Correlation between Hox code and vertebral morphology in archosaurs

The relationship between developmental genes and phenotypic variation is of central interest in evolutionary biology. An excellent example is the role of Hox genes in the anteroposterior regionalization of the vertebral column in vertebrates. Archosaurs (crocodiles, dinosaurs including birds) are highly variable both in vertebral morphology and number. Nevertheless, functionally equivalent Hox genes are active in the axial skeleton during embryonic development, indicating that the morphological variation across taxa is likely owing to modifications in the pattern of Hox gene expression. By using geometric morphometrics, we demonstrate a correlation between vertebral Hox code and quantifiable vertebral morphology in modern archosaurs, in which the boundaries between morphological subgroups of vertebrae can be linked to anterior Hox gene expression boundaries. Our findings reveal homologous units of cervical vertebrae in modern archosaurs, each with their specific Hox gene pattern, enabling us to trace these homologies in the extinct sauropodomorph dinosaurs, a group with highly variable vertebral counts. Based on the quantifiable vertebral morphology, this allows us to infer the underlying genetic mechanisms in vertebral evolution in fossils, which represents not only an important case study, but will lead to a better understanding of the origin of morphological disparity in recent archosaur vertebral columns.     

A regulatory 'landscape effect' over the HoxD cluster

Faithful expression of Hox genes in both time and space is essential for proper patterning of the primary body axis. Transgenic approaches in vertebrates have suggested that this collinear activation process is regulated in a largely gene cluster-autonomous manner. In contrast, more recently co-opted expression specificities, required in other embryonic structures, depend upon long-range enhancer sequences acting from outside the gene clusters. This regulatory dichotomy was recently questioned, since gene activation along the trunk seems to be partially regulated by signals located outside of the cluster. We investigated these alternative regulatory strategies by engineering a large inversion that precisely separates the murine HoxD complex from its centromeric neighborhood. Mutant animals displayed posterior transformations along with subtle deregulations of Hoxd genes, indicating an impact of the centromeric landscape on the fine-tuning of Hoxd gene expression. Proximal limbs were also affected, suggesting that this ‘landscape effect’ is generic and impacts upon regulatory mechanisms of various qualities and evolutionary origins.     

The ten Hox genes of the millipede Glomeris marginata

We have isolated the ten Hox genes from the pill millipede Glomeris marginata (Myriapoda:Diplopoda). All ten genes are expressed in characteristic Hox-gene-like expression patterns. The register of Hox gene expression borders is conserved and the expression profiles show that the anterior-most limb-bearing segment in arthropods (antennal/cheliceral segment) does not express any Hox gene, while the next segment (intercalary/second-antennal/premandibular/pedipalpal segment) does express Hox genes. The Hox expression patterns in this millipede thus support the conclusion that all arthropods possess a deuterocerebral segment. We find that there is an apparent posterior shift of Hox gene expression domains dorsally relative to their ventral patterns, indicating that the decoupling of dorsal and ventral segmentation is not restricted to the level of segment polarity genes but apparently includes the Hox genes. Although the mechanism for the decoupling of dorsal and ventral segmentation remains unsolved, the decoupling must be at a level higher in the hierarchy than that of the segment polarity and Hox genes. The expression patterns of Ultrabithorax and abdominal-A suggest a correlation between the function of these genes and the delayed outgrowth of posterior trunk appendages. This delay may be caused by an assumed repressor function of Ultrabithorax, which might partially repress the activation of the Distal-less gene. The Glomeris fushi tarazu gene is expressed in a Hox-like domain and in the developing central nervous system, but not in segmental stripes such as has been reported in another myriapod species, the centipede Lithobius. In contrast to the Lithobius fushi tarazu gene, there is no indication for a role in segment formation for the millipede fushi tarazu gene, suggesting that fushi tarazu first acquired its segmentation function in the lineage of the insects.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Hoxc Gene Collinear Expression and Epigenetic Modifications Established during Embryogenesis Are Maintained until after Birth

The Hox genes, which are organized into clusters on different chromosomes, are key regulators of embryonic anterior-posterior (A-P) body pattern formation and are expressed at specific times and in specific positions in developing vertebrate embryos. Previously, we have shown that histone methylation patterns are closely correlated with collinear Hox gene expression patterns along the A-P axis of E14.5 mouse embryos. Since histone modification is thought to play a crucial mechanistic role in the highly coordinated pattern of collinear Hox gene expression, we examined the maintenance of the spatial collinear expression pattern of Hoxc genes and the corresponding histone modifications during embryogenesis and in early postnatal mice. Hox expression patterns and histone modifications were analyzed by semi-quantitative RT-PCR and chromatin immunoprecipitation (ChIP)-PCR analyses, respectively. The spatiotemporal expression patterns of Hoxc genes in a cluster were maintained until the early postnatal stage (from E8.5 through P5). Examination of histone modifications in E14.5 and P5 tissues revealed that level of H3K27me3 is only a weak correlation with collinear Hoxc gene expression in the trunk regions although diminished in general, however the enrichment of H3K4me3 is strongly correlated with the gene expression in both stages. In summary, the initial spatiotemporal collinear expression pattern of Hoxc genes and epigenetic modifications are maintained after birth, likely contributing to the establishment of the gene expression code for position in the anatomic body axis throughout the entire life of the organism.     

Hox gene expression patterns in Lethenteron japonicum embryos--insights into the evolution of the vertebrate Hox code

The Hox code of jawed vertebrates is characterized by the colinear and rostrocaudally nested expression of Hox genes in pharyngeal arches, hindbrain, somites, and limb/fin buds. To gain insights into the evolutionary path leading to the gnathostome Hox code, we have systematically analyzed the expression pattern of the Hox gene complement in an agnathan species, Lethenteron japonicum (Lj). We have isolated 15 LjHox genes and assigned them to paralogue groups (PG) 1-11, based on their deduced amino acid sequences. LjHox expression during development displayed gnathostome-like spatial patterns with respect to the PG numbers. Specifically, lamprey PG1-3 showed homologous expression patterns in the rostral hindbrain and pharyngeal arches to their gnathostome counterparts. Moreover, PG9-11 genes were expressed specifically in the tailbud, implying its posteriorizing activity as those in gnathostomes. We conclude that these gnathostome-like colinear spatial patterns of LjHox gene expression can be regarded as one of the features already established in the common ancestor of living vertebrates. In contrast, we did not find evidence for temporal colinearity in the onset of LjHox expression. The genomic and developmental characteristics of Hox genes from different chordate species are also compared, focusing on evolution of the complex body plan of vertebrates.     

PI3K/Akt pathway regulates retinoic acid‐induced Hox gene expression in F9 cells

Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA‐induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA‐induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA‐induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time‐course gene expression profiles for all 39 Hox genes located in four different clusters—Hoxa, Hoxb, Hoxc, and Hoxd—were analyzed. Collinear expression of Hoxa and ‐b cluster genes was initiated earlier than that of the ‐c and ‐d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA‐induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.     

Evolutionary Constraints Favor a Biophysical Model Explaining Hox Gene Collinearity

The Hox gene collinearity enigma has often been approached using models based on biomolecular mechanisms. The biophysical model is an alternative approach based on the hypothesis that collinearity is caused by physical forces pulling the Hox genes from a territory where they are inactive to a distinct spatial domain where they are activated in a step by step manner. Such Hox gene translocations have recently been observed in support of the biophysical model. Genetic engineering experiments, performed on embryonic mice, gave rise to several unexpected mutant expressions that the biomolecular models cannot predict. On the contrary, the biophysical model offers convincing explanation. Evolutionary constraints consolidate the Hox clusters and as a result, denser and well organized clusters may create more efficient physical forces and a more emphatic manifestation of gene collinearity. This is demonstrated by stochastic modeling with white noise perturbing the expression of Hox genes. As study cases the genomes of mouse and amphioxus are used. The results support the working hypothesis that vertebrates have adopted their comparably more compact Hox clustering as a tool needed to develop more complex body structures. Several experiments are proposed in order to test further the physical forces hypothesis.     

Molecular mechanisms of segmental patterning in the vertebrate hindbrain and neural crest

Recent work has shown that segmentation underlies the patterning of the vertebrate hindbrain and its neural crest derivatives. Several genes have been identified with segment-restricted expression, and evidence is now emerging regarding their function and regulatory relationships. The expression patterns of Hox genes and the phenotype of null mutants indicate roles in specifying segment identity. A zinc finger gene Krox-20 is a segment-specific regulator of Hox expression, and it seems probable that retinoic acid receptors also regulate Hox genes in the hindbrain. The receptor tyrosine kinase gene Sek may mediate cell-cell interactions that lead to segmentation. These studies provide a starting point for understanding the molecular basis of segmental patterning in the hindbrain.     

Lizards possess the most complete tetrapod Hox gene repertoire despite pervasive structural changes in Hox clusters

Hox genes are a remarkable example of conservation in animal development and their nested expression along the head‐to‐tail axis orchestrates embryonic patterning. Early in vertebrate history, two duplications led to the emergence of four Hox clusters (A‐D) and redundancy within paralog groups has been partially accommodated with gene losses. Here we conduct an inventory of squamate Hox genes using the genomes of 10 lizard and 7 snake species. Although the HoxC1 gene has been hypothesized to be lost in the amniote ancestor, we reveal that it is retained in lizards. In contrast, all snakes lack functional HoxC1 and ‐D12 genes. Varying levels of degradation suggest differences in the process of gene loss between the two genes. The vertebrate HoxC1 gene is prone to gene loss and its functional domains are more variable than those of other Hox1 genes. We describe for the first time the HoxC1 expression patterns in tetrapods. HoxC1 is broadly expressed during development in the diencephalon, the neural tube, dorsal root ganglia, and limb buds in two lizard species. Our study emphasizes the value of revisiting Hox gene repertoires by densely sampling taxonomic groups and its feasibility owing to growing sequence resources in evaluating gene repertoires across taxa.     

Hox genes: Downstream “effectors” of retinoic acid signaling in vertebrate embryogenesis

One of the major regulatory challenges of animal development is to precisely coordinate in space and time the formation, specification, and patterning of cells that underlie elaboration of the basic body plan. How does the vertebrate plan for the nervous and hematopoietic systems, heart, limbs, digestive, and reproductive organs derive from seemingly similar population of cells? These systems are initially established and patterned along the anteroposterior axis (AP) by opposing signaling gradients that lead to the activation of gene regulatory networks involved in axial specification, including the Hox genes. The retinoid signaling pathway is one of the key signaling gradients coupled to the establishment of axial patterning. The nested domains of Hox gene expression, which provide a combinatorial code for axial patterning, arise in part through a differential response to retinoic acid (RA) diffusing from anabolic centers established within the embryo during development. Hence, Hox genes are important direct effectors of retinoid signaling in embryogenesis. This review focuses on describing current knowledge on the complex mechanisms and regulatory processes, which govern the response of Hox genes to RA in several tissue contexts including the nervous system during vertebrate development.     

Multiple Chromosomal Rearrangements Structured the Ancestral Vertebrate Hox-Bearing Protochromosomes

While the proposal that large-scale genome expansions occurred early in vertebrate evolution is widely accepted, the exact mechanisms of the expansion—such as a single or multiple rounds of whole genome duplication, bloc chromosome duplications, large-scale individual gene duplications, or some combination of these—is unclear. Gene families with a single invertebrate member but four vertebrate members, such as the Hox clusters, provided early support for Ohno's hypothesis that two rounds of genome duplication (the 2R-model) occurred in the stem lineage of extant vertebrates. However, despite extensive study, the duplication history of the Hox clusters has remained unclear, calling into question its usefulness in resolving the role of large-scale gene or genome duplications in early vertebrates. Here, we present a phylogenetic analysis of the vertebrate Hox clusters and several linked genes (the Hox “paralogon”) and show that different phylogenies are obtained for Dlx and Col genes than for Hox and ErbB genes. We show that these results are robust to errors in phylogenetic inference and suggest that these competing phylogenies can be resolved if two chromosomal crossover events occurred in the ancestral vertebrate. These results resolve conflicting data on the order of Hox gene duplications and the role of genome duplication in vertebrate evolution and suggest that a period of genome reorganization occurred after genome duplications in early vertebrates.     

Hox and ParaHox genes: A review on molluscs

Hox and ParaHox genes are involved in patterning the anterior‐posterior body axis in metazoans during embryo development. Body plan evolution and diversification are affected by variations in the number and sequence of Hox and ParaHox genes, as well as by their expression patterns. For this reason Hox and ParaHox gene investigation in the phylum Mollusca is of great interest, as this is one of the most important taxa of protostomes, characterized by a high morphological diversity. The comparison of the works reviewed here indicates that species of molluscs, belonging to different classes, share a similar composition of Hox and ParaHox genes. Therefore evidence suggests that the wide morphological diversity of this taxon could be ascribed to differences in Hox gene interactions and expressions and changes in the Hox downstream genes rather than to Hox cluster composition. Moreover the data available on Hox and ParaHox genes in molluscs compared with those of other Lophotrochozoa shed light on the complex and controversial evolutionary histories that these genes have undergone within protostomes. genesis 52:935–945, 2014. © 2014 Wiley Periodicals, Inc.     

Transformations in null mutants of hox genes: Do they represent intercalary regenerates?

In the minds of many, Hox gene null mutant phenotypes have confirmed the direct role that these genes play in specifying the pattern of vertebrate embryos. The genes are envisaged as defining discrete spatial domains and, subsequently, conferring specific segmental identities on cells undergoing differentiation along the antero-posterior axis. However, several aspects of the observed mutant phenotypes are inconsistent with this view. These include: the appearance of other, unexpected transformations along the dorsal axis; the occurrence of mirror-image duplications; and the development of anomalies outside the established domains of normal Hox gene expression. In this paper, Hox gene disruptions are shown to elicit regeneration-like responses in tissues confronted with discontinuities in axial identity. The polarities and orientations of transformed segments which emerge as a consequence of this response obey the rules of distal transformation and intercalary regeneration. In addition, the incidence of periodic anomalies suggests that the initial steps of Hox-mediated patterning occurs in Hensen's node. As gastrulation proceeds, mesoderm cell cycle kinetics impose constraints upon subsequent cellular differentiation. This results in the delayed manifestation of transformations along the antero-posterior axis. Finally, a paradigm is sketched in which temporal, rather than spatial axial determinants direct differentiation. Specific, testable predictions are made about the role of Hox genes in the establishment of segmental identity.     

Coordinated spatial and temporal expression ofHox genes during embryogenesis in the acoelConvolutriloba longifissura

Background  

Hox genes are critical for patterning the bilaterian anterior-posterior axis. The evolution of their clustered genomic arrangement and ancestral function has been debated since their discovery. As acoels appear to represent the sister group to the remaining Bilateria (Nephrozoa), investigatingHox gene expression will provide an insight into the ancestral features of theHox genes in metazoan evolution.