Family By Fate V0.3

During this time, Kent found the helmet becoming more and more possessive of him, so rather than abandoning his battle against chaos, Kent created the half helm. Although Kent's powers were severely limited, he still had the ability of flight, invulnerability and super strength.[29][30] However, Kent donned the helmet one last time in order to find the missing Spectre. Dr. Fate discovered that the Spectre was under the control of Kulak and in a titanic battle with the Spectre, he was defeated and the Helmet of Nabu was lost somewhere in the netherverse. It would not be until the early 1960s that Kent Nelson would somehow recover the helmet of Nabu and become Dr. Fate again. In the early 1950s Kent Nelson had retired his fate persona and became a physician, but some time later he had recovered the Helmet of Nabu under never explained events, and in 1955 he fought Khalis, the mad Egyptian priest who once used his Amulet of Anubis.[5] It was not until the 1963 that Kent would rejoin the Justice Society of America[31]. As their Justice Society comrades aged, Kent and Inza seemed immortal. The magic fate held over them, virtually stopped their aging process. Inza and Kent also received a small portion of Ian Karkull's power which gave them even more vitality.

Dbx1 is a homeodomain transcription factor involved in neuronal fate specification belonging to a widely conserved family among bilaterians. In mammals, Dbx1 was proposed to act as a transcriptional repressor by interacting with the Groucho corepressors to allow the specification of neurons involved in essential biological functions such as locomotion or breathing.

Sequence alignments of Dbx1 proteins from different species allowed us to identify two conserved domains related to the Groucho-dependent Engrailed repressor domain (RD), as well as a newly described domain composed of clusterized acidic residues at the C-terminus (Cter) which is present in tetrapods but also several invertebrates. Using a heterologous luciferase assay, we showed that the two putative repressor domains behave as such in a Groucho-dependent manner, whereas the Cter does not bear any intrinsic transcriptional activity. Consistently with in vitro data, we found that both RDs are involved in cell fate specification using in vivo electroporation experiments in the chick spinal cord. Surprisingly, we show that the Cter domain is required for Dbx1 function in vivo, acting as a modulator of its repressive activity and/or imparting specificity.

Here, we analyzed a multiple alignment of Dbx protein family members found in a representative range of bilaterians. In addition to previously suggested putative repressor domains (RDs), we identified a novel domain enriched in acidic residues at the C-terminus (Cter domain) highly conserved among tetrapods, but also found in several lineages among bilaterians, suggesting it yields an evolutionary conserved crucial function. We implemented in vitro luciferase reporter assays to assess the intrinsic transcriptional activity of Dbx1 domains and further tested their contribution to the in vivo function of the protein using chick in ovo electroporation. These experiments allowed us to demonstrate that the newly identified Cter domain is critical to regulate fate specification properties of Dbx1. We propose that the strong conservation of the Cter domain of Dbx1 among tetrapods reveals its contribution to the regulation of neuronal diversity and nervous system complexification during evolution.

To begin investigating the function of each Dbx protein domain, we analyzed their conservation during evolution by multiple alignment of protein sequences available for the Dbx family in metazoans. Protein sequences unambiguously belonging to the Dbx family were found in several protostomes, indicating that a dbx ancestral gene was already present in the common ancestor of all bilaterians (Fig. 3). Since dbx1 and dbx2 genes can be found in chondrichthyes, actinopterygians and sarcopterygians, compared to a single gene in petromyzontides, tunicates and cephalochordates, the divergence between both genes likely occurred in the gnathostomes lineage (Fig. 3b). Alternatively, if generated by a whole-genome duplication event, those two paralogs might have been present before the cyclostome/gnathostome split and lost secondarily in cyclostomes [42, 43]. In addition, the supplementary whole-genome duplication in teleost resulted in the presence of two Dbx1 paralogs, namely Dbx1a and Dbx1b. For greater clarity, only the latter was used when considering teleosts since it shows a slightly higher identity with mouse Dbx1 [6]. A multiple alignment of Dbx proteins selected in order to have a significant, although not exhaustive, representation of metazoan organisms, is available (see Additional file 1).

Multiple alignment of the functional domains in the Dbx protein family. a Multiple alignment of representative sequences of the Dbx family allowed the identification of a RD1 (blue) in all species with the exception of the tunicate Ciona intestinalis and the crustacean Daphnia pulex. RD2 (red) is found in all Dbx and Dbx1 sequences, but not in Dbx2 proteins. A specific enrichment and clusterization of acidic residues (green) is found at the C-terminus of Dbx1 proteins of tetrapods as well as in Saccoglossus kowalevskii, Patiria miniata, Lingula anatina and Capitella teleta. b Phylogenetic tree of the species used in a. The orange box indicates species containing both Dbx1 and Dbx2 sequences, suggesting that the Dbx duplication occurred in the common ancestor to all gnathostomes. Pink boxes indicate species in which it was possible to find a stretch of 10 amino acids containing at least 80 % of D or E within the C-terminus

Interestingly, the RD1, RD2 and Cter domains found in mouse Dbx1 were differentially conserved among species. The RD1 domain showed a high conservation level in all proteins of the Dbx family, with the noticeable exception of Ciona intestinalis and Daphnia pulex Dbx representatives (Fig. 3a), suggesting a specific loss of this domain in tunicates (the sequence of Ciona savignyi also lacks the RD1 domain) and crustaceans. The RD2 domain was well conserved in all species but, as already observed in mouse (Fig. 1) and Xenopus [5], it was absent in all Dbx2 sequences (Fig. 3a), indicating that a loss of this domain most probably occurred very soon after the divergence between Dbx1 and Dbx2. The evolution of the Cter domain appeared more complex as the alignment did not allow us to simply discriminate between the presence or absence of the domain, but rather gave us indications on the enrichment and clusterization of acidic residues. For better clarity, we decided to subsequently refer to species bearing a Cter as those in which it was possible to identify a stretch of ten amino acids containing at least 80 % Asp or Glu residues. We found Dbx1 proteins from all tetrapods as well as Dbx sequences from Saccoglossus kowalevskii, Patiria miniata, Lingula anatina and Capitella teleta to match such a criteria (Fig. 3). By contrast, Dbx2 sequences did not display any specific enrichment in acidic residues at the C-terminus (Fig. 3a).

We performed a maximum likelihood phylogenetic reconstruction of the Dbx protein family. As indicated in Additional file 2, Dbx and Dbx1 sequences of all invertebrates and vertebrates were grouped together with a strong statistical support. A second clade grouped all the Dbx2 sequences of vertebrates. This result can be easily correlated with the various modifications and losses that have been sustained by Dbx2 proteins (i.e., presence/absence of RD2 and Cter domains).

Taken together with our electroporation experiments using mDbx1 deletion constructs, these results indicate that domain composition of Dbx family proteins is critical for the promotion of v0 fate. Furthermore, we found a striking correlation between the ability of Dbx proteins to induce Evx1/2+ interneurons and the enrichment and clusterization of acidic residues at the C-terminus.

Dbx1 is a HD TF playing crucial roles in dorsoventral patterning of the spinal cord. It promotes v0/Evx1+ and inhibits v1/En1+ interneurons fates [10]. Until now, both functions were thought to be coupled, as Dbx1 and Evx1 gain- and loss-of-function was shown to prevent and activate En1 expression, respectively [10, 45], whereas Prdm12, a gene expressed in the p1 progenitor domain, had opposite effect through cross-repressive interactions with Dbx1 [46]. In addition, the precise contribution of Dbx1 conserved domains to its biological activity remained elusive: Although Muhr et al. [16] showed that Dbx1 RD2 binds Groucho, they did not assess the consequences of such an interaction. Our results make the picture more complex and interesting from an evolutionary point of view. Luciferase assays and in ovo electroporation experiments demonstrated that both RD1 and RD2 are genuine Groucho-dependent repressor domains. RD1 and RD2 are individually dispensable for the inhibition of alternative v1 cell fate in vivo, suggesting that they play redundant roles. By contrast, they appear to be both required, suggesting that they act synergistically, for the induction of v0 fate. These observations also argue that distinct molecular pathways underlie these two activities.

We have identified a novel acidic-rich C-terminal domain within the Dbx1 transcription factor that is conserved among tetrapods as well as several invertebrates. We have shown that this domain is required for Dbx1-induced neuronal fate specification in the developing spinal cord. Our data are consistent with the idea that the acquisition of this domain during evolution is linked to increased neuronal diversity and nervous system complexity.

Additional file 1. Full alignment of Dbx family proteins. Protein sequence alignment of all Dbx sequences used in this study obtained using the MUSCLE 3.6 software and manually improved. The functional and hypothetical domains are located at the following positions: RD1: 159-169, RD2: 538-548, HD: 824-883 and Cter: 1117-1134. 59ce067264

https://www.thesparklediva.com/forum/questions-answers/jazda-samochodem-miejskim-torrent-do-pobrania