Changes in Gene Expression at the Precursor Stem Cell Transition in Leech - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:37 |只看该作者 |倒序浏览 |打印

Biology Department, Rutgers, The State University of New Jersey, Camden, New Jersey, USA

Key Words. Differential display ? Embryonic stem cell ? Multipotency ? Invertebrate Teloblast ? Mesoderm ? Neuroectoderm
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Correspondence: Daniel H. Shain, Ph.D. Biology Department, Rutgers, The State University of New Jersey, 315 Penn Street, Camden, NJ 08102. Telephone: 856-225-6144; Fax: 856-225-6312; e-mail: dshain@camden.rutgers.edu4 d( w% L, Q3 ~

ABSTRACT
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Fundamental properties shared by all stem cells (e.g., self-renewal, cell type–specific propagation) are likely to be regulated by overlapping molecular pathways. To date, however, relatively few genes have been identified that are associated with stem cell formation or maintenance (e.g., esg1 , nanog , oct 4 , piwi ). Current studies are limited in part due to technical difficulties associated with purifying stem cells to homogeneity and maintaining pure populations in culture . While contemporary research is focused primarily on mammalian stem cells (SCs), we have examined gene expression profiles of stem cells in an invertebrate model, the glossiphoniid leech, Theromyzon trizonare.
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At the onset, it is important to establish the similarities between leech and mammalian embryonic stem (ES) cells, and also their differences, lest there be confusion about the semantics of two currently disparate research fields. Embryonic stem cells from both phyla (Annelida and Chordata) appear transiently during the early stages of embryogenesis, both display the potential for self-renewal under appropriate conditions , and both generate multiple cell types during development. There are clear differences, however, in the "potency" of each cell type. Thus, while mammalian ES cells display pluripotency in the embryo, descendants of leech stem cells are more restricted in cell fate. Five bilateral pairs of stem cells (M, N, O, P, Q, also known as teloblasts) generate chains of segmental founder cells that give rise to mesodermal (M), neuroectodermal (N), and ectodermal (O, P, Q) tissue (Fig. 1A); leech endoderm arises by a stem cell–independent process . M, O, and P stem cells produce two cell types, primary daughter cells and micromeres, while N and Q generate three distinct cell types, two different primary daughter cells that arise in alternation and micromeres. An O/P stem cell produces four primary daughter cells before dividing into equivalent O and P cells, whose lineages are specified by cell-cell interactions with other ectodermal progeny . Although bilateral M, N, O, P, and Q cells produce mainly germ-specific cell types, each contributes progeny to multiple germ layers (e.g., M-derived progeny appear in mesoderm and neuroectoderm; N-derived progeny appear in neuroectoderm and ectoderm, etc.) and displays the capacity to change fate .$ j3 u  r4 e3 f0 F7 V

Figure 1. Schematic of stem cell lineages and early development in leech. (A): Stem cells generate chains of daughter cells (bandlets) that coalesce and differentiate into segmental tissue. N and Q produce two distinct daughter cells in alternation (black and white cells); M, O, and P produce only one daughter cell type (white cells). (B): Precursors DM and NOPQ are born during stages 4 and 5, respectively, and give rise to 5 bilateral pairs of stem cells (M, N, O, P, and Q) by stage 7. Blackened cells were dissected from appropriate stages (~100 of each cell type).

On the basis of cell potency, stem cells in leech are more similar to mammalian adult SCs (e.g., hematopoietic, multi-potent) since their descendant progeny are restricted in cell fate. However, leech stem cells are expressed at the early stages of embryogenesis and give rise to most adult cell types, including the germ line , similar to the role of mammalian ES cells. We therefore propose the terminology LES (leech embryonic stem) cells to identify their functional role in developing embryos and to distinguish them from the unique properties that have been designated for mammalian ES cells (e.g., pluripotency).
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Embryogenesis in leech (Fig. 1B) begins with an unequal, meridional cleavage that divides the fertilized egg into a smaller AB and larger CD macromere. A second meridional cleavage forms three smaller macromeres (A, B, and C), which later fuse to form the gut , and a larger D macromere that gives rise to the segmental mesoderm and ectoderm. At around 12 hours postfertilization, the D macromere generates two precursor cells in succession, DM and NOPQ, respectively. Precursors undergo a series of highly unequal and stereotyped cell divisions giving rise to five bilateral pairs of LES cells (M, N, O, P, and Q) that are asymmetrically positioned on the embryo’s surface. LES cells divide repeatedly at the rate of about one division every hour, generating chains of segmental founder cells (bandlets; Fig. 1) that coalesce along the longitudinal axis while dividing and differentiating to form the segmental tissue .

Theromyzon offers several experimental advantages in comparison with other model systems. Most important, perhaps, is that LES cells and their respective precursors (i.e., founder cells) are among the largest cells in the animal kingdom (50–300 μm in diameter); moreover, their asymmetric position on the surface of developing embryos permits their identification and homogeneous isolation. The availability of these two identifiable cell populations (i.e., precursors and LES cells) permitted us to examine the molecular events leading up to stem cell formation, which previously have not been investigated in mammalian ES cells due largely to technical limitations. We report here dynamic changes in a novel set of genes that are turned on and off, respectively, upon the birth of embryonic SCs in leech.# R! I/ k. c0 V

MATERIALS AND METHODS
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Homogeneous populations of LES cells (M and N) and their respective precursors (DM and NOPQ) were manually dissected from appropriately staged Theromyzon embryos (~100 cells of each type; Fig. 1B). These cells were targeted based on their accessibility during development and the degree to which their lineages have been characterized . Following total RNA purification and cDNA synthesis, differential display-PCR  was conducted with around 150 primer combinations to generate a series of gene expression profiles for each cell type (Fig. 2). Each primer set generated around 70 distinct bands, resulting in the screening of more than 10,000 cDNAs, the estimated number of genes in a leech genome .5 S6 w5 a: z# O* }7 b- ~" T
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Figure 2. Differentially expressed cDNAs in Theromyzon embryonic cells. Representative autoradiograms of precursor-specific (A) and LES-cell-specific (B) cDNAs following differential display-PCR analysis; arrows identify respective bands. Bands appearing in precursor (DM, NOPQ) and LES-cell (M, N) lanes were designated "housekeeping" genes.- I$ A' x, Z& G7 S" q0 ]

Examination of DD profiles revealed that about 98% of cDNA fragments were identical between cell types (i.e., "housekeeping" genes), while 236 (~2%) were differentially expressed. Among the latter, eight categories were resolved and are presented in Figure 3. DD fragments that were expressed only in precursor cells (DM and NOPQ) were designated as precursor-specific (Fig. 2A), while those present only in M and N cells became LES cell–specific candidates (Fig. 2B). In total, 29 precursor-specific and 27 LES cell–specific cDNAs were identified.
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Figure 3. Categories of differentially displayed cDNAs. Those cDNAs expressed in both DM and NOPQ precursors but not M or N stem cells (29, dark gray) were designated as precursor-specific; cDNAs in both M and N cells but not precursors DM or NOPQ were designated as LES cell-specific (27, light gray). Note that DM, which gives rise to the bilateral M cells, contained only one differentially expressed cDNA while NOPQ, which gives rise to four stem cell types (N, O, P, and Q), contained 39 differentially expressed cDNAs; these latter cDNAs are likely to be a mixture of O, P, and Q determinants.

DD cDNAs were cloned, sequenced, and subjected to GenBank (BLAST) Basic Local Alignment Search Tool searches (Tables 1, 2). Collectively, 19 (34%) of the cDNAs were similar to reported genes, 27 (48%) produced no significant match, and 10 (18%) matched hypothetical sequences (expressed sequence tags or poorly characterized proteins). Among the putative homologues in DM and NOPQ precursors were CCR4-NOT subunit (an antiproliferation gene ), beta dynein heavy chain, a G-protein, ubiquitin-related genes, a transcriptional regulator, and an uncharacterized progenitor cell protein (Table 1). LES cell–specific homologues included Rad family members, a transcriptional regulator, a TATA-binding-protein (TBP)-associated factor, and proteins induced by either fibroblast growth factor or retinoic acid (Table 2).
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Table 1. Precursor-specific cDNAs4 \9 T, H/ u9 b3 I) G

Table 2. LES cell–specific cDNAs

To verify precursor-specific and LES cell–specific cDNAs, Northern blot analyses were performed using RNA from two distinct embryonic stages: stage 4, which contains precursors DM and DNOPQ, and stage 7, which contains all 10 LES cells (Fig. 1B). Representative Northern blots using cDNAs K224 (precursor-specific) and K243 (LES cell–specific) are shown in Figure 4, and Northern blot data is summarized in Tables 1 and 2. Although we examined all cDNAs reported here by Northern blot analysis, only 14 displayed detectable bands, suggesting that expression levels of the remaining cDNAs were below the sensitivity limits of the assay. Based on comparative analyses of gene expression in mammalian SCs, it has been proposed that stem cell–specific genes may be expressed at particularly low levels . Precursor-specific cDNAs that were confirmed by Northern blot analysis included beta dynein heavy chain, a G-protein, and an uncharacterized progenitor cell protein. Northern blots also verified LES cell–specific transcripts Rad21, a metal response transcription factor, TBP-associated factor, and a retinoic acid–inducible protein. Several novel, differentially expressed cDNAs were also corroborated by Northern blots on staged embryos. We observed no erroneous bands in the Northern blot data set (e.g., precursor-specific probe hybridizing with stage 7 RNA).7 B1 Z" z+ C$ z! D5 C7 N$ \
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Figure 4. Representative Northern blots of precursor- and LES cell–specific cDNAs. (A): Precursor cDNA K224 annealed to a ~2,300 bp transcript (left arrow) in total RNA from stage 4 embryos (i.e., containing precursors DM and DNOPQ). (B): LES cell–specific cDNA K243 annealed to ~2,600 and ~1,500 bp transcripts (right arrows) in total RNA from stage 7 embryos (containing M, N, O, P, and Q). Arrowheads indicate rRNA and demonstrate that approximately equal amounts of RNA were loaded in each lane.

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This work was supported by the Rutgers Life Science Fellowship to K.A.H. and Busch Biomedical Research Grant 6–49167 to D.H.S.

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