Hehmeyer, Jenks 2020 Biology Thesis
Title: Evolution of a transcriptional regulator of cup cell differentiation in Dictyostelium discoideum: Advisor: Robert Savage Advisor is Co-author/Adviser Restricted Data Used: None of the above Second Advisor: Release: release now Authenticated User Access (does not apply to released theses): Contains Copyrighted Material: No
EVOLUTION OF A TRANSCRIPTIONAL REGULATOR OF CUP CELL DIFFERENTIATION IN DICTYOSTELIUM DISCOIDEUM
by
JENKS HEHMEYER
Robert Savage, Advisor
A thesis submitted in partial fulfillment of the requirements for the Degree of Bachelor of Arts with Honors in Biology
WILLIAMS COLLEGE
Williamstown, Massachusetts
May 31, 2020 2
Abstract
The evolution of novel cell types contributed extensively to the emergence of new organismal functions in animals, plants, and other multicellular lineages. However, very little is currently understood about cell type evolution. A major challenge is understanding how transcription factor evolution contributes to the origination of new cell types. I characterized the developmental roles of two paralogous transcription factor genes, fasA and fasB, in the slime mold Dictyostelium discoideum, a Eukaryote that forms simple multicellular fruiting bodies consisting of only three terminal cell types. fasA is essential for the generation of the cups, structures that play critical somatic roles in Dictyostelium discoideum fruiting bodies.
Specifically, fasA initiates the sorting of precup cells to their ultimate locations, a process that seems to occur upon cup cell differentiation. fasB plays a minor role in the formation of the stalk.
The cup cell is a genus-specific cell type that evolved from the stalk cell, and the segmental duplication that gave rise to fasA and fasB also took place at the base of Dictyostelium. However, sequence and expression pattern analyses demonstrate that the evolution of fasA is unlikely to have contributed to the origination of cup cells.
3
Introduction
Cell differentiation is a critical process in unicellular and multicellular organisms alike.
During differentiation, a cell changes from one stable state to another. These states, or cell types, differ in form and function, a reflection of differences in the underlying patterns of gene expression. Specifically, the enactment of differentiation involves suppression of the one gene expression program and activation of another, through changes in the set of transcription factors
(TFs) interacting with the chromatin. Often, this transition involves multiple signaling and TF cascades, resulting in the passage through transient cell states before the activation of a battery of
TFs that is able to maintain its own expression—the “core regulatory complex” of “terminal selectors” [1-3].
In unicellular organisms, differentiation generally occurs in response to changes in environmental conditions. In multicellular organisms, differentiation may be induced by extracellular molecules, physical cell-cell or cell-surface interactions, or asymmetric cell division. The spatiotemporal patterning of these signals ensures the correct arrangement of cells and tissues. Generally, multicellular organisms have highly proliferative stem cells that are specifically responsible for differentiation; the “terminal cell types” that constitute the majority of a mature organism and carry out its metabolic, structural, and behavioral functions, have very limited capacity for differentiation.
The evolution of certain multicellular lineages has involved great increases in the number of terminal cell types. For example, histological and transcriptomic data indicate that adult mammals possess hundreds of cell types [4, 5]; however, the earliest branching metazoans, the sponges, ctenophores, placozoans, and cnidarians, have far fewer cell types [6-10], indicating that, similarly, the common ancestor of all metazoans had relatively little cell specialization. The 4 diversification of cell types over the course of metazoan evolution had critical functional ramifications. For example, the origination of neurons likely had significant consequences for the coordination of movement [11].
The current paradigm for cell type origination is the “sister cell type hypothesis” [1].
According to this hypothesis, the evolution of a new cell type is most likely to occur through the divergence of an existing cell type into two distinct “sister” cell types. This cell type duplication involves maintenance of the parent cell type’s function by one daughter cell type and adoption of a slightly distinct function by the other. Such “cell type neofunctionalization” requires differentiation of the cell type to occur by its own independent process—individualization—so that a change in the gene expression program—specialization—may occur. While individualization may occur by multiple mechanisms, specialization necessarily involves change in the core regulatory complex. Recruitment of an existent TF to the core regulatory complex could suffice for specialization (Figure 1A). This could occur, for example, if extracellular conditions change in a particular tissue of an organism, causing some, but not all, cells of one type to express a general reaction TF [12]. The duplication and neofunctionalization of a terminal selector is another likely mechanism for cell type neofuntionalization, as mutations in the DNA-binding domain and promoter of the neofunctionalized terminal selector could cause both cell type specialization and individualization, respectively (Figure 1B). Such a TF duplication event was responsible for the evolution of the vertebrate hair cell from an axoned mechanosensory cell [1]. 5
A. B. Transcription factor introduction Transcription factor duplication
Ancestral cell type Ancestral cell type
TS-A TS-A tf-c TS-B TS-B
TS-C TS-C
TS-D tf-c duplication to tf-c1 and tf-c2 Expression of tf-d in subset of cells Change in TF-C2 regulation Integration of TF-D into state regulation Change in TF-C2 binding site preference
Novel cell type Ancestral-like cell type Ancestral-like cell type Novel cell type
TS-A TS-A TS-A TS-A TS-B TS-B TS-B TS-B TS-C TS-C TS-C1 TS-C1 TS-D TS-D TS-C2 TS-C2
tf-c1 tf-c2
Further changes Further changes
Figure 1: Two mechanisms of cell type origination. TS stands for “terminal selector”; TS-A through D represent transcription factors regulating cell state. A. The expression of a preexisting transcription factor in a subset of cells of one type causes these cells to adopt a distinct identity.
B. Duplication of a terminal selector, followed by neofunctionalization through both DNA- binding domain amino acid sequence change and change in expression regulation, causes a new cell type to evolve.
This example, however, represents one of very few cell type origination events for which the molecular changes responsible have been inferred [1, 12, 13]. Indeed, the evolutionary relationships between the cell types of different species are poorly understood. While recent advances in single cell RNA sequencing and the interspecific analysis of this data mean that we could be entering an era of “cell type phylogenetics” soon [11, 14], understanding cell type evolution will likely still require biochemical and genetic experiments to identify the genes responsible for regulating differentiation of each cell type in each species. 6
One group of protistan Eukaryotes, the dictyostelids, show promise as a simple “model clade” for studying the mechanisms of cell type evolution. The dictyostelids—also referred to as the “social amoebae”—are in the class Eumycetozoa (the true slime molds) of the supergroup
Amoebozoa, the sister clade to the Animals and Fungi (Figure 2A) [15]. Dictyostelids are free- living amoeboid organisms that inhabit forest soils worldwide. They are generally found as mitotically-dividing populations of single cells that feed on bacteria. Under conditions of starvation, these amoebae localize and aggregate into a multicellular unit. This multicellular unit adopts an elongated form, the sorogen, that ultimately develops into a fruiting body, or sorocarp, consisting of a stalk bearing a round mass of spores. Fruiting body formation—sorocarpy—is largely independent of cell division, instead requiring organized movement of amoebae followed by their differentiation into terminal cell types [16].
Dictyostelid sorocarpic development is best known in the model species Dictyostelium discoideum (Figure 2B). In this species, the sorogen may migrate freely prior to fruiting body formation, a process that, in nature, allows cells to reach the surface of the soil. The migrating sorogen, or slug, is made up of several precursor cell populations (Figure 2C) [17, 18]. The anterior of the slug consists of the prestalk A (“PstA”) cells at the tip and the prestalk O (“PstO”) cells just behind them. The posterior two-thirds of the slug consists primarily of the prespore cells. Scattered amongst the prespore cells are the anterior-like cells (ALCs), so called because they, like the PstA and PstO cells, possess large lysosomal vacuoles; in addition, many ALCs express either ecmA or ecmB—extracellular matrix protein genes associated with the PstA and
PstO lineages—though some express neither. Several extracellular signals contribute to this precursor patterning [19]. Extracellular cAMP is critical for prespore differentiation [20], while 7 the polyketide DIF-1 is responsible for the differentiation of the PstO cells and some, but not all, of the ALCs [18, 21]. Another polyketide or polyketides contribute to PstA differentiation [22].
Eventually, the sorogen ceases slug migration and initiates fruiting body formation, or culmination (Figure 2D). A subset of ALCs differentiate en masse into stalk cells—unviable, encapsulated cells—to form the basal disc, a structure that anchors the fruiting body onto the substrate [23]. The sorogen moves upwards, the cells attracted to an extracellular cAMP gradient emanating in the PstA zone [19]. The PstA cells at the tip secrete a stalk tube composed of extracellular matrix before entering this tube and differentiating into a scaffold of stalk cells [24,
25]. These tip cells are replaced by the constant upwards flow of PstA cells from below [26].
Prespore cells gradually differentiate into spores, dormant cells with complex walls [26]. The
ALCs sort to the top and base of the prespore zone; those that migrate upwards join with the
PstO cells to form a structure called the upper cup [27, 28], and those that move downwards forming the lower cup [23, 28]. As the differentiating spores become walled and immobile, the cells of the upper cup are responsible for their continued ascendance by pulling the spore mass, or sorus, upwards [29]. The lower cup plays a role in holding up the sorus at the top of the mature fruiting body once all PstA cells have entered the stalk tube [21].
Given the relative simplicity of their terminal structures, the dictyostelids represent a promising model phylogenetic group for the study of the evolution of cell types. Indeed, recent research has begun to elucidate the details of the evolution of dictyostelid multicellular development [19]. In prespore cells, the cAMP-dependent kinase, Protein Kinase A (PKA), activates the transcription factor spaA, which directly or indirectly turns on all spore differentiation genes [30]. PKA is a critical regulator of Dictyostelium stalk cell differentiation as well [31, 32]. Comparative work has demonstrated that the PKA-based regulatory network is 8
A. Metazoa
Fungi
Dictyostelia
Eumycetozoa
Myxogastria
Amoebozoa Protosporangiida
other Amoebozoans
Plantae
Other Eukaryotes
0.6 B.
aggregating amoebae tipped mound slug culminant mature sorocarp
stalk-producing cells C. D. upper cup
PstA prespore + ALCs PstO differentiating spores stalk cells in stalk tube
lower cup
basal disc (stalk cells) E. F.
G.
9
Figure 2: Evolutionary developmental biology of Dictyostelium discoideum. A. Placement of the dictyostelids in Eukaryota. Tree is simplified, and branches are not to scale. B. Five major stages of sorocarpic development in Dictyostelium discoideum. This process is initiated when starvation causes thousands or even millions of cells (http://www.dictybase.org/
Bonner%20paper.pdf) to chemotactically localize (“aggregating amoebae”, far left). These aggregated cells then undergo sorting (“tipped mound”), mass migration (“slug”), and morphogenesis (“culmination”), resulting in the generation of a fruiting body (“mature sorocarp”, far right). Diagram based on [26]. C. Migrating slug of Dictyostelium discoideum with major precursor populations labelled as per [33]. D. Dictyostelium discoideum early/mid culminant. Colors of cell types in this figure correspond to colors of their precursor populations in C., with the exception of stalk cells (brown; originate from PstA cells in if the tube, from
ALCs if part of the basal disc) and the stalk tube (green; produced by the stalk-producing cells that are derived from PstA cells). Diagram based on [23]. E. Terminal cell types that comprise the fruiting bodies of most species of dictyostelids, illustrated here next to a small cluster of sorocarps. See [16] for macro- and micro- photographic imagery of the dictyostelids. F. Terminal cell types that comprise the fruiting bodies of Dictyostelium, shown here next to a sorocarp of
Dictyostelium discoideum. Amoeboid form of cells in the cups observed by [29, 34]. G.
Hypothesized stepwise evolution of dictyostelid terminal cell types.
involved in spore and stalk cell differentiation in other dictyostelids and was ancestrally involved controlling encystation, the unicellular differentiation process by which Amoebozoans enter the encapsulated state of the microcyst [35-40]. Despite their position around the sorus,
Dictyostelium discoideum cup cells are more transcriptionally similar to stalk cells than to spores 10
[41]. Cup cells, which are inconspicuous, have been definitively observed only in Dictyostelium discoideum. However, other members of the genus Dictyostelium demonstrate significant populations of non-sporulating cells at the top and bottom of the sorogen; this is not observed in other genera [42]. Thus, cup cells are believed to have evolved at the base of Dictyostelium, and perhaps allowed for the evolution of the larger sori of this group [43]. Altogether, the current data suggest a step-wise evolution of Dictyostelium terminal developmental cell types: spores evolved from microcysts first, through co-option of the PKA-based differentiation pathway; stalk cells evolved next, from spores, by co-option of the same PKA-based regulatory network; and cup cells evolved from stalk cells (Figure 2E-G). The specific molecular changes responsible for the initial specialization of each of these cell types remains unknown, especially because very little is known about the regulation of differentiation in the somatic cell types, the stalk and cup cells.
The goal of this project was to identify a transcriptional regulator of cup cell differentiation in Dictyostelium discoideum. I identified a transcription factor gene, fasA, that is required for the migration of ALCs from amongst the prespore cells to the poles of the sorus, and is essential for the cup-dependent ascendance of spores. This gene evolved in a gene duplication event at the base of the genus Dictyostelium, and its paralog plays a minor role in stalk formation. However, I find no indication that the evolution of this gene contributed to the initial evolution of the cup cell.
11
Methods
Computational analyses
All nucleotide and protein analyses used Amoebozoan sequences from the genomic and transcriptomic sources listed in Table 1. Unavailable transcriptomes were assembled from
Sequence Read Archive (SRA) [44] data using the Galaxy platform [45] and CD-HIT server
[46]. Specifically, RNA-seq data was downloaded in the FASTQ format using fasterq-dump
(https://github.com/ncbi/sra-tools). Reads were trimmed with fastp [47], and assembled using
Trinity [48]. Redundant transcripts removed with CD-HIT-EST, and CDSs were identified within transcripts using TransDecoder (https://github.com/TransDecoder/TransDecoder).
OrthoVenn2 [49] was utilized to group translated CDSs into gene orthologous clusters, while protein or translated nucleotide BLAST was utilized to identify more distant gene relationships. Reciprocal tblastn searches of translated nucleotide sequences were used to identify partial gene sequences in unannotated genomes.
Product descriptions for annotated Dictyostelium discoideum genes were extracted from dictybase [50] and from Schaap et al. [51]. Genes were considered putative transcriptional regulators if they were identified as such by Schaap et al [51].
For phylogenetic analyses, translated CDSs or genomic sequences were aligned in
MEGAX [52] using MUSCLE [53] . Model selection was carried out using ModelTest-NG [54].
Trees were inferred using GeneRax [55], and visualized in FigTree (http://tree.bio.ed.ac.uk/ software/figtree/). The aBSREL test [56] in Datamonkey 2.0 [57] was used to test for positive selection on branches of the gene phylogeny.
Synteny of a genomic region containing a gene of interest was assessed by a reciprocal
BLAST method. Specifically, for a given species, the closest homologs of genes neighboring the 12
Species Source Sequence tag Developmental or cell type Notes expression data Dictyostelium discoideum Dictyostelium discoideum AX4 annotated genome DDB Dictyostelium discoideum AX4 [58, 59] cup, stalk, vegetative, and spore cell transcriptomes [41]
Dictyostelium discoideum NC4 prespore and presomatic cell transcriptomes [60]
Dictyostelium discoideum AX4 sorocarpy transcriptome [61] Heterostelium album Heterostelium album PN500 annotated genome [58, PPL Heterostelium album PN500 stalk 62] and spore cell transcriptomes [63]
Heterostelium album PN500 sorocarpy transcriptome [63] Physarum polycephalum Physarum polycephalum LU352 genome and multi- Phypoly Non-dictyostelid strain transcriptomes [64] Eumycetozoan
A Physarum transcript, Phypoly_04771, was generated by manually aligning two redundant transcripts, 25582 and 04771. Dictyostelium purpureum Dictyostelium purpureum QSDP1/DpAX1 DICPUDRAFT Dictyostelium purpureum DpAX1 annotated genome [58, 65] prespore and presomatic cell transcriptomes [60]
Dictyostelium purpureum DpAX1 sorocarpy transcriptome [60] Acytostelium subglobosum Acytostelium subglobosum LB1/A1 annotated Asub genome [66] Ceratiomyxa fruticulosa SRA Experiment SRR5396448 from Ceratiomyxa Cfru Non-dictyostelid fruticulosa Pit14 [67] Eumycetozoan Tieghemostelium lacteum Tieghemostelium lacteum TK annotated genome DLA Tieghemostelium lacteum TK [63, 68] stalk, vegetative, and spore cell transcriptomes [51]
Tieghemostelium lacteum TK sorocarpy transcriptome [63] Cavenderia fasciculata Cavenderia fasciculata SH3 annotated genome [58, DFA Cavenderia fasciculata SH3 62] sorocarpy transcriptome [63]
Polysphondylium violaceum Polysphondylium violaceum QSvi11 annotated CYY Polysphondylium violaceum genome [69] QSvi11 stalk, vegetative, and spore cell transcriptomes [69] Dictyostelium giganteum SRA experiments SRR042784 and SRR042785 from Dictyostelium giganteum WS589 [70] Acytostelium leptosomum Acytostelium leptosomum FG12A unannotated Alep genome [71] Rostrostelium ellipticum Rostrostelium ellipticum AE2 uannotated genome Rell [71] Cavenderia deminutiva Cavenderia deminutiva MexM19A unannotated Cdem genome [71] Dictyostelium intermedium Dictyostelium intermedium PJ11 unannotated Dint genome [70] Dictyostelium firmibasis Dictyostelium firmibasis TNS-C-14 unannotated Dfir genome [70] Dictyostelium citrinum Dictyostelium citrinum OH494 unannotated Dcit genome [70] Speleostelium caveatum Speleostelium caveatum WS695 unannotated Scav genome [70] Coremiostelium Coremiostelium polycephalum MY1-1 unannotated Cpol polycephalum genome [71] Synstelium polycarpum Synstelium polycarpum OhioWilds unannotated Spol genome [71] Heterostelium Heterostelium multicystogenum AS2 unannotated multicystogenum genome [70] Planoprotostelium Planoprotostelium fungivorum Jena Gg-2016a Non-Eumycetozoan fungivorum annotated genome [72] Amoebozoan
Entamoeba invadens Entamoebae invadens IP1 annotated genome [73] Non-Eumycetozoan Amoebozoan Entamoeba histolytia Entamoeba histolytia HM-1:IMSS annotated genome [74, 75] Acanthamoeba castellanii Acanthamoeba castellanii Neff annotated genome Non-Eumycetozoan [76] Amoebozoan Table 1: Sources of gene and genomic sequences and quantitative transcriptomic data. 13 gene of interest were identified using a protein BLAST against another species: a closest homolog, if present, was the best return with an alignment score of 80 or greater. This was repeated for all species combinations. Synteny was established if the closest homologs of nearby genes were also adjacent; and results were consistent across species.
Transcriptomic analyses utilized available RNA-seq data, as listed in Table 1. For RNA- seq experiments for which mapped read data is not publically available, raw data from the SRA was downloaded in the FASTQ format using fasterq-dump. Then, reads were trimmed with fastp, and mapped to CDSs using Kallisto [77]. Normalized reads measured in transcripts per kilobase million (TPM) were utilized for all purposes; for public datasets that provided raw or RPKM
(reads per kilobase of transcript, per million mapped reads) counts, these values were converted to TPM using an R script [78]. Expression time courses were graphed using Microsoft Excel
(https://www.microsoft.com/en-us/microsoft-365/excel).
Predicted binding site preferences of Dictyostelium discoideum transcription factors were downloaded from CIS-BP [79]. The matrix-scan tool on the RSAT server was used to identify binding sites in genomic sequences located upstream of genes of interest [80].
Laboratory Experiments
HL5 medium [81] was prepared by the standard recipe (http://dictybase.org/techniques/ media/media.html). Specifically, 10g proteose peptone #2, 10 g glucose, 5 g yeast extract, 0.185g
Na2HPO4 anhydrous, and 0.35 g KH2PO4 anhydrous were combined, and volume was brought up to 1 liter; and HCl was used to adjust pH to 6.4 - 6.7 prior to autoclaving. 1 mL of 300mg/mL streptomycin-sulfate and 1 mL of 100mg/mL ampicillin sodium salt were added just prior to use.
A sterile 5x phosphates solution was prepared by adding Na2HPO4 to 25mM and KH2PO4 to 14
25mM, adjusting the pH to 6.5 with HCl, and autoclaving. 1 liter of Developmental Buffer (DB)
(http://dictybase.org/techniques/media/media.html) was prepared by combining 797 ml of distilled, autoclaved water with 200 ml of the 5x phosphates solution, 1mL of sterile CaCl2 solution, and 2mL of sterile MgCl2 solution. KK2 agar plates were made by mixing 1L of KK2
(2.2 grams KH2PO4 and 0.7grams K2HPO4 per L of distilled water) with 15 grams agar, autoclaving, and pouring into sterile petri dishes. Water agar, containing 15 grams agar per liter of distilled water, was made by the same method.
Dictyostelium discoideum strain AX4 [59], which is adapted for growth in axenic liquid culture, was purchased from the Dictyostelium Stock Center (DSC) [50]. Mutants of AX4, generated as part of the Genome-Wide Dictyostelium Insertion (GWDI) Project [82], were also purchased from the DSC. DDB_G0286351 is disrupted in GWDI_69_H_1 (Figure 3), and
DDB_G0270306 is disrupted in GWDI_410_H_7 (Figure 4). Amoebae were cultivated by standard axenic methods [83]. Specifically: Dictyostelium cells were isolated from the edge of colonies growing on bacterial plates or thawed from frozen stocks. Cells were transferred to sterile falcon tubes containing 1 mL of HL5, and allowed to adjust to axenic conditions for four days, with the medium replaced after 2 days. Axenically growing amoebae were maintained in
250, 500, 1000 or 2000 mL sterile flasks containing HL5 up to 20% of flask volume, at most.
Flasks were kept in a shaking incubator, set to 22°C and 180 r.p.m. Cell density was assessed by transferring 10 uL of amoebae to a hemocytometer. Density was maintained at 2x104-2x107/mL by transferring as necessary, generally every 2 to 3 days.
Frozen stocks were generated by one of two methods. After assessing cell density, amoebae were transferred into 50mL centrifuge tubes, and centrifuged at 500g for 4 minutes to pellet. The pellet was resuspended in 2mL eppendorf tubes at 5 x 107 cells/mL in HL5 containing 15
10% EDTA [83]. Tubes were then cooled incrementally by incubation at 0°C for several minutes and -20°C for several hours, before storage at -80°C. Or, mature fruiting bodies were harvested from plates (see below) using a sterile metal spatula, and mixed with 1 mL of sterile KK2 and
0.33mL of 60% glycerol in a 2mL eppendorf tube [84]
(https://strassmannandquellerlab.files.wordpress.com/2011/07/freezing-dictyostelium- strains1.pdf). These spore stocks were frozen gradually as with amoebal stocks. Stocks were used to generate fresh cultures every few weeks, to minimize genetic drift.
Cells were prepared for sorocarpic development using methods modified from several sources [83, 85, 86]. After assessing cell density, amoebae were collected in 50mL centrifuge tubes and centrifuged to pellet. The supernatant was poured off, and cells were resuspended in
50mL of DB by vigorously shaking the tubes. Tubes were centrifuged again. Cells were then resuspended at 107 cells/mL DB, transferred to sterile flasks, and allowed to starve for 4 hours at
22°C and 180 r.p.m. This starvation step was not necessary for cells that were not to be stained, and so was sometimes skipped; however, cells were not impacted if they were starved. Cells to be stained were separated into 15mL units in 50mL centrifuge tubes. These cells were pelleted down, resuspended in 2mL DB, and 4 drops of 0.2 mg/mL vital dye (Neutral Red or Nile Blue) were added to each tube. These vital dyes selectively stain the lysosomal vacuoles found in PstA,
PstO, ALC, and rearguard cells—the “presomatic cells”. Upon addition of the dye, tubes were gently tapped and swirled to mix. Cells were allowed to stain for 4 minutes, before the dye was diluted out with the addition of 48mL of DB. Whether stained or not, cells were washed one more time in DB. After the final wash, cells were resuspended at high density (approximately
109 cells/mL) by mixing the pellet in a very small volume of DB; care had to be taken to account for any residual DB that clung to the pellet after pouring. 16
To induce direct development, with limited slug migration, 75 uL of cell suspension was pipetted onto a 60 mm KK2 plate, or 200 uL of cell suspension was pipetted onto a 100 mm KK2 plate. Cells were spread evenly over the surface of the plate using a glass spreader sterilized in
80% ethanol. KK2 plates were incubated at 22C under overhead light until fruiting bodies formed (up to 24 hours). Mature fruiting bodies were imaged on plates with a dissecting microscope, or were transferred to distilled water on a glass slides for imaging under a compound microscope.
To induce extended slug migration, 50uL of cell suspension was plated as a streak along one end of a 100mm water agar plate. Water agar plates were incubated at 22C in the dark. Slugs appeared within 18 hours and were imaged with a dissecting microscope.
17
Results
Identification of a putative Dictyostelium-specific cup-enriched transcription factor and a paralogous gene
I sought to identify possible transcriptional regulators of cup cell state by identifying genes with higher expression in cup cells than in spores, stalk cells, or vegetative amoebae.
Available cell type transcriptomic data was used to identify transcription factors with at least two times greater expression in cup cells than in other purified terminal cell types. The ten cup- enriched transcription factor genes with the highest cup cell expression levels are listed in Table
2. While this list may not include some critical genes due to low expression or expression in multiple cell types, these transcription factors likely play roles in the regulation of certain aspects of the cup cell state.
Transcription factor duplication can contribute to cell type evolution. Thus, to see if this may have contributed to the evolution of cup cells, I used OrthoVenn2 to identify the number of homologs in 6 dictyostelid species with well-annotated genomes. One of the genes has two homologs in Dictyostelium but one in other genera: DDB_G0286351, a putative Cud-like transcription factor. The Cud family of DNA-binding proteins are specific to the supergroup
Amoebozoa [87]. The two characterized genes in this family possess critical roles in
Dictyostelium discoideum development: cudA (culmination deficient A) plays essential, cell- autonomous roles in stalk tube secretion and spore differentiation [88], while spaA (spores absent A) is the master regulator of spore differentiation [30]. The DDB_G0286351 contains a highly conserved region found to be involved in the DNA binding activity of Cud-type proteins
[87] (Figure 3). The identified DDB_G0286351 homolog in D. discoideum, DDB_G0270306, also contains an identical DNA-binding region (Figure 4), suggesting that it is also a 18
Dictyostelium discoideum transcription factor gene Homologs of gene
Gene ID Gene Predicted product description Cell type expression (TPM) Fold Number of homologs per species Dd homolog name increase identities in cup cells Cup Stalk Spores Vegetative Dd Dp Tl Ha Pv Cf cells cells amoebae
DDB_G0286351 fasA CudA-like transcription factor 101.03 40.22 0.38 0.42 2.51 2 2 1 1 1 1 DDB_G0270306 (fasB) CudA-like transcription factor DDB_G0275333 - ARID/BRIGHT DNA binding domain - 281.15 60.17 24.13 16.63 4.67 3 2 2 2 2 2 DDB_G0273097 containing protein (ptpA-1) protein tyrosine phosphatase A-1
DDB_G0273817 (ptpA-2) protein tyrosine phosphatase A-2 DDB_G0278077 crtf carA promoter -binding transcription factor 1147.54 322.78 140.86 55.91 3.56 1 1 1 1 1 1 -
DDB_G0277681 bzpH basic-leucine zipper transcription factor 146.30 27.20 20.34 5.81 5.38 1 1 1 0 1 1 -
DDB_G0278379 bzpD basic-leucine zipper transcription factor 82.323 32.42 15.51 18.67 2.54 1 1 1 1 1 1 -
DDB_G0268502 - C2H2 zinc finger domain -containing protein 16.88 0.57 0.20 0.35 29.62 1 0 0 0 0 0 -
DDB_G0272048 - C2H2 zinc finger domain -containing protein 44.76 6.38 2.11 17.05 7.01 1 1 1 1 1 1 -
DDB_G0284255 - C2H2 zinc finger domain -containing protein 85.42 32.50 27.41 1.43 2.63 1 1 1 1 1 1 -
DDB_G0290665 gtaX GATA zinc finger domain -containing 7.12 0.61 0.58 0.72 9.96 1 0 0 0 0 0 transcription factor
DDB_G0280547 comH GATA zinc finger domain -containing 37.21 9.49 3.34 17.54 2.12 1 0 0 0 0 0 transcription factor
Table 2: Ten Dictyostelium discoideum cup-enriched transcription factor genes with the highest expression. Abbreviations: Dd = Dictyostelium discoideum; Dp = Dictyostelium purpureum; Tl = Tieghemostelium lacteum; Ha = Heterostelium album; Pv = Polysphondylium violaceum; Cf = Cavenderia fasciculata. Note: Several homologs of DDB_G0275333 identified using OrthoVenn2 do not contain ARID domains; these include the D. discoideum protein tyrosine phosphatases ptpA1-1 and ptpA1-2. This artifactual result was found to be a consequence of a hybrid ARID/protein tyrosine phosphatase gene in the
Ceratiomyxa transcriptome (data not shown). 19
1 ATG ATT TAT AAT CAA GCT TTA ATT GAA ATT ACA AAA CAA GGT GCG GTG GCT GTA GAA GAG ATT AAA TTT TCT CCA CCA AAG TTA CAA ACA M I Y N Q A L I E I T K Q G A V A V E E I K F S P P K L Q T
91 TTA ATA AGT TCA ATT CAA AAT GGA GGT TCA CCA ATT ATA AAT AAA AAT GGT AGT CCA GCT GCA AAT GCT TTA AAA AAT AAA CAA ATT TTA L I S S I Q N G G S P I I N K N G S P A A N A L K N K Q I L
181 CAA TTA CAA GGT CAA CAA CAA CAG CAA CAA CAA CAA CAA CAA AAT CAT TCA CAA CAA CAA CAC AAT AAC CAA TCG AGT GTT TAT TCT TTA Q L Q G Q Q Q Q Q Q Q Q Q Q N H S Q Q Q H N N Q S S V Y S L
271 AAT TCT TTA AAT CCT TAT TAT GTA CAA CAA GTT CTT TGT CAA CTT CAA AAA CCA CAT AAT TTT ATA AAA CAA GTT CAT GTT GTA GTT AAA N S L N P Y Y V Q Q V L C Q L Q K P H N F I K Q V H V V V K V 361 AAC ACA CCA TTT GGT ATT TCA ATG AAA TCA AAC GAT CCA CAA TTC AAT TTT CAT AAT TAT GTT ATT AAA GCT ACA CTT CTC TAT GAT TGT N T P F G I S M K S N D P Q F N F H N Y V I K A T L L Y D C
451 GAT CCA CCA AAA ATG GTA GAT TTT ATT CAT AAT GAA CCA TTA CAA TAT GTT GCA ACC GTT AGT GAA GAT GGT ACT GAA GTT GTT GTT GAT D P P K M V D F I H N E P L Q Y V A T V S E D G T E V V V D
541 GTA AAG GTT GGT ATT CTT TCA TCA CAA CAT CAA GGC TCA ATG TTT CTT GCA GTT TTA CAT ATA AGT CAT ACA AGT GTA CCA TCC CCA TCA V K V G I L S S Q H Q G S M F L A V L H I S H T S V P S P S
631 CCT AAT GAA CCA ATT ATG ACC ATC TTG AAT AAT ATA GGT GGT AAT TCA ATA GCA AAT TTA AAT TCC CAA TTA CCA AAT CCA ATC TAT AAT P N E P I M T I L N N I G G N S I A N L N S Q L P N P I Y N
721 CTT TGT GTT GTT TCT CAT CCA ATT AGA ATC GTT TCA AAA GTT GAT CAT GTT AAA AAA GAA GGT ATA CCA ATA TTA AAG AAA AAG ACA TTT L C V V S H P I R I V S K V D H V K K E G I P I L K K K T F
811 CAT GAA ATT CTT ACA GAT AAA TTA AAG AAA TTA CAA AAG TTT CAA GAT AGT CAA AGT AAA TGG ATT AAA AAT TTA TAT CAA CAA CAT TTA H E I L T D K L K K L Q K F Q D S Q S K W I K N L Y Q Q H L
901 ATT GAA TTC GAT ATG GAA CCT TAT TGT TCA AAA AAA GAT CAA AAT AAT AAT AAT AAT AAT AAT AAT AGT AAT TCC AAT TGT CAA AAT GGT I E F D M E P Y C S K K D Q N N N N N N N N S N S N C Q N G
991 GGT GGT TCA ATT TGT GAT GAT TCT TCA AAT TCT TCT ACC CCT TCA TTA AGT TCT TAT TCA AAT GGT AAT AAT AAA TAT AAT AAT AAT AAT G G S I C D D S S N S S T P S L S S Y S N G N N K Y N N N N
1081 AAT GAT TCA AGT GAA TCC GAT GAA TCT GAT GAC GAT GAT AAC AAT GAT GAT GAC GAT AAT GAT TCA ATC GAT TTC AAT ATT TCA AAA CAG N D S S E S D E S D D D D N N D D D D N D S I D F N I S K Q
1171 AAA AAT CAA CAA CAT TTA CAA CAA CAA TTA TTA AAT CAT CAA TCA AAA CAA CAA AAA GTA TCT TCG ACT AAT AAT AAT ACT ACC ACT ACT K N Q Q H L Q Q Q L L N H Q S K Q Q K V S S T N N N T T T T
1261 ACT TCC TCT TCT TCT GCT TCT TCT ATA CAA CAA AAA CAA CAA CCT GTA CAA CCA CAA CAA CAA AAA CAA CAA AAT CAA TCG AGT AAT TTT T S S S S A S S I Q Q K Q Q P V Q P Q Q Q K Q Q N Q S S N F
1351 CAA AAT TCA TTT AAT AGA GTT GTC GAA GCA TTT AAA TAC GTA CCA GAA TCT GAA AGA AAA GAT ATT ATT ACG AAA ATG GTT GAA CAA TTA Q N S F N R V V E A F K Y V P E S E R K D I I T K M V E Q L
1441 CGT TCT GAT GAT TTA GAG CAA TTG GTT GCT ACA TTT ATG GAT GAA CTT GGT GTT GGT GAT GCA AAT AGT GAT GTA TCA TCA ACA AAA GGA R S D D L E Q L V A T F M D E L G V G D A N S D V S S T K G
1531 GGT AAC AAT AAT AAT AAC AAT AGT ATT GGT TGC TTC TGT GAG AAT TGT CCA TCC AAG AAA GAA TTA GAA AGG TTT CAA GGT TTA TGT ATG G N N N N N N S I G C F C E N C P S K K E L E R F Q G L C M
1621 AAC TTT TTC GTT CCT CAA TCA ACT TTA AAT CCA ATG AAT AAT CAA CAA CTT TTA TAT TCT TCA TTA TTT TCA TCA TCA AAT GAT AAC AAT N F F V P Q S T L N P M N N Q Q L L Y S S L F S S S N D N N
1711 ACG CAA AGT CAA ATG AAT AAT TCA ATC TCT GTA AAT AAT CAT CAC CAT CAT CAT CAC CAT CAA CCA AAT AAT TCA AAT CTA ACA AAT GAT T Q S Q M N N S I S V N N H H H H H H H Q P N N S N L T N D
1801 TTA TTA CAA TTA CCA ATA GTT TAA L L Q L P I V *
Figure 3: Sequence of DDB_G0286351 (fasA). Putative DNA binding domain is highlighted in yellow. Location of insertion in gene disruption mutant is indicated with a red arrow. 20
1 ATG ATA GTA AAT CAA GCA TTA ATT GAA TTA ACA AAA CAA GTT GCA GTT GCG GTT GAA GAA ATT AAA TTT TCA CCC AAC TCA ACT AGT AAT M I V N Q A L I E L T K Q V A V A V E E I K F S P N S T S N
91 AAT TCA ACA CCA ACA AAT AAT AAA TTA AAA TCA TCA TCG TCA TCA ATT TCA AAT TGT GAT TCG CCA TCA TCA AAA TCG AAA TCG AAT AGT N S T P T N N K L K S S S S S I S N C D S P S S K S K S N S
181 TCA ACT TCA ACC CCA ACA TCA CAA CCA CAA ACA CCA TCA CAA TTA CCA CAA CAA GCA CAA CAA CAA GCA CAA CAA CAA CAT TAT TCA GCA S T S T P T S Q P Q T P S Q L P Q Q A Q Q Q A Q Q Q H Y S A
271 AAT TCA ATG AAT CCG TAT TAT GCA CAA CAA GTT TTA TTA CAA TTA CAA AAA CCA TCA ACA TTT GTA AAA CAC GTT CAT GTC GTT GTA AAG N S M N P Y Y A Q Q V L L Q L Q K P S T F V K H V H V V V K
361 AAT ACA CCA TTT GGA ATT ACA TTA AGA TCT AAA GAA CCA TTA CAA TTT AAT TTT CAA AAT TAT GTT ATA AAA GCC ACC CTA CTC TAT GAT N T P F G I T L R S K E P L Q F N F Q N Y V I K A T L L Y D
451 AGT GAC CCA CCA AAA ATG GTT GAT TTC ATT CAT AAT GAG CCA TTA CAA TAT GTT GCA ACG GTA TCT GAA GAT GGG TCA GAG GTT TGT GTT S D P P K M V D F I H N E P L Q Y V A T V S E D G S E V C V
541 GAT GTT AAA GTT GGT ATT TTG TCA AGT CAA CAT CAA GGA TCA ATG TTT TTA GTT GTA TTA CAT ATT AGT CAT TGC TCA GCA CCA ACA CCA D V K V G I L S S Q H Q G S M F L V V L H I S H C S A P T P
631 TCA AAT AGT GAA CCA ATT AGT ACA ATT CTA ACA AAT ATT GGT AAT AAC TCT ATA CAC AGT TTA AAC GTT ACA AAT ATT TGT GTT GTA TCA S N S E P I S T I L T N I G N N S I H S L N V T N I C V V S
721 CAT CCA ATT AGA ATT GTT TCA AAA TTA GAT CAC GTT AAA AAA GAA GGT ATA CCA ATT TTA AAG AAA AGA ACA TTT CAC GAA ATT TTA ACT H P I R I V S K L D H V K K E G I P I L K K R T F H E I L T V 811 GAT AAA TTA AAA AAA TTA CAA AAA TCA CAA GAT AGT CAA AGT AAA TGG ATT AAA AAT CTA TAT CAA CAA CAT GGT GCT CAA TAT GAT ATG D K L K K L Q K S Q D S Q S K W I K N L Y Q Q H G A Q Y D M
901 GAA CCT TAT AAT AGT ACT TTA CAT TCT CAA AAA ACT GAT TCA CTC TGT TCT TCT TCA ACT TCC ACA CCA TCA TTT AAT TCA ACC TCC TCC E P Y N S T L H S Q K T D S L C S S S T S T P S F N S T S S
991 TCC TCA AAG AAT CAA TCC CAA TCA ATT AAA AAT GAA GAA GAA GAA GAT GGT GGT GAA GAC GAA GAA GAA GAA GGA GGC GAA GAT AAT GAT S S K N Q S Q S I K N E E E E D G G E D E E E E G G E D N D
1081 AAT GAA AGT GAA TCA AGT AAT ACC AAT AGT ACA CAA TTA ATT GGG AAA AAA AGT ATA AAT AAA TTA CCA ATT TCA ACA ACA ACT TCA TCT N E S E S S N T N S T Q L I G K K S I N K L P I S T T T S S
1171 TCA AAT TTT AAT AAT TTA ATG GTT AAT AAT AAT ATA AAT AAT AAT AAT AAT AAT AAT AAT AAT AAT CAT TTA ATT CAA AAT ACA ACT TTT S N F N N L M V N N N I N N N N N N N N N N H L I Q N T T F
1261 TCA TCA ACT AGT CAT TTT CAA AAT TCT TTT AAT AGA GTA GTT GAA GTT TAT AAT TTA ATT CCA GAA TAT GAA AGA GTT GAA GTT ATT AGA S S T S H F Q N S F N R V V E V Y N L I P E Y E R V E V I R
1351 AAA ATG GTT CAA CAA TTA AAA TCT TAT GAT TTA GAA CAA TTG GTT TCA ACA TTT ATT GAT GAA TTA AAA TGT GGT TAT GAA TCA ACT ATA K M V Q Q L K S Y D L E Q L V S T F I D E L K C G Y E S T I
1441 ATT TCT TAA I S *
Figure 4: Sequence of DDB_G0270306 (fasB). Annotated as in Figure 3.
transcription factor and is a bonafide DDB_G0286351 paralog.
DDB_G0286351 was previously mutated in an unpublished, high-throughput gene disruption experiment by W. Loomis and N. Iranfar and named “Developmental Gene 1062”
(dg1062). An image from this project of the DDB_G0286351 mutant shows a mature fruiting body with a sorus only partway up the stalk (http://wiki.dictybase.org/dictywiki/index.php/ 21
DDB_G0286351). Given the roles of the cups in elevating and supporting the sorus, this phenotype is consistent with defects in the upper or lower cup. Though phenotypic data is not available for DDB_G0270306, it is enriched in stalk cells, the sister cell type of the cup cells
(Table 3).
The expression of paralogous transcription factor genes in sister cell types (the stalk and cup cells) could be indicative of a role of transcription factor duplication in the origination of the neofunctionalized cell type (the cup cell). I sought to explore this possibility further by studying the developmental roles and molecular evolution of DDB_G0286451 and DDB_G270306.
Characterization of fasA- and fasB- mutant phenotypes reveals roles in somatic development
I characterized the roles of DDB_G0286351 and DDB_G0270306 in Dictyostelium discoideum using mutants with 1.5 kb insertions in these genes (Figures 3, 4), which were produced and identified as part of the GWDI Project [82]. Cells of these strains, referred to here as DDB_G0286351- and DDB_G0270306-, as well as the “wildtype” parental strain AX4, were grown in axenic liquid culture, and plated at high density on nutrient-free agar to induce sorocarpic development.
Figures 5-8 show macroscopic and microscopic imagery of AX4, DDB_G0286351-, and
DDB_G0270306- fruiting bodies. The sorus is often found only part-way up the stalk in
DDB_G0286351- fruiting bodies (Figure 5B, 6A-B), like what was observed by Loomis and
Iranfar. Instead, only a very small mass of cells is found at the top of the stalk (Figure 5, asterisks). However, besides these defects, the DDB_G0286351- fruiting bodies match those of
AX4 (Figure 5). Spores appear normal (Figure 8B), and the stalk consists of a typical multi- layered scaffold of stalk cells enclosed in a tube (Figure 8B). The size and proportions of the 22
A BB
Figure 5: Comparison of gross morphology of AX4 and DDB_G0286351- (fasA-) mature sorocarps. The sori are the largest round masses in each image; smaller round masses are droplets of condensed water. Scale bar 0.5 mm.
23
* *
A
*
B C
Figure 6: DDB_G0286351- (fasA-) mature sorocarps. Arrows point to basal discs; asterisks indicate small masses of cells found at the tops of the stalks. Scale bar 0.5 mm. 24
A B
D
C
Figure 7: DDB_G0270306- (fasB-) mature fruiting bodies. Scale bars 0.5mm; scale bar in C applies to A and B as well. 25
A B
C D
E F
Figure 8: Stalk cells and spores of AX4, DDB_G0286351- (fasA-), and DDB_G0270306-
(fasB-). Matured fruiting bodies were placed on glass slides in distilled water for imaging of these cells. A. AX4 stalk cells. B. fasA- stalk cells and spores. C. fasA- basal disc. D. AX4 spores. E. fasB- stalk cells. F. fasB- spores. Scale bar 10 µm.
26 fruiting bodies are normal, and, the basal disc is present in DDB_G0286351- (Figure 6, arrows;
Figure 8C).
DDB_G0270306- also demonstrates phenotypic defects at the fruiting body stage. Stalks are generally twisted (Figure 7A), and often contain kinks (Figure 7B) or segments of variable thickness (Figure 7C). Some fruiting bodies are found partially or entirely collapsed onto the agar (Figure 7D). The basal disc is present but is sometimes small (Figure 7A-B, arrows).
Microscopic observation confirms that stalks are variable in thickness; segments of some stalks consist of a single layer of elongated stalk cells within a normal tube (Figure 8E).
DDB_G0270306- spores appear normal (Figure 8F), and typically proportioned sori are almost always found at the ends of the stalks of DDB_G0270306- fruiting bodies (Figure 7).
Even though the defects in DDB_G0286351- and DDB_G0270306- are different, they both negatively impact the ascendance of the sorus above the substrate. Keeping with the precedent of naming Dictyostelium Cud-type transcription factor genes after their developmental roles, I have selected the names fallen sorus A (fasA) and fallen sorus B (fasB) for
DDB_G0286351 and DDB_G0270306, respectively.
The vital dye neutral red (NR) stains Dictyostelium lysosomal vacuoles, which are absent in prespore cells; thus, NR-stained cells correspond to the presomatic cells in the slug stage
(Figure 9A). Stalk cells lose their stain, so during culmination, NR staining is observed in the upper cup (Figure 9B, arrow), lower cup (Figure 9B, arrowhead), and the stalk-producing cells found above the upper cup (Figure 9B). fasA- and fasB- were stained to follow these cells during sorocarpic development. fasA- appears to possess normal precursor proportioning: like in AX4
(Figure 9A), the entire neck of the slug stained with NR, as did scattered areas of the posterior, indicative of normal PstA, PstO, and ALC differentiation (Figure 10A). For fasA-, perturbations 27
slime prespore prestalk trail and ALCs
A
B
Figure 9: NR staining in AX4. A. Slug, with major populations labeled. Only the prespore cells do not stain with NR. B. Culminating sorogen. Arrows point to the upper and lower cup. Stained cells above the upper cup are the stalk producing cells. Scale bar 1 mm. 28
A B
* *
C D
Figure 10: NR staining in fasA-. A. Slugs, showing typical staining patterns. B-C. Sorogens, showing scattered staining in posterior (arrows), and staining in anterior. Anterior in C is in the process of separating from the sorus (asterisk). D. Mature fruiting body with collapsed sorus.
Asterisk is adjacent to apical mass, which is strongly stained. Scale bar in B 0.1 mm; scale bar in
D 0.5 mm. Scale bar in D applies to A and C as well. 29
A
B B
C
Figure 11: NR staining in fasB-. A. Slugs. Note decreased staining in anterior and increased staining in posterior. B-C. Culminating sorogens that have collapsed on agar such that their typical patterns of staining (defined anterior and cup zones) are readily visible. Scale bars
0.5mm; Scale bar in B applies to A as well. 30 in staining patterns first appear during the culmination. In fasA-, NR-staining cells are found scattered throughout the prespore region (Figure 10B-C, arrows), and a zone of high staining is not present in the rear. This indicates that ALCs remain scattered amongst the prespore cells and do not sort to the lower cup. Midway through culmination, the NR-dotted sorus tends to cease upwards movement and separate from the well-stained cells of the tip, which continue to rise and contribute to the stalk (Figure 10C, asterisk). NR staining is observed in the small apical mass of the mature fruiting body (Figure 10D, asterisk). Thus, this mass presumably corresponds to PstO cells, indicating that these cells do not pull the sorus upwards in fasA-. This failure in upper cup activity, and the apparent lack of a lower cup, provide a basis for the suspended sorus phenotype.
Unlike fasA- cells, fasB- cells do show pertrubations in staining at the slug stage. fasB- slugs demonstrate weaker NR staining in the anterior and increased NR staining in the rear (Figure
11A). However, during culmination, NR staining patterns are identical to those of AX4, with defined anterior, rear, and prespore zones (Figure 11B-C). The precise implications of these staining patterns are unclear.
fasA- and fasB- phenotypes are predicted to be cell autonomous
β-galactosidase reporter or in situ hybridization experiments are commonly used to assess spatiotemporal patterns of gene expression in Dictyostelium discoideum. These experiments were not possible within the timeframe of this project. Instead, available transcriptomic data (Table 1) was utilized to identify the precursor populations and terminal cell types that express fasA and fasB (Table 3). Expression data of purified precursor populations from slugs indicate that both fasA and fasB are preferentially expressed in presomatic cells. As observed in the cup cell gene computational screen, fasA is highly expressed in cup cells, but is also expressed in stalk cells. 31 fasB shows highest expression in stalk cells out of all terminal cell types, although fasA stalk cell expression is actually greater. Transcriptomic developmental time course data indicates that expression of fasA and fasB spike at 18 hours (Figure 12), precisely when culmination initiates during synchronous sorocarpic development. Though these data do not indicate which presomatic populations express fasA and fasB, they are consistent with the mutant phenotypes observed; they suggest that fasA and fasB play cell autonomous roles in the activity of precup/cup cells and PstA/stalk cells, respectively, during culmination.
vegetative presomatic stalk cup prespore amoebae cells cells cells cells spores DDB_G0286351 (fasA) 0.42 706.34 40.22 101.03 41.14 0.38 DDB_G0270306 (fasB) 1.04 25.52 15.02 1.13 2.79 0.86
Table 3: Dictyostelium discoideum AX4 cell type fasA and fasB cell type expression data.
Data from [41, 60].
32
Figure 12: fasA and fasB expression levels over the course of sorocarpic development in
Dictyostelium discoideum AX4. Cells were induced to develop synchronously; culmination lasts from approximately 18 to 24 hours. Data from [61].
The fas and spa gene families share an origin in Eumycetozoa
To better understand the evolution of fasA and fasB, OrthoVenn2 and BLAST were used to identify related gene sequences in other Amoebozoans with annotated genomes (Table 1); the relationships of species for which related genes were identified are illustrated in Figure 13A.
OrthoVenn2 identified one fasA/B ortholog in all non-Dictyostelium dictyostelid genomes analyzed, except Acytostelium subglobosum. Gene clustering also identified one ortholog in the representatives of the two other major branches of Eumycetozoa : Physarum polycephalum
(Myxogastria) and Ceratiomyxa fruticulosa (Protosporangiida). OrthoVenn2 did not identify any fasA/B orthologs outside of Eumycetozoa. 33
Figure 13: fas gene phylogeny. A. Phylogenetic relationships between Eumycetozoan species with CDS sequences available. Based on [67, 70]. B. Unrooted phylogeny of spa and fas genes.
Inferred using the VT+I+G4 model of protein evolution. C. Phylogenetic relationships between all dictyostelids with publicly available sequenced genomes. Based on [70]. D. Phylogeny of fas conserved core sequence. Inferred using the LG+I+G4 model of protein evolution. fas sequences for Cavenderia fasciculata, Dictyostelium giganteum, and Heterostelium multicystogenum were not included because they are too short. 34
BLAST searches also identified additional CDSs with similarity to fas genes: spaA and its dictyostelid orthologs. No additional genes returned alignment scores above 80, consistent with the results of a previous inference of Cud transcription factor phylogeny [89], which similarly failed to find homologs of spa or fas genes outside of Eumycetozoa. Maximum likelihood phylogenetic inference was used to generate the spa/fas tree in Figure 13B. This tree indicates that the proto-spa/fas gene originated in a Eumycetozoan ancestor after its divergence from other Amoebozoans, and then underwent a duplication early in Eumycetozoan evolution.
The lack of a fas gene in A. subglobosum is intriguing, given that stalk cells were lost in the genus Acytostelium: these dictyostelids form stalks consisting of only the secreted stalk tube material. However, reciprocal translated nucleotide BLAST searches against available unannotated dictyostelid genomes identified fas orthologs in all dictyostelid species (Figure 13C-
D), including A. leptosomum and Rostrostelium ellipticum—a species that lost its stalk cells in an independent event. Thus, fas genes are conserved across Dictyostelia.
fasA and fasB evolved through a segmental duplication
To identify the type of duplication that gave rise to fasA and fasB, we compared the genes neighboring fas genes in D. discoideum, D. purpureum, and P. violaceum (Table 4). As shown in
Figure 14, there is extensive synteny between the Dictyostelium discoideum and Dictyostelium purpureum fasA regions, as is consistent with the results of Sucgang et al. [65]. In addition, the gene neighboring P. violaceum fas is homologous to the gene adjacent to fasB in D. discoideum and D. purpureum; however, beyond this pair, there is no further synteny between any of these genomic regions. This data indicates that there was a segmental duplication of the proto-fasA/B
35
Locus of interest Gene at or near locus Putative orthologs in other Dictyosteliaceae species DDB_G0286443 DICPUDRAFT_159871 CYY_001647 Chromosome 4, 4513947-4513273 Scaffold 633, 7285-6645 Contig 141, 6096-5497 DDB_G0286445 DICPUDRAFT_158742, CYY_001579 Chromosome 4, 4518430-4519368 Scaffold 469, 12439-11557 Contig 136, 51880-55631 DDB_G0286447 DICPUDRAFT_42559, CYY_001605 Chromosome 4, 4522001-4520436 Scaffold 469, 10036-10485 Contig 137, 45962..47413 DDB_G0286449 DICPUDRAFT_158740, CYY_001606 Dictyostelium Chromosome 4, 4526125-4523376 Scaffold 469, 6817..9683 Contig 137, 50958-48476 discoideum DDB_G0286351 (fasA) DICPUDRAFT_ 95900 (fasA) CYY_001607 fasA locus region Chromosome 4, 4526380-4528203 Scaffold 469, 3846-5765 Contig 137, 52763-51216 Chromosome 4, DDB_G0295785 DICPUDRAFT_100068 CYY_001609 4513270 - 4545410 Chromosome 4, 4536016-4537272 Scaffold 633, 447-2356 Contig 137, 60012-60898 DDB_G0295783 None None Chromosome 4, 4538199-4538839 DDB_G0295781 DICPUDRAFT_85249 CYY_001301 Chromosome 4, 4543476-4542261 Scaffold 633, 5,291-6,373 Contig 126, 18126-19200 DDB_G0295787 (hbx11) DICPUDRAFT_154980 CYY_001080 Chromosome 4, 4545405-4544537 Scaffold 223, 51506-50533 Contig 119, 43681-42673 DICPUDRAFT_99765 DDB_G0283787 CYY_000380 Scaffold 469, 19698-15737 Chromosome 4, 1103072-1106881 Contig 88, 24718-21001 DICPUDRAFT_5561 DDB_G0286621 (fncJ) CYY_001302 Scaffold 469, 15137-12915 Chromosome 4, 4729555-4732791 Contig 126, 22167-19265 DICPUDRAFT_158742 DDB_G0286445 CYY_001579 Scaffold 469, 12439-11557 Chromosome 4, 4518430-4519368 Contig 136, 51880-55631 Dictyostelium DICPUDRAFT_14104 DDB_G0286447 CYY_001605 purpureum Scaffold 469, 10840-10995 Chromosome 4, 4522001-4520436 Contig 137, 45962..47413 fasA locus region DICPUDRAFT_42559 DDB_G0286447 CYY_001605 Scaffold 469, Scaffold 469, 10036-10485 Chromosome 4, 4522001-4520436 Contig 137, 45962..47413 19700-0 DICPUDRAFT_158740 DDB_G0286449 CYY_001606 Scaffold 469, 6817-9683 Chromosome 4, 4526125-4523376 Contig 137, 50958-48476 DICPUDRAFT_84337 DDB_G0286449 CYY_001606 Scaffold 469, 5920-6315 Chromosome 4, 4526125-4523376 Contig 137, 50958-48476 DICPUDRAFT_ 95900 (fasA) DDB_G0286351 (fasA) CYY_001607 Scaffold 469, 3846-5765 Chromosome 4, 4526380-4528203 Contig 137, 52763-51216 DDB_G0270312 DICPUDRAFT_27790 CYY_006096 Chromosome 1, 4616748-4618103 Scaffold 26, 48805-49888 Contig 416, 28727-29729 DDB_G0270310 NONE NONE Chromosome 1, 4614754-4615947 DDB_G0270308 DICPUDRAFT_77192 NONE Chromosome 1, 4613378-4611669 Scaffold 66 , 57467-58999 Dictyostelium DDB_G0270306 (fasB) DICPUDRAFT_78149 (fasB) CYY_001607 discoideum Chromosome 1, 4610085-4611533 Scaffold 95, 6699-8036 Contig 137, 52763-51216 fasB locus region DDB_G0270304 DICPUDRAFT_32295 CYY_001608 Chromosome 1, Chromosome 1, 4608218-4607306 Scaffold 95, 5170-4498 Contig 137, 54966-56318 4616750-4603680 DDB_G0270302 DICPUDRAFT_78876 NONE Chromosome 1, 4606819-4605944 Scaffold 120, 44856-44177 DDB_G0270300 NONE NONE Chromosome 1, 4605654-4604758 DDB_G0269234 (act8) DICPUDRAFT_91068 CYY_009488 Chromosome 1, 4602557-4603687 Scaffold 5, 198752-150019 Contig 1013, 611-3463 DICPUDRAFT_151387 NONE NONE Scaffold 95, 10360-10603 DICPUDRAFT_151386 DDB_G0269658 CYY_009471 Scaffold 95, 9936-9045 Chromosome 1, 3247871-3248759 Contig 1004, 9521-8615 Dictyostelium DICPUDRAFT_151385 DDB_G0270360 (bud31) CYY_003508 purpureum Scaffold 95, 8297-8801 Chromosome 1, 4710295-4711059 Contig 224, 37173-36586 fasB locus region DICPUDRAFT_78149 (fasB) DDB_G0270306 (fasB) CYY_001607 Scaffold 95, Scaffold 95, 6699-8036 Chromosome 1, 4610085-4611533 Contig 137, 52763-51216 10620-0 DICPUDRAFT_32295 DDB_G0270304 CYY_001608 Scaffold 95, 5170-4498 Chromosome 1, 4608218-4607306 Contig 137, 54966-56318 DICPUDRAFT_47276 DDB_G0290825 (chlA) CYY_002059 Scaffold 95, 2165-4421 Chromosome 5, 4647665-4649476 Contig 159, 24978-23176 CYY_001605 DDB_G0286447 DICPUDRAFT_42559 Contig 137, 45962..47413 Chromosome 4, 4522001-4520436 Scaffold 469, 10036-10485 CYY_001606 DDB_G0286449 DICPUDRAFT_158740 Polysphondylium Contig 137, 50958-48476 Chromosome 4, 4526125-4523376 Scaffold 469, 6817..9683 violaceum CYY_001607 DDB_G0286351 (fasA) DICPUDRAFT_ 95900 (fasA) fasA locus region Contig 137, 52763-51216 Chromosome 4, 4526380-4528203 Scaffold 469, 3846-5765 Contig 137, 45950-60900 CYY_001608 DDB_G0270304 DICPUDRAFT_32295 Contig 137, 54966-56318 Chromosome 1, 4608218-4607306 Scaffold 95, 5170-4498 CYY_001609 DDB_G0295785 DICPUDRAFT_100068 Contig 137, 60012-60898 Chromosome 4, 4536016-4537272 Scaffold 633, 447-2356 Table 4: Results of reciprocal homology searches for genes neighboring fas genes in the
Dictyosteliaceae. 36
Figure 14: Synteny of fas genomic regions in the Dictyosteliaceae. Shaded regions connect homologous genes.
gene and its neighbor, the DDB_G0270304 gene, in a common ancestor of all species of
Dictyostelium. This was immediately followed by the loss of the DDB_G0270304 copy at the ancestral locus. Later genomic rearrangements resulted in the in different locations of the fasB+DDB_G0270304 segment in the D. discoideum and D. purpureum genomes.
Evolution of fas gene sequence and regulation
To attempt to understand the roles of fas genes in other species, I used compared available developmental and cell type expression data. As shown in Table 5, fas genes are enriched in stalk cells of all dictyostelid species for which data is available; however, stalk cell expression levels are far lower in P. violaceum than in the other species. fas genes are upregulated during sorocarpic development across Dictyostelia (Figure 10). fasA and fasB expression are similar in Dictyostelium discoideum and Dictyostelium purpureum, while proto- fasA/B genes show, overall, a pattern of expression similar to that of Dictyostelium fasB.
Cell type expression data are available for Dictyostelium purpureum prespore and presomatic cells (Table 6). While D. purpureum fasB is enriched in presomatic cells, much like 37
Cell type expression (TPM) Gene vegetative amoebas Stalk cells Spores CYY_001607 3.93 6.46 0.24 DLA_05575 0.16 373.43 4.33 PPL_04431 0 27.90 0.00 Table 5: Cell type expression of fas genes in non-Dictyostelium dictyostelids. Data from multiple sources; see Table 1.
Precursor cell expression (TPM) Gene Presomatic cells Prespore cells DICPUDRAFT_ 95900 (fasA) 272.9 218.52 DICPUDRAFT_78149 (fasB) 26.06 2.05 Table 6: Precursor cell expression of fas genes in Dictyostelium purpureum. Data from [60].
Figure 15: Expression of fas genes during sorocarpic development in a range of dictyostelids. Data from multiple sources; see Table 1. 38 its D. discoideum ortholog, high expression of fasA is observed in both presomatic and prespore cells in this species. These results call into question whether fasA plays a role in cup cell differentiation in all members of Dictyostelium. Altogether, these data suggest that fas plays a role in sorocarpic development across the Dictyostelia, but expression patterns appear to have changed at various points in the Dictyosteliaceae.
The varying expression patterns observed in Dictyostelium and Polysphondylium may be indicative of changes in the roles of fas genes. Changes in protein structure often contribute to such events. To look or evidence of significant amino acid sequence change, I used aBSREL to infer the ratio of nonsynonymous to synonymous mutations (ω) over all of the branches in a gene tree of complete, conserved fas sequences. The test detected statistically significant ω along three branches: D. discoideum fasA, D. discoideum fasB, and Dictyostelium fasB (Figure 16).
Regulatory changes can also contribute to the changing expression patterns. Attempts to identify the transcription factor binding sites upstream of fas genes were unsuccessful; predicted binding sites are available for only 40 Dictyostelium transcription factors (out of hundreds), as relatively few display high similarity to metazoan, yeast, or plant transcription factors that have been thoroughly characterized.
Overall, these results indicate that fas gene evolution has involved changes in gene expression patterns and function, but these modification events do not seem to correspond to the single predicted origin of cup cells at the base of Dictyostelium. 39
DICPUDRAFT_95900
DDB_G0286351
DICPUDRAFT_78149
DDB_G0270306
CYY_001607
DLA_05575
PPL_04431
0.09
Figure 16: Phylogenetic tree of results of branch test for selection. Tree inferred using the
LG+I+G4 model of protein evolution. Evidence of statistically-significant positive selection on the coding sequence, assessed using aBSREL [90], was found for branches shaded in red.
40
Discussion
The fasA- phenotype is distinct from that caused by perturbed DIF-1 signaling
The results of this project indicate that a spaA-like transcription factor, fasA, is required for proper cup development. fasA- ALCs fail to sort to the bottom of the sorus (Figure 10B-C).
In addition, the anterior cells separate from the top of the sorus (Figure 10C-D). These perturbations result in the sorus failing to rise up the entirety of the stalk (Figure 5B, 6), and, in some cases, sliding down to the substrate (Figure 10D). Several Dictyostelium mutants have previously been identified that show a “cupless” phenotype. Most of these mutants have perturbed DIF-1 signaling. The polyketide DIF-1 plays a critical role in ALC differentiation.
Biosynthesis of DIF-1 in D. discoideum requires the polyketide synthase Steely B (StlB) and an o-methyltransferase (DmtA). The lack of DIF-1 in stlB- and dmtA- results in a loss of many
ALCs in these strains [21]; the accumulation of a chemical intermediate in dmtA- causes the additional loss of PstO cells. Thus, these strains lack lower cups and basal discs, two structures derived entirely from ALCs. The presence of an functioning upper cup in dmtA- is likely due to the persistence of the PstU cells, a subpopulation of ALCs that can differentiate in the absence of
DIF-1 [18]. Multiple transcription factor genes have been identified that, when knocked out, also result in fruiting bodies without lower cups or basal discs. These include the basic leucine zipper transcription factors DimA and DimB [91-94]; the myb domain -containing protein MybE [95,
96]; and the GATA-binding transcription factor GtaC [91]. Like stlB- and dmtA- strains, strains lacking these genes have fewer ALCs; however, their phenotype cannot be rescued by treatment with DIF-1—these transcription factors are responsible for activating ALC differentiation in response to DIF-1. Thus, all previously identified components of the ALC-to-cup lineage are 41 involved in the initial generation of ALCs, and not in ALC activity during culmination, which is the stage at which fasA plays a role.
Another difference between previous “cupless” mutants perturbed in DIF-1 signaling and fasA- is the presence of a basal disc. The generation of a normal basal disc but absence of precup cell sorting is unexpected, given that a close relationship between the formation of the lower cup and basal disc has long been inferred. The ALCs that become the basal disc and those that become the lower cup are derived from some of the same ALC subpopulations; cells that will contribute to the lower cup and some of the cells that will contribute to the basal disc sort down simultaneously during early culmination [23]. There may be cells within these subpopulations with a “disc fate”, the downward migration of which is distinct from that of similar cells with the alternate “cup fate”. It is also possible that in fasA-, none of the cells in these subpopulations migrate, and the ALC subpopulation that contributes exclusively to the basal disc—the rearguard cells—are the source of the fasA- basal disc.
There may be one previously characterized strain that, like fasA-, lacks cups but has a basal disc: regA-. RegA is the intracellular cAMP phosphodiesterase in Dictyostelium discoideum. RegA breaks down cAMP through interactions with PKA [97]. Since cAMP and
PKA are critical regulators of multiple steps of Dictyostelium development, regA- develops precociously, forming a fruiting body more rapidly than wildtype cells [98]. The mature fruiting body of regA- consists of a sorus sitting at the base of the stalk, with a small mass of cells found at the tip of the stalk: a gross phenotype similar to that seen in Figure 10D. In situ hybridization reveals that expression of ecmA and ecmB is missing in the regA- cups, but not the basal disc, during culmination, and that regA is highly expressed in the cups [99]. A β-galactosidase reporter assay reveals that ecmA-expressing cells are found scattered within the fallen sorus of the mature 42 fruiting body [98], not unlike NR-stained cells in fasA-. This suggests an autonomous role for
RegA in cup cell differentiation, likely in suppressing the stalk or spore differentiation pathways downstream of PKA. However, more work with regA- is certainly needed to assess its role in cup cells; in spite of the recognition of the central role of RegA in regulating Dictyostelium development, little attention has been paid to this aspect of its function.
fasA in the cup cell pathway
The results of this project make it clear that fasA is not involved in ALC differentiation, but a later event. However, my experiments provide no further information on the molecular context of FasA. What are the targets of FasA? And what signals induce fasA activation?
Previous studies provide some clues as to genes that may be dependent on fasA for expression. For decades developmental biologists have recognized that two cell types will spontaneously sort if mixed together. Differential adhesion results in the one cell type with lower adhesiveness moving to the outside of cell mass and entirely enveloping the other cell type—the most thermodynamically stable arrangement [100]. In D. discoideum, the secreted protein
AmpA, which has negative effects on adhesion, demonstrates high expression in the cups and low expression in the prespore/spore zone during culmination [101, 102]. When ampA- and wildtype cells are mixed, ampA- cells are excluded from the cups, indicating that ampA expression contributes to the sorting of cells to these zones [103]. However, ampA- is not necessary for cup formation. In addition to differential AmpA production, other differences in
ALC/cup and prespore/spore adhesion, caused by unequal expression of classical transmembrane cell-cell adhesion proteins, likely contribute to sorting. The expression of ampA and the suppression of adhesion genes in sorting ALCs is probably due to FasA activity. 43
Given that the two cups are always found precisely at the poles, chemotaxis likely also plays a role in cup formation: orientation of sorting cells towards the top and bottom of the sorogen could readily account for their localization. Thus, FasA may additionally activate the expression of genes involved in chemotaxis. The expression of genes with predicted roles in taxis has been detected in cup cells [41].
Though this project only demonstrated essentiality for sorting, FasA may also be a transcriptional effector of the differentiation of cup cells. Distinct cell states for the ALCs and cup cells has been inferred, based on their distinct patterns of gene expression [96, 104], and the unusual tendency of the cups to carry upwards any object placed between them, whether the spores or a small piece of agar [29]. However, it is not clear exactly when the transition from
ALC to cup cell occurs, or what causes it. It could be that cell-cell interactions of localized ALCs induce their differentiation; or, that, cup cell differentiation occurs independently of their migration, and cup cells were present in fasA-, but were ineffective in raising the sorus because of their separation and incorrect location. However, NR-stained cells were still present at the site of the upper cup in fasA- (Figure 10C). While I did not determine whether or not PstU cells sort upwards in fasA-, at the very least, PstO cells are present in the upper cup zone; yet the sorus does not rise. This observation suggests that cup cell differentiation is also perturbed in fasA-, and that the expression of genes essential for sorting may be a component of the differentiation of the cup cells. If this is the case, than the transcriptional targets of fasA may include genes involved in the elevatory role of the upper cup and supportive role of the lower cup. FasA is unlikely to regulate the cup maturation genes that are apparently expressed only after development has ceased [41, 105]. 44
The signal regulating fasA expression is a similarly open question. ALCs are found scattered amongst spores, and so would be exposed to the same extracellular signals as spores.
The peptide SDF-2 and cytokinin discadenine contribute spore formation [106, 107], but neither could be the inducer of cup cell differentiation: an undifferentiated sorus ascends the stalk normally in the dhkA-/dhkB- strain that is unable to respond to these extracellular signals [108].
SDF-1, another sporulation-inducing signal produced at culmination [109], or c-di-GMP, the signal that induces stalk cell differentiation [105], are possible cup cell inducers. This process also could be moderated by a currently unidentified signal. However, amongst known dictyostelid signals, the most likely to regulate cup cell differentiation is ammonia.
Dictyostelid slugs show the capacity for regeneration: if the anterior a slug is removed, it is rapidly replaced. It was long thought that this is possible because ALCs can migrate forward and become the new PstAs. The regulation of this process was explored by Feit et al. [110]. They found that extracellular ammonia suppresses the chemotaxis of isolated ALCs to cAMP to a greater degree than it suppresses chemotaxis of isolated PstA cells. In addition, they found that if thin sections were isolated from the posterior of slugs, squashed, and exposed to cAMP, the
ALCs would sort to the periphery of the mass; however, if the squashes were exposed to both cAMP and ammonia, the ALCs remain scattered amongst the prespore cells. Feit et al. hypothesized that ammonia emanating from the tip suppresses the forward movement of ALCs when the tip is still present, and that, upon amputation of the tip and a drop in ammonia levels,
ALCs are able to move forward and form a new tip. In addition, they hypothesized that the drop in extracellular ammonia levels that initiates culmination also causes ALC-to-cup sorting by the same chemotactic mechanism involved in PstA replacement. Since there are two organizing centers during early culmination—the major PstA tip organizing center, as well as a transient 45 organizing center located around the differentiating basal disc [28]—their model could account for the formation of both the upper and lower cups.
However, a recent application of high definition imaging and fluorescent labeling has disproven the hypothesis of ALC-based anterior regeneration by demonstrating that the tip is regenerated through the transdifferentiation of prespore cells [111]. ALCs do not sort in response to the loss of the tip—they remain exactly where they are. This suggests that there is no drop in ammonia levels upon tip amputation; only upon the initiation of culmination—when ALCs not just sort but also differentiate—do ammonia levels drop. Thus, there is no indication that ALCs are simply suppressed PstA cells, an old assumption that was partially based on the incorrect belief that the same signal, DIF-1, is involved in the differentiation of both of these populations.
Thus, it is unlikely that ammonia moderates in ALCs a chemotactic process common to ALCs and PstA cells. Rather, it may be that a drop in ammonia levels activates a differentiation process in ALCs that involves an increase in their individual chemotactic process. Feit et al.’s in vitro slug slice assays support this model: in positive samples (+cAMP, -ammonia), ALCs were not evenly distributed along the periphery of the cell mass, but clumped on one side—in a crescent shape highly reminiscent of a cup—suggesting that not just chemotaxis, but also differential adhesion occurred. Further research, perhaps utilizing this very assay, is necessary to identify the signal inducing cup differentiation, and assess whether ALCs differentiate into cup cells upon a drop in ammonia levels.
fasB plays a role in stalk formation
The precise function of fasB remains an enigma. Some aspect of the prestalk/stalk pathway is evidently perturbed is fasB-, although stalk cells appear indistinguishable from those 46 of the parental strain. Given that fasB- sorogens often produce long stretches of thin stalk, followed by short thick, stalk segments (Figure 7D), variable flow of prestalk cells into the tube could be the cause of the fasB- defect. Disruption of the adhesion protein gene dtfA- results in the formation of fruiting bodies with twisted stalks like those of fasB- sorocarps. Thus, movement of prestalk cells into the stalk tube may be mediated by changes in cell-cell adhesion at the tip; like fasA, fasB might regulate expression of adhesion genes. However, phenomena besides aberrant adhesion dynamics could also be responsible for the fasB- phenotype. Diminishment of the PstA population through treatment with the polyketide synthase inhibitor cerulenin results in some sorogens producing very thin stalks, so perhaps the PstA population is impacted in fasB-, which could be related to its slightly aberrant NR-staining patterns. Further work is necessary to clarify the role of fasB-.
No relationship between fas gene duplication and the origination of the cup cell?
Given that transcription factor duplication events can contribute to cell type evolution, I sought to characterize the function and evolution of fasA, a TF highly expressed in a lineage- specific cell type, and its paralog, which demonstrates enriched expression in the sister cell type.
However, a high relative rate of nonsynonymous mutations (ω>1) is not predicted to have acted on the fasA gene following the duplication. ω is often utilized as a proxy for assessing neofunctionalization; however, the lack of a high ω does not prove that neofunctionalization did not occur. Changes in gene regulation can also contribute to neofunctionalization, especially in the case of transcription factors, which can target different genes in different molecular contexts
[112]. Whether there are conserved differences in the transcriptional regulation of fasA and fasB could not be assessed due to the shortcomings of my enhancer screen. However, the differences 47 in precursor cell expression levels between D. discoideum (Table 3) and D. purpureum (Table 6) suggest that changes in fasA regulation may have occurred after the divergence of these species, and, therefore, significantly after the fas duplication event. These results leave open the possibility that fasA was only recruited to a role in the regulation of the cup differentiation late in its evolution.
Similarly, I found evidence that fasB function has undergone changes with no evident relation to cup cell origination. Fas genes show stalk cell specificity in species in both major branches of Dictyostelia (Table 5), and fasB is enriched in stalk cells in D. discoideum (Table 3) and D. purpureum (Table 6). However, the positive selection on fasB sequence following the fas gene duplication (Figure 16), as well as the low expression of fas in Polysphondylium stalk cells
(Table 5), suggests that fas may not have played a role in stalk formation at the base of the
Dictyosteliaceae.
It is not uncommon for transcription factor roles to change over time. Through the stepwise replacement of transcription factors, expression of a target gene can remain largely unchanged even as the underlying transcriptional network is entirely modified [113]. Thus, the phenotypic roles of TFs are generally not conserved over large evolutionary distances. Despite the high phenotypic similarity of dictyostelids, they are an ancient and genetically diverse group: the genus Dictyostelium alone is estimated to have as much genomic diversity as the jawed vertebrates (Gnathostomata) [60].
Recent advances have made genetic transformation possible in non-model dictyostelid strains [69, 114]. In the future, the role of fas genes, and other cup-expressed transcription factors, should be characterized in a range of species to understand how these regulators were recruited to the new cell type and how their roles changed over time. In addition, the regulation 48 of cup cell differentiation should be compared to the also poorly understood regulation of the differentiation of the stalk cell—the proposed sister cell type to the cup cell—as well as other cell types that may be related to cup cells. For example, Polysphondylium violaceum possesses
NR-staining cells within its prespore zone [115]. Like Dictyostelium ALCs, they apparently move to the bottom of the rising sorogen, where they contribute to the prewhorl cell masses that give rise to the lateral branches characteristic of the genus. The sorting of this transient cell type
(all “Polysphondylium ALCs” end up as stalk cells or spores) may be homologous to cup cell sorting. Overall, piecing together the evolution of a new cell type requires data from a range of species; however, this type of work promises to allow us to understand the evolution of development on a far finer scale than ever before.
49
Sources
1. Arendt, D., Musser, J. M., Baker, C. V., Bergman, A., Cepko, C., Erwin, D. H., Pavlicev, M.,
Schlosser, G., Widder, S. & Laubichler, M. D. (2016) The origin and evolution of cell types,
Nature Reviews Genetics. 17, 744.
2. Wagner, G. P. (2014) Homology, Genes, and Evolutionary Innovation, Princeton University
Press.
3. Hobert, O. & Kratsios, P. (2019) Neuronal identity control by terminal selectors in worms, flies, and chordates, Current opinion in neurobiology. 56, 97-105.
4. Han, X., Wang, R., Zhou, Y., Fei, L., Sun, H., Lai, S., Saadatpour, A., Zhou, Z., Chen, H. &
Ye, F. (2018) Mapping the mouse cell atlas by microwell-seq, Cell. 172, 1091-1107. e17.
5. Vickaryous, M. K. & Hall, B. K. (2006) Human cell type diversity, evolution, development, and classification with special reference to cells derived from the neural crest, Biological reviews. 81, 425-455.
6. Eitel, M., Francis, W. R., Varoqueaux, F., Daraspe, J., Osigus, H.-J., Krebs, S., Vargas, S.,
Blum, H., Williams, G. A. & Schierwater, B. (2018) Comparative genomics and the nature of placozoan species, PLoS biology. 16.
7. Sebé-Pedrós, A., Chomsky, E., Pang, K., Lara-Astiaso, D., Gaiti, F., Mukamel, Z., Amit, I.,
Hejnol, A., Degnan, B. M. & Tanay, A. (2018) Early metazoan cell type diversity and the evolution of multicellular gene regulation, Nature ecology & evolution. 2, 1176-1188.
8. Sebé-Pedrós, A., Saudemont, B., Chomsky, E., Plessier, F., Mailhé, M.-P., Renno, J., Loe-
Mie, Y., Lifshitz, A., Mukamel, Z. & Schmutz, S. (2018) Cnidarian cell type diversity and regulation revealed by whole-organism single-cell RNA-Seq, Cell. 173, 1520-1534. e20. 50
9. Siebert, S., Farrell, J. A., Cazet, J. F., Abeykoon, Y., Primack, A. S., Schnitzler, C. E. &
Juliano, C. E. (2019) Stem cell differentiation trajectories in Hydra resolved at single-cell resolution, Science. 365, eaav9314.
10. Smith, C. L., Varoqueaux, F., Kittelmann, M., Azzam, R. N., Cooper, B., Winters, C. A.,
Eitel, M., Fasshauer, D. & Reese, T. S. (2014) Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens, Current Biology. 24, 1565-1572.
11. Arendt, D., Bertucci, P. Y., Achim, K. & Musser, J. M. (2019) Evolution of neuronal types and families, Current opinion in neurobiology. 56, 144-152.
12. Wagner, G. P., Erkenbrack, E. M. & Love, A. C. (2019) Stress-induced evolutionary innovation: A mechanism for the origin of cell types, BioEssays. 41, 1800188.
13. Brunet, T., Fischer, A. H., Steinmetz, P. R., Lauri, A., Bertucci, P. & Arendt, D. (2016) The evolutionary origin of bilaterian smooth and striated myocytes, Elife. 5, e19607.
14. Shafer, M. E. R. (2019) Cross-species analysis of single-cell transcriptomic data, Frontiers in cell and developmental biology. 7, 175.
15. Adl, S. M., Bass, D., Lane, C. E., Lukeš, J., Schoch, C. L., Smirnov, A., Agatha, S., Berney,
C., Brown, M. W. & Burki, F. (2019) Revisions to the classification, nomenclature, and diversity of eukaryotes, Journal of Eukaryotic Microbiology. 66, 4-119.
16. Raper, K. B. (2014) The Dictyostelids, Princeton University Press.
17. Jermyn, K. & Williams, J. (1991) An analysis of culmination in Dictyostelium using prestalk and stalk-specific cell autonomous markers, Development. 111, 779-787.
18. Yamada, Y., Kay, R. R., Bloomfield, G., Ross, S., Ivens, A. & Williams, J. G. (2010) A new
Dictyostelium prestalk cell sub-type, Developmental biology. 339, 390-397. 51
19. Kawabe, Y., Du, Q., Schilde, C. & Schaap, P. (2019) Evolution of multicellularity in
Dictyostelia, International Journal of Developmental Biology. 63, 359-369.
20. Alvarez-Curto, E., Saran, S., Meima, M., Zobel, J., Scott, C. & Schaap, P. (2007) cAMP production by adenylyl cyclase G induces prespore differentiation in Dictyostelium slugs,
Development. 134, 959-966.
21. Saito, T., Kato, A. & Kay, R. R. (2008) DIF-1 induces the basal disc of the Dictyostelium fruiting body, Developmental biology. 317, 444-453.
22. Sato, Y. G., Suarez, T. & Saito, T. (2016) Stalk cell differentiation without polyketides in the cellular slime mold, Bioscience, biotechnology, and biochemistry. 80, 1368-1374.
23. Jermyn, K., Traynor, D. & Williams, J. (1996) The initiation of basal disc formation in
Dictyostelium discoideum is an early event in culmination, Development. 122, 753-760.
24. Grimson, M. J., Haigler, C. H. & Blanton, R. L. (1996) Cellulose microfibrils, cell motility, and plasma membrane protein organization change in parallel during culmination in
Dictyostelium discoideum, Journal of cell science. 109, 3079-3087.
25. George, R., Hohl, H. & Raper, K. (1972) Ultrastructural development of stalk-producing cells in Dictyostelium discoideum, a cellular slime mould, Microbiology. 70, 477-489.
26. Bonner, J. T. (1944) A descriptive study of the development of the slime mold
Dictyostelium discoideum, American Journal of Botany, 175-182.
27. Early, A. E., Gaskell, M. J., Traynor, D. & Williams, J. G. (1993) Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination, Development. 118, 353-362.
28. Dormann, D., Siegert, F. & Weijer, C. J. (1996) Analysis of cell movement during the culmination phase of Dictyostelium development, Development. 122, 761-769. 52
29. Sternfeld, J. (1998) The anterior-like cells in Dictyostelium are required for the elevation of the spores during culmination, Development genes and evolution. 208, 487-494.
30. Yamada, Y., Cassidy, A. & Schaap, P. (2018) The transcription factor spores absent a is a
PKA dependent inducer of Dictyostelium sporulation, Scientific reports. 8, 1-13.
31. Hopper, N. A., Anjard, C., Reymond, C. D. & Williams, J. G. (1993) Induction of Terminal
Differentiation of Dictyostelium by Camp-Dependent Protein-Kinase and Opposing Effects of
Intracellular and Extracellular Camp on Stalk Cell-Differentiation, Development. 119, 147-154.
32. Maeda, M. (1988) Dual Effects of Camp on the Stability of Prespore Vesicles and 8-Bromo
Camp-Enhanced Maturation of Spore and Stalk Cells of Dictyostelium discoideum, Dev Growth
Differ. 30, 573-587.
33. Williams, J. G. (2006) Transcriptional regulation of Dictyostelium pattern formation, EMBO reports. 7, 694-698.
34. HAYASHI, M. & TAKEUCHI, I. (1981) Differentiation of various cell types during fruiting body formation of Dictyostelium discoideum, Development, Growth & Differentiation. 23, 533-
542.
35. Du, Q., Kawabe, Y., Schilde, C., Chen, Z. H. & Schaap, P. (2015) The Evolution of
Aggregative Multicellularity and Cell-Cell Communication in the Dictyostelia, J Mol Biol. 427,
3722-33.
36. Funamoto, S., Anjard, C., Nellen, W. & Ochiai, H. (2003) cAMP-dependent protein kinase regulates Polysphondylium pallidum development, Differentiation. 71, 51-61.
37. Kawabe, Y., Schilde, C., Du, Q. & Schaap, P. (2015) A conserved signalling pathway for amoebozoan encystation that was co-opted for multicellular development, Sci Rep. 5, 9644. 53
38. Schaap, P. (2011) Evolution of developmental cyclic adenosine monophosphate signaling in the Dictyostelia from an amoebozoan stress response, Dev Growth Differ. 53, 452-62.
39. Schaap, P. (2011) Evolutionary crossroads in developmental biology: Dictyostelium discoideum, Development. 138, 387-96.
40. Schaap, P. & Schilde, C. (2018) Encystation: the most prevalent and underinvestigated differentiation pathway of eukaryotes, Microbiology. 164, 727-739.
41. Kin, K., Forbes, G., Cassidy, A. & Schaap, P. (2018) Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells, BMC
Genomics. 19, 764.
42. Schilde, C., Skiba, A. & Schaap, P. (2014) Evolutionary reconstruction of pattern formation in 98 Dictyostelium species reveals that cell-type specialization by lateral inhibition is a derived trait, EvoDevo. 5, 34.
43. Romeralo, M., Skiba, A., Gonzalez-Voyer, A., Schilde, C., Lawal, H., Kedziora, S.,
Cavender, J. C., Glöckner, G., Urushihara, H. & Schaap, P. (2013) Analysis of phenotypic evolution in Dictyostelia highlights developmental plasticity as a likely consequence of colonial multicellularity, Proceedings of the Royal Society B: Biological Sciences. 280, 20130976.
44. Kodama, Y., Shumway, M. & Leinonen, R. (2012) The Sequence Read Archive: explosive growth of sequencing data, Nucleic acids research. 40, D54-D56.
45. Afgan, E., Baker, D., Batut, B., Van Den Beek, M., Bouvier, D., Čech, M., Chilton, J.,
Clements, D., Coraor, N. & Grüning, B. A. (2018) The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update, Nucleic acids research. 46,
W537-W544. 54
46. Huang, Y., Niu, B., Gao, Y., Fu, L. & Li, W. (2010) CD-HIT Suite: a web server for clustering and comparing biological sequences, Bioinformatics. 26, 680-682.
47. Chen, S., Zhou, Y., Chen, Y. & Gu, J. (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics. 34, i884-i890.
48. Grabherr, M. G., Haas, B. J., Yassour, M., Levin, J. Z., Thompson, D. A., Amit, I., Adiconis,
X., Fan, L., Raychowdhury, R. & Zeng, Q. (2011) Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data, Nature biotechnology. 29, 644.
49. Xu, L., Dong, Z., Fang, L., Luo, Y., Wei, Z., Guo, H., Zhang, G., Gu, Y. Q., Coleman-Derr,
D. & Xia, Q. (2019) OrthoVenn2: a web server for whole-genome comparison and annotation of orthologous clusters across multiple species, Nucleic acids research. 47, W52-W58.
50. Fey, P., Dodson, R. J., Basu, S., Hartline, E. C. & Chisholm, R. L. (2019) dictyBase and the
Dicty Stock Center (version 2.0)-a progress report, International Journal of Developmental
Biology. 63, 563-572.
51. Forbes, G., Chen, Z.-h., Kin, K., Lawal, H. M., Schilde, C., Yamada, Y. & Schaap, P. (2019)
Phylogeny-wide conservation and change in developmental expression, cell-type specificity and functional domains of the transcriptional regulators of social amoebas, BMC genomics. 20, 890.
52. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms, Molecular biology and evolution. 35,
1547-1549.
53. Edgar, R. C. (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity, BMC bioinformatics. 5, 113. 55
54. Darriba, D., Posada, D., Kozlov, A. M., Stamatakis, A., Morel, B. & Flouri, T. (2020)
ModelTest-NG: a new and scalable tool for the selection of DNA and protein evolutionary models, Molecular biology and evolution. 37, 291-294.
55. Morel, B., Kozlov, A. M., Stamatakis, A. & Szöllősi, G. J. (2019) GeneRax: A tool for species tree-aware maximum likelihood based gene tree inference under gene duplication, transfer, and loss, BioRxiv, 779066.
56. Smith, M. D., Wertheim, J. O., Weaver, S., Murrell, B., Scheffler, K. & Kosakovsky Pond,
S. L. (2015) Less is more: an adaptive branch-site random effects model for efficient detection of episodic diversifying selection, Mol Biol Evol. 32, 1342-53.
57. Weaver, S., Shank, S. D., Spielman, S. J., Li, M., Muse, S. V. & Kosakovsky Pond, S. L.
(2018) Datamonkey 2.0: A Modern Web Application for Characterizing Selective and Other
Evolutionary Processes, Mol Biol Evol. 35, 773-777.
58. Fey, P., Dodson, R. J., Basu, S. & Chisholm, R. L. (2013) One stop shop for everything
Dictyostelium: dictyBase and the Dicty Stock Center in 2012, Methods Mol Biol. 983, 59-92.
59. Eichinger, L., Pachebat, J., Glöckner, G., Rajandream, M.-A., Sucgang, R., Berriman, M.,
Song, J., Olsen, R., Szafranski, K. & Xu, Q. (2005) The genome of the social amoeba
Dictyostelium discoideum, Nature. 435, 43-57.
60. Parikh, A., Miranda, E. R., Katoh-Kurasawa, M., Fuller, D., Rot, G., Zagar, L., Curk, T.,
Sucgang, R., Chen, R. & Zupan, B. (2010) Conserved developmental transcriptomes in evolutionarily divergent species, Genome Biol. 11, R35.
61. Rosengarten, R. D., Santhanam, B., Fuller, D., Katoh-Kurasawa, M., Loomis, W. F., Zupan,
B. & Shaulsky, G. (2015) Leaps and lulls in the developmental transcriptome of Dictyostelium discoideum, BMC genomics. 16, 294. 56
62. Heidel, A. J., Lawal, H. M., Felder, M., Schilde, C., Helps, N. R., Tunggal, B., Rivero, F.,
John, U., Schleicher, M., Eichinger, L., Platzer, M., Noegel, A. A., Schaap, P. & Glockner, G.
(2011) Phylogeny-wide analysis of social amoeba genomes highlights ancient origins for complex intercellular communication, Genome Res. 21, 1882-91.
63. Glöckner, G., Lawal, H. M., Felder, M., Singh, R., Singer, G., Weijer, C. J. & Schaap, P.
(2016) The multicellularity genes of dictyostelid social amoebas, Nature communications. 7, 1-
11.
64. Glockner, G. & Marwan, W. (2017) Transcriptome reprogramming during developmental switching in Physarum polycephalum involves extensive remodeling of intracellular signaling networks, Sci Rep. 7, 12304.
65. Sucgang, R., Kuo, A., Tian, X., Salerno, W., Parikh, A., Feasley, C. L., Dalin, E., Tu, H.,
Huang, E., Barry, K., Lindquist, E., Shapiro, H., Bruce, D., Schmutz, J., Salamov, A., Fey, P.,
Gaudet, P., Anjard, C., Babu, M. M., Basu, S., Bushmanova, Y., van der Wel, H., Katoh-
Kurasawa, M., Dinh, C., Coutinho, P. M., Saito, T., Elias, M., Schaap, P., Kay, R. R., Henrissat,
B., Eichinger, L., Rivero, F., Putnam, N. H., West, C. M., Loomis, W. F., Chisholm, R. L.,
Shaulsky, G., Strassmann, J. E., Queller, D. C., Kuspa, A. & Grigoriev, I. V. (2011) Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum,
Genome Biol. 12, R20.
66. Urushihara, H., Kuwayama, H., Fukuhara, K., Itoh, T., Kagoshima, H., Shin, I. T., Toyoda,
A., Ohishi, K., Taniguchi, T., Noguchi, H., Kuroki, Y., Hata, T., Uchi, K., Mohri, K., King, J. S.,
Insall, R. H., Kohara, Y. & Fujiyama, A. (2015) Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation, BMC Genomics. 16, 80. 57
67. Kang, S., Tice, A. K., Spiegel, F. W., Silberman, J. D., Panek, T., Cepicka, I., Kostka, M.,
Kosakyan, A., Alcantara, D. M. C., Roger, A. J., Shadwick, L. L., Smirnov, A., Kudryavtsev, A.,
Lahr, D. J. G. & Brown, M. W. (2017) Between a Pod and a Hard Test: The Deep Evolution of
Amoebae, Molecular Biology and Evolution. 34, 2258-2270.
68. Singh, R., Lawal, H. M., Schilde, C., Glockner, G., Barton, G. J., Schaap, P. & Cole, C.
(2017) Improved annotation with de novo transcriptome assembly in four social amoeba species,
BMC Genomics. 18, 120.
69. Narita, T. B., Kawabe, Y., Kin, K., Gibbs, R. A., Kuspa, A., Muzny, D. M., Richards, S.,
Strassmann, J. E., Sucgang, R. & Worley, K. C. (2020) Loss of the polyketide synthase StlB results in stalk cell overproduction in Polysphondylium violaceum, Genome Biology and
Evolution.
70. Schilde, C., Lawal, H. M., Kin, K., Shibano-Hayakawa, I., Inouye, K. & Schaap, P. (2019)
A well supported multi gene phylogeny of 52 dictyostelia, Molecular phylogenetics and evolution. 134, 66-73.
71. Singh, R., Schilde, C. & Schaap, P. (2016) A core phylogeny of Dictyostelia inferred from genomes representative of the eight major and minor taxonomic divisions of the group, BMC evolutionary biology. 16, 251.
72. Hillmann, F., Forbes, G., Novohradská, S., Ferling, I., Riege, K., Groth, M., Westermann,
M., Marz, M., Spaller, T. & Winckler, T. (2018) Multiple roots of fruiting body formation in
Amoebozoa, Genome biology and evolution. 10, 591-606.
73. Ehrenkaufer, G. M., Weedall, G. D., Williams, D., Lorenzi, H. A., Caler, E., Hall, N. &
Singh, U. (2013) The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation, Genome biology. 14, R77. 58
74. Loftus, B., Anderson, I., Davies, R., Alsmark, U. C. M., Samuelson, J., Amedeo, P.,
Roncaglia, P., Berriman, M., Hirt, R. P. & Mann, B. J. (2005) The genome of the protist parasite
Entamoeba histolytica, Nature. 433, 865-868.
75. Lorenzi, H. A., Puiu, D., Miller, J. R., Brinkac, L. M., Amedeo, P., Hall, N. & Caler, E. V.
(2010) New assembly, reannotation and analysis of the Entamoeba histolytica genome reveal new genomic features and protein content information, PLoS neglected tropical diseases. 4.
76. Clarke, M., Lohan, A. J., Liu, B., Lagkouvardos, I., Roy, S., Zafar, N., Bertelli, C., Schilde,
C., Kianianmomeni, A. & Bürglin, T. R. (2013) Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling, Genome biology.
14, R11.
77. Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. (2016) Near-optimal probabilistic RNA- seq quantification, Nature biotechnology. 34, 525-527.
78. Ihaka, R. & Gentleman, R. (1996) R: a language for data analysis and graphics, Journal of computational and graphical statistics. 5, 299-314.
79. Weirauch, M. T., Yang, A., Albu, M., Cote, A. G., Montenegro-Montero, A., Drewe, P.,
Najafabadi, H. S., Lambert, S. A., Mann, I. & Cook, K. (2014) Determination and inference of eukaryotic transcription factor sequence specificity, Cell. 158, 1431-1443.
80. Nguyen, N. T. T., Contreras-Moreira, B., Castro-Mondragon, J. A., Santana-Garcia, W.,
Ossio, R., Robles-Espinoza, C. D., Bahin, M., Collombet, S., Vincens, P. & Thieffry, D. (2018)
RSAT 2018: regulatory sequence analysis tools 20th anniversary, Nucleic acids research. 46,
W209-W214.
81. Ashworth, J. & Watts, D. (1970) Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture, Biochemical Journal. 119, 175-182. 59
82. Gruenheit, N., Baldwin, A., Stewart, B., Jaques, S., Keller, T., Parkinson, K., Chisholm, R.,
Harwood, A. & Thompson, C. (2019) REMI-seq: Development of methods and resources for functional genomics in Dictyostelium, bioRxiv, 582072.
83. Fey, P., Kowal, A. S., Gaudet, P., Pilcher, K. E. & Chisholm, R. L. (2007) Protocols for growth and development of Dictyostelium discoideum, Nature protocols. 2, 1307.
84. Queller, D. C. & Strassmann, J. E. (2014) Fruiting bodies of the social amoeba
Dictyostelium discoideum increase spore transport by Drosophila, BMC evolutionary biology.
14, 105.
85. Weijer, C. J., David, C. N. & Sternfeld, J. (1987) Vital staining methods used in the analysis of cell sorting in Dictyostelium discoideum in Methods in cell biology pp. 449-459, Elsevier.
86. Wong, E., Yang, C., Wang, J., Fuller, D., Loomis, W. F. & Siu, C.-H. (2002) Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell- type proportioning during Dictyostelium development, Development. 129, 3839-3850.
87. Yamada, Y., Wang, H. Y., Fukuzawa, M., Barton, G. J. & Williams, J. G. (2008) A new family of transcription factors, Development. 135, 3093-3101.
88. Fukuzawa, M., Hopper, N. & Williams, J. (1997) cudA: a Dictyostelium gene with pleiotropic effects on cellular differentiation and slug behaviour, Development. 124, 2719-2728.
89. Forbes, G., Chen, Z. H., Kin, K., Lawal, H. M., Schilde, C., Yamada, Y. & Schaap, P.
(2019) Phylogeny-wide conservation and change in developmental expression, cell-type specificity and functional domains of the transcriptional regulators of social amoebas, BMC
Genomics. 20, 890. 60
90. Smith, M. D., Wertheim, J. O., Weaver, S., Murrell, B., Scheffler, K. & Kosakovsky Pond,
S. L. (2015) Less is more: an adaptive branch-site random effects model for efficient detection of episodic diversifying selection, Molecular biology and evolution. 32, 1342-1353.
91. Keller, T. & Thompson, C. R. (2008) Cell type specificity of a diffusible inducer is determined by a GATA family transcription factor, Development. 135, 1635-1645.
92. Thompson, C. R., Fu, Q., Buhay, C., Kay, R. R. & Shaulsky, G. (2004) A bZIP/bRLZ transcription factor required for DIF signaling in Dictyostelium, Development. 131, 513-523.
93. Huang, E., Blagg, S. L., Keller, T., Katoh, M., Shaulsky, G. & Thompson, C. R. (2006) bZIP transcription factor interactions regulate DIF responses in Dictyostelium, Development. 133,
449-458.
94. Zhukovskaya, N. V., Fukuzawa, M., Yamada, Y., Araki, T. & Williams, J. G. (2006) The
Dictyostelium bZIP transcription factor DimB regulates prestalk-specific gene expression,
Development. 133, 439-448.
95. Fukuzawa, M., Zhukovskaya, N. V., Yamada, Y., Araki, T. & Williams, J. G. (2006)
Regulation of Dictyostelium prestalk-specific gene expression by a SHAQKY family MYB transcription factor, Development. 133, 1715-1724.
96. Tsujioka, M., Zhukovskaya, N., Yamada, Y., Fukuzawa, M., Ross, S. & Williams, J. G.
(2007) Dictyostelium Myb transcription factors function at culmination as activators of ancillary stalk differentiation, Eukaryotic cell. 6, 568-570.
97. Moorthy, B. S., Gao, Y. & Anand, G. S. (2011) Phosphodiesterases catalyze hydrolysis of cAMP-bound to regulatory subunit of protein kinase A and mediate signal termination,
Molecular & Cellular Proteomics. 10. 61
98. Shaulsky, G., Fuller, D. & Loomis, W. F. (1998) A cAMP-phosphodiesterase controls PKA- dependent differentiation, Development. 125, 691-699.
99. Shaulsky, G., Escalante, R. & Loomis, W. F. (1996) Developmental signal transduction pathways uncovered by genetic suppressors, Proceedings of the National Academy of Sciences.
93, 15260-15265.
100. Foty, R. A. & Steinberg, M. S. (2013) Differential adhesion in model systems, Wiley
Interdisciplinary Reviews: Developmental Biology. 2, 631-645.
101. Varney, T. R., Casademunt, E., Ho, H. N., Dolman, J. & Blumberg, D. D. (2002) A novel
Dictyostelium gene encoding multiple repeats of adhesion inhibitor-like domains has effects on cell–cell and cell–substrate adhesion, Developmental biology. 243, 226-248.
102. Casademunt, E., Varney, T. R., Dolman, J., Petty, C. & Blumberg, D. D. (2002) A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior- like cells during development of Dictyostelium, Differentiation. 70, 23-35.
103. Varney, T. R., Ho, H. & Blumberg, D. D. (2002) A novel disintegrin domain protein affects early cell type specification and pattern formation in Dictyostelium, Development. 129,
2381-2389.
104. Maeda, M., Sakamoto, H., Iranfar, N., Fuller, D., Maruo, T., Ogihara, S., Morio, T.,
Urushihara, H., Tanaka, Y. & Loomis, W. F. (2003) Changing patterns of gene expression in
Dictyostelium prestalk cell subtypes recognized by in situ hybridization with genes from microarray analyses, Eukaryotic cell. 2, 627-637.
105. Chen, Z.-H., Singh, R., Cole, C., Lawal, H. M., Schilde, C., Febrer, M., Barton, G. J. &
Schaap, P. (2017) Adenylate cyclase A acting on PKA mediates induction of stalk formation by 62 cyclic diguanylate at the Dictyostelium organizer, Proceedings of the National Academy of
Sciences. 114, 516-521.
106. Anjard, C. & Loomis, W. F. (2005) Peptide signaling during terminal differentiation of
Dictyostelium, Proceedings of the National Academy of Sciences. 102, 7607-7611.
107. Anjard, C. & Loomis, W. F. (2008) Cytokinins induce sporulation in Dictyostelium,
Development. 135, 819-827.
108. Wang, N., Söderbom, F., Anjard, C., Shaulsky, G. & Loomis, W. F. (1999) SDF-2 induction of terminal differentiation in Dictyostelium discoideum is mediated by the membrane- spanning sensor kinase DhkA, Molecular and cellular biology. 19, 4750-4756.
109. Anjard, C., Su, Y. & Loomis, W. F. (2011) The polyketide MPBD initiates the SDF-1 signaling cascade that coordinates terminal differentiation in Dictyostelium, Eukaryotic cell. 10,
956-963.
110. Feit, I. N., Medynski, E. J. & Rothrock, M. J. (2001) Ammonia differentially suppresses the cAMP chemotaxis of anterior-like cells and prestalk cells indictyostelium discoideum,
Journal of biosciences. 26, 157-166.
111. Mohri, K., Tanaka, R. & Nagano, S. (2020) Live cell imaging of cell movement and transdifferentiation during regeneration of an amputated multicellular body of the social amoeba
Dictyostelium discoideum, Developmental Biology. 457, 140-149.
112. Reiter, F., Wienerroither, S. & Stark, A. (2017) Combinatorial function of transcription factors and cofactors, Current Opinion in Genetics & Development. 43, 73-81.
113. Dalal, C. K. & Johnson, A. D. (2017) How transcription circuits explore alternative architectures while maintaining overall circuit output, Genes & development. 31, 1397-1405. 63
114. Paschke, P., Knecht, D. A., Silale, A., Traynor, D., Williams, T. D., Thomason, P. A.,
Insall, R. H., Chubb, J. R., Kay, R. R. & Veltman, D. M. (2018) Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum, PloS one. 13.
115. Fukushima, S. & Maeda, Y. (1991) Whorl Formation in Polysphondylium violaceum:
Relevance to Organization by Cyclic AMP: (cellular slime mold/Polysphondylium violaceum/pattern formation/cAMP/anterior-like cell), Development, growth & differentiation.
33, 525-533.