Hand2 inhibits kidney specification while promoting vein formation within the posterior mesoderm
- University of California, San Diego, United States
- School of Medicine, University of California, San Diego, United States
- Universidad de los Andes, Colombia
Figures
Figure 1
hand2 inhibits pronephron formation.
(A–P) Dorsal views, anterior to the left, of pronephron schematics (A, E, I, M), wild-type embryos (B, F, J, N), hans6 mutant embryos (C, G, K, O), and hand2-overexpressing embryos (D, injected with hand2 mRNA; H, L, P, carrying Tg(hsp70-hand2-2A-mcherry), abbreviated hs:hand2) at 24 hpf. In schematics (A, E, I, M), colored regions correspond to area of pronephron gene expression. In situ hybridization demonstrates that atp1a1a.4 (A–D) and cdh17 (E–H) are expressed throughout the pronephron tubules, slc12a3 (I–L) is expressed in the distal late segments of the pronephron tubules, and lhx1a (M–P) is expressed in the glomerular precursors (arrows, N), as well as overlying spinal neurons (asterisks, N). Compared to wild-type (B, F, J, N), gene expression is expanded in hans6 mutants (C, G, K, O) and reduced in hand2-overexpressing embryos (D, H, L, P). Of note, injection of a hand2 translation-blocking morpholino caused effects on pronephron formation similar to those seen in hans6 mutants (data not shown). Scale bars represent 100 μm. (Q, R) Transverse sections through wild-type (Q) and hans6 mutant (R) pronephron tubules at 24 hpf. Dashed lines outline the tubule and asterisks indicate individual tubule cells. (S, T) Bar graphs indicate the average number of tubule cells per cross-section (S) and the average tubule area per cross-section (T) in wild-type and hans6 mutant embryos; error bars indicate standard deviation. Asterisks indicate statistically significant differences compared to wild-type (p<0.0001, Student’s t test; n = 18).
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Figure 1—source data 1
Number of tubule cells per cross-section.
Number of tubule cells observed in transverse sections from embryos at 24 hpf were quantified at three different anterior-posterior levels along the pronephron, with two sections analyzed per level in each embryo. Three animals for each genotype (wild-type and hans6) were examined. Average number of cells and standard deviation are represented in Figure 1S.
- https://doi.org/10.7554/eLife.19941.004
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Figure 1—source data 2
Area of pronephron tubule in cross-section.
Pronephron tubules in transverse sections from embryos at 24 hpf were measured at three different anterior-posterior levels along the pronephron, with two sections analyzed per level in each embryo. Three animals for each genotype (wild-type and hans6) were examined. Average tubule area and standard deviation are represented in Figure 1T.
- https://doi.org/10.7554/eLife.19941.005
Figure 2 with 2 supplements
hand2 inhibits IM production.
(A–C) Dorsal views, anterior to the left, of the posterior mesoderm at the 11 somite stage. In situ hybridization depicts normal expression of pax2a in the IM (arrows) of wild-type embryos (A), widened expression in hans6 mutants (B), and lack of expression in hand2-overexpressing embryos (hs:hand2) (C). Expression in the spinal neurons (asterisk) is unaffected by altered hand2 function. Scale bar represents 100 μm. (D–F) Pax2a immunofluorescence in the posterior mesoderm of wild-type (D), hans6 mutant (E), and hs:hand2 (F) embryos at the 12 somite stage. Dorsal views, anterior to the left, are three-dimensional reconstructions of flat-mounted embryos from which the yolk and anterior tissues have been dissected away. Scale bar represents 100 μm. (D'–F') Magnification of 250 µm long regions from (D–F) used for quantification of the number of Pax2a+ cells in wild-type (D'), hans6 mutant (E'), and hs:hand2 (F') embryos. White dots indicate Pax2a+ nuclei. Intensity of staining varied from strong (for example, green arrows) to weak (for example, yellow arrows). Scale bar represents 25 μm. (G) Bar graph indicates the average number of Pax2a+ cells per 100 µm of IM in wild-type, hans6, and hs:hand2 mutant embryos; error bars indicate standard deviation. Asterisks indicate a statistically significant difference compared to wild-type (p<0.0001, Student’s t test; n=13 for wild-type, n=10 for hans6, and n=19 for hs:hand2).
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Figure 2—source data 1
Pax2a+ cells in wild-type, hans6, and hs:hand2 intermediate mesoderm.
The number of Pax2a+ cells was quantified on the indicated dates of analysis. For wild-type and hans6 embryos, representative 250 μm long regions of IM were analyzed, while for hs:hand2 embryos, 500 μm long regions of IM were analyzed. For hs:hand2 embryos, the IM on both the left and right sides of the embryo were analyzed independently when the dissection and preparation of the sample allowed. All values were normalized to represent the number of cells per 100 μm. Average number of Pax2a+ cells per 100 μm and standard deviation are represented in Figure 2G.
- https://doi.org/10.7554/eLife.19941.007
Figure 2—figure supplement 1
hand2 inhibits IM production.
(A–C) Dorsal views, anterior to the top, of the posterior mesoderm at the 10 somite stage. In situ hybridization depicts normal expression of lhx1a in the IM (arrows) of wild-type embryos (A), widened expression in hans6 mutants (B), and reduced expression in hand2-overexpressing embryos (C). Expression in the notochord (asterisk) is unaffected by altered hand2 function. Scale bar represents 100 μm.
Figure 2—figure supplement 2
Comparable proliferation in the IM of wild-type and hans6 mutant embryos.
(A, B) Immunofluorescence for Pax2a and phospho-Histone H3 (pH3) in wild-type (A) and hans6 mutant (B) embryos at the 6 somite stage; dorsal views, anterior to the left, are three-dimensional reconstructions of 400 µm long regions used for quantification of the numbers of Pax2a+ (A', B') and pH3+ (A'', B'') cells. Arrows indicate Pax2a+ pH3+ cells. Scale bar represents 50 μm. (C) Table compares the number of Pax2a+ pH3+ cells per 100 µm of IM, the number of Pax2a+ cells per 100 µm of IM, and the proliferation index (Pax2a+ pH3+ cells / Pax2a+ cells X 100) in wild-type and hans6 mutant embryos. Data shown represent the average and standard deviation for each value. While there was a statistically significant increase in the number of Pax2a+ cells in hans6 mutants compared to wild-type (p<0.0001), no significant differences were observed for the number of Pax2a+ pH3+ cells (p=0.1461) or the proliferation index (p=0.0654).
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Figure 2—figure supplement 2—source data 1
Pax2a+ pH3+ cells in wild-type and hans6 intermediate mesoderm.
The numbers of Pax2a+ cells and Pax2a+ pH3+ cells were quantified on the indicated dates of analysis. 400 μm long regions of IM were analyzed, and values were normalized to represent the number of cells per 100 μm. A proliferation index (percentage of Pax2a+ pH3+ cells per Pax2a+ cells) was calculated. Average numbers with standard deviation and proliferation index are represented in Figure 2—figure supplement 2C.
- https://doi.org/10.7554/eLife.19941.010
Figure 3
Pronephron development is susceptible to hand2 overexpression prior to the 10 somite stage.
(A–E) Dorsal views, anterior to the left, of in situ hybridization for atp1a1a.4 at 24 hpf depict a range of severity of pronephron defects, ranging from absence of the pronephron (A) to unaffected (E). Tg(hsp70:FLAG-hand2-2A-mCherry) embryos were subjected to heat shock at the tailbud, 2 somite, 6 somite, or 10 somite stages, and the consequences on pronephron development were scored at 24 hpf. Percentages indicate the distribution of phenotypes produced by each treatment; the number of embryos examined is in the right-hand column. Heat shock at later stages resulted in more mild loss of atp1a1a.4 expression in the tubule, and heat shock at the 10 somite stage did not disrupt atp1a1a.4 expression in the tubule. Scale bar represents 100 μm.
Figure 4
hand2 expression in the posterior lateral mesoderm.
(A–D, F–I) Two-color fluorescent in situ hybridization (A, C, F, H) and immunofluorescence (B, D, G, I) label components of the posterior mesoderm in dorsal views, anterior to the left, of three-dimensional reconstructions, as in Figure 2D–E. In embryos containing transgenes in which GFP expression is driven by the regulatory elements of hand2 (B, G) or etv2 (D, I), anti-GFP immunofluorescence was used to enhance visualization. Tg(etv2:egfp) expression was also observed in the midline neural tube, as previously reported (Proulx et al., 2010). Scale bar represents 100 μm. (E, J) Schematics depict posterior mesoderm territories, dorsal views, anterior to the left; hand2-expressing cells, IM, and medial vessel/blood progenitors are shown at 2–10 somites (E) and at 11–13 somites (J), together with lateral vessel progenitors. (A–D) hand2 is expressed lateral to the IM at the 2–10 somite stages. Embryos shown are at the 10 somite stage; similar expression patterns were seen at earlier stages. (A, B) hand2 is expressed lateral to the IM markers lhx1a (A) and Pax2a (B). (C) hand2 is expressed lateral to tal1, a marker of blood and vessel progenitors; note the unlabeled gap between expression territories. (D) A marker of vessel progenitors, Tg(etv2:egfp), lies medially adjacent to Pax2a. (F–I) Vessel progenitors arise at the interface between hand2-expressing cells and the IM at the 11–13 somite stages. (F, G) hand2 is expressed lateral to the IM marker pax2a. (H) hand2 is expressed lateral to a second territory of tal1 expression; note the presence of lateral tal1-expressing cells (arrows) immediately adjacent to hand2 expression. (I) The IM lies between two territories of etv2 expression; the more lateral etv2-expressing cells (arrows) are lateral to the IM.
Figure 5 with 1 supplement
hand2 promotes vessel progenitor development.
(A–C, F–H, K–M) In situ hybridization depicts etv2 (A–C), tal1 (F–H) and gata1 (K–M) expression in wild-type (A, F, K), hans6 mutant (B, G, L) and hand2-overexpressing (C, H, M) embryos; dorsal views, anterior to the left, at the 12 somite stage. (A, F) etv2 and tal1 are expressed in relatively medial and lateral (arrows) territories on each side of the wild-type embryo. In hans6 embryos (B, G), only the medial territory is present. In hand2-overexpressing embryos (C, H), expression of both etv2 and tal1 is increased, but it is not possible to distinguish whether this represents an increase in the medial or the lateral vessel progenitor populations, since no markers exist that distinguish these two groups of progenitors. (K–M) gata1 expression is equivalent in wild-type (K) and hans6 (L) embryos, but it is decreased in hand2-overexpressing embryos (M). Scale bar represents 100 μm. (D, E, I, J) Fluorescent in situ hybridization depicts the relationship of etv2 and pax2a expression in wild-type (D, D') and hand2 morphant (hand2 MO) embryos (E, E'), and the relationship of tal1 and hand2 expression in wild-type (I, I') and hand2 MO (J, J') embryos; dorsal views, anterior to the left, at the 12 somite stage. Medial and lateral (arrows) territories of etv2 expression flank pax2a in wild-type embryos (D, D'). The lateral territory of expression is absent in hand2 morphants (E, E'). The lateral territory (arrows) of tal1 expression is located at the medial border of hand2 expression in wild-type embryos (I, I'), but is absent in hand2 morphants (J, J'). Note that we observed variable thickness of tal1 expression in its medial territory of expression.
Figure 5—figure supplement 1
Presence of medial vessel and blood progenitors is unaffected in hans6 mutants.
(A–F) In situ hybridization depicts expression of etv2 (A, B), tal1 (C, D), and gata1 (E, F) in wild-type (A, C, E) and hans6 mutant (B, D, F) embryos; dorsal views, anterior to the top, of the posterior mesoderm at the 6 somite stage. Expression of all three markers appears unperturbed by loss of hand2 function at this stage. Scale bar represents 100 μm.
Figure 6
hand2 promotes proper vein formation.
(A–I) In situ hybridization depicts expression of mrc1a (A–C), flt4 (D–F) and efnb2a (G–I) in wild-type (A, D, G), hans6 mutant (B, E, H) and hand2-overexpressing (C, F, I) embryos; lateral views, anterior to the left, at 24 hpf (A–C, G–I) and the 20 somite stage (D–F). (A–C) mrc1a expression in the posterior cardinal vein (blue arrow) was present in wild-type (A), absent in hans6 mutant (B), and increased in hand2-overexpressing (C) embryos. Expression in the posterior blood island (red arrowhead) was grossly unaffected. (D–F) flt4 expression in the posterior cardinal vein (blue arrow) was present in wild-type (D), absent in hans6 mutant (E), and increased in hand2-overexpressing (F) embryos. Expression in the dorsal aorta (red arrow) was grossly unaffected. (G–I) efnb2a expression in the dorsal aorta (red arrow) was present in wild-type (G), grossly unaffected in hans6 mutant (H), and slightly increased in hand2-overexpressing (I) embryos. (J–O) Lateral views of three-dimensional reconstructions of Tg(flk1:ras-mcherry) expression in the vasculature of wild-type (J, M) and hans6 mutant (K, N) embryos at 28 hpf (J, K) and 32 hpf (M, N). Pictured region in (J, K) is the area of the trunk boxed in the schematic (L); pictured region in (M, N) is the area of the tail boxed in the schematic (O). Both posterior cardinal vein (blue arrow) and dorsal aorta (red arrow) were present in wild-type (J) and hans6 mutant (K) embryos. In contrast to wild-type (M), the caudal venous plexus (arrowhead) fails to undergo proper remodeling in hans6 mutants (N). Scale bars represent 100 μm.
Figure 7 with 1 supplement
Increased presence of Pax2a in hand2-expressing cells of hand2 mutants.
(A–B) In situ hybridization depicts presence of hand2 expression in hand2 morphants (B); dorsal views, anterior to the left, at the 10 somite stage. There is no evident loss of hand2-expressing cells in hand2 morphants; moreover, hand2 expression levels appear higher in the context of hand2 loss-of-function. Scale bar represents 100 μm. (C–D) Immunofluorescence for Pax2a and GFP in wild-type (C) and hans6 mutant (D) embryos, both carrying Tg(hand2:EGFP); dorsal views, anterior to the left, of three-dimensional reconstructions at the 12 somite stage, as in Figure 4G. (C'–D''') Magnification of 250 µm long regions from (C) and (D) used for quantification of the numbers of GFP+ and Pax2a+ cells in wild-type and hans6 mutant embryos. Yellow dots indicate GFP+ cells, white dots indicate Pax2a+ nuclei, and examples of GFP+ Pax2a+ cells are indicated by white arrows. Intensity of Pax2a+ staining varied from strong (for example, green arrows) to weak (for example, yellow arrows). Scale bars represent 100 μm (C, D) and 25 μm (C'–D'''). (E) Bar graph indicates the average numbers of GFP+ cells and GFP+ Pax2a+ cells per 100 µm of IM in wild-type and hans6 mutant embryos; error bars indicate standard deviation. Asterisk indicates a statistically significant difference compared to wild-type (p<0.0001, Student’s t test; n = 13 for wild-type and n = 10 for hans6).
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Figure 7—source data 1
GFP+ and Pax2a+ GFP+ cells in wild-type and hans6 intermediate mesoderm.
The numbers of GFP+ and Pax2a+ GFP+ cells were quantified on the indicated dates of analysis. Representative 250 μm long regions of IM were analyzed, and values were normalized to represent the number of cells per 100 μm. Average numbers and standard deviation are represented in Figure 7E.
- https://doi.org/10.7554/eLife.19941.017
Figure 7—figure supplement 1
Increased hand2:gfp expression in hans6.
Immunofluorescence for GFP in wild-type (left) and hans6 mutant (right) embryos, both carrying Tg(hand2:EGFP); dorsolateral views, anterior to the top, at 12 somite stage. Levels of hand2:gfp expression appear higher in the context of hand2 loss-of-function.
Figure 8 with 1 supplement
hand2 and osr1 act in opposing, parallel pathways to regulate pronephron development.
(A–A'') Fluorescent in situ hybridization depicts overlap (A) of hand2 (A') and osr1 (A'') expression in wild-type embryos; dorsal views, anterior to the left, of three-dimensional reconstructions at the 6 somite stage. (B–I) In situ hybridization depicts pax2a (B–E) and atp1a1a.4 (F–I) expression at 24 hpf in wild-type embryos (B, F), hans6 mutant embryos (C, G), osr1 morphant (osr1 MO) embryos (D, H) and hans6 mutant embryos injected with osr1 morpholino (hans6 + osr1 MO) (E, I); dorsal views, anterior to the left. (B–E) Compared to wild-type (B), pax2a expression in the glomerular and neck precursors (arrow) was expanded in 100% of hans6 mutants (C, n=11), absent (48%) or reduced (48%) in osr1 morphants (D, n=25), and relatively normal in hans6 + osr1 MO embryos (E, n=11). While the extent of marker expression was generally comparable to wild-type in the double loss-of-function embryos, the stereotypic patterning of this population was often somewhat disrupted. Expression in overlying spinal neurons (asterisk) was unaffected. (F–I) Compared to wild-type (F), atp1a1a.4 expression in the pronephric tubules was wide in 85% of hans6 mutants (G, n=13), while many osr1 morphants (H, n=133) had tubules with shortened anterior expression (18%) or tubules with segmental losses (35%), and 47% of osr1 morphants had a wild-type appearance. 85% of hans6 + osr1 MO embryos (I, n=46) resembled wild-type, whereas 11% had a shortened anterior tubule and 4% had segmental losses in the tubules. Scale bars represent 100 μm.
Figure 8—figure supplement 1
Expression patterns of osr1 and hand2 appear unaffected by loss of each other's function.
(A–D) In situ hybridization depicts expression of osr1 (A, B) and hand2 (C, D) in the posterior mesoderm of wild-type (A, C), hans6 mutant (B), and osr1 MO (D) embryos; dorsal views, anterior to the top, of the posterior portion of the embryo at the 4 somite (A, B) and 6 somite (C, D) stages. (A, B) Loss of hand2 function does not appear to affect osr1 expression. (C, D) Loss of osr1 function does not appear to affect hand2 expression. Scale bar represents 100 µm.
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Hand2 inhibits kidney specification while promoting vein formation within the posterior mesoderm
eLife 5:e19941.
https://doi.org/10.7554/eLife.19941
