2 Results
2.1 Gene expression in the pelvic fin bud during early development
In the first part of this thesis, the expression patterns of selected genes were analysed during the early stages of pelvic fin development. The aim of the project was to get an overview of the most important signalling pathways taking part in this developmental process and to map the expression domains of the respective genes. Most of the molecular processes in hindlimb development, explained in the introduction, are based on studies in mice or chicken embryos and it is not known if these findings are transferable to zebrafish.
Previous expression studies on zebrafish pelvic fins were carried out by Emily Don, investigating the genes Pitx1, Tbx4, Tbx5, Fgf10a, Fgf8a, Sp8 and Shh in larvae aged 21 and 28 dpf (Don, 2013). Here in this study, the scope was expanded and based on the classification of the pelvic fin developmental stages, defined by Lisa Marzi (Marzi, 2015) (Fig. 10, Fig. S1).
For this, fli:eGFP larvae were grown until an age of 3 - 4 weeks and then sorted according to their current pelvic fin developmental stage. The focus was on the Stages 1 - 6. Thereafter, whole-mount in situ hybridisation (WISH) was performed using specific RNA antisense probes detecting transcripts of Pitx1, Tbx4, Prrx1a, Prrx1b, Rdh10a, Aldh1a2, Aldh1a3, Cyp26a1, Cyp26b1, Cyp26c1, Fgf8a, Fgf10a and Shh. For a high-resolution imaging of the expression site, the stained pelvic fin buds were dissected (Fig. 13 - 15) (Eberlein, 2018a;
Weber, 2020).
First of all, the fin-specific genes Pitx1, Tbx4 as well as Prrx1a and Prrx1b were examined (Fig. 13). Pitx1 expression was observed from S2 - S6 (Fig. 13A-E). In the early Stages 2, 3 and 4, the WISH staining extended over almost the entire fin bud, with exception of the most distal edge (Fig. 13A-C). Later, as the fin edge elongates, the expression area was restricted to the proximal part of the outgrowing pelvic fin bud, leaving the fin edge clear of Pitx1 transcripts (Fig. 13D,E) (Weber, 2020). WISH staining for Tbx4 was obtained from Stages 2 - 5. It was located in the mesenchymal area of the pelvic fin bud, excepting the most distal edge and the most proximal region (Fig. 13F-I). Consequently, it is overlapping with the Pitx1 expression domain. In S2, Tbx4 expression was evenly distributed over this area, while from
staining got more faint in Stage 5 and was no longer detectable in Stage 6 (Weber, 2020). For the two homolougous genes Prrx1a and Prrx1b, intense WISH staining was detected from S2 - S6 (Fig. 13J-N and 13O-S, respectively). The expression patterns of these two genes were very similar, expanding over the entire pelvic fin bud, except of the most distal edge. The intensity of WISH staining increased from S2 to S3 and remained at this level in all developmental stages that were further examined. In addition, it was observed in S6 that the edges of Prrx1a and Prrx1b expression domains in the anterior half of the fin bud were clearly defined, while in contrast to that, in the posterior fin bud, the transition of the stained mesenchymal area to the clear fin edge was blurred (Fig. 13N,S) (Eberlein, 2018a).
Fig. 13 Expression patterns of Pitx1, Tbx4, Prrx1a and Prrx1b during the early stages of pelvic fin development. WISH was performed on fli:eGFP zebrafish larvae of pelvic fin developmental stages 2 - 6 at an age of approximately 3 - 4 wpf. Stained pelvic fin buds were dissected and subsequently imaged by means of bright field microscopy. A-E: Pitx1. Expression from S2 - S6 in the fin bud mesenchyme. F-I:
Tbx4. Expression from S2 - S5 in the fin bud mesenchyme, excepting the most proximal part. From S3 - S6 it concentrates in the posterior region (arrows). J-N: Prrx1a. O-S: Prrx1b. Strong expression of both Prrx1 genes in the fin bud mesenchyme in all stages. Arrowheads are pointing to the sharp border between the Prrx1a/1b expression domain and the fin fold, while brackets indicate an area of blurred transition. The cross on the right shows the orientation of the pelvic fin buds. Scale bars: 50 µm. Pictures A-I by Sophia Weber, J-S by Jean Eberlein. Taken and modified from Weber, 2020 and Eberlein, 2018a.
Stage 5 Stage 4 Stage 3 Stage 2
Pitx1
Stage 6
Tbx4 Prrx1a Prrx1b
B
C A
D
E
G
H F
I
K
L J
M
N
P
Q O
R
S
anterior posterior distal
proximal
Next, important genes of the RA signalling pathway - Rdh10a, Aldh1a2, Aldh1a3, Cyp26a1, Cyp26b1 and Cyp26c1 - were examined for their expression during early pelvic fin development. Rdh10a, encoding the enzyme that transforms ROL to RAL, was detected earliest at S2, showing weak WISH staining in the posterior pelvic fin bud. The staining intensity increased steadily as the bud grows out, while the expression area remained restricted to the proximal, mesenchymal region (Fig. 14A-E) (Weber, 2020).
The expression of Aldh1a2 was detected from S3 - S6 (Fig. 14F-I). It showed a conspicuous expression pattern, which was restricted to a certain area in the posterior part of the outgrowing pelvic fin bud. In S5, the Aldh1a2 expression domain began to expand anteriorly (Fig. 14H). In S6, it extended over the entire length of the pelvic fin bud, with the area of highest expression still localizing posteriorly (Fig. 14I). Across all examined developmental stages, Aldh1a2 expression was limited to the proximal region of the fin bud (Weber, 2020).
Similarly, Cyp26b1 expression first occurred at S3 and localized in a defined spot in the centre of the pelvic fin bud, with a slight tendency to the posterior region (Fig. 14J).
Consequently, it localizes right next to the Aldh1a2 expression domain in this developmental stage. In the following Stages 4 and 5, WISH staining of Cyp26b1 expanded more anteriorly, always being limited to the fin bud mesenchyme. Additionally, high Cyp26b1 expression was detected in the lateral plate mesoderm just beneath the outgrowing fin bud, which is seen in the preparations of the S4 and S5 fin buds (Fig. 14K,L) (Eberlein, 2018a; Weber, 2020).
In contrast to that, Cyp26c1 showed a completely different expression pattern. This gene was detected only in Stages 2 and 3. The WISH staining localized in the most distal part of the pelvic fin bud, extending in anteroposterior orientation along the entire fin edge (Fig. 14M,N) (Weber, 2020). For the remaining two genes, Aldh1a3 and Cyp26a1, no expression could be detected in the pelvic fin bud in any of the investigated developmental stages (Weber, 2020) (data not shown).
The next genes to be examined were Fgf8a, Fgf10a and Shh that are all known to play a key role during limb development. The gene Shh is expressed in a defined area in the posterior margin of the pelvic fin bud from S2 - S4 (Fig. 15K-M). In S5, the expression pattern changes completely and WISH staining is now found in five distinct stripes, orientated proximodistally, presumably defining the places of fin ray formation (Fig. 15N). The expression pattern of Shh in S6 was not determined (Eberlein, 2018a).
Fig. 14 Expression patterns of Rdh10a, Aldh1a2, Cyp26b1 and Cyp26c1 during the early stages of pelvic fin development. WISH was performed on fli:eGFP zebrafish larvae of pelvic fin developmental stages 2 - 6 at an age of approximately 3 - 4 wpf. Stained pelvic fin buds were dissected and subsequently imaged by means of bright field microscopy. A-E: Rdh10a. Expression from S2 - S6 in the fin bud mesenchyme. F-I: Aldh1a2. Expression from S3 - S6, localized posteriorly in S3 and S4, from S5 on it expanded in anterior direction. J-L: Cyp26b1. Expression from S3 - S5 in the fin bud mesenchyme (arrows), in S3 restricted to a distinct spot in the centre of the fin bud. Brackets mark the tissue of the LPM beneath the fin bud showing Cyp26b1 expression. M-N: Cyp26c1. Expression in S2 and S3, restricted to the most distal fin edge (arrowheads). The cross shows the orientation of the pelvic fin buds. Scale bars: 50 µm. All pictures by Sophia Weber, taken and modified from Weber, 2020.
Surprisingly, Fgf8a expression was detected in the fin bud mesenchyme, right in the centre of the outgrowing fin bud (Fig. 15A-E). The intense WISH staining was first detected in S2 and remained at least until S6. These observations were made twice, in two independent WISH experiments, carried out by two different students (Eberlein, 2018a; Weber, 2020).
These results are contradictory to the knowledge gained from studies on mouse and chicken embryos that unanimously identified Fgf8 as a marker for the apical ectodermal ridge (see Fig. 4; Fernandez-Teran & Ros, 2008). Moreover, they do not reproduce the data of Don, 2013, where an expression along the distal edge of the pelvic fin bud was described.
Therefore the identity of the WISH probe was additionally checked by Sanger sequencing
Stage 5 Stage 4 Stage 3 Stage 2 Stage 6
Rdh10a Aldh1a2 Cyp26b1 Cyp26c1
B
C A
D
E
F
G
H
J
K
L
N M
I
posterior proximal anterior
distal
and confirmed as Fgf8a (Fig. S2). The expression pattern of Fgf10a is very similar to that of Fgf8a. Fgf10a is expressed from S2 - S6 in the fin bud mesenchyme. However, unlike than Fgf8a, it expands more distally, forming a defined border to the overlying ectoderm (Fig. 15F-J) (Eberlein, 2018a). The Fgf10a probe was also checked by sequencing to avoid confusion (Fig. S3). These results on the expression of Fgf8a and Fgf10a suggest a completely different mechanism for the outgrowth of pelvic fins compared to what is known from the hindlimbs of mice or chickens.
Fig. 15 Expression patterns of Fgf8a, Fgf10a and Shh during the early stages of pelvic fin development.
WISH was performed on fli:eGFP zebrafish larvae of pelvic fin developmental stages 2 - 6 at an age of approximately 3 - 4 wpf. Stained pelvic fin buds were dissected and subsequently imaged by means of bright field microscopy. A-E: Fgf8a. Expression in the fin bud mesenchyme from S3 - S6. No WISH staining was detected in the distal edge of the fin bud. F-J: Fgf10a. Expression in the fin bud mesenchyme from S2 - S6, forming a defined border to the overlying ectoderm in S2 and S3 (arrowheads). K-N: Shh.
Expression in a defined posterior area from S2 - S4; in S5 in five separate, proximodistally orientated stripes. The cross shows the orientation of the pelvic fin buds. Scale bars: 50 µm. Pictures A-E by Sophia Weber, F-N by Jean Eberlein. Taken and modified from Weber, 2020 and Eberlein, 2018a.
Fgf8a Fgf10a Shh
Stage 5 Stage 4 Stage 3 Stage 2 Stage 6
B
C A
D
E
G
H F
I
J
K
L
M
N
anterior posterior distal
proximal
2.2 Manipulation of the retinoic acid pathway during pelvic fin