An archetype and scaling of developmental tissue dynamics across species.

Morphometric studies have revealed the existence of simple geometric relationships among various animal shapes. However, we have little knowledge of the mathematical principles behind the morphogenetic dynamics that form the organ/body shapes of different species. Here, we address this issue by focusing on limb morphogenesis in Gallus gallus domesticus (chicken) and Xenopus laevis (African clawed frog). To compare the deformation dynamics between tissues with different sizes/shapes as well as their developmental rates, we introduce a species-specific rescaled spatial coordinate and a common clock necessary for cross-species synchronization of developmental times. We find that tissue dynamics are well conserved across species under this spacetime coordinate system, at least from the early stages of development through the phase when basic digit patterning is established. For this developmental period, we also reveal that the tissue dynamics of both species are mapped with each other through a time-variant linear transformation in real physical space, from which hypotheses on a species-independent archetype of tissue dynamics and morphogenetic scaling are proposed.

Appendage-restricted gene induction using a heated agarose gel for studying regeneration in metamorphosed Xenopus laevis and Pleurodeles waltl.

Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage-specific gene induction using a simplified protocol named the "agarose-embedded heat shock (AeHS) method" involving the combination of a heat-shock-inducible system and insertion of an appendage in a temperature-controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord.

Infrared Laser-Mediated Gene Induction at the Single-Cell Level in the Regenerating Tail of Xenopus laevis Tadpoles.

We describe a precise and reproducible gene-induction method in the amphibian, Xenopus laevis Tetrapod amphibians are excellent models for studying the mechanisms of three-dimensional organ regeneration because they have an exceptionally high regenerative ability. However, spatial and temporal manipulation of gene expression has been difficult in amphibians, hindering studies on the molecular mechanisms of organ regeneration. Recently, however, development of a Xenopus transgenic system with a heat-shock-inducible gene has enabled the manipulation of specific genes. Here, we applied an infrared laser-evoked gene operator (IR-LEGO) system to the regenerating tail of Xenopus tadpoles. In this method, a local heat shock by laser irradiation induces gene expression at the single-cell level. After amputation, Xenopus tadpoles regenerate a functional tail, including spinal cord. The regenerating tail is flat and transparent enabling the targeting of individual cells by laser irradiation. In this protocol, a single neural progenitor cell in the spinal cord of the regenerating tail is labeled with heat-shock-inducible green fluorescent protein (GFP). Gene induction at the single-cell level provides a method for rigorous cell-lineage tracing and for analyzing gene function in both cell-autonomous and noncell-autonomous contexts. The method can be modified to study the regeneration of limbs or organs in other amphibians, including Xenopus tropicalis, newts, and salamanders.

Morphometric staging of organ development based on cross sectional images.

An objective, continuous, and robust method for staging developing embryos or organs is essential for providing a common measure of time when studying quantitative/systems developmental biology. However, classical methods based on factors such as somite number or qualitative visual attributes are discrete and/or ambiguous due to observers' subjectivity. Thus, an alternative staging method based on an explicit and continuous description of developmental states over time, such as anatomy/morphology, is needed. Here, we briefly propose a novel staging method as a natural extension of classical staging based on cross sectional images of organs, which are more accessible than full 3D structures. The contours are represented as 2D closed curves and quantified using elliptic Fourier descriptors. Treating the ambiguity in classical staging as a statistical model, the relationship between the novel morphometric staging and classical staging can be determined. This method was validated by applying it to two different sets of data: chick forebrain and Xenopus hindlimb development. Using this method, it is also possible to reconstruct the time evolution of the average morphology, which would be useful for quantitatively comparing morphologies between embryos or between normal and abnormal conditions.

Cells from subcutaneous tissues contribute to scarless skin regeneration in Xenopus laevis froglets.

BACKGROUND: Mammals cannot regenerate the dermis and other skin structures after an injury and instead form a scar. However, a Xenopus laevis froglet can regenerate scarless skin, including the dermis and secretion glands, on the limbs and trunk after skin excision. Subcutaneous tissues in the limbs and trunk consist mostly of muscles. Although subcutaneous tissues beneath a skin injury appear disorganized, the cellular contribution of these underlying tissues to skin regeneration remains unclear. RESULTS: We crossed the inbred J strain with a green fluorescent protein (GFP)-labeled transgenic Xenopus line to obtain chimeric froglets that have GFP-negative skin and GFP-labeled subcutaneous tissues and are not affected by immune rejection after metamorphosis. We found that GFP-positive cells from subcutaneous tissues contributed to regenerating the skin, especially the dermis, after an excision injury. We also showed that the skin on the head, which is over bone rather than muscle, can also completely regenerate skin structures. CONCLUSIONS: Cells derived from subcutaneous tissues, at least in the trunk region, contribute to and may be essential for skin regeneration. Characterizing the subcutaneous tissue-derived cells that contribute to skin regeneration in amphibians may lead to the induction of cells that can regenerate complete skin structures without scarring in mammals. Developmental Dynamics 246:585-597, 2017. © 2017 Wiley Periodicals, Inc.

Application of local gene induction by infrared laser-mediated microscope and temperature stimulator to amphibian regeneration study.

Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three-dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the single-cell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR-LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature-controlled metal probe (temperature stimulator). The local gene manipulation systems, the IR-LEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre-LoxP system as well as to analyze gene function in a target region or cells with less off-target effects in the study of amphibian regeneration.

Epigenetic modification maintains intrinsic limb-cell identity in Xenopus limb bud regeneration.

Many amphibians can regenerate limbs, even in adulthood. If a limb is amputated, the stump generates a blastema that makes a complete, new limb in a process similar to developmental morphogenesis. The blastema is thought to inherit its limb-patterning properties from cells in the stump, and it retains the information despite changes in morphology, gene expression, and differentiation states required by limb regeneration. We hypothesized that these cellular properties are maintained as epigenetic memory through histone modifications. To test this hypothesis, we analyzed genome-wide histone modifications in Xenopus limb bud regeneration. The trimethylation of histone H3 at lysine 4 (H3K4me3) is closely related to an open chromatin structure that allows transcription factors access to genes, whereas the trimethylation of histone H3 at lysine 27 (H3K27me3) is related to a closed chromatin state that blocks the access of transcription factors. We compared these two modification profiles by high-throughput sequencing of samples prepared from the intact limb bud and the regenerative blastema by chromatin immunoprecipitation. For many developmental genes, histone modifications at the transcription start site were the same in the limb bud and the blastema, were stable during regeneration, and corresponded well to limb properties. These results support our hypothesis that histone modifications function as a heritable cellular memory to maintain limb cell properties, despite dynamic changes in gene expression during limb bud regeneration in Xenopus.