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HEMATOPOIESIS
From the Howard Hughes Medical Institute, Children's
Hospital, Boston, MA; Ludwig Institute for Cancer Research, Melbourne
Tumour Biology Branch, Royal Melbourne Hospital, Victoria, Australia;
Department of Molecular Biology, Princeton University, Princeton, NJ;
Department of Genetics, Medical School, Washington University, St.
Louis, MO; and Institute of Neurosciences, University of Oregon,
Eugene.
The Krüppel-like factor
(KLF) family of genes encodes
transcriptional regulatory proteins that play roles in differentiation of a diverse set of cells in mammals. For instance, the founding member
KLF1 (also known as EKLF) is required for
normal globin production in mammals. Five new KLF genes
have been isolated from the zebrafish, Danio rerio, and the
structure of their products, their genetic map positions, and their
expression during development of the zebrafish have been characterized.
Three genes closely related to mammalian KLF2 and
KLF4 were found, as was an ortholog of mammalian
KLF12. A fifth gene, apparently missing from the genome of
mammals and closely related to KLF1 and KLF2,
was also identified. Analysis demonstrated the existence of novel
conserved domains in the N-termini of these proteins. Developmental
expression patterns suggest potential roles for these zebrafish genes
in diverse processes, including hematopoiesis, blood vessel function, and fin and epidermal development. The studies imply a high degree of
functional conservation of the zebrafish genes with their mammalian homologs. These findings further the understanding of the
KLF genes in vertebrate development and indicate an ancient
role in hematopoiesis for the Krüppel-like factor
gene family.
(Blood. 2001;98:1792-1801) The Krüppel gene of
Drosophila is required for the correct formation of the
embryonic body plan through its activity as a transcriptional
repressor.1-3 Members of the Krüppel-like
factor (KLF) gene family have been discovered in
mammals; they are expressed in a wide variety of tissues and encode
both transcriptional activator and repressor proteins.4
Recent targeted-deletion experiments in mice have revealed roles for
members of the KLF family in erythroid cell
maturation,5,6 T-cell activation,7 and blood
vessel stability,8 as well as skin
permeability.9 These results indicate that the mammalian
KLF gene family plays an important role in the control of
hematopoietic and other cell type differentiation.
Although the DNA-binding activity of the highly conserved,
C-terminal zinc finger domain of KLF proteins is clear, the function of
diverged, N-terminal structures remains under investigation. Membership
in the KLF family is defined by the presence of 3 tandem zinc finger
motifs at the C-terminus of the protein with a
F/Y-X-C-X2-4-C-X3-F-X5-L-X2-H-X-R/K-X-H consensus that are connected by a characteristic linker:
T/S-G-E-R/K-P.10 This domain confers sequence-specific DNA
binding to GC-rich elements of the general structure CCN CNC
CCN,11 such as the CACCC-box,12-16 and in
vitro site selection has suggested the consensus sequence NNR CRC
CYY.17 The N-termini of KLF proteins are highly variable, and several are characterized by the presence of domains with transcriptional activator and/or repressor functions in
vitro.14-16,18-23 In addition, the N-terminus of
some KLF proteins contains sites of phosphorylation and acetylation
that may contribute to regulation of KLF activity.24,25
Despite these studies, it remains an important task to identify
functionally significant sites in KLF protein N-termini. Zebrafish is
sufficiently diverged from mammals that amino acid sites unimportant
for protein function will have diverged, leaving functionally important
sites as islands of sequence conservation. Thus, the isolation of
zebrafish homologs of the mammalian KLF genes and comparison
of the N-terminal regions would allow the demarcation and functional
testing of evolutionarily conserved domains.
Loss of Klf1 (erythroid Krüppel-like factor,
EKLF) gene function in mice results in a lethal
Isolation of zebrafish klf cDNA
Phylogenetic analysis
Phylograms were built using the output.dnd and .phb files from ClustalX in Treeview PPC.70 Genetic mapping Gene mapping on the mother of pearl (MOP) cross-meiotic panel was done as described,32-34 as was typing on the Goodfellow hybridoma panel.35In situ hybridization Riboprobes were prepared and hybridization was performed as described previously,36 with modifications.37 For each klf gene, full-length cDNA was used to generate riboprobe by cutting with EcoR1 and transcribing with T7 RNA polymerase.
To isolate the Klf1 homolog in zebrafish, we hybridized a fragment of the cDNA of mouse Klf112 to a zebrafish adult kidney cDNA library because the kidney is the site of definitive hematopoiesis in the zebrafish. Sequence analysis of 32 positive clones demonstrated that these cDNAs represented 5 independent genes, each containing Krüppel-like zinc finger motifs. In 4 cases, the assignment of probable orthologies required information from both protein phylogeny and chromosomal localization (see below, klf2a, klf2b, klf4, and klf12). For the fifth gene, these data suggested that no ortholog exists in modern mammalian genomes, and this gene is designated klfd, consistent with zebrafish nomenclature procedures. Phylogenetic and structural analysis of zebrafish klf genes In an amino acid comparison with other KLF family members, the C-terminal regions of the 5 zebrafish genes were highly conserved (Figure 1A), consisting of 3 tandem zinc fingers. However, the regions outside the finger domain strongly diverged and cannot be reliably aligned across the entire KLF family. Of 83 amino acid positions in the conserved zinc finger domain, only 19 were phylogenetically informative, which limits the resolution of phylogenetic reconstruction. A probable phylogeny of this gene family based on the relations between the zinc finger domains is presented in Figure 1B.
Zebrafish Klf2a, Klf2b, and Klf4 proteins were most similar to mammalian Klf2 (lung Krüppel-like factor, Lklf38) and Klf4 (gut Krüppel-like factor, Gklf39) (Figure 1B,C). They were more closely related to each other than to either Klf2 or Klf4, however, although low bootstrap values precluded the assignment of direct orthologies to either of these mammalian genes on the basis of sequence alone. Examination of an amino acid alignment of the N-termini of this group of proteins revealed 5 highly conserved blocks of residues separated by divergent regions (numbered brackets in Figure 1C). These are potentially functional domains and include (1) the N-terminal-most 16 amino acids, (2) an acidic domain (D20 to T37 [single-letter amino acid code] of Klf2a), (3) a proline-rich domain (P75 to R106), and (4) a conserved block (F218 to D246) immediately N-terminal to (5) a basic domain (K254 to R266). The Klf12 protein was more similar to mouse Klf12 (AP2-rep) and human KLF12 than to any other protein, including the closely related KLF3/Klf3 (BKLF/Bklf) and KLF8, which together form a clade (Figure 1B). Thus, it appears that zebrafish klf12 is the ortholog of the mammalian KLF12 genes (Figure 1B,D). Comparison of the amino acid sequences of the N-termini of these proteins revealed that they are less conserved than those of the KLF2/4 subfamily, with 2 regions of high similarity (indicated with brackets and numbers in Figure 1D): (1) from Q65 to K75 of Klf12, and (2) another cluster immediately N-terminal to the zinc finger domain (E221 to I242). Although klfd was placed at the base of the KLF2/4 subfamily by virtue of its zinc finger domain, it lies close to the stem of the Klf1-Klf2/4 divergence in Figure 1B. We note that the bootstrap replication values for the nodes in the tree at this point were sufficiently low to regard this placement as unresolved with respect to membership in the Klf1 or Klf2/4 family. To place these putatively novel zebrafish klf genes more precisely in the evolutionary history of the KLF family, we determined their chromosomal positions and potential syntenic relations to mammalian chromosomes. Genetic mapping of klf family members Each of the klf genes was genetically mapped to the zebrafish genome (Table 1). klfd, klf12, klf2b, and klf4 were analyzed by meiotic mapping on the MOP-cross DNA panel.32-34 Both klf2b and klf4 are found on linkage group 2 (LG2), whereas klf12 is found on LG1 and klfd on LG6. klf2a was mapped using a radiation hybrid panel,35 placing it on LG22. None of these positions correspond to the 6 published hematopoietic mutant positions: sauternes,40 weissherbst,41 dracula,42 yquem,43 riesling,44 or spadetail.45
The orthology assignments of the zebrafish klf genes based on amino acid sequences were reassessed and refined using information about syntenic chromosomal regions between fish and mammals34,46-48 and are described in Table 1. This analysis will be presented in depth elsewhere (manuscript in preparation), but is beyond the scope of the current report. The syntenic relations between the chromosomes containing the zebrafish klf2a, klf2b, and klf4 genes and their closest known mammalian homologs provide support for the notion that klf2a and klf2b are collectively orthologous to KLF2 and that klf4 is orthologous to the mammalian KLF4 gene. The syntenic relations between the chromosomes containing klf12 and human KLF12 support the phylogenetic analysis showing that these genes are likely orthologous. Finally, klfd is found in a chromosomal region that shares conserved synteny with both Klf1 and Klf2 genes in mammals. Although sequence analysis indicates that klfd does not have a known ortholog in mammalian genomes, it is most closely related to Klf1 and Klf2 (Figure 1B). Combined, these data suggest that klfd may be related to an ancestor of these genes. Expression of the klf genes during development While the structural comparisons, phylogenetic analyses and conserved syntenies presented above argue gene histories, expression analysis can reveal potential developmental properties of the zebrafish klf genes. The expression of each of the klf genes reported here was determined during embryonic and larval stages by whole-mount in situ hybridization.klf12, klf4, and klfd expression patterns klf12, klf4, and klfd were characterized by high-level expression in the hematopoietic system, as illustrated in Figure 2. Transcripts from each of these genes were detected in a line of cells immediately below the notochord along the ventral aspect of the trunk at 24 hours past fertilization (hpf) (Figure 2A,E,H). This region is known as the intermediate cell mass (ICM) and is the site of primitive erythropoiesis.49 The pattern of expression along the anterior/posterior axis was similar to that seen with erythrocyte markers gata1,50,51 jak2,37 and embryonic globins40,52 and did not extend into the posterior tail as seen with the stem cell marker scl,53 consistent with expression in primitive erythrocytes. To confirm that the signal was produced in hematopoietic cells, we assayed the expression of each of these genes in the cloche mutant background, which exhibits a profound defect in the hematopoietic and vascular lineages.54-56 Staining was absent from the ICM in one quarter of embryos in clutches from cloche+/ parents (Figure 2A,E,H; Figure
3D), verifying a hematopoietic site of
expression for klf12, klf4, and klfd.
Expression of klf4 and klfd in circulating
primitive erythrocytes was detected at 48 hpf (Figure 2F,I), whereas
expression of klf12 in these cells was absent by this time
(Figure 2B). Only klfd expression was detected in definitive
hematopoietic cells. By 8 days past fertilization (dpf),
transcripts were detected in the site of definitive hematopoiesis, the
pronephros, and in circulating definitive erythrocytes in the heart
lumen and vessels of the tail (Figure 2J).
The cells of the hatching gland expressed both klf12 and klf4 at 24 to 36 hpf (Figure 2A,B,E), an organ derived from the prechordal plate mesoderm, or polster, which also showed high levels of klf4 at 12 hpf (Figure 2D). By 36 hpf, klf12 was also detected in cells of the pronephric duct, being at highest levels in 2 bilaterally symmetrical clusters of cells of unknown identity at an axial position level with somite 14 (Figure 2B,C). At 24 hpf, klf4 was present in 2 paired cell clusters lateral to the hindbrain (Figure 2E), consistent with the location of the cranial ganglia of the anterior and posterior lateral line.57 This was confirmed by observations at 36 hpf of klf4 expression in the migrating lateral line primordia and in the clusters of neuromast cells deposited in its wake (Figure 2F). By 4 dpf, the full array of lateral line organs was marked by klf4 expression (Figure 2G). klf2a and klf2b expression patterns The closely related klf2a and klf2b genes were expressed during gastrula stages in the early epidermis in a distinctive, partially overlapping pattern (Figure 3). Beginning at approximately 70% epiboly, klf2a transcripts were detected in the ventral, animal portion of the epiblast (Figure 3A,B), and as epiboly progressed, this expression domain extended vegetally (Figure 3C,D). At 70% epiboly, klf2b was also expressed in the ventral, animal portion of the epiblast, but in a more extensive domain than klf2a (Figure 3E,F), extending further across the gastrula both dorsally and vegetally. In addition, a thin line of cells along the dorsal margin also expressed this gene. By the end of epiboly, klf2b expression was lost in the animal-most one third of the epiblast and was expressed at higher levels in a lateral band extending around all but the dorsal midline of the embryo (Figure 3G,H). Thus, the domain of klf2a expression in the epiblast was nested concentrically within the domain of klf2b expression. klf2b expression was maintained in the dorsal marginal cells.klf2a expression was detected at 24 hpf in the anus and in
small clusters of superficial cells lateral to the most posterior notochord (Figure 4A), as well as in
scattered cells closely associated with the axial and head vessels and
in the heart (Figure 4A,B). These expression patterns persisted until
48 hpf, and at this time a faint signal was detected from the distal
margin of the caudal fin and in the pectoral fins (Figure 4C; also see
below).
klf2b was expressed in the superficial layer of the
epidermis in large squamous cells more populous dorsally (Figure
5A). At 36 hpf, this expression was
dramatically decreased; klf2b transcripts at high level were
instead present in 2 cords of cells in the body wall immediately
anterior and ventral to the pectoral fin buds (Figure 5B). By 48 hpf,
the mesenchymal interior of each fin bud expressed klf2b
strongly (Figure 5C).
Both klf2a and klf2b were expressed in a dynamic
manner in the developing pectoral fins; the klf2b riboprobe
gave a clearer and more intense signal, but we could not otherwise
distinguish between the 2 patterns in the limb. The expression of
klf2b is illustrated in Figure
6 from 2 until 5 dpf. At 2 dpf,
klf2b is expressed in the mesenchymal interior of the fin
bud (Figure 6A). By 3 dpf, expression had decreased in the proximal
portion of the fin concomitant with the emergence of striated muscle
(Figure 6B), but was maintained in the distal portion of the
mesenchymal part of the fin. Expression was confined to a thin row
of cells at the distal tip of the striated muscles at 4 dpf (Figure
6C), and by 5 dpf, klf2b was found only in scattered
epidermal cells over the fin (Figure 6D).
In this report, we characterize KLF gene family members from a nonmammalian vertebrate. The sequences, genetic map positions, and developmental expression patterns of 5 new KLF genes represent an opportunity to analyze the evolution of the roles played by this gene family in vertebrate development. Potential structural domains in the N-termini of the KLF family proteins In contrast to the highly conserved C-termini of the KLF family, which contain 3 tandem DNA-binding zinc fingers, the N-termini are significantly diverged.4 Determination of activator and repressor functions in the N-termini of KLF proteins has required the generation of extensive deletion series through these regions.14-16,18-23 Our finding of domains of high amino acid sequence conservation between zebrafish and mammalian KLF proteins offers the possibility of guiding these searches and potentially extending them to detect previously unsuspected activities. We have delineated 5 regions of high sequence conservation in the N-terminus of Klf2/4 subfamily proteins. Three of these correspond to domains previously defined to possess activities essential for transcription factor function in mouse and human KLF4. Domain 2 is an acidic domain conferring transcriptional activation via the recruitment of p300/CBP15,23; domain 3 is a proline-rich domain containing sequences required for transcriptional repression by KLF415; and domain 5 is a basic nuclear localization signal.58 The function of the 2 novel conservation domains, including the N-terminal-most 16 amino acids and the conserved block immediately N-terminal to the potential nuclear localization signal, is currently unknown.Similarly, the alignment between zebrafish Klf12 and its closest mammalian relatives revealed 2 domains of high sequence conservation N-terminal to the zinc fingers: (1) a region that includes the mCtBP2 interaction domain as defined for Klf3,22 and (2) a short block neighboring the DNA-binding domain. This subfamily does not appear to possess the extended basic sequence immediately N-terminal to the zinc finger domain seen in the KLF2/4 group, and we speculate that this short C-terminal homology domain may confer nuclear localization instead. The N-terminus of Klfd was sufficiently diverged from the members of the KLF2/4 and KLF1 families to preclude a meaningful assessment of blocks of conserved amino acid similarity. Thus, we have identified 7 conserved domains in the N-termini of KLF2/4 and KLF3/12/8 family proteins. The close correspondence between those domains previously defined by functional testing in cell culture and those identified by evolutionary conservation suggests that the 3 novel conserved domains we have defined by comparison between zebrafish and mammalian KLF proteins may also possess important conserved functions. The differential conservation of residues within these blocks likely reflects requirements for specific amino acids in their functions. These domains would make suitable target sequences for deletion analysis or use in a yeast 2-hybrid experiment,59 for example; and because members of both protein groups are expressed in hematopoietic cells, these domains may therefore allow the characterization of previously unrecognized activities important in hematopoiesis. Comparison of developmental expression patterns of zebrafish klf genes with mammalian KLF genes The mRNA expression patterns of the zebrafish klf genes not only suggest possible functions for these genes in the development of zebrafish, but, by comparison with the expression patterns and loss-of-function phenotypes of their murine homologs, also reveal the likely expression patterns of ancestral vertebrate KLF genes. To date, loss-of-function phenotypes are known only for the Klf1, Klf2, and Klf4 genes of mice.Removing Klf1 function in the mouse results in a lethal
Additional roles can be postulated for klf4 and klf12 in the development of the hatching gland, but the identity of a homologous structure in mammals is unknown. klf4 is also expressed in the cranial ganglia of the zebrafish and in the migrating and differentiating neuromasts of the lateral line that are derived from these ganglia. The expression of murine Klf4 in cranial sensory ganglia has not been reported, suggesting that this pattern of expression may be a novel acquisition in the zebrafish lineage or has been lost in mouse. The expression of Klf4 in the mouse is notable for the high levels seen in the differentiated epithelial cells of the gastrointestinal tract39,63 and epidermis.9,14 Loss-of-function mutation in this gene results in death of mice shortly after birth from dehydration because of an impaired epidermal barrier function of the skin.9 There is no evidence of zebrafish klf4 expression in the epidermis, or any other epithelium, suggesting that the functions of these genes during embryogenesis have diverged significantly. Although no additional KLF4-related genes were detected in BLAST searches of the zebrafish EST database, we cannot rule out the existence of another zebrafish KLF4 ortholog, and we note that any gene expressed only during embryogenesis would not have been recovered in our screen of an adult kidney library. The spatial expression patterns of klf2a and klf2b in the zebrafish gastrula cannot yet be compared with those of murine Klf2 during gastrulation because the latter has not been determined, although Klf2 mRNA is detectable in the midgastrula by Northern blotting at 7 dpc.38 At later stages in mouse development, Klf2 is expressed in the vertebra and the endothelium of developing blood vessels of all sizes.8 A loss-of-function mutation in Klf2 in mouse results in the failure to form stable tunica media in the embryonic blood vessels, and the affected mice die in utero from massive hemorrhage.8 Zebrafish klf2a is expressed in cells associated with blood vessels in the head, trunk, and tail, although not with the intersegmental vessels, indicating that a function in blood vessel formation is likely to be ancestral for KLF2 genes in vertebrates. The expression of the paralogous klf2b gene is interesting because it appears to be expressed in the differentiating epidermis of the zebrafish, like Klf4 in mouse. It is possible, therefore, that epidermal expression was a feature of the ancestral vertebrate KLF2/4 gene, but was lost from klf4 and retained in klf2 in the zebrafish lineage, whereas in the mammalian lineage, epidermal expression was retained by Klf4 and lost by Klf2. This change in epidermal expression patterns may also be related to the aquatic habitat of the zebrafish, where dehydration is not such a problem and the requirements for the barrier function of the skin are likely to be different.64 The expression of Klf2 is also observed in the spleen of mice and rats,38 and mice without Klf2 function suffer additionally from hyperactivation of their single-positive T cells, which appear to die in the spleen and lymph nodes.7 We do not observe the expression of either zebrafish klf2 gene in the developing thymus; this function may be a mammalian innovation or an ancestral character retained in mammals and lost in zebrafish. Finally, we observe a striking expression pattern of klf2a and klf2b in the mesenchyme of developing pectoral fin buds, and in underlying mesenchyme of the body wall, suggesting that the klf2 orthologs may play some role in the differentiation of limb muscle.65 Expression of mouse Klf2 has not been reported in the developing limbs; therefore, the ancestry of this and other divergent expression patterns mentioned above cannot be resolved until the expression patterns of KLF2 genes in additional vertebrates are known. In summary, we have isolated the first vertebrate KLF genes known outside the mammals and have characterized the structure of their protein products, their genetic map positions, and expression patterns in the developing zebrafish. Our results identify novel conserved structural domains in the vertebrate KLF2/4 and KLF3/12 subfamilies and describe the probable evolutionary histories of these subfamilies. Furthermore, we present evidence suggesting that developmental function in epidermis formation is ancestral to the vertebrate KLF2/4 subfamily and that a function for the KLF2 gene family in blood vessel formation has been conserved in fish and mammals. Expression during erythrocyte development is seen in fish and mammalian genes throughout the KLF family, suggesting that a function in hematopoiesis is likely ancestral to all vertebrate KLF genes. The developmental roles of these genes can now be tested in the zebrafish using overexpression and antisense loss-of-function approaches.66 It will be of particular interest to determine whether any of the zebrafish klf genes expressed during erythropoiesis, in particular klfd, perform Klf1-like functions in the zebrafish. Note added in proof. During the preparation of this manuscript, Kawahara and Dawid67 reported the independent cloning of klf4 as biklf.
We thank Tracy Roskoph for fish care, Ilya Ruvinsky for discussion and advice on phylogenetic methods, and Ashley Bruce for critical comments on the manuscript.
Submitted March 19, 2001; accepted May 18, 2001.
Supported by a Postdoctoral Fellowship from the Ludwig Institute for Cancer Research (A.C.O.), National Institutes of Health (NIH) grants RO1RR10715 and 5-PO1-HD22486 (J.H.P.), the W. M. Keck Foundation 961582, M. J. Murdoch Charitable Trust 96127, National Science Foundation ST19602828 and NIH 1-G20-RR11724 (renovation of the University of Oregon zebrafish facility), and Associate Scholar of the Howard Hughes Medical Institute and NIH (L.I.Z.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Leonard I. Zon, Howard Hughes Medical Institute, Children's Hospital, Enders Building, Room 761, 320 Longwood Avenue, Boston, MA 02115; e-mail: zon{at}smtp.ne.mediaone.net.
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Abstract
Introduction
-thallassemia.
-globin to adult