Hoxd13 Contribution to the Evolution of Vertebrate Appendages Renata Freitas, Carlos Gómez-Marín, Jonathan Mark Wilson, Fernando Casares, José Luis Gómez-Skarmeta  Developmental Cell  Volume 23, Issue 6, Pages 1219-1229 (December 2012) DOI: 10.1016/j.devcel.2012.10.015 Copyright © 2012 Elsevier Inc. Terms and Conditions

Developmental Cell 2012 23, 1219-1229DOI: (10.1016/j.devcel.2012.10.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Effects of hoxd13a Overexpression in Zebrafish Fins (A) Schemes based on Grandel and Schulte-Merker (1998). Endoskeletal disc (Ed) in blue, distal undifferentiated mesenchyme (Dm) in light blue, marginal blood vessel (Mbv) in yellow, ectodermal finfold (Ff) in gray, cleithrum (Cle). Black arrows indicate the distal mesenchymal expansion, taking place during zebrafish fin development. Left scheme (1) shows morphology of a zebrafish pectoral fin at ∼96 hpf. Dashed line indicates the approximate place where length measurements were performed for statistical analyses. Central (2) and right (3) schemes represent longitudinal sections through fins at two stages of the mesenchymal expansion. Undifferentiated mesenchymal cells (2) are detected at the proximal vicinity of the Mbv at 4 dpf and extend further distally later (3). (B–D) Left and right columns show control wild-type (WT) and hoxd13a-GR injected fins, respectively. Embryonic stages are indicated on the upper left corners of each panel. (B) Different degrees of finfold truncation (severe and mild) are found in hoxd13a-overexpressing fins from 96 hpf onward. (C) Histological sections stained with AB, PAS, and Gill's hematoxylin (Hematox) show expansion of chondrogenic tissue distally to the Mbv in hoxd13a-overexpressing fins (arrows). Dashed yellow lines represent the length of the chondrogenic tissue measured for statistical evaluations. (D) AB skeleton preparations show staining extending distally in hoxd13a-overexpressing fins (arrowhead) when compared with the controls (arrow). (E and F) col2a1a (E) and sox9a (F) expressions expand further distally toward the area affected by hoxd13a overexpression (arrowheads) when compared with wild-type fins equally treated with Dex (WT + Dex) at the same stage (arrows). (G and H) Fin alterations caused by hoxd13a overexpression controlled by hsp70 and col2a1a promoters. Stages of development are indicated in left up corner for each panel. Left, middle, and right column show wild-type (WT), hsp70:hoxd13a and col2a1a:hoxd13a transgenic fins, respectively. Ed, endoskeletal disc; Ff, finfold. Fins overexpressing hoxd13a show finfold reduction (G, arrows) and show distally expanded sox9a expression domains (H), compare arrowheads in transgenics with arrows in controls fins). See also Figures S1 and S3. Developmental Cell 2012 23, 1219-1229DOI: (10.1016/j.devcel.2012.10.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Gene Expression Dynamics after hoxd13a Overexpression in Zebrafish Fins Each panel shows expression dynamics of a gene from 48 to 120 hpf, in which wild-type controls (WT + Dex) and Dex-treated hoxd13a-GR injected fins are on left or right columns, respectively. Stages of development are indicated in each row at left side of the figure. Arrows point to the WT expression and arrowheads indicate expression induced by hoxd13a-GR injections. (A–C) hoxd13a (A), hoxa13b (B), and cyclin d1 (C) expression is located throughout most fin mesenchyme after injection (48 hpf). At 60 hpf, these genes show slightly higher levels in the injected fins. At 90 hpf, their expression domains start to be expanded distally. This expansion becomes very clear at 120 hpf. (D) No differences of and1 expression are observed between injected and control fins at 48 hpf. However, between 60 and 120 hpf, injected fins present a progressive reduction of and1 expression in the finfold. (E) Gene expression changes caused by hoxd13a overexpression controlled by hsp70 and col2a1a promoters. Transgenic fins show distally expanded hoxa13b and cyclin d1 expression 3 to 4 dpf and reduced and1 expression (arrowheads) when compared with controls (arrows). See also Figure S2. Developmental Cell 2012 23, 1219-1229DOI: (10.1016/j.devcel.2012.10.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Other Distal Markers Are Also Affected by hoxd13a Overexpression Each panel shows expression pattern of a gene at 120 hpf, in which wild-type controls (WT + Dex) and Dex-treated hoxd13a-GR injected fins are on left and right columns, respectively. Stages of development are indicated at left of each panel. Arrows point to the WT expression and arrowheads indicate expression induced by hoxd13a-GR injections. Ed, endoskeletal disc; Ff, finfold. (A and B) cyp26b1 (A) and pea3 (B) is extended distally in hoxd13a overexpressing fins. Dashed lines in (A) indicate the approximate plane of the sections shown bellow. In these sections distal cyp26b1 expression can be observed surrounding the differentiated chondrocytes (yellow dashed line) in injected fins. (C) fgf8a expression is confined to a narrower distal domain in injected fins. Dashed lines indicate the approximate plane of the section shown bellow. (D) shha is upregulated in hoxd13a overexpressing fins. (E) Schematic representations of the observed phenotypes. Blocks of graded blue represent the domain where characteristic distal limb markers (hox13b, pea3, and cyp26b1) are expressed in wild-type and in hoxd13a overexpressing fins. Developmental Cell 2012 23, 1219-1229DOI: (10.1016/j.devcel.2012.10.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 hoxd13a Overexpression Cause Increased Proliferation Left column show wild-type controls (WT) and central and right columns show hoxd13a-overexpressing fins in two distinct dorsal/ventral planes. Ed, endoskeletal disc; Ff, finfold. (A) cyclin d1 expression is restricted to the distal end of the endoskeletal plate in WT (arrow). In hoxd13a-overexpressing fins, cyclin d1is observed throughout the expanded endoskeleton (arrowheads) and in the mass of cells folding from it (arrowheads). (B) Blue (DAPI) corresponds to cell nucleus and pink are proliferating cells (anti-BrdU). In WT fins, BrdU labeled cells are detected mainly at the distal end of the endoskeletal disc (arrow). Confocal planes throughout hoxd13a overexpressing fins show proliferation in the distal endoskeletal territory (white arrowheads) and in the mass of cells folding from it (arrowheads). Note similarities of the cyclin d1 expression and BrdU labeled cells distribution (yellow arrows mark equivalent domains). (C) Schematic representation summarizing cell proliferation levels in wild-type and hoxd13a-overexpressing fins. Chondrocytes are represented in blue and BrdU staining in red. Developmental Cell 2012 23, 1219-1229DOI: (10.1016/j.devcel.2012.10.015) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Enhancer Activity of Mouse CsC in Zebrafish Fins Developmental stages are on upper left corners in each panel. (A) Enhancer activity of mouse CsC (mCsC) shown by the expression of GFP in the posterior half of the fin buds (Fb) at 30 hpf and surrounding the distal margin of the endoskeletal disc 4 dpf. Bracket line delimits the distal border of this territory. The scheme on the right represents the main domain of mCsC activity in zebrafish fins at 4 dpf (green). (B) Transverse sections throughout a mCsC transgenic fin expressing GFP at 4 dpf. Left panel shows restriction of GFP mRNA to the distal tip of the endoskeletal territory (arrowheads). Central panel shows a higher magnification of the expressing region (arrowheads). Note GFP expression in chondrocyte-like cells (delimited by a white dashed line) and in surrounding cells. The scheme on the right represents the inferred localization of the expression pattern in the endoskeletal territory (green: chondrocytes and undifferentiated distal mesenchyme). (C) Comparison between mCsC activity and hoxd13 expression in zebrafish and mouse. Dashed lines indicate the distal limits of the endoskeletal territory. Left panel shows that expression of GFP in mCsC transgenic zebrafish fin is nested within hoxd13a expressing domain at 60 hpf (arrowheads). Central panel shows simultaneous anterior expansion of mCsC activity and hoxd13a expression throughout the distal portion of the endoskeletal territory at 4 dpf (arrowheads). Note that mCsC activity is detected also within a subdomain of hoxd13a expression at this stage. Schemes on the right are based on Gonzalez et al. (2007). Left scheme indicates relative position of mCsC upstream of Hoxd13 in mice. Schemes on the right represent CsC and Hoxd13 expression in mouse limbs (blue and pink respectively). Note that mCsC is also active in a subdomain of Hoxd13 expression in this organism. Developmental Cell 2012 23, 1219-1229DOI: (10.1016/j.devcel.2012.10.015) Copyright © 2012 Elsevier Inc. Terms and Conditions