Evolution of Highly Conserved Non-coding Sequences in ParaHox Clusters and their Function

　　　Cis-regulatory element is a functional unit controlling gene expression spatially and temporarily. Acquirement, retention and/or loss of cis-regulatory elements during evolution can alter gene expression patterns. In particular, cis-elements of transcription factors expressed in developmental process, as typified by tool-kit genes, would be a key to understand evolutionary novelty that emerged through vertebrate evolution.　　　ParaHox genes are tool-kit genes that are controlling anterior-posterior body axis determination in vertebrates. ParaHox genes play important roles for digestive gut differentiation in both vertebrates and invertebrates. The ParaHox gene clusters consist of three classes of homeobox containing genes, Gsh1/2, Pdx1, and Cdx1/2/4. There are four ParaHox clusters in a mammalian genomes, most likely as a consequence of two round whole genome duplications. Despite their importance in development, cis-regulatory mechanisms of ParaHox gene clusters, particularly Gsh genes that function in segmented anterior brain in vertebrates, have not been studied well. My analysis and previous studies suggested that evolutionarily conserved sequences in ParaHox non-coding regions are strong candidates as tissue specific cis-regulatory elements. In this study, to search for cis-regulatory elements in ParaHox gene clusters, genomic sequence comparison was conducted by using diverse species of vertebrates. Furthermore cis-regulatory candidates were functionally tested by transgenic mice experiments to clarify their enhancer activities. 　　　I compared genomic sequences of the vertebrate Gsh1-Pdx1-Cdx2 cluster. Multiple con served non-coding sequences are identified in ParaHox clusters by this analysis, and they were termed as CNSPs. The 1012bp CNSP#1, the most evolutionarily conserved element within the Gsh1-Pdx1-Cdx2 cluster, showed the highly reproducible reporter gene expression pattern in central nervous system that was similar to endogenous mRNA expression pattern of Gsh1 gene. 　　　 　　　Another highly conserved sequence among vertebrates was found in the paralogous Gsh2 gene cluster, too. One of the conserved sequences termed CNSG2#2/#4was found in similar relative position to the Gsh gene coding region. The 1246bpCNSG2#2/#4 drove lacZ expression largely similar to Gsh2 endogenous expression in anterior brain.　　　Interestingly the expression pattern of CNSG2#2/#4 was more similar to that ofCNSP#1 than its neighboring Gsh2 gene. This prompted me to search for any sequence similarity between CNSP#1 and CNSG2#2/#4, and then I found an approximately 200bpconserved core sequence between these elements. Based on the synteny and the phylogenetic relationship between these clusters, I conclude that CNSP#1 andCNSG2#2/#4 were generated by whole genome duplication event during the ancestral vertebrate era and the core homologous 200bp sequence have been preserved for some functional reason. Paralogous conservation between CNSP#1 and CNSG2#2/#4 strongly suggests that the core conserved 200bp element already existed in the ancestral primitive vertebrate. If we assume that the core sequence is directly related to the cis-regulatory function in segmented anterior brain, we could suppose that the ancestral primitive vertebrate already had a segmented brain. Function of the shared 200bp element betweenCNSP#1 and CNSG2#2/#4 should be tested in the future, and that will not only clarify its biological function in extant vertebrates but also will give a hint to understand the timing of emergence of evolutionary novelty in ancestral vertebrates

Pax4 is not essential for beta-cell differentiation in zebrafish embryos but modulates alpha-cell generation by repressing arx gene expression.

BACKGROUND: Genetic studies in mouse have demonstrated the crucial function of PAX4 in pancreatic cell differentiation. This transcription factor specifies beta- and delta-cell fate at the expense of alpha-cell identity by repressing Arx gene expression and ectopic expression of PAX4 in alpha-cells is sufficient to convert them into beta-cells. Surprisingly, no Pax4 orthologous gene can be found in chicken and Xenopus tropicalis raising the question of the function of pax4 gene in lower vertebrates such as in fish. In the present study, we have analyzed the expression and the function of the orthologous pax4 gene in zebrafish. RESULTS: pax4 gene is transiently expressed in the pancreas of zebrafish embryos and is mostly restricted to endocrine precursors as well as to some differentiating delta- and epsilon-cells but was not detected in differentiating beta-cells. pax4 knock-down in zebrafish embryos caused a significant increase in alpha-cells number while having no apparent effect on beta- and delta-cell differentiation. This rise of alpha-cells is due to an up-regulation of the Arx transcription factor. Conversely, knock-down of arx caused to a complete loss of alpha-cells and a concomitant increase of pax4 expression but had no effect on the number of beta- and delta-cells. In addition to the mutual repression between Arx and Pax4, these two transcription factors negatively regulate the transcription of their own gene. Interestingly, disruption of pax4 RNA splicing or of arx RNA splicing by morpholinos targeting exon-intron junction sites caused a blockage of the altered transcripts in cell nuclei allowing an easy characterization of the arx- and pax4-deficient cells. Such analyses demonstrated that arx knock-down in zebrafish does not lead to a switch of cell fate, as reported in mouse, but rather blocks the cells in their differentiation process towards alpha-cells. CONCLUSIONS: In zebrafish, pax4 is not required for the generation of the first beta- and delta-cells deriving from the dorsal pancreatic bud, unlike its crucial role in the differentiation of these cell types in mouse. On the other hand, the mutual repression between Arx and Pax4 is observed in both mouse and zebrafish. These data suggests that the main original function of Pax4 during vertebrate evolution was to modulate the number of pancreatic alpha-cells and its role in beta-cells differentiation appeared later in vertebrate evolution

Tau isoform expression and phosphorylation in marmoset brains

Tau is a microtubule-associated protein expressed in neuronal axons. Hyperphosphorylated tau is a major component of neurofibrillary tangles, a pathological hallmark of Alzheimer’s disease (AD). Hyperphosphorylated tau aggregates are also found in many neurodegenerative diseases, collectively referred to as “tauopathies,” and tau mutations are associated with familiar frontotemporal lobar degeneration (FTLD). Previous studies have generated transgenic mice with mutant tau as tauopathy models, but non-human primates, which are more similar to humans, may be a better model to study tauopathies. For example, the common marmoset is poised as a nonhuman primate model for investigating the etiology of age-related neurodegenerative diseases. However, no biochemical studies of tau have been conducted in marmoset brains. Here, we investigated several important aspects of tau, including expression of different tau isoforms and its phosphorylation status, in the marmoset brain. We found that marmoset tau does not possess the “primate unique motif” in its N-terminal domain. We also discovered that the tau isoform expression pattern in marmosets is more similar to that of mice than of humans, with adult marmoset brains expressing only four-repeat tau isoforms as in adult mice but unlike in adult human brains. Of note, tau in brains of marmoset newborns was phosphorylated at several sites associated with AD pathology. However, in adult marmoset brains, much of this phosphorylation was lost, except for Ser-202 and Ser-404 phosphorylation. These results reveal key features of tau expression and phosphorylation in the marmoset brain, a potentially useful non-human primate model of neurodegenerative diseases

Copy number variants in patients with intellectual disability affect the regulation of ARX transcription factor gene

Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease