Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Loss-of-Function Mutations in ELMO2 Cause Intraosseous Vascular Malformation by Impeding RAC1 Signaling.

Vascular malformations are non-neoplastic expansions of blood vessels that arise due to errors during angiogenesis. They are a heterogeneous group of sporadic or inherited vascular disorders characterized by localized lesions of arteriovenous, capillary, or lymphatic origin. Vascular malformations that occur inside bone tissue are rare. Herein, we report loss-of-function mutations in ELMO2 (which translates extracellular signals into cellular movements) that are causative for autosomal-recessive intraosseous vascular malformation (VMOS) in five different families. Individuals with VMOS suffer from life-threatening progressive expansion of the jaw, craniofacial, and other intramembranous bones caused by malformed blood vessels that lack a mature vascular smooth muscle layer. Analysis of primary fibroblasts from an affected individual showed that absence of ELMO2 correlated with a significant downregulation of binding partner DOCK1, resulting in deficient RAC1-dependent cell migration. Unexpectedly, elmo2-knockout zebrafish appeared phenotypically normal, suggesting that there might be human-specific ELMO2 requirements in bone vasculature homeostasis or genetic compensation by related genes. Comparative phylogenetic analysis indicated that elmo2 originated upon the appearance of intramembranous bones and the jaw in ancestral vertebrates, implying that elmo2 might have been involved in the evolution of these novel traits. The present findings highlight the necessity of ELMO2 for maintaining vascular integrity, specifically in intramembranous bones.

Defective neural crest migration revealed by a Zebrafish model of Alx1-related frontonasal dysplasia.

Frontonasal dysplasia (FND) refers to a class of midline facial malformations caused by abnormal development of the facial primordia. The term encompasses a spectrum of severities but characteristic features include combinations of ocular hypertelorism, malformations of the nose and forehead and clefting of the facial midline. Several recent studies have drawn attention to the importance of Alx homeobox transcription factors during craniofacial development. Most notably, loss of Alx1 has devastating consequences resulting in severe orofacial clefting and extreme microphthalmia. In contrast, mutations of Alx3 or Alx4 cause milder forms of FND. Whilst Alx1, Alx3 and Alx4 are all known to be expressed in the facial mesenchyme of vertebrate embryos, little is known about the function of these proteins during development. Here, we report the establishment of a zebrafish model of Alx-related FND. Morpholino knock-down of zebrafish alx1 expression causes a profound craniofacial phenotype including loss of the facial cartilages and defective ocular development. We demonstrate for the first time that Alx1 plays a crucial role in regulating the migration of cranial neural crest (CNC) cells into the frontonasal primordia. Abnormal neural crest migration is coincident with aberrant expression of foxd3 and sox10, two genes previously suggested to play key roles during neural crest development, including migration, differentiation and the maintenance of progenitor cells. This novel function is specific to Alx1, and likely explains the marked clinical severity of Alx1 mutation within the spectrum of Alx-related FND.

Differentiation stage-specific expression of transcriptional regulators for epithelial mesenchymal transition in dentate granule progenitors.

During the development of the mouse dentate gyrus (DG), granule neuronal progenitors (GNPs) arise from glial fibrillary acidic protein (GFAP)-expressing neural stem cells in the dentate notch. However, the transcriptional regulators that control their stepwise differentiation remain poorly defined. Since neurogenesis involves epithelial-to-mesenchymal transition (EMT)-like processes, we investigated the spatio-temporal expression profiles of the EMT transcription factors Zeb1, Scratch2 (Scrt2) and Nkx6-2 in relation to known GNP markers. Our results show that Zeb1 and Scrt2 exhibit sequential, but partially overlapping expression across embryonic and postnatal stages of GNP differentiation. Zeb1 is highly enriched in gfap-GFP+/Sox2+ neural stem/progenitor pools and subsets of Tbr2+/Prox1+/NeuroD+ intermediate GNPs, whereas Scrt2 predominates in Tbr2+/Prox1+/NeuroD+ GNPs. Strikingly, the neuronal EMT regulator Nkx6-2 shows selective expression in postnatal Tbr2+/Prox1+ GNPs, but it is excluded from embryonic counterparts. This temporally coordinated yet distinct expression of Zeb1, Scrt2 and Nkx6-2 reveals discrete transcriptional programs orchestrating GNP differentiation and neurogenic progression at embryonic versus postnatal stages of DG neurogenesis.

Olig2+/NG2+/BLBP+ astrocyte progenitors: a novel component of the neurovascular unit in the developing mouse hippocampus.

Astrocytes are key components of the neurovascular unit. While we have recently identified Olig2+ astrocyte progenitors (ASPs) in the developing mouse dentate gyrus (DG), their molecular signature remains incompletely characterized. Here we demonstrate that Olig2+ ASPs predominantly express brain lipid-binding protein (BLBP), while only a small population of them expresses gfap-GFP. These Olig2+/BLBP+ ASPs co-express the transcription factors Sox3, Sox9 and the proteoglycan NG2 but not Sox10, a marker for oligodendrocyte progenitors (OLPs). Olig2+ ASPs appear from embryonic day 18 (E18) onwards and decline at postnatal day 14 (P14). Consistent with the proliferation of both Olig2+ and NG2+ glial cells after brain injury, intrauterine intermittent hypoxia (IH) led to an increase in Olig2+/NG2+/BLBP+ ASPs in the postnatal DG. IH also promoted both angiogenesis and vascular coupling of Olig2+/NG2+ ASPs. Our data suggest that IH-induced expression of HIF1a increases Olig2+/NG2+/BLBP+ ASPs in a cell non-autonomous manner. Our data also revealed increased vascular coupling of GFAP+ astrocytes following IH, while the number of GFAP+ astrocytes remains unchanged. Given that BLBP, Olig2 and NG2 are expressed in reactive astrocytes, our findings suggest that Olig2+/NG2+/BLBP+ ASPs represent a subtype of reactive astrocyte progenitors. Furthermore, the enhanced vascular coupling of Olig2+/NG2+/BLBP+ ASPs appears to be an adaptive response to hypoxic brain injury. This study provides new insights into the molecular characteristics of Olig2+/NG2+/BLBP+ ASPs and their potential role in the brain's response to hypoxic injury, contributing to our understanding of neurovascular unit dynamics in both development and pathological conditions.

Evolution of the Alx homeobox gene family: parallel retention and independent loss of the vertebrate Alx3 gene.

The Alx gene family is implicated in craniofacial development and comprises two to four homeobox genes in each vertebrate genome analyzed. Using phylogenetics and comparative genomics, we show that the common ancestor of jawed vertebrates had three Alx genes descendent from the two-round genome duplications (Alx1, Alx3, Alx4), compared with a single amphioxus gene. Later in evolution one of the paralogues, Alx3, was lost independently from at least three different vertebrate lineages, whereas Alx1 and Alx4 were consistently retained. Comparison of spatial gene expression patterns reveals that the three mouse genes have equivalent craniofacial expression to the two chick and frog genes, suggesting that redundancy compensated for gene loss. We suggest that multiple independent loss of one Alx gene was predisposed by extensive and persistent overlap in gene expression between Alx paralogues. Even so, it is unclear whether it was coincidence or evolutionary bias that resulted in the same Alx gene being lost on each occasion, rather than different members of the gene family.

Anatomical variations of the thyroid gland: An experimental cadaveric study.

INTRODUCTION: The thyroid gland displays numerous variations in its anatomy. Understanding the variations that occur can benefit diagnosis of thyroid disorders and improve management. The aim of this study was to investigate how factors such as age and sex may influence variations in the thyroid. METHODS: Twenty cadavers (10 males & 10 females) with a mean age of 78 were dissected. Variations in anatomy and vasculature were examined. Correlation between age and thyroid size was tested for significance using GraphPad prism 7. RESULTS: Most cadavers, 65%, had the superior thyroid artery originating from the external carotid artery, while 25% were from the bifurcation and 10% from the common carotid. The average weight for thyroids was 19.9 g in males and 13.9 g in females. A significant negative correlation was found between age and thyroid size. DISCUSSION: Thyroid gland variations, such as pyramidal lobes which affected 30% of cadavers, could impact medical interventions. Evidence from this study has confirmed the high incidence of such variations emphasising the requirement for preoperative imaging.

An EST screen from the annelid Pomatoceros lamarckii reveals patterns of gene loss and gain in animals.

BACKGROUND: Since the drastic reorganisation of the phylogeny of the animal kingdom into three major clades of bilaterians; Ecdysozoa, Lophotrochozoa and Deuterostomia, it became glaringly obvious that the selection of model systems with extensive molecular resources was heavily biased towards only two of these three clades, namely the Ecdysozoa and Deuterostomia. Increasing efforts have been put towards redressing this imbalance in recent years, and one of the principal phyla in the vanguard of this endeavour is the Annelida. RESULTS: In the context of this effort we here report our characterisation of an Expressed Sequence Tag (EST) screen in the serpulid annelid, Pomatoceros lamarckii. We have sequenced over 5,000 ESTs which consolidate into over 2,000 sequences (clusters and singletons). These sequences are used to build phylogenetic trees to estimate relative branch lengths amongst different taxa and, by comparison to genomic data from other animals, patterns of gene retention and loss are deduced. CONCLUSION: The molecular phylogenetic trees including the P. lamarckii sequences extend early observations that polychaetes tend to have relatively short branches in such trees, and hence are useful taxa with which to reconstruct gene family evolution. Also, with the availability of lophotrochozoan data such as that of P. lamarckii, it is now possible to make much more accurate reconstructions of the gene complement of the ancestor of the bilaterians than was previously possible from comparisons of ecdysozoan and deuterostome genomes to non-bilaterian outgroups. It is clear that the traditional molecular model systems for protostomes (e.g. Drosophila melanogaster and Caenorhabditis elegans), which are restricted to the Ecdysozoa, have undergone extensive gene loss during evolution. These ecdysozoan systems, in terms of gene content, are thus more derived from the bilaterian ancestral condition than lophotrochozoan systems like the polychaetes, and thus cannot be used as good, general representatives of protostome genomes. Currently sequenced insect and nematode genomes are less suitable models for deducing bilaterian ancestral states than lophotrochozoan genomes, despite the array of powerful genetic and mechanistic manipulation techniques in these ecdysozoans. A distinct category of genes that includes those present in non-bilaterians and lophotrochozoans, but which are absent from ecdysozoans and deuterostomes, highlights the need for further lophotrochozoan data to gain a more complete understanding of the gene complement of the bilaterian ancestor.

Gene profiling of head mesoderm in early zebrafish development: insights into the evolution of cranial mesoderm.

BACKGROUND: The evolution of the head was one of the key events that marked the transition from invertebrates to vertebrates. With the emergence of structures such as eyes and jaws, vertebrates evolved an active and predatory life style and radiated into diversity of large-bodied animals. These organs are moved by cranial muscles that derive embryologically from head mesoderm. Compared with other embryonic components of the head, such as placodes and cranial neural crest cells, our understanding of cranial mesoderm is limited and is restricted to few species. RESULTS: Here, we report the expression patterns of key genes in zebrafish head mesoderm at very early developmental stages. Apart from a basic anterior-posterior axis marked by a combination of pitx2 and tbx1 expression, we find that most gene expression patterns are poorly conserved between zebrafish and chick, suggesting fewer developmental constraints imposed than in trunk mesoderm. Interestingly, the gene expression patterns clearly show the early establishment of medial-lateral compartmentalisation in zebrafish head mesoderm, comprising a wide medial zone flanked by two narrower strips. CONCLUSIONS: In zebrafish head mesoderm, there is no clear molecular regionalisation along the anteroposterior axis as previously reported in chick embryos. In contrast, the medial-lateral regionalisation is formed at early developmental stages. These patterns correspond to the distinction between paraxial mesoderm and lateral plate mesoderm in the trunk, suggesting a common groundplan for patterning head and trunk mesoderm. By comparison of these expression patterns to that of amphioxus homologues, we argue for an evolutionary link between zebrafish head mesoderm and amphioxus anteriormost somites.

Positive Feedback Defines the Timing, Magnitude, and Robustness of Angiogenesis.

Angiogenesis is driven by the coordinated collective branching of specialized leading "tip" and trailing "stalk" endothelial cells (ECs). While Notch-regulated negative feedback suppresses excessive tip selection, roles for positive feedback in EC identity decisions remain unexplored. Here, by integrating computational modeling with in vivo experimentation, we reveal that positive feedback critically modulates the magnitude, timing, and robustness of angiogenic responses. In silico modeling predicts that positive-feedback-mediated amplification of VEGF signaling generates an ultrasensitive bistable switch that underpins quick and robust tip-stalk decisions. In agreement, we define a positive-feedback loop exhibiting these properties in vivo, whereby Vegf-induced expression of the atypical tetraspanin, tm4sf18, amplifies Vegf signaling to dictate the speed and robustness of EC selection for angiogenesis. Consequently, tm4sf18 mutant zebrafish select fewer motile ECs and exhibit stunted hypocellular vessels with unstable tip identity that is severely perturbed by even subtle Vegfr attenuation. Hence, positive feedback spatiotemporally shapes the angiogenic switch to ultimately modulate vascular network topology.

The evolutionary origins of vertebrate midbrain and MHB: insights from mouse, amphioxus and ascidian Dmbx homeobox genes.

Comparative studies on developmental gene expression suggest that the ancestral chordate central nervous system comprised anterior, midbrain-hindbrain boundary (MHB) and posterior regions. The most anterior region consists of both forebrain and midbrain in vertebrates. It remains, however, unresolved when or how the vertebrate midbrain was established from this anterior zone. I previously reported a mouse PRD-class homeobox gene, Dmbx1, expressed in the presumptive midbrain at early developmental stages, and in the hindbrain at later stages, with exclusion from the MHB. To investigate the evolution of midbrain development, I have cloned Dmbx genes from amphioxus and from Ciona, representing the two most closely related lineages to the vertebrates, and examined embryonic Dmbx expression in these species. In amphioxus, no Dmbx expression is observed in the neural tube, supporting previous arguments that the MHB equivalent region has been secondarily lost in evolution. In Ciona, the CiDmbx gene is detected in neural cells posterior to Pax-2/5/8-positive cells (MHB homologue), but not in any cells anterior to them. These results support the lack of a midbrain homologue in Ciona, and suggest that midbrain development is a vertebrate innovation. Here, I report the full sequences of these genes and discuss the evolution of midbrain development in relation to the tripartite neural ground plan and the origin of the MHB organizer.

The evolution of homeobox genes: Implications for the study of brain development.

The homeobox gene superfamily includes many genes implicated in brain development in vertebrates; for example, the Otx, Emx, Dmbx, Gbx, En and Hox gene families. We describe the evolutionary history of the homeobox gene superfamily, as inferred from molecular phylogenetics and chromosomal mapping. Studies of amphioxus, a close relative of vertebrates, have proven particularly informative because it has a genome uncomplicated by recent lineage-specific gene duplications and because in situ hybridisation techniques exist for mapping gene positions and gene expression patterns. We describe an ancient subdivision into gene classes (ANTP, PRD, LIM, POU, SIN, TALE), each containing multiple gene families. The original ANTP class gene duplicated to give distinct NK-like and Hox/ParaHox-related genes, both of which underwent tandem duplication, before the expanding Hox gene cluster duplicated to give Hox and ParaHox clusters. A chromosomal breakage event probably occurred to separate the NK-like and extended Hox genes. Finally, there was additional and extensive gene duplication and gene loss in the vertebrate lineage. We argue that understanding evolutionary history is important for establishing consistent gene nomenclature, and for comparing gene expression patterns and gene functions between species and between gene families.

Expression of Notch ligands, Jagged1, 2 and Delta1 in antigen presenting cells in mice.

Notch1 is indispensable for T cell development. It is anticipated that Notch1 and other Notch receptors expressed on the surface of thymic T cell precursors are activated by ligands present on environmental cells, including antigen presenting cells (APCs), and involved in positive and negative selections. Notch receptors on peripheral T cells may also be activated by ligands on APCs. Here, we examined the expression pattern of three Notch ligands, Jagged1, 2 and Delta1 in APCs by an immunofluorescence cell staining method and a reverse transcriptase-polymerase chain reaction (RT-PCR) method. Peritoneal macrophages were strongly positive for Jagged1 staining. In contrast, macrophages separated from spleen and dendritic cells (DCs) separated from spleen and thymus showed positive staining for all the three ligands at a similar intensity. An analysis by RT-PCR revealed that peritoneal and splenic macrophages and splenic and thymic DCs, show a distinct pattern in Notch ligand expression. These findings may represent that expression of various Notch ligands in APCs has a physiological relevance in each organ.

Generation of Fas-independent CD4+ cytotoxic T-cell clone specific for p190 minor bcr-abl fusion peptide.

In the majority of Ph+ALL patients, p190 bcr-abl fusion protein is generated in the Philadelphia chromosome. The fusion protein may serve as a leukemia antigen because it is not expressed in normal cells and hardly in any other malignancy. From a healthy donor, we have established a p190 bcr-abl fusion peptide-specific CD4+ cytotoxic T-cell clone, activation of which depends on HLA-DRB1*1501. This T-cell clone has a strong cytotoxic activity against autologus MoDCs pulsed with e1a2 peptide and its cytotoxicity is not mediated by Fas/Fas ligand or perforin pathway. Success in establishment of the p190 bcr-abl fusion peptide-specific T-cell clone encourages us to develop a new approach to an effective immunotherapy for Ph+ALL.

Evolution and functional divergence of enzymes involved in sesquiterpenoid hormone biosynthesis in crustaceans and insects.

Juvenile hormone (JH) and methyl farnesoate (MF) play well-known roles in the development and reproduction of insects and crustaceans. Juvenile hormone acid O-methyltransferase (JHAMT) and farnesoic acid O-methyltransferase (FAMeT) are the enzymes responsible for catalyzing the biosynthesis of JH and MF, respectively. It is not clear whether the genes that encode these enzymes are present in animal lineages outside of the arthropods. Based on DNA sequence similarity, the literature suggests that an FAMeT ortholog is present in humans. However, vertebrates do not appear to produce JH or MF. To help unravel the evolution of hormonal systems in animals we have carried out the first comparative genomic analysis of JHAMT and FAMeT. We identify the first JHAMT ortholog in a crustacean genome, and FAMeT orthologs in annelid and cephalochordate genomes. Moreover, phylogenetic analyses suggest that there is no true homolog of FAMeT in humans contrary to previous hypotheses. Our analyses suggest that the presence of multiple FAMeT isoforms in arthropods may be a consequence of different evolutionary mechanisms. The genes responsible for hormone biosynthesis in extant insects and crustaceans appear to have been present at least in the Pancrustacea. Different selective forces appear to have subsequently acted on the two lineages, leading to the present functional divergence. Our use of comparative genomics and phylogenetic analysis advance knowledge of the relationships of the hormonal enzyme genes in question, and provide new insights into the evolution of hormonal systems in the largest animal phylum, the Arthropoda.

Comparative genomic and phylogenetic analysis of vitellogenin and other large lipid transfer proteins in metazoans.

Vitellogenins and other large lipid transfer proteins (LLTP) are well known to play significant roles in the development, metabolism and reproduction of animals. Comparative genomics and phylogenetic analyses of LLTPs using the most comprehensive dataset in metazoans to date are carried out. Our analyses demonstrate that LLTP genes arose significantly earlier, and are more widespread than previously proposed - being present in numerous additional bilaterian and non-bilaterian lineages. A hypothesis is advanced that the most ancestral animal LLTP gene is Vtg, while loss of domains occurred at the bilaterians stem giving rise to apolipoprotein and microsomal triglyceride transfer proteins genes.

Amphioxus and ascidian Dmbx homeobox genes give clues to the vertebrate origins of midbrain development.

The ancestral chordate neural tube had a tripartite structure, comprising anterior, midbrain-hindbrain boundary (MHB) and posterior regions. The most anterior region encompasses both forebrain and midbrain in vertebrates. It is not clear when or how the distinction between these two functionally and developmentally distinct regions arose in evolution. Recently, we reported a mouse PRD-class homeobox gene, Dmbx1, expressed in the presumptive midbrain at early developmental stages, and the hindbrain at later stages, with exclusion from the MHB. This gene provides a route to investigate the evolution of midbrain development. We report the cloning, genomic structure, phylogeny and embryonic expression of Dmbx genes from amphioxus and from Ciona, representing the two most closely related lineages to the vertebrates. Our analyses show that Dmbx genes form a distinct, ancient, homeobox gene family, with highly conserved sequence and genomic organisation, albeit more divergent in Ciona. In amphioxus, no Dmbx expression is observed in the neural tube, supporting previous arguments that the MHB equivalent region has been secondarily modified in evolution. In Ciona, the CiDmbx gene is detected in neural cells caudal to Pax2/5/8-positive cells (MHB homologue), in the Hox-positive region, but, interestingly, not in any cells rostral to them. These results suggest that a midbrain homologue is missing in Ciona, and argue that midbrain development is a novelty that evolved specifically on the vertebrate lineage. We discuss the evolution of midbrain development in relation to the ancestry of the tripartite neural ground plan and the origin of the MHB organiser.

Integrity of intracellular domain of Notch ligand is indispensable for cleavage required for release of the Notch2 intracellular domain.

The biological activity of the soluble form of the Notch ligand (sNL) and requirement of the intracellular domain (ICD) of the Notch ligand have been debated. Here we show that soluble Delta1 (sD1) activates Notch2 (N2), but much more weakly than full-length Delta1 (fD1). Furthermore, tracing the N2 molecule after sD1 stimulation revealed that sD1 has a defect in the cleavage releasing ICD of N2 (intracellular cleavage), although it triggers cleavage in the extracellular domain of N2. This represents the molecular basis of the lower activity of sD1 and suggests the presence of an unknown mechanism regulating activation of the intracellular cleavage. The fact that Delta1 lacking its ICD (D1Delta(ICD)) exhibits the phenotype similar to that exhibited by sD1 indicates that the ICD of D1 (D1(ICD)) is involved in such an as yet unknown mechanism. Furthermore, the findings that D1Delta(ICD) acts in a dominant-negative fashion against fD1 and that the signal-transducing activity of sD1 is enhanced by antibody-mediated cross-linking suggest that the multi merization of Delta1 mediated by D1(ICD) may be required for activation of the N2 intracellular cleavage.

An orphan PRD class homeobox gene expressed in mouse brain and limb development.

We report the cDNA sequence and expression of a mouse homeobox gene, Dmbx1, from the PRD class and comparison to its human orthologue. The gene defines a new homeobox gene family, Dmbx, phylogenetically distinct from the Ptx, Alx, Prx Otx, Gsc, Otp and Pax gene families. The Dmbx1 gene is expressed in the developing mouse diencephalon, midbrain and hindbrain, and has dynamic expression during forelimb and hindlimb development. Unusually for homeobox genes, there is no orthologue in the Drosophila or Caenorhabditis genomes; we argue this reflects secondary loss.

Notch1 but not Notch2 is essential for generating hematopoietic stem cells from endothelial cells.

Hematopoietic stem cells (HSCs) are thought to arise in the aorta-gonad-mesonephros (AGM) region of embryo proper, although HSC activity can be detected in yolk sac (YS) and paraaortic splanchnopleura (P-Sp) when transplanted in newborn mice. We examined the role of Notch signaling in embryonic hematopoiesis. The activity of colony-forming cells in the YS from Notch1(-/-) embryos was comparable to that of wild-type embryos. However, in vitro and in vivo definitive hematopoietic activities from YS and P-Sp were severely impaired in Notch1(-/-) embryos. The population representing hemogenic endothelial cells, however, did not decrease. In contrast, Notch2(-/-) embryos showed no hematopoietic deficiency. These data indicate that Notch1, but not Notch2, is essential for generating hematopoietic stem cells from endothelial cells.