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1.
Figure 5

Figure 5. From: Smad1 and Smad5 differentially regulate embryonic hematopoiesis.

Early mesoderm markers are not affected by Smad1 or Smad5 knockdown. Shown are representative embryos following in situ hybridization for no tail (A-C) or pax2.1 (D-F) at the 5-somite stage in control (A,D), smad1 morphants (B,E), and piggytail mutant embryos (C,F). Pgy/pgy embryos were identified by genotyping after hybridization. Neither Smad1 nor Smad5 loss causes a significant change in the expression pattern of these markers. However, in piggytail embryos, the expression of the hematopoietic marker gata1 is expressed in a compacted pattern that forms a loop bisecting the myod-expressing domain (G,H). A total of 50 embryos per sample were analyzed.

Lisa J. McReynolds, et al. Blood. 2007 December 1;110(12):3881-3890.
2.
Figure 7

Figure 7. From: Smad1 and Smad5 differentially regulate embryonic hematopoiesis.

Smad1 can rescue the hematopoietic defect in smad5 morphants. (A) In vitro transcription-translation of 35S-methionine–labeled Smad proteins. Translation of wobbled-changed Smad1 and Smad5 (Smad1* or S1* and Smad5* or S5*) are not inhibited by their own MO or the opposite Smad MO (Smad1*, lanes 1-3; Smad5*, lanes 4-6). (B-S) Embryos were either uninjected control (left column) (B,E,H,K,N,Q), coinjected with Smad1 MO plus Smad5 mRNA (middle column) (C,F,I,L,O,R), or Smad5 MO plus Smad1 mRNA (right column) (D,G,J,M,P,S) and processed at 24 hpf for in situ hybridization to identify hematopoietic transcripts: gata1 (B-D), lmo2 (E-G), pu.1 (15 somites, panels H- J; 24 hpf, panels K-M), l-plastin (N-P), and c-myb (Q-S). Smad1 is capable of rescuing gata1 (D) (◀), lmo2 (G), pu.1 (J) (◀), and c-myb (S) (small arrow) in smad5 morphants. However, Smad5 is unable to rescue the loss of l-plastin expression in smad1 morphants (O) (long arrow). Each panel is a representative embryo of a 25-embryo sample. All images are shown whole mount. In panels B-G and L-S, anterior is to the left; in panels H-J, anterior is to the top.

Lisa J. McReynolds, et al. Blood. 2007 December 1;110(12):3881-3890.
3.
Figure 6

Figure 6. From: Smad1 and Smad5 differentially regulate embryonic hematopoiesis.

Smad1 morphants have an increase in gata1 expression, but lack l-plastin, while piggytail embryos lose most posterior hematopoietic marker expression at 24 hpf. Shown are representative embryos, 50 to 75 embryos per sample, following in situ hybridization. Uninjected control embryos (left column) (A,D,G,J,M,P,S) were compared with smad1 morphants (S1MO; middle column) (B,E,H,K,N,Q,T), and pgy/pgy mutant embryos (right column) (C,F,I,L,O,R,U) for primitive and definitive hematopoietic transcripts: gata1 (A-C), lmo2 (D-F), pu.1 (G-I), l-plastin (J-L), myeloperoxidase (mpo) (M-O), c-myb (P-R), and runx1 (S-U). At 24 hpf, smad1 morphants have an increase in gata1 expression in the tail (◀ in panel B), while pgy/pgy embryos have a significant decrease (◀ in panel C). In contrast, pgy/pgy have normal l-plastin (long arrow, panel L) and mpo (long arrow, panel O) expression, while smad1 morphants lack all l-plastin expression (long arrow, panel K) and most mpo expression (long arrow, panel N). Loss of either Smad1 or Smad5 leads to a lack of the definitive markers c-myb and runx1 (noted by ). In all images, the embryos are shown whole mount with anterior to the left.

Lisa J. McReynolds, et al. Blood. 2007 December 1;110(12):3881-3890.
4.
Figure 4

Figure 4. From: Smad1 and Smad5 differentially regulate embryonic hematopoiesis.

Early hematopoietic markers are expressed in smad1 morphants and piggytail mutants at the 5-somite stage. Shown are representative embryos following in situ hybridization for various hematopoietic markers at 5 somites using control (uninjected) embryos (A,D,G,J), smad1 morphants (B,E,H,K), and piggytail (pgy/pgy) mutants (C,F,I,L). Transcripts were analyzed for gata1 (A-C), gata2 (D-F), lmo2 (G-I), and scl (J-L). Pgy/pgy embryos were identified by genotyping after hybridization. Smad1 morphants have a modest increase in gata1, lmo2, and scl expression, while pgy/pgy embryos have more caudal expression (◀) of these as well as gata2. In panels A and B, note the increase in gata1 expression in smad1 morphants; the length of the gata1 stripe is longer (B), indicated by the bars that denote the extent of expression in wt. In contrast, pgy/pgy embryos have a shorter region of gata1 expression (C). The anterior lateral plate mesoderm expression of these markers is not effected. In situ hybridization was also performed at the 15-somite stage to detect pu.1 transcripts. (M-O) Levels are equivalent in control embryos (M) and smad1 morphants (N), while pgy/pgy (O) do not express pu.1 (noted by ). Each panel is a representative of 50 to 75 embryos analyzed per sample. Embryos are oriented: panels A-L are anterior to the left; panels A-F are whole mount; panels G-L are flat mount; and panels M-O are anterior to the top, whole mount.

Lisa J. McReynolds, et al. Blood. 2007 December 1;110(12):3881-3890.
5.
Figure 2

Figure 2. From: Smad1 and Smad5 differentially regulate embryonic hematopoiesis.

Depletion of Smad1 or Smad5 causes distinct embryonic phenotypes. (A) Embryos were either control (wild-type [wt]; row 1) or injected with 2 ng translation-blocking Smad1 MO (S1MO), which causes a phenotype first visible at the bud stage by elongation of the embryo (row 2). This phenotype is more severe starting around 18 hpf, with a ragged dorsal edge, neural degeneration, and a widened notochord (row 2; ). By 24 hpf, heart, brain, and endoderm defects are visible ( indicate neural degeneration and yolk stalk regression). These embryos go on to die at 3 to 4 dpf. Alternatively, embryos were injected with 1 ng translation-blocking Smad5 MO (S5MO), which causes a known dorsalization phenotype (row 3) similar to that of characterized smad5 mutants. This phenotype is first visible around the 5-somite stage with a tail defect () and a more caudal arrangement of somites. By the 18 hpf stage, the curvature of the tail is visible, and by 24 hpf, the lack of ventral tissues is evident (). The Smad5 MO phenocopies the piggytail mutant (row 4). Each panel is a representative photograph of phenotype and stage shown. More than 200 embryos were observed for each panel. (B-E) Double knockdown of both Smad1 and Smad5 at half-dose causes lethality at 15 hpf. Shown are wt (B,D) and double-morphant (S1MO + S5MO) (C,E) embryos at 15 hpf and 24 hpf, as indicated. The phenotype just prior to death has combined features of each individual knockdown. Less than 1% of double morphant embryos survive to 24 hpf, as in panel E. Approximately 150 double-injected embryos were observed, and a representative is shown.

Lisa J. McReynolds, et al. Blood. 2007 December 1;110(12):3881-3890.
6.
Figure 3

Figure 3. From: Smad1 and Smad5 differentially regulate embryonic hematopoiesis.

Smad1 knockdown enhances erythropoiesis. (A-C) Fertilized eggs derived from the gata1:DsRed reporter line were either uninjected controls (A) or injected with S1MO (B) or S5MO (C). There is an increase in erythrocyte production in the tail of smad1 morphants at 48 hpf (B), while smad5 morphants have a significant decrease in DsRed+ cells (C). Representative embryos are shown, and approximately 200 of each type of embryo were observed. (D) These phenotypes are quantified by flow cytometry of cells derived from control or morphant embryos at 32 hpf. Shown is the percentage of DsRed+ cells compared with control wt. *The difference compared with wt is significant by Student t test; P < .001. For each of 4 experiments, 30 embryos of each phenotype were collected for flow cytometry. (E-G) In situ hybridization was carried out for βe3 globin on wild-type (E), smad1 morphants (F), and pgy/pgy embryos (G). Shown are representative embryos at 32 hpf, showing that smad1 morphants are increased in erythrocyte production at 32 hpf (F); in contrast, piggytail (Smad5) mutants have a decrease in βe3 globin expression (G). In situ hybridization was done on a sample of 25 embryos for each; panels E-G are representative of the phenotypes seen. (H) Quantitative real-time PCR for βe3 globin was carried out on smad1 (S1MO) and smad5 (S5MO) morphant embryos at 32 hpf. Shown is the average fold change in expression calculated from 4 independent experiments, with samples analyzed each time in triplicate. Samples were normalized to β-actin, and wt set to 1. *The difference compared with wt is significant by Student t test; P < .001. Error bars in D and H indicate the standard deviation from the mean.

Lisa J. McReynolds, et al. Blood. 2007 December 1;110(12):3881-3890.
7.
Figure 1

Figure 1. From: Smad1 and Smad5 differentially regulate embryonic hematopoiesis.

Smad1 and Smad5 are highly homologous, but can be individually targeted for loss of function studies using mutants and MO knockdown.(A) Comparison of zebrafish Smad1 and Smad5 protein structure. The MH1 and MH2 domains are highly similar, compared with the more divergent central linker region. The piggytail mutation (★) is located in the MH2 domain at amino acid 447 of Smad5. (B) In vitro transcription-translation of 35S-methionine–labeled Smad proteins. Smad1 (S1) can be efficiently and specifically targeted for knockdown by the addition of the Smad1 translation-blocking MO (S1MO; lanes 1 and 2). The reaction is not inhibited by the addition of the Smad5 MO (S5MO; lane 3). Smad5 (S5) can be targeted for inhibition by the addition of the Smad5 MO, but not the Smad1 MO (lanes 4-6). (C) In control reactions, a distinct MO (G4MO, specific for the gata4 gene) was used in equivalent reactions against Smad1 or Smad5. In each case, the control MO had no affect on translation, confirming that inhibition seen in panel B is specific. (D) RT-PCR analysis comparing RNA from uninjected embryos and embryos injected with the splice site–blocking MO (S1MO1) collected at 32 hpf. βactin serves as a positive control and generates a band of 277 bp. Primers that span exon2-intron2 generate for smad1 a 232-bp fragment. S1MO1 injection efficiently decreases the amount of properly spliced smad1 message. The smaller fragment at 170 bp was shown by sequencing to encode a misspliced smad1 isoform.

Lisa J. McReynolds, et al. Blood. 2007 December 1;110(12):3881-3890.

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