The genetics of niche-specific behavioral tendencies in an adaptive radiation of cichlid fishes.

Behavior is critical for animal survival and reproduction, and possibly for diversification and evolutionary radiation. However, the genetics behind adaptive variation in behavior are poorly understood. In this work, we examined a fundamental and widespread behavioral trait, exploratory behavior, in one of the largest adaptive radiations on Earth, the cichlid fishes of Lake Tanganyika. By integrating quantitative behavioral data from 57 cichlid species (702 wild-caught individuals) with high-resolution ecomorphological and genomic information, we show that exploratory behavior is linked to macrohabitat niche adaptations in Tanganyikan cichlids. Furthermore, we uncovered a correlation between the genotypes at a single-nucleotide polymorphism upstream of the AMPA glutamate-receptor regulatory gene cacng5b and variation in exploratory tendency. We validated this association using behavioral predictions with a neural network approach and CRISPR-Cas9 genome editing.

Direct reprogramming of non-limb fibroblasts to cells with properties of limb progenitors.

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.

Self-organized BMP signaling dynamics underlie the development and evolution of digit segmentation patterns in birds and mammals.

Repeating patterns of synovial joints are a highly conserved feature of articulated digits, with variations in joint number and location resulting in diverse digit morphologies and limb functions across the tetrapod clade. During the development of the amniote limb, joints form iteratively within the growing digit ray, as a population of distal progenitors alternately specifies joint and phalanx cell fates to segment the digit into distinct elements. While numerous molecular pathways have been implicated in this fate choice, it remains unclear how they give rise to a repeating pattern. Here, using single-cell RNA sequencing and spatial gene expression profiling, we investigate the transcriptional dynamics of interphalangeal joint specification in vivo. Combined with mathematical modeling, we predict that interactions within the BMP signaling pathway-between the ligand GDF5, the inhibitor NOGGIN, and the intracellular effector pSMAD-result in a self-organizing Turing system that forms periodic joint patterns. Our model is able to recapitulate the spatiotemporal gene expression dynamics observed in vivo, as well as phenocopy digit malformations caused by BMP pathway perturbations. By contrasting in silico simulations with in vivo morphometrics of two morphologically distinct digits, we show how changes in signaling parameters and growth dynamics can result in variations in the size and number of phalanges. Together, our results reveal a self-organizing mechanism that underpins amniote digit segmentation and its evolvability and, more broadly, illustrate how Turing systems based on a single molecular pathway may generate complex repetitive patterns in a wide variety of organisms.

Distinct patterning responses of wing and leg neuromuscular systems to different preaxial polydactylies.

The tetrapod limb has long served as a paradigm to study vertebrate pattern formation and evolutionary diversification. The distal part of the limb, the so-called autopod, is of particular interest in this regard, given the numerous modifications in both its morphology and behavioral motor output. While the underlying alterations in skeletal form have received considerable attention, much less is known about the accompanying changes in the neuromuscular system. However, modifications in the skeleton need to be properly integrated with both muscle and nerve patterns, to result in a fully functional limb. This task is further complicated by the distinct embryonic origins of the three main tissue types involved-skeleton, muscles and nerves-and, accordingly, how they are patterned and connected with one another during development. To evaluate the degree of regulative crosstalk in this complex limb patterning process, here we analyze the developing limb neuromuscular system of Silkie breed chicken. These animals display a preaxial polydactyly, due to a polymorphism in the limb regulatory region of the Sonic Hedgehog gene. Using lightsheet microscopy and 3D-reconstructions, we investigate the neuromuscular patterns of extra digits in Silkie wings and legs, and compare our results to Retinoic Acid-induced polydactylies. Contrary to previous findings, Silkie autopod muscle patterns do not adjust to alterations in the underlying skeletal topology, while nerves show partial responsiveness. We discuss the implications of tissue-specific sensitivities to global limb patterning cues for our understanding of the evolution of novel forms and functions in the distal tetrapod limb.

A method to investigate muscle target-specific transcriptional signatures of single motor neurons.

BACKGROUND: Motor neurons in the vertebrate spinal cord have long served as a paradigm to study the transcriptional logic of cell type specification and differentiation. At limb levels, pool-specific transcriptional signatures first restrict innervation to only one particular muscle in the periphery, and get refined, once muscle connection has been established. Accordingly, to study the transcriptional dynamics and specificity of the system, a method for establishing muscle target-specific motor neuron transcriptomes would be required. RESULTS: To investigate target-specific transcriptional signatures of single motor neurons, here we combine ex-ovo retrograde axonal labeling in mid-gestation chicken embryos with manual isolation of individual fluorescent cells and Smart-seq2 single-cell RNA-sequencing. We validate our method by injecting the dorsal extensor metacarpi radialis and ventral flexor digiti quarti wing muscles and harvesting a total of 50 fluorescently labeled cells, in which we detect up to 12,000 transcribed genes. Additionally, we present visual cues and cDNA metrics predictive of sequencing success. CONCLUSIONS: Our method provides a unique approach to study muscle target-specific motor neuron transcriptomes at a single-cell resolution. We anticipate that our method will provide key insights into the transcriptional logic underlying motor neuron pool specialization and proper neuromuscular circuit assembly and refinement.

Assessing evolutionary and developmental transcriptome dynamics in homologous cell types.

BACKGROUND: During development, complex organ patterns emerge through the precise temporal and spatial specification of different cell types. On an evolutionary timescale, these patterns can change, resulting in morphological diversification. It is generally believed that homologous anatomical structures are built-largely-by homologous cell types. However, whether a common evolutionary origin of such cell types is always reflected in the conservation of their intrinsic transcriptional specification programs is less clear. RESULTS: Here, we developed a user-friendly bioinformatics workflow to detect gene co-expression modules and test for their conservation across developmental stages and species boundaries. Using a paradigm of morphological diversification, the tetrapod limb, and single-cell RNA-sequencing data from two distantly related species, chicken and mouse, we assessed the transcriptional dynamics of homologous cell types during embryonic patterning. With mouse limb data as reference, we identified 19 gene co-expression modules with varying tissue or cell type-restricted activities. Testing for co-expression conservation revealed modules with high evolutionary turnover, while others seemed maintained-to different degrees, in module make-up, density or connectivity-over developmental and evolutionary timescales. CONCLUSIONS: We present an approach to identify evolutionary and developmental dynamics in gene co-expression modules during patterning-relevant stages of homologous cell type specification using single-cell RNA-sequencing data.

Extracellular matrix gene expression signatures as cell type and cell state identifiers.

Transcriptomic signatures based on cellular mRNA expression profiles can be used to categorize cell types and states. Yet whether different functional groups of genes perform better or worse in this process remains largely unexplored. Here we test the core matrisome - that is, all genes coding for structural proteins of the extracellular matrix - for its ability to delineate distinct cell types in embryonic single-cell RNA-sequencing (scRNA-seq) data. We show that even though expressed core matrisome genes correspond to less than 2% of an entire cellular transcriptome, their RNA expression levels suffice to recapitulate essential aspects of cell type-specific clustering. Notably, using scRNA-seq data from the embryonic limb, we demonstrate that core matrisome gene expression outperforms random gene subsets of similar sizes and can match and exceed the predictive power of transcription factors. While transcription factor signatures generally perform better in predicting cell types at early stages of chicken and mouse limb development, i.e., when cells are less differentiated, the information content of the core matrisome signature increases in more differentiated cells. Moreover, using cross-species analyses, we show that these cell type-specific signatures are evolutionarily conserved. Our findings suggest that each cell type produces its own unique extracellular matrix, or matreotype, which becomes progressively more refined and cell type-specific as embryonic tissues mature.

Development of the chick wing and leg neuromuscular systems and their plasticity in response to changes in digit numbers.

The tetrapod limb has long served as a paradigm to study vertebrate pattern formation. During limb morphogenesis, a number of distinct tissue types are patterned and subsequently must be integrated to form coherent functional units. For example, the musculoskeletal apparatus of the limb requires the coordinated development of the skeletal elements, connective tissues, muscles and nerves. Here, using light-sheet microscopy and 3D-reconstructions, we concomitantly follow the developmental emergence of nerve and muscle patterns in chicken wings and legs, two appendages with highly specialized locomotor outputs. Despite a comparable flexor/extensor-arrangement of their embryonic muscles, wings and legs show a rotated innervation pattern for their three main motor nerve branches. To test the functional implications of these distinct neuromuscular topologies, we challenge their ability to adapt and connect to an experimentally altered skeletal pattern in the distal limb, the autopod. Our results show that, unlike autopod muscle groups, motor nerves are unable to fully adjust to a changed peripheral organisation, potentially constrained by their original projection routes. As the autopod has undergone substantial morphological diversifications over the course of tetrapod evolution, our results have implications for the coordinated modification of the distal limb musculoskeletal apparatus, as well as for our understanding of the varying degrees of motor functionality associated with human hand and foot malformations.

A sense of place, many times over - pattern formation and evolution of repetitive morphological structures.

Fifty years ago, Lewis Wolpert introduced the concept of "positional information" to explain how patterns form in a multicellular embryonic field. Using morphogen gradients, whose continuous distributions of positional values are discretized via thresholds into distinct cellular states, he provided, at the theoretical level, an elegant solution to the "French Flag problem." In the intervening years, many experimental studies have lent support to Wolpert's ideas. However, the embryonic patterning of highly repetitive morphological structures, as often occurring in nature, can reveal limitations in the strict implementation of his initial theory, given the number of distinct threshold values that would have to be specified. Here, we review how positional information is complemented to circumvent these inadequacies, to accommodate tissue growth and pattern periodicity. In particular, we focus on functional anatomical assemblies composed of such structures, like the vertebrate spine or tetrapod digits, where the resulting segmented architecture is intrinsically linked to periodic pattern formation and unidirectional growth. These systems integrate positional information and growth with additional patterning cues that, we suggest, increase robustness and evolvability. We discuss different experimental and theoretical models to study such patterning systems, and how the underlying processes are modulated over evolutionary timescales to enable morphological diversification.

The constrained architecture of mammalian Hox gene clusters.

In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters toward a tighter structure during the evolution of the vertebrate lineage. Here, we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster, such as to invert one or several transcription units, with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis.

A single-cell transcriptomic atlas of the developing chicken limb.

BACKGROUND: Through precise implementation of distinct cell type specification programs, differentially regulated in both space and time, complex patterns emerge during organogenesis. Thanks to its easy experimental accessibility, the developing chicken limb has long served as a paradigm to study vertebrate pattern formation. Through decades' worth of research, we now have a firm grasp on the molecular mechanisms driving limb formation at the tissue-level. However, to elucidate the dynamic interplay between transcriptional cell type specification programs and pattern formation at its relevant cellular scale, we lack appropriately resolved molecular data at the genome-wide level. Here, making use of droplet-based single-cell RNA-sequencing, we catalogue the developmental emergence of distinct tissue types and their transcriptome dynamics in the distal chicken limb, the so-called autopod, at cellular resolution. RESULTS: Using single-cell RNA-sequencing technology, we sequenced a total of 17,628 cells coming from three key developmental stages of chicken autopod patterning. Overall, we identified 23 cell populations with distinct transcriptional profiles. Amongst them were small, albeit essential populations like the apical ectodermal ridge, demonstrating the ability to detect even rare cell types. Moreover, we uncovered the existence of molecularly distinct sub-populations within previously defined compartments of the developing limb, some of which have important signaling functions during autopod pattern formation. Finally, we inferred gene co-expression modules that coincide with distinct tissue types across developmental time, and used them to track patterning-relevant cell populations of the forming digits. CONCLUSIONS: We provide a comprehensive functional genomics resource to study the molecular effectors of chicken limb patterning at cellular resolution. Our single-cell transcriptomic atlas captures all major cell populations of the developing autopod, and highlights the transcriptional complexity in many of its components. Finally, integrating our data-set with other single-cell transcriptomics resources will enable researchers to assess molecular similarities in orthologous cell types across the major tetrapod clades, and provide an extensive candidate gene list to functionally test cell-type-specific drivers of limb morphological diversification.

Convergent origination of a Drosophila-like dosage compensation mechanism in a reptile lineage.

Sex chromosomes differentiated from different ancestral autosomes in various vertebrate lineages. Here, we trace the functional evolution of the XY Chromosomes of the green anole lizard (Anolis carolinensis), on the basis of extensive high-throughput genome, transcriptome and histone modification sequencing data and revisit dosage compensation evolution in representative mammals and birds with substantial new expression data. Our analyses show that Anolis sex chromosomes represent an ancient XY system that originated at least ≈160 million years ago in the ancestor of Iguania lizards, shortly after the separation from the snake lineage. The age of this system approximately coincides with the ages of the avian and two mammalian sex chromosomes systems. To compensate for the almost complete Y Chromosome degeneration, X-linked genes have become twofold up-regulated, restoring ancestral expression levels. The highly efficient dosage compensation mechanism of Anolis represents the only vertebrate case identified so far to fully support Ohno's original dosage compensation hypothesis. Further analyses reveal that X up-regulation occurs only in males and is mediated by a male-specific chromatin machinery that leads to global hyperacetylation of histone H4 at lysine 16 specifically on the X Chromosome. The green anole dosage compensation mechanism is highly reminiscent of that of the fruit fly, Drosophila melanogaster Altogether, our work unveils the convergent emergence of a Drosophila-like dosage compensation mechanism in an ancient reptilian sex chromosome system and highlights that the evolutionary pressures imposed by sex chromosome dosage reductions in different amniotes were resolved in fundamentally different ways.

On the Formation of Digits and Joints during Limb Development.

Critical steps in forming the vertebrate limb include the positioning of digits and the positioning of joints within each digit. Recent studies have proposed that the iterative series of digits is established by a Turing-like mechanism generating stripes of chondrogenic domains. However, re-examination of available data suggest that digits are actually patterned as evenly spaced spots, not stripes, which then elongate into rod-shaped digit rays by incorporating new cells at their tips. Moreover, extension of the digit rays and the patterning of the joints occur simultaneously at the distal tip, implying that an integrated model is required to fully understand these processes.

Deep homology in the age of next-generation sequencing.

The principle of homology is central to conceptualizing the comparative aspects of morphological evolution. The distinctions between homologous or non-homologous structures have become blurred, however, as modern evolutionary developmental biology (evo-devo) has shown that novel features often result from modification of pre-existing developmental modules, rather than arising completely de novo. With this realization in mind, the term 'deep homology' was coined, in recognition of the remarkably conserved gene expression during the development of certain animal structures that would not be considered homologous by previous strict definitions. At its core, it can help to formulate an understanding of deeper layers of ontogenetic conservation for anatomical features that lack any clear phylogenetic continuity. Here, we review deep homology and related concepts in the context of a gene expression-based homology discussion. We then focus on how these conceptual frameworks have profited from the recent rise of high-throughput next-generation sequencing. These techniques have greatly expanded the range of organisms amenable to such studies. Moreover, they helped to elevate the traditional gene-by-gene comparison to a transcriptome-wide level. We will end with an outlook on the next challenges in the field and how technological advances might provide exciting new strategies to tackle these questions.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer.

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.

A relative shift in cloacal location repositions external genitalia in amniote evolution.

The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.

The genetics of murine Hox loci: TAMERE, STRING, and PANTHERE to engineer chromosome variants.

Following their duplications at the base of the vertebrate clade, Hox gene clusters underwent remarkable sub- and neo-functionalization events. Many of these evolutionary innovations can be associated with changes in the transcriptional regulation of their genes, where an intricate relationship between the structure of the gene cluster and the architecture of the surrounding genomic landscape is at play. Here, we report on a portfolio of in vivo genome engineering strategies in mice, which have been used to probe and decipher the genetic and molecular underpinnings of the complex regulatory mechanisms implemented at these loci.

Development: facial makeup enhancing our looks.

A recent study in mice deciphers the complex genetic regulatory network underlying the morphogenesis of the face. The enhancer landscape underlying craniofacial development provides multiple entry points to understand what makes up the face, in natural variation or pathological conditions.

A genetic approach to the recruitment of PRC2 at the HoxD locus.

Polycomb group (PcG) proteins are essential for the repression of key factors during early development. In Drosophila, the polycomb repressive complexes (PRC) associate with defined polycomb response DNA elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. We have used an in vivo approach to identify DNA sequences of importance for the proper recruitment of polycomb proteins at the HoxD locus. We report that various genomic re-arrangements of the gene cluster do not strongly affect PRC2 recruitment and that relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. We discuss the importance of PRC2 recruitment over Hox gene clusters in embryonic stem cells, for their subsequent coordinated transcriptional activation during development.

Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations.

When positioned into the integrin α-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting.