thresholds of 4.6 and 3.9 eV corresponding to oxidation potentials of -0.2 and +0.5 V vs normal hydrogen electrode for eumelanosomes and pheomelanosomes, respectively. The difference in oxidation potential is attributed to the pigment content of the melanosome, namely whether it contains primarily eumelanin and pheomelanin. The effect of added melanosomes on the reduction of Fe(III)-cytochrome showed pheomelanosomes are stronger reducing agents than eumelanosomes, consistent with the measured oxidation potentials. The FEL-PEEM experiment offers to be an important new approach for quantifying the effects of age, oxidation and metal accumulation on the oxidation potentials of intact melanosomes.

Time-resolved, light scattering measurements of cartilage and cornea denaturation due to free electron laser radiation.

Light scattering is used to monitor the dynamics and energy thresholds of laser-induced structural alterations in biopolymers due to irradiation by a free electron laser (FEL) in the infrared (IR) wavelength range 2.2 to 8.5 microm. Attenuated total reflectance (ATR) Fourier-transform IR (FTIR) spectroscopy is used to examine infrared tissue absorption spectra before and after irradiation. Light scattering by bovine and porcine cartilage and cornea samples is measured in real time during FEL irradiation using a 650-nm diode laser and a diode photoarray with time resolution of 10 ms. The data on the time dependence of light scattering in the tissue are modeled to estimate the approximate values of kinetic parameters for denaturation as functions of laser wavelength and radiant exposure. We found that the denaturation threshold is slightly lower for cornea than for cartilage, and both depend on laser wavelength. An inverse correlation between denaturation thresholds and the absorption spectrum of the tissue is observed for many wavelengths; however, for wavelengths near 3 and 6 microm, the denaturation threshold does not exhibit the inverse correlation, instead being governed by heating kinetics of tissue. It is shown that light scattering is useful for measuring the denaturation thresholds and dynamics for different biotissues, except where the initial absorptivity is very high.

Complete canthi removal reveals that forces from the amnioserosa alone are sufficient to drive dorsal closure in Drosophila.

Drosophila's dorsal closure provides an excellent model system with which to analyze biomechanical processes during morphogenesis. During native closure, the amnioserosa, flanked by two lateral epidermal sheets, forms an eye-shaped opening with canthi at each corner. The dynamics of amnioserosa cells and actomyosin purse strings in the leading edges of epidermal cells promote closure, whereas the bulk of the lateral epidermis opposes closure. Canthi maintain purse string curvature (necessary for their dorsalward forces), and zipping at the canthi shortens leading edges, ensuring a continuous epithelium at closure completion. We investigated the requirement for intact canthi during closure with laser dissection approaches. Dissection of one or both canthi resulted in tissue recoil and flattening of each purse string. After recoil and a temporary pause, closure resumed at approximately native rates until slowing near the completion of closure. Thus the amnioserosa alone can drive closure after dissection of one or both canthi, requiring neither substantial purse string curvature nor zipping during the bulk of closure. How the embryo coordinates multiple, large forces (each of which is orders of magnitude greater than the net force) during native closure and is also resilient to multiple perturbations are key extant questions.

Drosophila morphogenesis: tissue force laws and the modeling of dorsal closure.

Dorsal closure, a stage of Drosophila development, is a model system for cell sheet morphogenesis and wound healing. During closure, two flanks of epidermal tissue progressively advance to reduce the area of the eye-shaped opening in the dorsal surface, which contains amnioserosa tissue. To simulate the time evolution of the overall shape of the dorsal opening, we developed a mathematical model, in which contractility and elasticity are manifest in model force-producing elements that satisfy force-velocity relationships similar to muscle. The action of the elements is consistent with the force-producing behavior of actin and myosin in cells. The parameters that characterize the simulated embryos were optimized by reference to experimental observations on wild-type embryos and, to a lesser extent, on embryos whose amnioserosa was removed by laser surgery and on myospheroid mutant embryos. Simulations failed to reproduce the amnioserosa-removal protocol in either the elastic or the contractile limit, indicating that both elastic and contractile dynamics are essential components of the biological force-producing elements. We found it was necessary to actively upregulate forces to recapitulate both the double and single-canthus nick protocols, which did not participate in the optimization of parameters, suggesting the existence of additional key feedback mechanisms.

Apoptotic force and tissue dynamics during Drosophila embryogenesis.

Understanding cell morphogenesis during metazoan development requires knowledge of how cells and the extracellular matrix produce and respond to forces. We investigated how apoptosis, which remodels tissue by eliminating supernumerary cells, also contributes forces to a tissue (the amnioserosa) that promotes cell-sheet fusion (dorsal closure) in the Drosophila embryo. We showed that expression in the amnioserosa of proteins that suppress or enhance apoptosis slows or speeds dorsal closure, respectively. These changes correlate with the forces produced by the amnioserosa and the rate of seam formation between the cell sheets (zipping), key processes that contribute to closure. This apoptotic force is used by the embryo to drive cell-sheet movements during development, a role not classically attributed to apoptosis.

Actomyosin purse strings: renewable resources that make morphogenesis robust and resilient.

Dorsal closure in Drosophila is a model system for cell sheet morphogenesis and wound healing. During closure two sheets of lateral epidermis move dorsally to close over the amnioserosa and form a continuous epidermis. Forces from the amnioserosa and actomyosin-rich, supracellular purse strings at the leading edges of these lateral epidermal sheets drive closure. Purse strings generate the largest force for closure and occur during development and wound healing throughout phylogeny. We use laser microsurgery to remove some or all of the purse strings from developing embryos. Free edges produced by surgery undergo characteristic responses as follows. Intact cells in the free edges, which previously had no purse string, recoil away from the incision and rapidly assemble new, secondary purse strings. Next, recoil slows, then pauses at a turning point. Following a brief delay, closure resumes and is powered to completion by the secondary purse strings. We confirm that the assembly of the secondary purse strings requires RhoA. We show that alpha-actinin alternates with nonmuscle myosin II along purse strings and requires nonmuscle myosin II for its localization. Together our data demonstrate that purse strings are renewable resources that contribute to the robust and resilient nature of closure.

The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface.

Neuromelanin (NM) isolated from the substantia nigra region of the human brain was studied by scanning probe and photoelectron emission microscopies. Atomic force microscopy reveals that NM granules are comprised of spherical structures with a diameter of approximately 30 nm, similar to that observed for Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy images were collected at specific wavelengths of UV light between 248 and 413 nm, using the spontaneous-emission output from the Duke OK-4 free electron laser. Analysis of the data establishes a threshold photoionization potential for NM of 4.5 +/- 0.2 eV, which corresponds to an oxidation potential of -0.1 +/- 0.2 V vs. the normal hydrogen electrode (NHE). The oxidation potential of NM is within experimental error of the oxidation potential measured for human eumelanosomes (-0.2 +/- 0.2 V vs. NHE), despite the presence of a significant fraction of the red pigment, pheomelanin, which is characterized by a higher oxidation potential (+0.5 +/- 0.2 V vs. NHE). Published kinetic studies on the early chemical steps of melanogenesis show that in the case of pigments containing a mixture of pheomelanin and eumelanin, of which NM is an example, pheomelanin formation occurs first with eumelanin formation predominantly occurring only after cysteine levels are depleted. Such a kinetic model would predict a structural motif with pheomelanin at the core and eumelanin at the surface, which is consistent with the measured surface oxidation potential of the approximately 30-nm constituents of NM granules.

Advantage of the Mark-III FEL for biophysical research and biomedical applications.

Although 6.45 micro m is not the strongest absorption band of biological tissues in the mid-infrared, a Mark-III free-electron laser (FEL) tuned to this wavelength can efficiently ablate tissue while minimizing collateral damage. A model has previously been presented that explains this wavelength dependence as a competition between two dynamic processes--explosive vaporization of saline and denaturation of structural proteins. Here it is shown that this model predicts a 'sweet-spot' for each wavelength, i.e. a region of parameter space (incident intensity and pulse width) in which explosive vaporization is preceded by substantial protein denaturation. This sweet-spot is much larger for wavelengths where protein is the dominant chromophore. At other wavelengths, collateral damage may be minimized within the sweet-spot, but the maximum intensities and pulse widths in these regions are insufficient to remove tissue at surgically relevant rates.

Temperature alterations of infrared light absorption by cartilage and cornea under free-electron laser radiation.

Like pure water, the water incorporated into cartilage and cornea tissue shows a pronounced dependence of the absorption coefficient on temperature. Alteration of the temperature by radiation with an IR free-electron laser was studied by use of a pulsed photothermal radiometric technique. A computation algorithm was modified to take into account the real IR absorption spectra of the tissue and the spectral sensitivity of the IR detector used. The absorption coefficients for several wavelengths within the 2.9- and 6.1-microm water absorption bands have been determined for various laser pulse energies. It is shown that the absorption coefficient for cartilage decreases at temperatures higher than 50 degrees C owing to thermal alterations of water-water and water-biopolymer interactions.

Forces for morphogenesis investigated with laser microsurgery and quantitative modeling.

We investigated the forces that connect the genetic program of development to morphogenesis in Drosophila. We focused on dorsal closure, a powerful model system for development and wound healing. We found that the bulk of progress toward closure is driven by contractility in supracellular "purse strings" and in the amnioserosa, whereas adhesion-mediated zipping coordinates the forces produced by the purse strings and is essential only for the end stages. We applied quantitative modeling to show that these forces, generated in distinct cells, are coordinated in space and synchronized in time. Modeling of wild-type and mutant phenotypes is predictive; although closure in myospheroid mutants ultimately fails when the cell sheets rip themselves apart, our analysis indicates that beta(PS) integrin has an earlier, important role in zipping.