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nature research | reporting summary
April 2018
Corresponding author(s):
Marianne E. Bronner
Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency
and transparency
in reporting. For further information on Nature Research policies, see
Authors & Referees
and the
Editorial Policy Checklist
.
Statistical parameters
When statistical analyses are reported, confirm that the following items are present in the relevant location (e.g. figure lege
nd, table legend, main
text, or Methods section).
n/a
Confirmed
The exact sample size (
n
) for each experimental group/condition, given as a discrete number and unit of measurement
An indication of whether measurements were taken from distinct samples or whether the same sample was measured repeatedly
The statistical test(s) used AND whether they are one- or two-sided
Only common tests should be described solely by name; describe
more complex techniques in the Methods section.
A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons
A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coeffici
ent) AND
variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
For null hypothesis testing, the test statistic (e.g.
F
,
t
,
r
) with confidence intervals, effect sizes, degrees of freedom and
P
value noted
Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes
Estimates of effect sizes (e.g. Cohen's
d
, Pearson's
r
), indicating how they were calculated
Clearly defined error bars
State explicitly what error bars represent (e.g. SD, SE, CI)
Our web collection on
statistics for biologists
may be useful.
Software and code
Policy information about
availability of computer code
Data collection
Zeiss Zen, Illumina HiSeq2500, WinList from Verity Software House
Data analysis
BLAT alignment software, R v3.6.1, Bowtie2, HTSeq-Count, DESeq2, TCoffee, Expresso, MegAlign Pro (DNAstar 15.0.0), MrBayes3.2.1
,
FigTree 1.4.2, Biotapestry, Adobe Illustrator CS5.1
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published lit
erature, software must be made available to editors/reviewers
upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research
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Data
Policy information about
availability of data
All manuscripts must include a
data availability statement
. This statement should provide the following information, where applicable:
- Accession codes, unique identifiers, or web links for publicly available datasets
- A list of figures that have associated raw data
- A description of any restrictions on data availability
All raw sequencing data for all RNAseq libraries (Figure 3) and merged reference transcriptomes are available online (NCBI BioP
roject# PRJNA497902). Sequences of
in situ probe templates for Figures 1B, 1C, 2A, and 2C are available through GenBank accession codes found in the methods.
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nature research | reporting summary
April 2018
Field-specific reporting
Please select the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
Life sciences
Behavioural & social sciences
Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see
nature.com/authors/policies/ReportingSummary-flat.pdf
Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size
For chicken RNAseq replicates, a pre-determined number of a cells was collected per kit instructions for the SMART-seq v4 ultra
low input
RNA kit (Cranial Rep 1= 1534 cells, Cranial Rep 2= 1530 cells, Cranial Rep 3= 1527 cells; Trunk Rep 1= 1500, Trunk Rep 2= 721,
Trunk Rep 3=
958). For lamprey dissections, approximately 100 embryos were dissected for each replicate of each axial level for RNAseq libra
ries. No
statistical tests were used to determine sample size. The sample size provided enough RNA for subsequent library preparation.
Data exclusions
No datasets were excluded from this analysis.
Replication
Chicken RNAseq libraries were collected in triplicate for biological replicates. Lamprey RNAseq libraries were collected with r
eplicates, as well,
for biological replicates. All attempts at replication were successful. For in situ hybridization, embryos were pooled from dif
ferent breeding
pairs (fish), brooding stocks (skates), or embryo batches (lamprey) to ensure replication of results in multiple fixed collecti
ons. All in situ
expression patterns were replicated 100% over multiple rounds of in situs.
Randomization
Chicken eggs obtained from a local chicken farm were incubated in multiple incubators over different days to account for inter-
batch
variability. Lamprey tissues were collected from different breeding events to account for inter-batch variability.
Blinding
Different batches of chicken embryos were separately incubated in different incubators and electroporated with fresh DNA report
er construct
solution. Different breeding pairs for lamprey were used for tissue collections to blind for batch variability. For all trunk
libraries, dissections
were made at the same somitic levels and not based on reporter expression to ensure blinding in sample collection.
Reporting for specific materials, systems and methods
Materials & experimental systems
n/a
Involved in the study
Unique biological materials
Antibodies
Eukaryotic cell lines
Palaeontology
Animals and other organisms
Human research participants
Methods
n/a
Involved in the study
ChIP-seq
Flow cytometry
MRI-based neuroimaging
Antibodies
Antibodies used
anti-Digoxigenin-Alkaline Phosphatase Fab Fragments (Roche ref#11093274910)
Validation
From the supplier: The polyclonal antibody from sheep is specific to digoxigenin and digoxin and shows no cross-reactivity with
other steroids, such as human estrogens and androgens. Concentrations of antibody used for each animal has been previously
reported and validated as cited in the methods section.
Animals and other organisms
Policy information about
studies involving animals
;
ARRIVE guidelines
recommended for reporting animal research
Laboratory animals
Adult (3 months - 2 years of age) Danio rerio, zebrafish, were maintained in the Beckman Institute Fish Facility at Caltech, an
d all
animal and embryo work was compliant under approved IACUC protocol 1346. Adult zebrafish are bred via natural spawning,
which is triggered by light-dark cycle. The fish are kept in a specialized recirculating aquatic system with mechanical, chemi
cal,
and biological filters to maintain water quality. The water is kept at 28 degrees C and highly oxygenated.
Wild animals
Gravid male and female sea lamprey (Petromyzon marinus) were caught in the wild and provided by the Great Lakes Fishery
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nature research | reporting summary
April 2018
Wild animals
Commission, in cooperation with its partners at the USGS Hammond Bay Biological Station, USFWS Marquette Biological Station,
and the Department of Fisheries and Oceans, Canada. They were sent overnight in chilled, oxygenated water to the lamprey
facility at the California Institute of Technology, where they were maintained under the parameters set in accordance with the
Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, with protocols approved by the
Institutional Animal Care and Use Committees of the California Institute of Technology (lamprey, Protocol #1436-17). After
spawning, the captive adult lamprey died of natural causes.
Field-collected samples
Lamprey embryos were produced by in vitro fertilization at the California Institute of Technology lamprey facility, using capti
ve
gravid lamprey (Petromyzon marinus) provided by the Great Lakes Fishery Commission, in cooperation with its partners at the
USGS Hammond Bay Biological Station, USFWS Marquette Biological Station, and the Department of Fisheries and Oceans,
Canada. Lamprey were maintained with a water tempature of 10-18 degrees C on a 15:9h light:dark cycle.
All skate embryos were collected from wild-caught brood stock housed at the Marine Resources Centre of the Marine Biological
Laboratory (MBL) in Woods Hole. Eggs were maintained in a flow-through seawater system with a water temp of 15 degrees C,
on a 12h:12h light:dark cycle. Prior to fixation, all embryos were euthanized with an overdose of buffered MS-222 (1g/L in
seawater). All embryos collection was performed in accordance with protocols approved by the MBL Institutional Animal Care
and Use committee.
Fertilized chicken eggs were obtained from a local farm in Sylmar, CA. Developing chicken embryos were maintained at a
temperature of 37 degrees C.
Flow Cytometry
Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of
identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
Methodology
Sample preparation
Chicken embryos were dissected in Ringers and washed thrice in chilled 1x PBS. The tissues were dissociated in Accumax
(Innovative Cell Technologies, Inc.) for 15 minutes at 37ºC.
Instrument
Sony SY3200 Cell Sorter
Software
WinList from Verity Software House
Cell population abundance
Collected cells were obtained and analyzed on a hemocytometer for fluorescence and viability to ensure 100% purity.
Gating strategy
Gating was assigned according to standard protocols. Clear differentials between GFP+ and GFP- populations were observed.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.