Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jeremy S. Dasen ( jeremy.dasen@nyumc.org ).

Leucoraja erinacea embryos of the desired sts were obtained from the Marine Resources Department of Woods Hole Marine Biology Laboratory (MBL). Animals at MBL are maintained in accordance with procedures approved by the MBL Institutional Animal Care and Use Committee (IACUC) following standards established by the NIH. Prior to use, egg cases were maintained in reconstituted Instant Ocean (Aquarium Systems) at 16°C with a standard aquarium filtration system. Before manipulation, embryos were anesthetized with MS-222 (0.17 g/L; Sigma-Aldrich) at room temperature for 10 min ().

Hoxc9 and Foxp1 conditional mutants and Mnx1::GFP zebrafish have been described previously (). Animal procedures were performed in accordance with the US National Institutes of Health Animal Protection Guidelines and approved by the Institutional Animal Care and Use Committee of the New York University School of Medicine. Animal ages are indicated in the main text and figure legends. No gender-specific differences in reported phenotypes are expected, but were not formally tested.

Methods Details

Video Imaging of Leucoraja Embryos and Hatchlings Skates were placed in a 25 cm square glass tank and imaged from the ventral surface using a CMOS camera (Basler Ace a1920-155) with an 8 mm lens (Computar M0814-MP2). The bottom of the tank was covered with a thin (2 mm) Sylgard layer that was prepared by curing on a sheet of Glad Press’n Seal to create a textured surface. The tank was partially surrounded by dark infrared transmitting plastic (CYRO ACRYLITE IR acrylic 11460) and illuminated obliquely and from below with multiple infrared (850 nm) illuminators (CMVIsion-IR200). Images were 8-bit, 960x600 pixels at 60 frames/sec. Data were analyzed in real-time with a custom program written in LabView 2014 (National Instruments). To estimate movement, we computed the number of pixels that changed above baseline noise (8/255) from frame to frame. Empirically, frames with changes greater than 0.7% of their pixels corresponded to movement of the skate’s body. These frames were timestamped and saved, uncompressed, for as long as the skate moved. Cessation of movement and video recording occurred once 150 consecutive frames had elapsed with < 0.7% pixel change.

Retrograde Labeling Retrograde tracing of MNs was performed through intramuscular injection of horseradish peroxidase (HRP, Sigma-Aldrich). Prior to injection, embryos were removed from egg cases with the yolk sac attached and placed in reconstituted Instant Ocean at room temperature. Retrograde labeling was performed by injection of 1-2 μl of a 10% HRP solution into the desired muscle of anesthetized skate embryos. Embryos were then removed from anesthetic and incubated for 6-8 hr at room temperature in aerated Instant Ocean. After incubation, skates were euthanized by rabid decapitation and processed for immunohistochemistry as described below. For each experiment, injections were performed on 4-6 embryos and data shown in figures are representative of results in which the labeling efficiency was > 50% in the targeted MN subtypes.

In ovo Chick Electroporations In ovo electroporation of Hox genes was performed on Hamburger Hamilton (HH) st 13-15 chick embryos and analyzed at HH st 26-27. Fertilized chicken eggs (Charles River) were incubated in a humidified incubator at 39°C for 40-48 hr until they reached HH12-15. The top of the egg shell was removed and a 1 mg/ml DNA solution containing ∼0.02% Fast green was injected into the central canal of the neural tube using a sharpened glass capillary tube. Electrodes (Platinum/Iridium (80%/20%), 250 μm diameter, UEPMGBVNXNND, FHC) were placed on both sides of the neural tube (4 mm separation) and DNA was electroporated using an ECM 830 electroporator (ECM 830, BTX; 25V, 4 pulses, 50 ms duration, 1 s interval). Mulley et al., 2009 Mulley J.F.

Zhong Y.F.

Holland P.W.H. Comparative genomics of chondrichthyan Hoxa clusters. In each electroporation, Hox expression constructs (pCAGGs) were used in the range of 0.1-0.2 mg/ml using pBKS as carrier DNA (0.8-0.9 mg/ml). Leucoraja Hox genes were isolated by RT-PCR, cloned into pCAGGs expression vectors, and confirmed by DNA sequencing. Sequence information on Leucoraja HoxA genes was obtained from GenBank: FJ944024 ).

In ovo Skate Electroporations St 27-28 Leucoraja embryos were used for electroporation. Embryos were removed from egg cases and kept in 35 mm culture dishes containing Instant Ocean. A raised platform was made using glass slides and playdough. Embryo yolk sacs were kept in the bottom of the dish and the embryo was placed on the glass slides. DNA solutions (1-2 mg/ml) containing ∼0.02% Fast green were injected into the central canal of the neural tube and directly into spinal tissue using sharpened glass capillary tubes. After DNA injection, the embryos were placed between microelectrodes. Electric pulses (ECM 830 electroporator, BTX; 20-25V, 5-8 pulses, 50 ms duration, 1 s interval) were given to the embryos in the anesthetic solution. After electroporation, embryos were returned to Instant Ocean; incubated for 11 days at room temperature; and analyzed at ∼st30.

In Situ Hybridization Embryos were fixed in 4% PFA for 1.5-2 hr at 4°C. Embryos were washed 5-6 times in cold PBS, 15-30 min for each wash, and incubated overnight in 30% sucrose. For skate tissue, the spinal cord was removed prior to sucrose treatment. Tissue was embedded in OCT, frozen in dry ice, and sectioned at 16 μm on a cryostat. Wyffels et al., 2014 Wyffels J.

King B.L.

Vincent J.

Chen C.

Wu C.H.

Polson S.W. SkateBase, an elasmobranch genome project and collection of molecular resources for chondrichthyan fishes. Antisense riboprobes were generated using the Digoxigenin-dUTP (SP6/T7) labeling kit (Sigma-Aldrich). To generate antisense riboprobes for Leucoraja genes, sequence information was obtained from SkateBase ( http://skatebase.org ) (). RNA was extracted from st30 skate embryos using TRIzol (Invitrogen) and cDNA was generated using SuperScript II Reverse Transcriptase (Invitrogen). Riboprobes were prepared by PCR using cDNA templates, incorporating a T7 polymerase promoter sequence in the antisense oligo. Oligonucleotide sequences used to generate in situ probes are listed in Table S2 For in situ hybridization sections were first dried for 10-15 min at room temperature, placed in 4% PFA, and fixed for 10 min at room temperature. Slides were then washed three times for 3 min each in PBS, and then placed in Proteinase K solution (1 μg/ml) for 5 min at room temperature. After an additional PFA fixation and washing step, slides were treated in triethanolamine for 10 min, to block positive charges in tissue. Slides were then washed three times in PBS and blocked for 2-3 hr in hybridization solution (50% formamide, 5X SSC, 5X Denhardt’s solution, 0.2 mg/ml yeast RNA, 0.1 mg/ml salmon sperm DNA). Prehybridization solution was removed, and replaced with 100 μL of hybridization solution containing 100 ng of DIG-labeled antisense probe. Slides were then incubated overnight (12-16 hr) at 72°C. 2 ) and incubated for 5 min. Slides were then developed in 0.75 ml/slide of B3 solution containing 3.5 μl/ml BCIP and 3.5 μl/ml NBT for 12-48 hr. After color development, slides were washed in ddH 2 0 and coverslipped in Glycergel (Agilent). A more detailed in situ hybridization protocol is available on our lab website ( After hybridization, slides were transferred to a container with 400 mL of 5X SSC and incubated at 72°C for 20 min. During this step, coverslips were removed using forceps. Slides were then washed in 400 mL of 0.2X SSC for 1 hr at 72°C. Slides were transferred to buffer B1 (0.1 M Tris pH 7.5, 150 mM NaCl) and incubated for 5 min at room temperature. Slides were then transferred to staining trays and blocked in 0.75 ml/slide of B1 containing 10% heat inactivated goat serum. The blocking solution was removed and replaced with antibody solution containing 1% heat inactivated goat serum and a 1:5000 dilution of anti-DIG-AP antibody (Roche). Slides were then incubated overnight at 4°C in a humidified chamber. The following day, slides were washed 3 times, 5 min each, with 0.75 ml/slide of buffer B1. Slides were then transferred to buffer B3 (0.1 M Tris pH 9.5, 100 mM NaCl, 50 mM MgCl) and incubated for 5 min. Slides were then developed in 0.75 ml/slide of B3 solution containing 3.5 μl/ml BCIP and 3.5 μl/ml NBT for 12-48 hr. After color development, slides were washed in ddH0 and coverslipped in Glycergel (Agilent). A more detailed in situ hybridization protocol is available on our lab website ( https://www.med.nyu.edu/dasenlab ).

Immunohistochemistry For antibody staining of sections, slides were first placed in PBS for 5 min to remove OCT. Sections were then transferred to humidified trays and blocked for 20-30 min in 0.75 ml/slide of PBT (PBS with 0.1% Triton) containing 1% Bovine serum albumin (BSA). The blocking solution was replaced with primary staining solution containing antibodies diluted in PBT with 0.1% BSA. Primary antibody staining was performed overnight at 4°C. Slides were then washed three times for 5 min each in PBT. Fluorophore-conjugated secondary antibodies were diluted 1:500-1:1000 in PBT and filtered through a 0.2 μm syringe filter. Secondary antibody solution was added to slides (0.75 ml/slide) and incubated at room temperature for 1 hr. Slides were washed three times in PBT, followed by a final wash in PBS. Coverslips were placed on slides using 110 μL of Vectashield (Vector Laboratories). For vibratome sectioning, embryos were embedded in 4% low melting point agarose and sectioned at 200 μm using a Leica VT1200 vibratome. Sections were individually collected and placed in multi-well plates containing PBS. For staining, sections were first blocked in PBT containing 1% BSA for 1 hr. Primary antibodies were diluted in PBT containing 0.1% BSA, 0.1% sodium azide, and sections were incubated overnight at room temperature. Sections were then washed 4-5 times in PBT; 20-30 min each wash. Secondary antibodies were diluted in PBT containing 0.1% BSA, 0.1% sodium azide, and incubated overnight at room temperature. Sections were then washed 4-5 times in PBT, 20-30 min each wash, followed by a final wash in PBS for 10 min. Sections were mounted between two coverslips containing 80-100 μL of Vectashield. 2 O 2 , 10% DMSO solution prepared in methanol. Embryos were washed three times for 10 min each in methanol, followed by five washes for 10 min in PBS. Primary antibodies were diluted in staining solution (5% BSA, 20% DMSO in PBS) and specimens were incubated in staining solution on a rotator overnight at room temperature. Samples were then washed three times for 5 min each in PBS, followed by four 1 hr washes in PBS. Specimens were then incubated in secondary antibodies diluted in staining solution overnight at room temperature. Samples were then washed three times for 5 min each in PBS, followed by four 1 hr washes in PBS, a single 10 min wash in 50% methanol, and three 20 minute washes in 100% methanol. Samples were transferred to glass depression slides and tissue was cleared by incubating samples in BABB solution (1-part benzyl alcohol: 2-parts benzyl benzoate). Confocal images of embryos were obtained from Z stacks using Zen software (Zeiss). Further details of immunohistochemistry protocols are available on our lab website: ( For whole-mount immunohistochemistry PFA fixed embryos were bleached for 24 hr at 4°C in a 10% H, 10% DMSO solution prepared in methanol. Embryos were washed three times for 10 min each in methanol, followed by five washes for 10 min in PBS. Primary antibodies were diluted in staining solution (5% BSA, 20% DMSO in PBS) and specimens were incubated in staining solution on a rotator overnight at room temperature. Samples were then washed three times for 5 min each in PBS, followed by four 1 hr washes in PBS. Specimens were then incubated in secondary antibodies diluted in staining solution overnight at room temperature. Samples were then washed three times for 5 min each in PBS, followed by four 1 hr washes in PBS, a single 10 min wash in 50% methanol, and three 20 minute washes in 100% methanol. Samples were transferred to glass depression slides and tissue was cleared by incubating samples in BABB solution (1-part benzyl alcohol: 2-parts benzyl benzoate). Confocal images of embryos were obtained from Z stacks using Zen software (Zeiss). Further details of immunohistochemistry protocols are available on our lab website: ( https://med.nyu.edu/dasenlab/protocols.html ).

Antibodies Dasen et al., 2005 Dasen J.S.

Tice B.C.

Brenner-Morton S.

Jessell T.M. A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity. Jung et al., 2010 Jung H.

Lacombe J.

Mazzoni E.O.

Liem Jr., K.F.

Grinstein J.

Mahony S.

Mukhopadhyay D.

Gifford D.K.

Young R.A.

Anderson K.V.

et al. Global control of motor neuron topography mediated by the repressive actions of a single hox gene. Tsuchida et al., 1994 Tsuchida T.

Ensini M.

Morton S.B.

Baldassare M.

Edlund T.

Jessell T.M.

Pfaff S.L. Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Antibodies against Hox proteins, LIM HD proteins, and other proteins were used as described (). Additional antibodies include monoclonal anti-HA (1:16K; Covance) and antibodies obtained from the Developmental Studies Hybridoma Bank (DSHB). Antibodies against Leucoraja Hox proteins were generated in rabbits and guinea pigs at Covance using the following peptide sequences, with an additional cysteine (C) residue incorporated for conjugation: LeHoxa5, DSATMHSSRYGYGYNC; LeHoxa7, TAGTSVFQNTSGFSEATSC; LeHoxa9, ENDDLLASRYASGPLAQASRC; LeRaldh2, CERAQRRTVGNPFDPATE. Antibody specificity was evaluated by comparison of immunoreactivity with Hox mRNA expression patterns in skate spinal cord.

MN Isolation, RNA Extraction, Library Preparation, RNA Sequencing and Bioinformatics Hempel et al., 2007 Hempel C.M.

Sugino K.

Nelson S.B. A manual method for the purification of fluorescently labeled neurons from the mammalian brain. St 30 skate embryos were removed from egg cases and kept in 35 mm culture dishes. Pectoral fins were injected multiple times with Dextran Rhodamine (RhD) (15% solution in ddH20; 3k MW lysine fixable, Molecular Probes, D-3308). During injection procedures embryos were kept in the anesthetic solution. After injections embryos were returned to reconstituted Instant Ocean and incubated for 1-3 days at room temperature. MN dissociation was performed as described () with a few modifications: 1. Before pronase incubation, isolated spinal cords were cut along the dorsal midline to make open-book preparations. 2. Spinal cords were incubated in pronase solution for 30 min. at room temperature. 3. Trituration was performed in 100 μL of ACSF containing 1% FBS. RNA was extracted from purified MNs, using the Arcturus Picopure RNA isolation kit. RNA quality and quantity were measured with an Agilent Picochip using a Bioanalyzer. Library preps for RNA-Seq were made from 500 pg of total RNA, as follows: cDNA was amplified using the Nugen Ovation RNA-Seq System V2 kit (Part 7102) and 100 ng of amplified cDNA were used to prepare Illumina libraries, using the Nugen Ovation Ultralow Library system (Parts No 0303-05 and 0330-31), and amplified by 7 cycles of PCR. The samples were mixed into one pool and run in one 50-nucleotide paired-end lanes on the HiSeq 2500 sequencer, using v4 chemistry. Bolger et al., 2014 Bolger A.M.

Lohse M.

Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Haas et al., 2013 Haas B.J.

Papanicolaou A.

Yassour M.

Grabherr M.

Blood P.D.

Bowden J.

Couger M.B.

Eccles D.

Li B.

Lieber M.

et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Patro et al., 2017 Patro R.

Duggal G.

Love M.I.

Irizarry R.A.

Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Robinson et al., 2010 Robinson M.D.

McCarthy D.J.

Smyth G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Because a high-quality annotated genome is not available for Leucoraja, we used a de novo assembly method to build a transcriptome and quantify gene expression with RNA-seq data. RNA-seq data from pectoral fin neurons and from tail neurons were cleaned with Trimmomatic (v0.33) (), then merged and assembled with Trinity (r2013-02-25) (). The resulting transcriptome assembly contained 82,429 contigs, of which 19,244 were similar to annotated zebrafish genes (BLAST e-value < 10e-4). We then quantified gene expression in each RNA-seq data file (3 replicates from pectoral fin and 3 replicates from tail) by quasi-mapping to the set of transcriptome contigs with Salmon (v0.8.2) (). The gene expression values were tested for differential expression using the Bioconductor package edgeR (v3.18.1) ().

Sequence Comparisons Jung et al., 2014 Jung H.

Mazzoni E.O.

Soshnikova N.

Hanley O.

Venkatesh B.

Duboule D.

Dasen J.S. Evolving Hox activity profiles govern diversity in locomotor systems. DNA sequences from vertebrate species were aligned using Multiz alignment in UCSC genome browser. A previously identified Hox binding element in the Foxp1 locus (chr6:99,190,051-99,190,212 in mm10 assembly) () was subjected to BLAT in UCSC genome browser and SkateBLAST to retrieve homologous genomic sequences from elephant shark (C. milii) and little skate (L. erinacea), respectively: elephant shark, GenBank: KI635892, 4582601- 4582762 (calMil1 assembly); little skate, LSb2-ctg161924 (Little skate Genomic Contigs Build 2). Identified genomic sequences were then re-subjected to BLAT to mouse genome assembly (mm10) for the sequence comparisons and manually added into the alignments.