Reconstruction and deconstruction of human somitogenesis in vitro.

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1-4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.

Intracellular pH controls WNT downstream of glycolysis in amniote embryos.

Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic ß-catenin acetylation downstream of WNT signalling. As acetylated ß-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.

In vitro characterization of the human segmentation clock.

The segmental organization of the vertebral column is established early in embryogenesis, when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock1,2. Although this oscillator has been well-characterized in model organisms1,2, whether a similar oscillator exists in humans remains unknown. Genetic analyses of patients with severe spine segmentation defects have implicated several human orthologues of cyclic genes that are associated with the mouse segmentation clock, suggesting that this oscillator might be conserved in humans3. Here we show that human PSM cells derived in vitro-as well as those of the mouse4-recapitulate the oscillations of the segmentation clock. Human PSM cells oscillate with a period two times longer than that of mouse cells (5 h versus 2.5 h), but are similarly regulated by FGF, WNT, Notch and YAP signalling5. Single-cell RNA sequencing reveals that mouse and human PSM cells in vitro follow a developmental trajectory similar to that of mouse PSM in vivo. Furthermore, we demonstrate that FGF signalling controls the phase and period of oscillations, expanding the role of this pathway beyond its classical interpretation in 'clock and wavefront' models1. Our work identifying the human segmentation clock represents an important milestone in understanding human developmental biology.

The genetic program for cartilage development has deep homology within Bilateria.

The evolution of novel cell types led to the emergence of new tissues and organs during the diversification of animals. The origin of the chondrocyte, the cell type that synthesizes cartilage matrix, was central to the evolution of the vertebrate endoskeleton. Cartilage-like tissues also exist outside the vertebrates, although their relationship to vertebrate cartilage is enigmatic. Here we show that protostome and deuterostome cartilage share structural and chemical properties, and that the mechanisms of cartilage development are extensively conserved--from induction of chondroprogenitor cells by Hedgehog and ß-catenin signalling, to chondrocyte differentiation and matrix synthesis by SoxE and SoxD regulation of clade A fibrillar collagen (ColA) genes--suggesting that the chondrogenic gene regulatory network evolved in the common ancestor of Bilateria. These results reveal deep homology of the genetic program for cartilage development in Bilateria and suggest that activation of this ancient core chondrogenic network underlies the parallel evolution of cartilage tissues in Ecdysozoa, Lophotrochozoa and Deuterostomia.

A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids.

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.

Transcriptome sequencing and annotation of the predatory mite Metaseiulus occidentalis (Acari: Phytoseiidae): a cautionary tale about possible contamination by prey sequences.

Next-generation sequencing was applied to the transcriptome of the phytoseiid Metaseiulus occidentalis to characterize gene expression in all life stages reared under different conditions to optimize the recovery of as many genes as possible. One production and one titration run produced a total of 862,069 reads (average size: 314.87 bp), which generated 255.6 Mbp of sequences on the GS-FLX Titanium sequencing platform. After removal of putative prey sequences 850,543 reads were used in NewBler and PTA assemblies to produce 74,172 non-redundant sequences, including 30,691 contigs and 43,481 singlets with 11,994 contigs consisting of more than 500 bp and 37,278 sequences >300 bp, constituting 48.7 % of all sequences. There were 25,888 hits with the NCBI non-redundant database and 15,376 unique transcripts. There were 26,225 hits with the Ixodes scapularis genome and 6,634 unique transcripts. There were 22,225 hits with the RefSeq of Homo sapiens with 6,465 unique transcripts, and 23,656 hits with the RefSeq of Drosophila melanogaster with 9,216 unique transcripts. Selected ESTs corresponding to genes of interest were analyzed including those related to transposable elements, GPCRs, Sox transcription factors, diapause and foraging behavior, and pesticide resistances. Novel and important genes appear to have been discovered that provide insight into the evolution, biology, and physiology of this important predator of pest mites in agriculture and will be useful in analyzing complete genome sequences of this natural enemy.

Exploring the Influence of Cell Metabolism on Cell Fate through Protein Post-translational Modifications.

The connection between cell fate transitions and metabolic shifts is gaining momentum in the study of cell differentiation in embryonic development, adult stem cells, and cancer pathogenesis. Here, we explore how metabolic transitions influence post-translational modifications (PTMs), which play central roles in the activation of transcriptional programs. PTMs can control the function of transcription factors acting as master regulators of cell fate as well as activation or repression of cell identity genes by regulating chromatin state via histone tail modifications. It now becomes clear that cell metabolism is an integral part of the complex landscape of regulatory mechanisms underlying cell differentiation.

The morphology and post-hatching development of the skull of Bolitoglossa nicefori (Caudata: Plethodontidae): developmental implications of recapitulation and repatterning.

The cranial morphology of the direct-developing salamander Bolitoglossa nicefori and its post-hatching development are described and compared with that of other urodeles. Four stages of cranial development are defined on the basis of conspicuous events that occur during post-hatching ontogeny. The adult skull morphology of B. nicefori is similar to that of other plethodontids; however, some regions show interspecific variation. The post-hatching ontogeny of the skull and the stage of ossification observed in the hatchlings of B. nicefori show two important ontogenetic features: (1) a mosaic of early larval, metamorphic and post-metamorphic skull features in hatchlings, and (2) absence of characteristic larval elements in skull and hyoid apparatus. The distinctive stage of ossification in the hatchlings of B. nicefori could be caused by heterochronic changes in the ossification sequence, compared to the ontogeny of metamorphic salamanders. The possible heterochronic changes and the absence of larval traits are perhaps due to ontogenetic repatterning, yet without an obvious impact on the adult skull morphology (absence of morphological novelties). This might indicate a compartmentalized development. Further studies should be performed in order to establish the possible occurrence of recapitulatory patterns or ontogenetic repatterning in the skull morphogenesis of B. nicefori during its embryonic development.

Evolution of limb development in cephalopod mollusks.

Cephalopod mollusks evolved numerous anatomical novelties, including arms and tentacles, but little is known about the developmental mechanisms underlying cephalopod limb evolution. Here we show that all three axes of cuttlefish limbs are patterned by the same signaling networks that act in vertebrates and arthropods, although they evolved limbs independently. In cuttlefish limb buds, Hedgehog is expressed anteriorly. Posterior transplantation of Hedgehog-expressing cells induced mirror-image limb duplications. Bmp and Wnt signals, which establish dorsoventral polarity in vertebrate and arthropod limbs, are similarly polarized in cuttlefish. Inhibition of Bmp2/4 dorsally caused ectopic expression of Notum, which marks the ventral sucker field, and ectopic sucker development. Cuttlefish also show proximodistal regionalization of Hth, Exd, Dll, Dac, Sp8/9, and Wnt expression, which delineates arm and tentacle sucker fields. These results suggest that cephalopod limbs evolved by parallel activation of a genetic program for appendage development that was present in the bilaterian common ancestor.

Limb development in the gekkonid lizard Gonatodes albogularis: A reconsideration of homology in the lizard carpus and tarsus.

Despite the attention squamate lizards have received in the study of digit and limb loss, little is known about limb morphogenesis in pentadactyl lizards. Recent developmental studies have provided a basis for understanding lizard autopodial element homology based on developmental and comparative anatomy. In addition, the composition and identity of some carpal and tarsal elements of lizard limbs, and reptiles in general, have been the theme of discussions about their homology compared to non-squamate Lepidosauromorpha and basal Amniota. The study of additional embryonic material from different lizard families may improve our understanding of squamate limb evolution. Here, we analyze limb morphogenesis in the gekkonid lizard Gonatodes albogularis describing patterns of chondrogenesis and ossification from early stages of embryonic development to hatchlings. Our results are in general agreement with previous developmental studies, but we also show that limb development in squamates probably involves more chondrogenic elements for carpal and tarsal morphogenesis, as previously recognized on the grounds of comparative anatomy. We provide evidence for the transitory presence of distal carpale 1 and intermedium in the carpus and tibiale, intermedium, distal centralia, and distal tarsale 2 in the tarsus. Hence, we demonstrate that some elements that were believed to be lost in squamate evolution are conserved as transitory elements during limb development. However, these elements do not represent just phylogenetic burden but may be important for the morphogenesis of the lizard autopodium.

Morphology and posthatching ontogeny of the autopodial skeleton of Bolitoglossa nicefori (Caudata: Plethodontidae).

Webbed foot morphology is a highly homoplastic character in species of Bolitoglossa and has been assumed to be pedomorphic. This study examines the morphology and posthatching ontogeny of the autopodial skeleton of Bolitoglossa nicefori and compares the descriptive and morphometric results with other species of the genus. We show that the autopodial morphology of B. nicefori coincides with the generalized pattern of the genus; webbed foot morphology is produced by pedomorphosis that affect the phalange length of the digits, resulting in a synchronized growth of digits (length and ossification rates) and the fleshy web. Although the webbed foot morphology of B. nicefori might be explained by the pervasive pedomorphic developmental trend observed in the genus, the large degree of variation encountered in the morphology of the distal phalanges indicates that the pedomorphic processes acting in this species are neither a simple truncation of the autopodial developmental program during early posthatching development nor a global process acting over the whole body plan. Instead, this morphological pattern is probably a result of the modular nature of limb development.

The unusual orbitosphenoid of the snakelike lizard Bachia bicolor.

We studied the morphology of the chondrocranial orbitotemporal region in the snakelike gymnophthalmid lizard Bachia bicolor and its relation to other structures such as the ophthalmic division of the trigeminal nerve and eye muscles, to show its particular morphology, and discuss the homology of some skeletal structures relative to other squamates. We used three-dimensional computer reconstructions from serial histological sections; additionally we studied the embryonic and postembryonic development of the orbitosphenoid bone using cleared and double-stained whole-mount skeletal material. The chondrocranial orbitotemporal morphology in B. bicolor was found to be severely reduced as described for other miniaturized snakelike squamates, but it was accompanied by extensive orbitosphenoid ossifications. Within squamates, only amphisbaenians showed an expanded orbitosphenoid, which originates from fused endochondral and membranous ossifications. In B. bicolor the orbitosphenoid was also found to be formed by endochondral and membranous ossifications, but contrary to the amphisbaenian condition the membranous ossifications were found to arise as membrane bone outgrowths from the perichondral ossification of the chondral core. Despite its derived morphologies, we argue that the orbitosphenoids in amphisbaenians and B. bicolor are homologous to the orbitosphenoids of other squamates. Thus, the expanded orbitosphenoid morphology is found to be achieved by different ontogenetic processes in amphisbaenians and B. bicolor, representing a case of independent evolution by convergence.