Spatial mapping of tissue properties in vivo reveals a 3D stiffness gradient in the mouse limb bud.

Numerous hypotheses invoke tissue stiffness as a key parameter that regulates morphogenesis and disease progression. However, current methods are insufficient to test hypotheses that concern physical properties deep in living tissues. Here we introduce, validate, and apply a magnetic device that generates a uniform magnetic field gradient within a space that is sufficient to accommodate an organ-stage mouse embryo under live conditions. The method allows rapid, nontoxic measurement of the three-dimensional (3D) spatial distribution of viscoelastic properties within mesenchyme and epithelia. Using the device, we identify an anteriorly biased mesodermal stiffness gradient along which cells move to shape the early limb bud. The stiffness gradient corresponds to a Wnt5a-dependent domain of fibronectin expression, raising the possibility that durotaxis underlies cell movements. Three-dimensional stiffness mapping enables the generation of hypotheses and potentially the rigorous testing of mechanisms of development and disease.

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage.

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.

Coordination between body growth and tissue growth: Wolffian duct elongation and somitogenesis proceed in harmony with axial growth.

During embryogenesis, different tissues develop coordinately, and this coordination is often in harmony with body growth. Recent studies allow us to understand how this harmonious regulation is achieved at the levels of inter-cellular, inter-tissue, and tissue-body relationships. Here, we present an overview of recently revealed mechanisms by which axial growth (tail growth) drives a variety of morphogenetic events, with a focus on the coordinated progression between Wolffian (nephric) duct elongation and somitogenesis. We also discuss how we can relate this coordination to the events occurring during limb bud outgrowth, since the limb buds and tail bud are appendage anlagen acquired during vertebrate evolution, both of which undergo massive elongation/outgrowth.

Biophysical regulation of early limb bud morphogenesis.

The physical basis of morphogenesis is a fascinating concern that has been a longstanding interest of developmental biologists. In this review, I attempt to incorporate earlier and recent biophysical concepts and data to explain basic features of early limb bud morphogenesis. In particular, I discuss the influence of mesenchymal cohesion and physical properties that might contribute to phase separation of the bud from the lateral plate, the possibility that the early dorsoventral limb bud axis is moulded by the surface ectoderm, and endogenous electric fields that might contribute to oriented cell movements which generate the early limb bud. A combination of quantitative biophysical experimentation and modelling will likely advance this field.

Different regulation of limb development by p63 transcript variants.

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

mTORC1 Signaling Promotes Limb Bud Cell Growth and Chondrogenesis.

mTORC1 signaling has been shown to promote limb skeletal growth through stimulation of protein synthesis in chondrocytes. However, potential roles of mTORC1 in prechondrogenic mesenchyme have not been explored. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in prechondrogenic limb mesenchymal cells. Deletion of Raptor reduced the size of limb bud cells, resulting in overall diminution of the limb bud without affecting skeletal patterning. We then examined the potential role of mTORC1 in chondrogenic differentiation in vitro. Both pharmacological and genetic disruption of mTORC1 significantly suppressed the number and size of cartilage nodules in micromass cultures of limb bud mesenchymal cells. Similarly, inhibition of mTORC1 signaling in chondrogenic ATDC5 cells greatly impaired cartilage nodule formation, and decreased the expression of the master transcriptional factor Sox9, along with the cartilage matrix genes Acan and Col2a1. Thus, we have identified an important role for mTORC1 signaling in promoting limb mesenchymal cell growth and chondrogenesis during embryonic development. J. Cell. Biochem. 118: 748-753, 2017. © 2016 Wiley Periodicals, Inc.

Positional plasticity in regenerating Amybstoma mexicanum limbs is associated with cell proliferation and pathways of cellular differentiation.

BACKGROUND: The endogenous ability to dedifferentiate, re-pattern, and re-differentiate adult cells to repair or replace damaged or missing structures is exclusive to only a few tetrapod species. The Mexican axolotl is one example of these species, having the capacity to regenerate multiple adult structures including their limbs by generating a group of progenitor cells, known as the blastema, which acquire pattern and differentiate into the missing tissues. The formation of a limb regenerate is dependent on cells in the connective tissues that retain memory of their original position in the limb, and use this information to generate the pattern of the missing structure. Observations from recent and historic studies suggest that blastema cells vary in their potential to pattern distal structures during the regeneration process; some cells are plastic and can be reprogrammed to obtain new positional information while others are stable. Our previous studies showed that positional information has temporal and spatial components of variation; early bud (EB) and apical late bud (LB) blastema cells are plastic while basal-LB cells are stable. To identify the potential cellular and molecular basis of this variation, we compared these three cell populations using histological and transcriptional approaches. RESULTS: Histologically, the basal-LB sample showed greater tissue organization than the EB and apical-LB samples. We also observed that cell proliferation was more abundant in EB and apical-LB tissue when compared to basal-LB and mature stump tissue. Lastly, we found that genes associated with cellular differentiation were expressed more highly in the basal-LB samples. CONCLUSIONS: Our results characterize histological and transcriptional differences between EB and apical-LB tissue compared to basal-LB tissue. Combined with our results from a previous study, we hypothesize that the stability of positional information is associated with tissue organization, cell proliferation, and pathways of cellular differentiation.

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.

Lhx9 gene expression during early limb development in mice requires the FGF signalling pathway.

Lhx9 is a member of the LIM-homeodomain gene family necessary for the correct development of many organs including gonads, limbs, heart and the nervous system. In the context of limb development, Lhx9 has been implicated as an integrator for Fibroblast growth factor (FGF) and Sonic hedgehog (Shh) signalling required for proximal-distal (PD) and anterior-posterior (AP) development of the limb. Three splice variants of the Lhx9 transcript are expressed during development, two of which are predicted to act in a dominant negative fashion, competing with the DNA binding version of Lhx9 for binding to cofactors via the LIM-domain. We examined the expression pattern for the three alternative splice forms of Lhx9; Lhx9α, Lhx9ß and Lhx9c during early limb development. We have found that of the three Lhx9 isoforms, only Lhx9α and Lhx9c (intact homeodomain) are expressed during early limb development, each with their own distinct expression pattern. Additionally we determined that Lhx9 expression overlaps with FGF10 expression in the developing limb bud mesenchyme. Limb bud explant cultures, in the presence of signalling pathway inhibitors, also indicated that Lhx9 mRNA expression in the limb bud was dependent on FGF signalling.

Anisotropic stress orients remodelling of mammalian limb bud ectoderm.

The physical forces that drive morphogenesis are not well characterized in vivo, especially among vertebrates. In the early limb bud, dorsal and ventral ectoderm converge to form the apical ectodermal ridge (AER), although the underlying mechanisms are unclear. By live imaging mouse embryos, we show that prospective AER progenitors intercalate at the dorsoventral boundary and that ectoderm remodels by concomitant cell division and neighbour exchange. Mesodermal expansion and ectodermal tension together generate a dorsoventrally biased stress pattern that orients ectodermal remodelling. Polarized distribution of cortical actin reflects this stress pattern in a ß-catenin- and Fgfr2-dependent manner. Intercalation of AER progenitors generates a tensile gradient that reorients resolution of multicellular rosettes on adjacent surfaces, a process facilitated by ß-catenin-dependent attachment of cortex to membrane. Therefore, feedback between tissue stress pattern and cell intercalations remodels mammalian ectoderm.

Embryonic origin and compartmental organization of the external genitalia.

Genital malformations occur at a high frequency in humans, affecting ~1:250 live births. The molecular mechanisms of external genital development are beginning to be identified; however, the origin of cells that give rise to external genitalia is unknown. Here we use cell lineage analysis to show that the genital tubercle, the precursor of the penis and clitoris, arises from two populations of progenitor cells that originate at the lateral edges of the embryo, at the level of the posterior hindlimb buds and anterior tail. During body wall closure, the left and right external genital progenitor pools are brought together at the ventral midline, where they form the paired genital swellings that give rise to the genital tubercle. Unexpectedly, the left and right external genital progenitor pools form two lineage-restricted compartments in the phallus. Together with previous lineage studies of limb buds, our results indicate that, at the pelvic level, the early lateral mesoderm is regionalized from medial to lateral into dorsal limb, ventral limb, and external genital progenitor fields. These findings have implications for the evolutionary diversification of external genitalia and for the association between external genital defects and disruption of body wall closure, as seen in the epispadias-extrophy complex.

Limb regeneration in salamanders and digital tip regeneration in experimental mice: implications for the hand surgeon.

UNLABELLED: Several lessons and observations from limb regeneration in animals could open new insights to direct related research in the field of hand surgery. This article briefly reviews the biology of limb regeneration in salamanders and experimental mice, with special emphasis on implications for hand surgery. LEVEL OF EVIDENCE: 5.

Yap1, transcription regulator in the Hippo signaling pathway, is required for Xenopus limb bud regeneration.

The Hippo signaling pathway is conserved from insects to mammals and is important for multiple processes, including cell proliferation, apoptosis and tissue homeostasis. Hippo signaling is also crucial for regeneration, including intercalary regeneration, of the whole body in the flatworm and of the leg in the cricket. However, its role in vertebrate epimorphic regeneration is unknown. Therefore, to identify principles of regeneration that are conserved among bilaterians, we investigated the role of Hippo signaling in the limb bud regeneration of an anuran amphibian, Xenopus laevis. We found that a transcription factor, Yap1, an important downstream effector of Hippo signaling, is upregulated in the regenerating limb bud. To evaluate Yap1׳s function in limb bud regeneration, we made transgenic animals that expressed a dominant-negative form of Yap under a heat-shock promoter. Overexpression of a dominant-negative form of Yap in tadpoles reduced cell proliferation, induced ectopic apoptosis, perturbed the expression domains of limb-patterning genes including hoxa13, hoxa11, and shh in the regenerating limb bud. Transient expression of a dominant-negative Yap in transgenic tadpoles also caused limb bud regeneration defects, and reduced intercalary regeneration. These results indicate that Yap1 has a crucial role in controlling the limb regenerative capacity in Xenopus, and suggest that the involvement of Hippo signaling in regeneration is conserved between vertebrates and invertebrates. This finding provides molecular evidence that common principles underlie regeneration across phyla, and may contribute to the development of new therapies in regenerative medicine.

Misexpression of Pknox2 in mouse limb bud mesenchyme perturbs zeugopod development and deltoid crest formation.

The TALE (Three Amino acid Loop Extension) family consisting of Meis, Pbx and Pknox proteins is a group of transcriptional co-factors with atypical homeodomains that play pivotal roles in limb development. Compared to the in-depth investigations of Meis and Pbx protein functions, the role of Pknox2 in limb development remains unclear. Here, we showed that Pknox2 was mainly expressed in the zeugopod domain of the murine limb at E10.5 and E11.5. Misexpression of Pknox2 in the limb bud mesenchyme of transgenic mice led to deformities in the zeugopod and forelimb stylopod deltoid crest, but left the autopod and other stylopod skeletons largely intact. These malformations in zeugopod skeletons were recapitulated in mice overexpressing Pknox2 in osteochondroprogenitor cells. Molecular and cellular analyses indicated that the misexpression of Pknox2 in limb bud mesenchyme perturbed the Hox10-11 gene expression profiles, decreased Col2 expression and Bmp/Smad signaling activity in the limb. These results indicated that Pknox2 misexpression affected mesenchymal condensation and early chondrogenic differentiation in the zeugopod skeletons of transgenic embryos, suggesting Pknox2 as a potential regulator of zeugopod and deltoid crest formation.

Isl1 and Ldb co-regulators of transcription are essential early determinants of mouse limb development.

BACKGROUND: The developing limb has served as an excellent model for studying pattern formation and signal transduction in mammalians. Many of the crucial genes that regulate growth and patterning of the limb following limb bud formation are now well known. However, details regarding the control of limb initiation and early stages of outgrowth remain to be defined. This report is focused on genetic events that pave the way for the establishment of a hindlimb bud. RESULTS: Fgf10 and Tbx are crucial for early phases of limb bud initiation. Here we show that in the absence of Isl1 or of Ldb1/2, there is no hindlimb bud development. Fgf10 expression in the bud mesenchyme is dependent on Isl1 and its Ldb co-regulators. CONCLUSIONS: Thus, Isl1 and the Ldb co-regulators of transcription are essential early determinants of mouse limb development. Isl1/Ldb complexes regulate Fgf10 to orchestrate the earliest stages of hindlimb formation.

Collagen reconstitution is inversely correlated with induction of limb regeneration in Ambystoma mexicanum.

Amphibians can regenerate missing body parts, including limbs. The regulation of collagen has been considered to be important in limb regeneration. Collagen deposition is suppressed during limb regeneration, so we investigated collagen deposition and apical epithelial cap (AEC) formation during axolotl limb regeneration. The accessory limb model (ALM) has been developed as an alternative model for studying limb regeneration. Using this model, we investigated the relationship between nerves, epidermis, and collagen deposition. We found that Sp-9, an AEC marker gene, was upregulated by direct interaction between nerves and epidermis. However, collagen deposition hindered this interaction, and resulted in the failure of limb regeneration. During wound healing, an increase in deposition of collagen caused a decrease in the blastema induction rate in ALM. Wound healing and limb regeneration are alternate processes.

Generation of mice with functional inactivation of talpid3, a gene first identified in chicken.

Specification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid(3) chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid(3) chicken has a mutation in KIAA0586, which encodes a centrosomal protein required for the formation of primary cilia, which are sites of vertebrate Hh signalling. The highly conserved exons 11 and 12 of KIAA0586 are essential to rescue cilia in talpid(3) chicken mutants. We constitutively deleted these two exons to make a talpid3(-/-) mouse. Mutant mouse embryos lack primary cilia and, like talpid(3) chicken embryos, have face and neural tube defects but also defects in left/right asymmetry. Conditional deletion in mouse limb mesenchyme results in polydactyly and in brachydactyly and a failure of subperisoteal bone formation, defects that are attributable to abnormal sonic hedgehog and Indian hedgehog signalling, respectively. Like talpid(3) chicken limbs, the mutant mouse limbs are syndactylous with uneven digit spacing as reflected in altered Raldh2 expression, which is normally associated with interdigital mesenchyme. Both mouse and chicken mutant limb buds are broad and short. talpid3(-/-) mouse cells migrate more slowly than wild-type mouse cells, a change in cell behaviour that possibly contributes to altered limb bud morphogenesis. This genetic mouse model will facilitate further conditional approaches, epistatic experiments and open up investigation into the function of the novel talpid3 gene using the many resources available for mice.

Spatiotemporal changes in cell adhesiveness during vertebrate limb morphogenesis.

During vertebrate limb development, various molecules are expressed in the presumptive limb field or the limb bud in a spatiotemporal-specific manner. The combination of these molecules regulates cellular properties that affect limb initiation and its morphogenesis, especially cartilage formation. Cell adhesiveness of the limb mesenchyme is a key factor in the regulation of cell distribution. Differential adhesiveness of mesenchymal cells is first observed between cells in the presumptive limb field and flank region, and the adhesiveness of the cells in the limb field is higher than that of cells in the flank region. In the limb bud, the adhesiveness of mesenchymal cells shows spatiotemporal difference, which reflects the positional identity of the cells. Position-dependent cell adhesiveness is also observed in blastema cells of the regenerating limb. Therefore, local changes in cell adhesiveness are observed during limb development and regeneration, suggesting significant roles for cell adhesiveness in limb morphogenesis.

A computational clonal analysis of the developing mouse limb bud.

A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis.

Gene silencing of Tead3 abrogates radiation-induced adaptive response in cultured mouse limb bud cells.

There is a crucial need to better understand the effects of low-doses of ionizing radiation in fetal models. Radiation-induced adaptive response (AR) was described in mouse embryos pre-exposed in utero to low-doses of X-rays, which exhibited lower apoptotic levels in the limb bud. We previously described AR-specific gene modulations in the mouse embryo. In this study, we evaluated the role of three candidate genes in the apoptotic AR in a micromass culture of limb bud cells: Csf1, Cacna1a and Tead3. Gene silencing of these three genes abrogated AR. Knowing that TEAD3 protein levels are significantly higher in adapted cells and that YAP/TAZ/TEAD are involved in the control of cell proliferation and apoptosis, we suggest that modulation of Tead3 could play a role in the induction of AR in our model, seen as a reduction of radiation-induced apoptosis and a stimulation of proliferation and differentiation in limb bud cells.