A Loss of Function Screen of Identified Genome-Wide Association Study Loci Reveals New Genes Controlling Hematopoiesis

The formation of mature cells by blood stem cells is very well understood at the cellular level and we know many of the key transcription factors that control fate decisions. However, many upstream signalling and downstream effector processes are only partially understood. Genome wide association studies (GWAS) have been particularly useful in providing new directions to dissect these pathways. A GWAS meta-analysis identified 68 genetic loci controlling platelet size and number. Only a quarter of those genes, however, are known regulators of hematopoiesis. To determine function of the remaining genes we performed a medium-throughput genetic screen in zebrafish using antisense morpholino oligonucleotides (MOs) to knock down protein expression, followed by histological analysis of selected genes using a wide panel of different hematopoietic markers. The information generated by the initial knockdown was used to profile phenotypes and to position candidate genes hierarchically in hematopoiesis. Further analysis of brd3a revealed its essential role in differentiation but not maintenance and survival of thrombocytes. Using the from-GWAS-to-function strategy we have not only identified a series of genes that represent novel regulators of thrombopoiesis and hematopoiesis, but this work also represents, to our knowledge, the first example of a functional genetic screening strategy that is a critical step toward obtaining biologically relevant functional data from GWA study for blood cell traits

Emotional behavior in aquatic organisms? Lessons from crayfish and zebrafish

Experimental animal models are a valuable tool to study the neurobiology of emotional behavior and mechanisms underlying human affective disorders. Mounting evidence suggests that various aquatic organisms, including both vertebrate (e.g., zebrafish) and invertebrate (e.g., crayfish) species, may be relevant to study animal emotional response and its deficits. Ideally, model organisms of disease should possess considerable genetic and physiological homology to mammals, display robust behavioral and physiological responses to stress, and should be sensitive to a wide range of drugs known to modulate stress and affective behaviors. Here, we summarize recent findings in the field of zebrafish- and crayfish-based tests of stress, anxiety, aggressiveness and social preference, and discuss further perspectives of using these novel model organisms in translational biological psychiatry. Outlining the remaining questions in this field, we also emphasize the need in further development and a wider use of crayfish and zebrafish models to study the pathogenesis of affective disorders. © 2019 Wiley Periodicals, Inc.MCS is currently supported by National Funds through FCT ‐ Foundation for Science and Technology. AVK is supported by the Russian Science Foundation grant 19‐15‐00053. KAD is supported by the Fellowship of the President of Russia and SPSU Rector Productivity Fellowship for PhD Students. CM is supported by CNPq/Brazil under Edital Universal 2016 (400726/2016‐5). PMA and FB are supported by the strategic plan of MARE ‐ Marine and Environmental Sciences Centre (UID/MAR/04292/2019)

Polyamine Sharing between Tubulin Dimers Favours Microtubule Nucleation and Elongation via Facilitated Diffusion

We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends). This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine) are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics

Distinct Functional Roles of β-Tubulin Isotypes in Microtubule Arrays of Tetrahymena thermophila, a Model Single-Celled Organism

<div><h3>Background</h3><p>The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, <em>Tetrahymena thermophila</em>. <em>Tetrahymena</em> forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of <em>Tetrahymena</em> demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (<em>BTU1</em> and <em>BTU2</em>) encode the canonical β-tubulin, BTU2, and six genes (<em>BLT1-6</em>) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.</p> <h3>Methodology/Principal Findings</h3><p>With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins <em>in vivo</em>, we transformed <em>Tetrahymena</em> with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.</p> <h3>Conclusion/Significance</h3><p>We conclude that <em>Tetrahymena</em> uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.</p> </div

Synchiropus valdiviae

&lt;p&gt;Synchiropus valdiviae (Trunov, 1981)&lt;/p&gt; &lt;p&gt;(Fig. 1)&lt;/p&gt; &lt;p&gt;Presently there are two classifications of dragonets, variously at odds with one another (Fricke 1982, 2002; Nakabo 1982, 1983). Although Nakabo (op. cit.) and Nakabo and Hartel (1999) placed this species in Foetorepus Whitley, we follow Fricke (2002) in placing it in Synchiropus Gill.&lt;/p&gt; &lt;p&gt;Two adult females and three juveniles of S. valdiviae were taken in the beam trawl at station 85 just north of Nightingale Island. Originally described from a single adult male from Walvis Ridge, a second male has been discovered, and both are from the same locality (Trunov, 1981; Fricke, 1985, but not a paratype as stated). Our specimens are identified on the basis of their agreement with the two males in several characters which separate the species from other Atlantic congeners (reviewed by Nakabo and Hartel, 1999): their coloration, bowed lateral line, counts and proportions, which separate the species from other Atlantic congeners.&lt;/p&gt; &lt;p&gt;Counts and measurements (ranges of the two adult females first, followed in parentheses by juveniles): Vertebrae 7 + 13-14 (7 + 14); D I V, 8 (IV, 8 9); A 7 (7); P 22 (21-22); pelv. I, 5 (I, 5); C ii -iii + 10 + ii (ii -iii + 10 + i -ii); gill rakers 0 + 7 (0 + 7-8); branchiostegal rays 6 (6); pseudobranch filaments 12 (6-7). Following proportions as percent SL: body width 16.8-18.3 (16.1-16.7); body depth 15.3-15.8 (14.9-15.0); caudal peduncle depth 5.9-6.5 (5.7-5.9); predorsal length 30.8-32.1 (30.7-32.1); preanal length 53.9-54.4 (52.4-52.6); caudal fin length 28.6- 30.9 (30.0- 31.2); head length 27.5-27.8 (29.6-30.4); head depth 15.1-17.1 (15.1-16.7); eye diameter 11.3-11.9 (13.1-13.3); snout length 6.5-7.4 (6.3-6.7); upper jaw length 9.7-10.1 (10.2-10.4); interorbital width 2.4-2.5 (1.5-2.2); 1st dorsal spine length 15.4-15.6 (15.3-15.4); 2nd dorsal spine length 11.1-11.9 (12.2); 3rd dorsal spine length 10.0- 0.1 (8.9); 4th dorsal spine length 6.8- 7.0 (7.0-7.1); 1st dorsal ray length 18.1-19.0 (20.5-23.2); last dorsal ray length 18.6- 19.8 (17.8-18.0); 1st anal ray length 9.2-9.4 (10.8-12.0); last anal ray length 19.3-20.0 (20.1- 23.0); 1st to 2nd dorsal distance 6.0-6.2 (6.5-7.0); 1st dorsal base 7.1-7.3 (6.5-6.7); 2nd dorsal base 30.3- 30.5 (28.4-31.3); anal base 23.2-24.0 (20.0-21.6); pectoral base depth 8.6-9.1 (7.8-8.2); pectoral fin length 21.2-21.9 (15.8-19.0); pelvic fin length 27.4-27.9 (29.2-30.7).&lt;/p&gt; &lt;p&gt;First dorsal spine not elongate, only very slightly so in juvenile male. Lateral line distinctly bowed on body, sharply descending to midline between third and fifth dorsal rays (Trunov, 1981, shows bow extending to last ray). Anal fin origin on vertical through fourth dorsal ray, not first as stated by Trunov, but illustrated by him under third. Preopercular spine with trifid tip in all. Body reddish with yellow variegations, first dorsal fin membrane dusky except in juvenile male which has a solid black membrane except proximally. Membrane of anal fin with yellowish distal streak. Membrane of second dorsal fin with indistinct yellowish areas.&lt;/p&gt; &lt;p&gt;Material: SAIAB 74966 (2 females, 52.7-54.5 mm SL; male, 61.6 mm SL); sta. 85/BT 44; 37&deg; 20.3'S, 12&deg; 30.0'W; 2 m beam trawl; 188-201 m; 12:59-13:11 hrs; 7 July 2004. SAIAB 74969 (2 females, 106.5-116.8 mm SL); sta. 85/BT 45; 37&deg; 19.2'S, 12&deg; 29.9'W; 2 m beam trawl; 210- 223 m; 15:29-15:50 hrs; 7 July 2004.&lt;/p&gt;Published as part of &lt;i&gt;M. E. Anderson, D. L. Stein &amp; H. W. Detrich, 2005, Additions to the ichthyofauna of the Tristan da Cunha Group, South Atlantic Ocean., pp. 27-33 in Zootaxa 1072&lt;/i&gt; on pages 28-3

Synchiropus phaeton

Synchiropus phaeton: CAS 56691 (1; 98 mm SL); GHANA: 05° 08'N, 01° 00'W; 70 m; 3 June 1969.SAIAB 67400 (1; 117 mm SL); ANGOLA: 06° 51'S, 11° 50'E; 255-267 m; 23 Mar. 2002.Published as part of M. E. Anderson, D. L. Stein & H. W. Detrich, 2005, Additions to the ichthyofauna of the Tristan da Cunha Group, South Atlantic Ocean., pp. 27-33 in Zootaxa 1072 on page 2

Sebastes oculatus

Sebastes oculatus: CAS 29887 (1; 22 mm SL); CHILE: off Valparaiso; 5-9 Feb. 1934.CAS 220224 (1; 181 mm SL); CHILE: 41° 33'S, 73° 01'W; 18-30 m; 16 Sept. 1967.CAS 17644 (9; 122-246 mm SL); CHILE: ELTANIN crew, 21 Sept. 1966.Published as part of M. E. Anderson, D. L. Stein & H. W. Detrich, 2005, Additions to the ichthyofauna of the Tristan da Cunha Group, South Atlantic Ocean., pp. 27-33 in Zootaxa 1072 on page 2

Sebastes sp.

Sebastes sp. (Fig. 2) A single juvenile rockfish was obtained from lobstermen by Mr. James Glass at Edinburgh and donated to us for description. It differs remarkably from the only known species at Tristan, Sebastes capensis (Gmelin), by its rusty red colour without a trace of white blotches on the dorsum typical of the large subgenus Sebastomus Gill, and its lack of supraocular spines. Rocha-Olivares et al. (1999) clarified the systematics of southern hemisphere Sebastes using mitochondrial DNA sequence variation. They concluded that S. capensis was widespread across the South Atlantic with an unsampled clade in the southeastern Pacific where it overlaps the range of S. oculatus. Both these species are clearly members of subgenus Sebastomus. Almost all juvenile specimens of S. capensis from Tristan known to date have been more orangish than our specimen, with the white blotches and with supraocular spines. Mr John Hyde, Scripps Institution of Oceanography, La Jolla, California, has sequenced tissue samples of this specimen for the cytochrome b gene. He reports that out of the 1141 base pairs for the gene, 1138 match with 10 S. capensis from South Africa which he puts well within normal intraspecific variation in Sebastes and less than the difference between S. capensis and S. oculatus. Three unique nucleotide differences were found in the red specimen, suggesting along with the morphological differences, that it may represent an undescribed, cryptic species. However, Mr. Hyde reports that three nucleotide differences from one fish, with the small sample size of S. capensis, make it difficult to consider that our specimen represents a distinct species. Also, the nucleotide differences would not cause a change in the protein encoded by the gene. More specimens for confirmation are sought. Dr. Paul Tyler, Darwin Project, U. K., sent the first author several underwater photographs of Sebastes capensis and Helicolenus mouchezi from Tristan taken during dives in late 2004 at depths of 15-25 m. One (Fig. 3) is of a red individual with poorly developed dorsal blotches and with small spots reminiscent of the northeastern Pacific S. constellatus Jordan and Gilbert. Thus, it appears that S. capensis at Tristan may exist as several morphotypes, possibly including the odd specimen in Fig. 2. Counts: Vertebrae 8 + 17 = 25; D XIII, 14; A III, 6; P 18; C (branched rays) 12; gill rakers 8 + 1 + 17 + 3 pads; lateral line pores 38; pyloric caeca 9; vertical scale rows above lateral line 66; scales between lateral line and D origin 12. Head spines present: nasal, preocular, postocular, tympanic, parietal, supracleithral and cleithral. Head spines absent: supraocular, coronal and nuchal. Proportions in percent SL: head length 37.7; snout length 5.9; orbit diameter 11.7; interorbital width 5.1; lacrimal width 4.6; body depth 33.0; predorsal length 29.2; preanal length 69.0; prepelvic length 38.5; caudal peduncle depth 9.1; pectoral fin length 25.2; pelvic fin length 20.4; length 4th D spine 14.7; length 2nd A spine 18.8; spinous D base 35.2; rayed D base 22.7; A base 13.8. Both jaws and branchiostegal membranes naked. Interorbital concave. Symphyseal knob weak, ventral margin evenly rounded. No dentigerous premaxillary knobs. Body colour uniformly dark rusty red without white blotches, ventral part of abdomen whitish; fin membranes reddishorange except pectoral fin which is more red; irregular, black humeral spot present. Material: SAIAB 74965 (male, 147 mm SL); Tristan da Cunha off Edinburgh; lobster trap; 50-70 m; 3-6 July 2004; J. Glass.Published as part of M. E. Anderson, D. L. Stein & H. W. Detrich, 2005, Additions to the ichthyofauna of the Tristan da Cunha Group, South Atlantic Ocean., pp. 27-33 in Zootaxa 1072 on pages 30-3