Digits and fin rays share common developmental histories

1Understanding the transformation of fish fins into tetrapod limbs is a fundamental problem in biology. The search for antecedents of tetrapod digits in fish has remained controversial because the distal skeletons of limbs and fins differ structurally, developmentally, and histologically,. Moreover, comparisons of fins with limbs have been limited by a relative paucity of data on the cellular and molecular processes underlying the development of the fin skeleton. Here, we provide the first functional analysis, using CRISPR/Cas9 and fate mapping, of 5′ hox genes and enhancers in zebrafish that are indispensable for the development of the wrists and digits of tetrapods,. We show that the fates of cells marked by the activity of an autopodial hoxa13 enhancer exclusively form elements of the fin fold, including the osteoblasts of the dermal rays. In hox13 knockout fish, we find that a dramatic reduction and loss of fin rays is associated with an increased number of endochondral distal radials. These discoveries reveal a deep cellular and genetic connection between fin rays of fish and the digits of tetrapods and suggest a mechanism of digit origins via the transition of distal cellular fates.

2The origin of tetrapod limbs involved profound changes to the distal skeleton of fins. Fin skeletons are composed mostly of fin rays, whereas digits are the major anatomical and functional components of the distal limb skeleton. One of the central shifts during the origin of limbs in the Devonian period involved the reduction of fin rays coincident with an expansion of the distal endochondral bones of the appendage,. Because the distal skeletons of fins and limbs are composed of different types of bone tissue (dermal and endochondral, respectively) it remains unclear how the terminal ends of fish and tetrapod appendages are related and, consequently, how digits arose developmentally. While an understanding of ectodermal signaling centers in fin buds and fin folds has advanced in recent years–, that of the cells that actually form the skeletal patterns has remained elusive.

3Hox genes, namely those of the HoxA and HoxD clusters, have figured prominently in discussions of limb development and origins,–. An “early” and “late” phase of HoxD and HoxA transcription play a role in specifying the proximal (arm and forearm) and distal (autopod) segments, respectively. Both fate map assays and knockout phenotypes in mouse limbs reveal an essential role for Hox13 paralogues in the formation of the autopod,. Mice engineered to lack Hoxa13 and Hoxd13 in limbs lack the wrists and digits exclusively. Moreover, the lineage of cells expressing Hoxa13 reside exclusively in the autopod of adult mice. Together, these lines of evidence reveal the extent to which 5′ Hox genes are involved in, and serve as markers for, the developmental pattern of wrist and digits. Unfortunately, since no such studies have yet been performed in fish, the means to find antecedents of autopodial development in fins has been lacking.

4Analyses of 5′ Hox expression in phylogenetically diverse wild-type fish– as well as experimental misexpression in teleosts reveal that 5′ Hox activity may be involved in patterning, and defining the extent of, the distal chondrogenic region of fish fins. Despite these advances, however, little is known about the contribution of different hox paralogues—individually and in combination— to the adult fin phenotype and the origin of cells that give rise to the distal fin skeleton. While previous investigations have shown that osteoblasts of the fin rays in the caudal fin of zebrafish are derived from either neural crest or from paraxial mesoderm, the source of osteoblasts in pectoral fin rays is currently unknown–. Consequently, it remains an open question where the cellular and genetic markers of the autopod of the tetrapod limb reside in fish fins.

5In order to bridge these gaps in knowledge, we followed the fates of cells marked by early and late phase hox enhancers to adult stages in pectoral fins. In addition, we engineered zebrafish that completely lack each individual hox13 gene, and bred stable lines with multiple gene knockout combinations of hox paralogues. The power of these experiments is twofold: 1) they represent the first functional analyses of hox activity in fins, and 2) invite a direct developmental comparison to experiments performed in tetrapod limbs.

6We performed in situ hybridization of hox13 a and d paralog group genes from 48-120 hours post fertilization (hpf) in zebrafish to determine if active hox expression has a role in the development of the pectoral fin fold. Hox13 a genes in zebrafish are expressed in the distal fin mesenchyme at 48 hpf and weakly in the proximal portion of the pectoral fin fold from 72-96 hpf, indicating that hox13 a genes are not actively expressed in the developing fold (Fig. 1a, b). Hoxd13a is expressed in the posterior half of the fin, but it becomes weak after 96 hpf (Fig. 1c). Hox expression is entirely absent in 10 days post fertilization (dpf) fins (Extended Data Fig. 1). As hox13 genes do not appear to have a main role in zebrafish fin fold development past 72-96 hpf, we sought to determine what structures hox positive cells populate in the developing and adult fold.

7To follow the fates of cells that experience early phase activity in the zebrafish fin, we modified our previously reported transgenesis vector to express Cre-recombinase driven by the zebrafish early phase enhancer CNS65. This enhancer activates expression throughout the endochondral disk of pectoral fins from 31 to ∼38 hpf (Fig. 2a, Extended Data Fig. 1). Stable lines expressing CNS65x3-Cre were crossed to the lineage-tracing zebrafish line Tg(ubi:Switch) fish, where cells are permanently labeled with mCherry. At 6 dpf mesenchymal cells that received expression driven by CNS65 at 38 hpf make up the entire endochondral disk of the pectoral fin (Fig. 2b). Surprisingly, we also found mCherry positive cells in the fin fold at 6 dpf and extensively at 20 dpf (Fig. 2b). These cells contained filamentous protrusions extending distally as well as nuclei positioned at the posterior side, both of which suggest that the cells are migrating distally out of the endochondral disk (Fig. 2b).

8To determine the fate of late phase cellular activity, we employed the same fate mapping strategy but used a late phase hoxa enhancer (e16) from the spotted gar genome. We chose a hoxa enhancer because lineage-tracing data in mouse has shown that late phase Hoxa13 cells in the limb make up the osteoblasts of the wrist and digits exclusively, making it a bona fide marker of the autopod. In addition, gar e16 (which has no sequence conservation in zebrafish) drives expression throughout the autopod in transgenic mice in a pattern that mimics the endogenous murine enhancer and Hoxa13 expression,. In transgenic zebrafish, gar e16 is active in the distal portion of the endochondral disk of the pectoral fin at 48 hpf, and ceases activity after approximately 55 hpf (Fig. 2a, Extended Data Fig. 1). When crossed to Tg(ubi:Switch), at 6 dpf we detected the majority of mCherry positive cells in the developing fin fold with a small number of cells lining the distal edge of the endochondral disk (Fig. 2c). At 20 dpf, the fin fold contains nearly all of the mCherry positive cells that have formed tube-like cells that are appear to be developing actinotrichia (Fig. 2c). In adult fish (90 dpf), late phase cells are restricted to the adult structures of the fin fold, where they compose osteoblasts that make up the fin rays, among other tissues (Fig. 2d). As the e16 enhancer is only active in the distal endochondral disk at 48 hpf, and the labeled cells end up in the fin rays of the adult, late phase hox-positive cells are likely migrating from the endochondral portion of the fin into the fin fold, a hypothesis supported by extensive filopodia in mCherry positive cells projecting in the direction of the distal edge of the fin (Fig. 2c).

9To explore the function of hox13 genes, we inactivated individual hox13 genes from the zebrafish genome by CRISPR/Cas9 and, in addition, made combinatorial deletions through genetic crosses of stable lines (Extended Data Fig. 2, Extended Data Table 2-4). Homozygous null embryos for individual hox13 gene exhibited embryonic pectoral fins that were comparable in size with wild-type at 72 hpf (Extended Data Fig. 3). The shape and size of the fin fold and endochondral disk were also assayed by in situ hybridization for and1 and shha genes, which serve as markers for the developing fin fold and endochondral disk, respectively (Extended Data Fig. 3). In adult fins (∼120 dpf), we observed no detectable difference in the length of fin rays of hoxd13a-/- mutants when compared to wild-type (Fig. 3d, Extended Data Fig. 5). However, both hoxa13a -/- and hoxa13b -/- single mutant fish retained fin rays that were shorter than wild-type, suggesting a role for hoxa13 genes in fin ray development (Fig. 3g, j, Extended Data Fig. 5). In order to determine the degree to which endochondral bones were affected, we utilized CT scanning technology for wild-type and mutant adult fish. Each single mutant, hoxa13a-/-,a13b-/- or d13a -/-, has four proximal radials and 6-8 distal radials with similar morphology to wild-type zebrafish (Fig. 3c, f, i, l, Extended Data Fig. 5). We crossed heterozygous mutants to obtain fish that completely lacked all hoxa13 genes (hoxa13a -/-,a13b-/-). The fin folds of hoxa13a -/-,a13b -/- embryos were ∼30% shorter at 72 and 96 hpf, whereas the number of cells in the endochondral disk was ∼10% greater than wild-type (Extended Data Fig. 4). Surprisingly, adult hoxa13a -/-,a13b -/- exhibited drastically reduced fin rays (Fig. 3m, Extended Data Fig. 5, Supplementary Information). In contrast to dermal reduction, the endochondral distal radials of double mutants were significantly increased to 10-13 in number, often stacked along the proximodistal axis (Fig. 3o, Extended Data Fig. 5, Supplementary Information). A similar pattern was seen in triple knockout fish (mosaic for hoxa13b and hoxd13a) (Fig. 3p-r, Extended Data Fig. 5), implying a role for late phase hox genes in patterning the proximal endochondral radials of fins that is unlike that of tetrapods (Fig. 3)

10Despite being composed of different kinds of skeletal tissues, fin rays and digits share a common population of distal mesenchymal cells that experience late phase Hox expression driven by shared regulatory architectures and enhancer activities. In addition, loss of 5′ Hox activity results in the deletion or reduction of both of these structures. Whereas phylogenetic evidence suggests that rays and digits are not homologous as morphological structures, the cells and regulatory processes in both the fin fold and the autopod share a deep homology that may be common for either bony fish or jawed vertebrates.

11Two major trends underlie the fin to limb transition--the elaboration of endochondral bones and the progressive loss of the extensive dermal fin skeleton,,. In the combinatorial knockouts of hox13 genes, which in tetrapods result in a loss of the autopod, distal endochondral radials were increased in number while fin rays were dramatically reduced. Since a common population of cells in the distal appendage is involved in the formation of rays and digits, the endochondral expansion in tetrapod origins may have occurred through the transition of distal cellular fates and differential allocation of cells from the fin fold to the fin bud (Fig. 4). The two major trends of skeletal evolution in the fin to limb transition may be linked at cellular and genetic levels.

12All zebrafish work was performed according to standard protocols approved by The University of Chicago (ACUP #72074).

13In situ hybridization for the hox13, Cre, and1 and shha genes were performed according to standard protocols after fixation in 4 % paraformaldehyde overnight at 4° C. Probes for hox13 and shha were as previously described. Primers to clone Cre and and1 into vectors can be found in Extended Data Table 1 and 2. Specimens were visualized on a Leica M205FA microscope.

14In order to create a destination vector for lineage tracing, we first designed a random sequence of 298 bp that contained a SmaI sites to be used in downstream cloning. This sequence was ordered as a gBlocks fragment (IDT) and ligated into the pCR8/GW/TOPO TA cloning vector (Invitrogen). We then performed a Gateway LR reaction according to the manufacturers specifications between this entry vector and pXIG-cFos-GFP, which abolished an NcoI site present in the gateway cassette and introduced an SmaI site. We then removed the GFP gene with NcoI and BglII of the destination vector and ligated in Cre with (primers in Extended Data Table 1), using the “pCR8GW-Cre-pA-FRT-kan-FRT” (kind gift of Maximiliano L. Suster, Sars International Center for Marine Molecular Biology, University of Bergen, Bergen, Norway) as a template for Cre PCR and Platinum Taq DNA polymerase High Fidelity (Invitrogen). In order to add a late phase enhancer to this vector, we first ordered four identical oligos (IDT gBlocks) of the core e16 sequence from gar, each flanked by different restriction sites. Each oligo was then ligated into pCR8/GW/TOPO, and sequentially cloned via restriction sites into a single pCR8/GW/TOPO vector. This entry vector was used a template to PCR the final Lo-e16x4 sequence and ligate into the Cre destination vector using XhoI and SmaI, creating Lo-e16x4-Cre. The early phase enhancer Dr-CNS65x3 was cloned into the destination vector using the same strategy. Final vectors were confirmed by sequencing. A full list of sequences and primers used can be found in Extended Data Table 1.

15*AB zebrafish embryos were collected from natural spawning and injected according to the Tol2 system as described previously. Transposase RNA was synthesized from the pCS2-zT2TP vector using the mMessage mMachine SP6 kit (Ambion). All injected embryos were raised to sexual maturity according to standard protocols. Adult F0 fish were outcrossed to wild-type *AB, and the total F1 clutch was lysed and DNA isolated at 24 hpf for genotyping (see Extended Data Table 1 for primers) to confirm germline transmission of Cre plasmids in the F0 founders. Multiple founders were identified and tested for the strongest and most consistent expression via antibody staining and in situ hybridization. One founder fish was identified as best, and all subsequent experiments were performed using these individual fish.

16Founder Lo-e16x4-Cre and Dr-CNS65x3-Cre fish were crossed to the Tg(ubi:Switch) line (kind gift of Leonard I. Zon, Stem Cell Program, Children's Hospital Boston, Boston, MA). Briefly, this line contains a construct where a constitutively active promoter (ubiquitin) drives expression of a loxP flanked GFP protein in all cells assayed of the fish. When Cre is introduced, the GFP gene is removed and the ubiquitin promoter is exposed to mCherry, thus permanently labeling the cell. We crossed our founder Cre fish to Tg(ubi:Switch) and fixed progeny at different time points to track cell fate. In order to detect mCherry signal, embryos or adults were fixed overnight in 4 % paraformaldehyde and subsequently processed for whole-mount antibody staining according to standard protocols using the following antibodies and dilutions: 1st rabbit anti-mCherry/DsRed (Clontech #632496) at 1:250, 1st mouse anti-zns-5 (Zebrafish International Resource Center, USA) at 1:200, 2nd goat anti-rabbit Alexa Fluor 546 (Invitrogen #A11071) at 1:400, 2nd goat anti-mouse Alexa 647 (Invitrogen #A21235) at 1:400. Stained zebrafish were mounted under a glass slide and visualized using an LSM 710 confocal microscope (Organismal Biology and Anatomy, the University of Chicago). Antibody stains on adult zebrafish (90 dpf) fins were imaged on a Leica SP5 II tandem scanner AOBS Laser Scanning Confocal (the University of Chicago Integrated Light Microscopy Core Facility).

17Two mutations were simultaneously introduced into the first exon of each hox13 gene by CRISPR/Cas9 system as previously described in Xenopus tropicalis. Briefly, two gRNAs that match the sequence of exon 1 of each hox13 gene were designed by ZiFiT (http://zifit.partners.org/ZiFiT/). To synthesize gRNAs, forward and reverse oligonucleotides that are unique for individual target sequences were synthesized by Integrated DNA Technologies, Inc. (IDT). Each oligonucleotide sequence can be found in Extended Data Table 2. Subsequently, each forward and reverse oligonucleotide were hybridized, and double stranded products were individually amplified by PCR with primers that include a T7 RNA promoter sequence, followed by purification by NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). Each gRNA was synthesized from the purified PCR products by in vitro transcription with the MEGAscript T7 Transcription kit (Ambion). Cas9 mRNA was synthesized by mMESSAGE mMACHINE SP6 Transcription Kit according to the manufacturer's instructions (Ambion).

18Two gRNAs targeting exon 1 of each hox gene were injected with Cas9 mRNA into zebrafish eggs at the one cell stage. We injected ∼2 nL of the injection solution (5 μl solution containing 1000 ng of each gRNA and 500 ng Cas9 diluted in nuclease free water) into the single cell of the embryo. Injected embryos were raised to adulthood, and at three months were genotyped by extracting DNA from tail clips. Briefly, zebrafish were anesthetized by Tricaine (0.004 %) and tips of the tail fin (2-3 mm square) were removed and placed in an eppendorf tube. The tissue was lysed in standard lysis buffer (10 mM Tris pH 8.2, 10 mM EDTA, 200 mM NaCl, 0.5 % SDS, 200 ug/mL proteinase K) and DNA recovered by ethanol precipitation. Approximately 800-1100 bp of exon 1 from each gene was amplified by PCR according to primers found in Extended Data Table 2. To determine if mutations were present, PCR products were subjected to T7E1 (T7 endonuclease1) assay as previously reported. After identification of mutant fish by T7E1 assay, detailed analysis of mutation patterns were performed by sequencing at the Genomics Core at the University of Chicago.

19Identified mutant fish were outcrossed to wild-type to select frameshift mutations from mosaic mutational patterns and establish single heterozygous lines. Obtained embryos were raised to adults (∼ three months), then analyzed by T7E1 assay and sequenced. Among a variety of mutational patterns, fish that have frameshift mutations were used for assays as single heterozygous fish. We obtained several independent heterozygous mutant lines for each hox13 gene to compare the phenotype among different frameshift mutations. For obtaining hoxa13a +/-,hoxa13b +/- double heterozygous mutant fish, each single heterozygous mutant line were crossed each other. Offspring were analyzed by T7E1 assay and sequenced after three months, and double heterozygous mutant fish were selected. For generating double homozygous hoxa13 mutant embryos and adult fish (hoxa13a-/-, hoxa13b-/-), double heterozygous fish (hoxa13a +/-, hoxa13b +/-) were crossed each other. Ratio of each genotype from crossing heterozygous fish are summarized in Extended Data Table 4.

20After establishing mutant lines, single (hoxa13a or hoxa13b) or double (hoxa13a, hoxa13b) mutant embryos and adult fish were genotyped by PCR for each analysis. Primer sequence for PCR are listed in Extended Data Table 2. To identify 8 bp deletion in exon1 of hoxa13a, PCR product was treated by Ava1 at 37 °C for two hours, because 8 bp deletion produces new Ava1 site in PCR product (“zebra hoxa13a_8 bp del” primers, wild-type; 231 bp, mutant; 111 bp + 119 bp). Final product size was confirmed by 3 % agarose gel electrophoresis. To identify 29 bp deletion in exon1 of hoxa13a, PCR product was confirmed by gel electrophoresis (“zebra hoxa13a_29 bp del” primers, wild-type; 110 bp, mutant; 81 bp). To identify 13 bp insertion in exon1 of hoxa13b, PCR product was treated by Bcc1 at 37 °C for two hours, because 13 bp insertion produces new Bcc1 site in PCR product (“zebra hoxa13b_13 bp ins” primers, wild-type; 98 bp, mutant; 53 bp + 57 bp). Final product size was confirmed by 3 % agarose gel electrophoresis. The detail of mutant sequence are summarized in Extended Data Table 3 a-c.

21Two gRNAs targeting exon 1 of hoxa13b and two gRNAs targeting exon 1 of hoxd13a were injected with Cas9 mRNA into zebrafish one cell eggs that were obtained from crossing hoxa13a+/- and hoxa13a+/-, hoxa13b+/-, hoxd13a+/- (gRNAs were same as that were used to establish single hox13 knockout fishes and found in Extended Data Table 2). Injected eggs were raised to adult fish and genotyped by extracting DNA from tail fins. PCR products of each hox13 gene were cloned into PCRIITOPO (Invitrogen) and deep sequencing was performed (Genomic Core, the University of Chicago). At four months old, skeletal staining and CT scanning were performed to analyze the effect of triple gene deletions. The knockout ratios of each hox13 allele were calculated from the results of deep sequencing.

22Embryos were obtained by crossing hoxa13a +/-, hoxa13b +/- to each other and raised to 72 hpf or 96 hpf. After fixation by 4 % PFA for 15 hours, caudal halves were used for PCR genotype. Pectoral fins of wild-type and hoxa13a -/-, hoxa13b -/- were detached from embryonic body and placed hotizontally on a glass slide. The fins were photographed with a Leica M205FA microscope, and the fin fold length along the proximodistal axis at the center of the fin was measured by ImageJ. The resulting data was subjected to a standard t-test.

23Embryos were obtained by crossing hoxa13a +/-, hoxa13b +/- to each other and raised to 96 hpf. After fixation by 4 % PFA for 15 hours, caudal halves were used for PCR genotype. Wild-type and hoxa13a -/-, hoxa13b -/- embryos were stained by DAPI (1/4000 in PBS-0.1 %Triton) for three hours and washed for three hours by PBS–0.1 % Triton. Pectoral fins were detached from embryonic body, placed on slide glasses and covered by a coverslip. DAPI signal was detected by Zeiss LSM 710 (Organismal Biology and Anatomy, the University of Chicago). Individual nuclei were manually marked using Adobe Illustrator and the number was counted. The data was subjected to a standard t-test.

24Skeletal staining was performed as previously described. Briefly, fish were fixed by 10 % neutral-buffered formalin overnight. After washing by milli-Q water, solutions were substituted to 70 % EtOH in a stepwise fashion and then to 30 % acetic acid / 70 % EtOH. Cartilage were stained by 0.02 % alcian blue in 30 % acetic acid / 70 % EtOH overnight. Washed by milli-Q water, solution was changed to 30 % saturated sodium borate solution and incubated overnight. Then, specimens were immersed in 1 % trypsin / 30 % saturated sodium borate and incubated at room temperature overnight. Following milli-Q water wash, specimens were transferred into 1 % KOH solution including 0.005 % Alzarin Red S. Next day, specimens were washed by milli-Q water and had been subjected to glycerol substitution. Three replicates for each genotype were investigated.

25After skeletal staining, girdles and pectoral fins were manually separated from the body. Girdles and fins were stained by 0.5 % PMA (Phosphomolybdic Acid) in milli-Q water for 16 hours and followed by washes of milli-Q water. Specimens were placed into 1.5 ml eppendorf tubes with water and kept overnight to settle in the tubes. The next day, tubes containing specimens were set and scanned with the UChicago PaleoCT scanner (GE Phoenix v/tome/x 240kv/180kv scanner) (http://luo-lab.uchicago.edu/paleoCT.html), at 50 kVp, 160 μA, no filtration, 5x-averaging, exposure timing of 500 ms per image, and a resolution of 8.000 μm per slice (512 μm3 per voxel). Scanned images were analyzed and segmented using Amira 3D Software 6.0 (FEI). Three replicates for single and double homozygotes and five for mosaic triple knockout were investigated.