An ancient role for Gata-1/2/3 and Scl transcription factor homologs in the development of immunocytes

AbstractAlthough vertebrate hematopoiesis is the focus of intense study, immunocyte development is well-characterized in only a few invertebrate groups. The sea urchin embryo provides a morphologically simple model for immune cell development in an organism that is phylogenetically allied to vertebrates. Larval immunocytes, including pigment cells and several blastocoelar cell subtypes, emerge from a population of non-skeletal mesodermal (NSM) precursors that is specified at the blastula stage. This ring of cells is first partitioned into oral and aboral fields with distinct blastocoelar and pigment cell gene regulatory programs. The oral field is subsequently specified into several distinct immune and non-immune cell types during gastrulation. Here we characterize the oral NSM expression and downstream function of two homologs of key vertebrate hematopoietic transcription factors: SpGatac, an ortholog of vertebrate Gata-1/2/3 and SpScl, an ortholog of Scl/Tal-2/Lyl-1. Perturbation of SpGatac affects blastocoelar cell migration at gastrulation and later expression of immune effector genes, whereas interference with SpScl function disrupts segregation of pigment and blastocoelar cell precursors. Homologs of several transcription regulators that interact with Gata-1/2/3 and Scl factors in vertebrate hematopoiesis are also co-expressed in the oral NSM, including SpE-protein, the sea urchin homolog of vertebrate E2A/HEB/E2-2 and SpLmo2, an ortholog of a dedicated cofactor of the Scl–GATA transcription complex. Regulatory analysis of SpGatac indicates that oral NSM identity is directly suppressed in presumptive pigment cells by the transcription factor SpGcm. These findings provide part of a comparative basis to understand the evolutionary origins and regulatory biology of deuterostome immune cell differentiation in the context of a tractable gene regulatory network model

Major changes in the Ni-Sp185/333 protein repertoire may not occur until after the second challenge with <i>V</i>. <i>diazotrophicus</i>.

<p>(A) Sea urchin 106 has a limited array of Ni-Sp185/333 proteins prior to immune challenge, with a few high MW proteins of basic pI (arrow). (B) After the first challenge with <i>V</i>. <i>diazotrophicus</i>, the Ni-Sp185/333 proteins appear as more acidic (arrows) plus proteins of ~60 kDa appear (arrow head). (C) After the second challenge with <i>V</i>. <i>diazotrophicus</i>, a significant change in the repertoire of Ni-Sp185/333 proteins shows a shift to more basic pI for both the large MW proteins (arrows), and the ~60 kDa proteins (arrowhead). These images were cropped on the bottom because they do not show spots of less than 50 kDa.</p

Immune challenge increases the intensity of some Ni-Sp185/333 proteins and decreases the intensity of others.

<p>(A) Prior to immune challenge, sea urchin 105 shows a range of Ni-Sp185/333 proteins ranging from ~35 kDa (likely monomers, black arrow) to ~150 to 250 kDa that are all within the pI range of ~7 to 10 (black boxes). (B) After challenge with <i>V</i>. <i>diazotrophicus</i>, larger MW proteins decrease in intensity (compare white arrows in A and B), whereas the monomers increase in intensity (compare black arrows in A and B).</p

Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

<div><p>Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The <i>Sp185/333</i> gene family, which is expressed by the California purple sea urchin <i>Strongylocentrotus purpuratus</i> in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity.</p></div

Ni-Sp185/333 proteins of a single MW by 1DE/Western blots resolve to multiple spots and trains in the basic pI range by 2DE/Western blots.

<p>The sizes of the Ni-Sp185/333 proteins from sea urchin 101 and that were isolated under optimized protocols (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138892#pone.0138892.s005" target="_blank">S1 Protocol</a>) and analyzed by 2DE/Western blot, range in MW from ~40 kDa to over 200 kDa. Most MW sizes are composed of multiple pI variants of which most have a basic pI. (A) A complex of variants with different pI are present within the pI range of 3 to 10, and appear as multiple spots and trains of proteins of ~60 to 80 kDa (white arrows) and >150 kDa (black arrows). (B) The same sample is evaluated in basic pI range of 7 to 10, which is the location on 2DE gels to which most of the Sp185/333 charge variants migrate. Protein trains of similar size and pI as that in A are identified by corresponding arrows.</p

<i>Sp185/333</i> mRNA editing decreases in response to immune challenge.

<p><i>Sp185/333</i> cDNA sequences reported by Terwilliger et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138892#pone.0138892.ref007" target="_blank">7</a>] were evaluated for the change in the number sequences encoding truncated vs. full-length Sp185/333 proteins post-challenge compared to pre-challenge with LPS, β,1–3,glucan (glucan), double stranded RNA (dsRNA), or artificial coelomic fluid (aCF, buffer, sham injection control). Bars that appear to be missing indicate 0 change. Nine sea urchins (indicated by number) were used in the analysis and animal 2 was evaluated for multiple types of challenges.</p

A wide range of changes in the MW/pI of Ni-Sp185/333 proteins can occur over time and in response to multiple challenges with different types of microbes.

<p>(A) Prior to immune challenge, sea urchin 107 shows two major trains of Ni-Sp185/333 proteins of ~80 and ~150 kDa (arrows). (B) After the first challenge with <i>V</i>. <i>diazotrophicus</i>, the majority of the proteins in the trains shift to more basic (arrows), with an expansion of acidic proteins within the same trains (black arrowheads). (C) Prior to challenge with <i>Bacillus sp</i>, sea urchin 107 shows a wide repertoire of Sp185/333 proteins, particularly those of ~60 to 80 kDa and pI of ~7 to 8 (white box). (D) After challenge with <i>Bacillus sp</i>, there is a slight decrease in the intensity of the Ni-Sp185/333 proteins, particularly those of ~60 to 80 kDa/pI ~6 to 8 (white box). (E) The second challenge with <i>Bacillus sp</i> results in a decrease in the intensity of the repertoire of Sp185/333 proteins. (F) The third challenge with <i>Bacillus sp</i> further decreases the repertoire and intensity of the Ni-Sp185/333 proteins. (G) Two weeks after challenge with <i>Bacillus sp</i> the Ni-Sp185/333 protein repertoire shows a further decrease in the number of spots and their intensity. (H) Subsequent challenge with <i>V</i>. <i>diazotrophicus</i> does not induce an increase in the Ni-Sp185/333 protein repertoire, but shows further decreases in diversity. Images (A, B, E-H) were cropped at the bottom because they do not show spots of less than 40 kDa.</p

Challenge with <i>Bacillus sp</i> does not induce increased expression of Ni-Sp185/333 proteins.

<p>(A) Sea urchin 108 shows a repertoire of Ni-Sp185/333 proteins that range in size from ~45 to 200 kDa prior to challenge. (B) After challenge with <i>Bacillus sp</i> there is a decrease in the array and intensity of the Ni-Sp185/333 proteins, with only three short trains (arrow heads) matching in size and pI to those observed prior to challenge.</p

Bacterial isolates¹ from a dissected sea urchin that were available for immune challenges.

<p>¹Only colonies that grew on Marine Broth plates at room temperature were evaluated.</p><p>²Searches were done with Geneious R6 ver. 6.1.8.</p><p>Bacterial isolates<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138892#t001fn001" target="_blank">¹</a> from a dissected sea urchin that were available for immune challenges.</p

Some sea urchins do not express many Ni-Sp185/333 proteins.

<p>A small repertoire of Ni-Sp185/333 proteins is expressed by sea urchin 102 before (A) and after (B) challenge with <i>V</i>. <i>diazotrophicus</i>. White arrows indicate short trains of proteins with the same MW that are present both before and after challenge. A limited array of more basic proteins appears after challenge (Box in B). These images were cropped on the bottom because they do not show spots of less than 40 kDa.</p