Hypoxia inducible factor‐2α importance for migration, proliferation, and self‐renewal of trunk neural crest cells

RESEARCH ARTICLE

Hypoxia inducible factor-2

α

importance for migration,

proliferation, and self-renewal of trunk neural crest cells

Camilla U. Niklasson

| Elina Fredlund

1,2

| Emanuela Monni

3,4

Jessica M. Lindvall

| Zaal Kokaia

3,4

| Emma U. Hammarlund

Marianne E. Bronner

| Sofie Mohlin

1,2,6

Translational Cancer Research, Lund

University Cancer Center at Medicon

Village, Lund University, Lund, Sweden

Division of Pediatrics, Department of

Clinical Sciences, Lund University, Lund,

Sweden

Laboratory of Stem Cells and Restorative

Neurology, University Hospital, Lund,

Sweden

Lund Stem Cell Center, Lund University,

Lund, Sweden

National Bioinformatics Infrastructure

Sweden (NBIS), Science for Life

Laboratory, Department of Biochemistry

and Biophysics, Stockholm University,

Stockholm, Sweden

Division of Biology and Biological

Engineering, California Institute of

Technology, Pasadena, California

Correspondence

Sofie Mohlin, Translational Cancer

Research, Lund University Cancer Center

at Medicon Village, Lund University,

Lund 223 63, Sweden.

Email: sofie.mohlin@med.lu.se

Funding information

Barncancerfonden; Cancerfonden;

Gunnar Nilssons Cancerstiftelse;

Gyllenstierna Krapperup's Foundation;

Royal Physiographic Society of Lund;

Hans von Kantzow's Foundation; Thelma

Zoéga Foundation; Magnus Bergvall's

Foundation; Mary Bevé Foundation; NIH:

R01HL14058; NIH: DE027568; Ollie and

Elof Ericsson's Foundation; Jeansson

Foundations; Crafoord Foundation

Abstract

Background:

The neural crest is a transient embryonic stem cell population.

Hypoxia inducible factor (HIF)-2

is associated with neural crest stem cell

appearance and aggressiveness in tumors. However, little is known about its

role in normal neural crest development.

Results:

Here, we show that HIF-2

is expressed in trunk neural crest cells of

human, murine, and avian embryos. Knockdown as well as overexpression of

HIF-2

in vivo causes developmental delays, induces proliferation, and self-

renewal capacity of neural crest cells while decreasing the proportion of neural

crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the

in vivo phenotype, transcriptome changes after loss of HIF-2

reveal enrich-

ment of genes associated with cancer, invasion, epithelial-to-mesenchymal

transition, and growth arrest.

Conclusions:

Taken together, these results suggest that expression levels of

HIF-2

must be strictly controlled during normal trunk neural crest develop-

ment and that dysregulated levels affects several important features connected

to stemness, migration, and development.

KEYWORDS

embryogenesis, HIF-2

, migration, neural crest, stem cells, trunk neural crest

Camilla U. Niklasson and Elina Fredlund contributed equally to this work.

Received: 29 July 2020

Revised: 2 September 2020

Accepted: 11 September 2020

DOI: 10.1002/dvdy.253

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any me

dium, provided

the original work is properly cited.

Developmental Dynamics

published by Wiley Periodicals LLC on behalf of American Association of Anatomists.

Developmental Dynamics.

2021;250:191

–

236.

wileyonlinelibrary.com/journal/dvdy

191

INTRODUCTION

The neural crest is a multipotent stem cell population that

is unique to vertebrate embryos. Originating from the ecto-

dermal germ layer, premigratory neural crest cells arise in

the dorsal neural tube during neurulation and are charac-

terized by expression of transcription factors like

FOXD3

TFAP2

,and

SOXE

Neural crest cells subsequently

undergo an epithelial-to-mesenchymal transition (EMT) to

delaminate from the neuroepithelium, then migrate exten-

sively throughout the embryo, populating distant sites.

Upon reaching their final destinations, neural crest cells

form a large variety of cell types, as diverse as elements of

the craniofacial skeleton, melanocytes of the skin, adrenal

chromaffin cells, and sympathetic neurons and glia.

2-5

Under normal conditions, hypoxia inducible factor

(HIF)-2

is stabilized at low oxygen levels and responds

to hypoxia by initiating a transcriptional program for cel-

lular adaptation to changes in energy demand. Tumor

cells that express high levels of HIF-2

together with

numerous neural crest markers have been detected in

perivascular niches despite the access to oxygen in these

areas.

6-8

Accordingly, HIF-2

can become abnormally

stabilized at physiological oxygen tensions (

5% O

)

in vitro.

6,9

Previous studies in chick, quail and Xenopus embryos

have shown that related

HIF1A

(encoding HIF-1

) and

ARNT

(encoding HIF-1

, transcriptional binding partner

of both HIF-

isoforms) genes co-localize and are ubiqui-

tously expressed within the developing embryo, as investi-

gated at time points up to HH14 (HH stages in chick

embryos).

10-14

EPAS1

(encoding HIF-2

) is however

expressed in a more distinct pattern and in tissues not

expressing

HIF1A

(extraembryonic and endothelial

cells).

Embryos experience a milieu with low oxygena-

tion (

5% O

), particularly before the blood circulation is

fully functional, which starts at stage HH14.

Despite

this, HIF-2

is not ubiquitously expressed. In addition,

trunk neural crest cells form mainly after commencement

of vasculogenesis and hence are not affected by high

(20%-40%) oxygen.

This is in concordance with data

from Barriga et al, suggesting that HIF-

stability in neu-

ral crest cells can be controlled by both oxygen-dependent

as well as oxygen-independent mechanisms

as suggested

in other systems, including neuroblastoma.

Here, we explore the role of HIF-2

during normal

development up to the time point when trunk neural

crest cells have completed emigration and begin to

populate sympathetic ganglia. We show that HIF-2

expressed in migrating trunk neural crest and sympa-

thetic neuroblasts in human, murine, and avian embryos.

RNA sequencing of trunk neural crest cells with dys-

regulated HIF-2

levels demonstrates a shift in the global

transcriptional program, resulting in enrichment of genes

associated with tumor morphology, invasion, EMT, and

arrested embryo growth. Knockdown and overexpression

experiments in chick embryos in vivo result in a delay in

embryonic growth, altered expression of trunk neural

crest genes, increased proliferation and disrupted trunk

neural crest cell migration. Consistent with this, in vitro

HIF-2

knockout crestospheres display increased self-

renewal capacity. The results suggest that expression

levels of HIF-2

must be strictly controlled for proper

neural crest development. These findings enhance our

understanding of how genes dysregulated in normal

development and tumor cells connect, and how oxygen

sensing HIF-2

plays noncanonical roles during trunk

neural crest development.

RESULTS

2.1

HIF-2

α

is expressed in migratory

trunk neural crest cells in chick embryos

The presence of neuroblastoma cells expressing HIF-2

in perivascular tumor niches indicates poor prognosis.

That these cells express stem cell- and neural crest associ-

ated proteins raises the intriguing possibility that they

may constitute a tumor-initiating subpopulation resem-

bling embryonic neural crest cells. As a first step in

exploring the role of HIF-2

in the embryo, we examined

its spatiotemporal expression during normal trunk neural

crest development. To this end, we performed immuno-

cytochemistry in transverse sections through the trunk

axial level of stage HH11, HH13, and HH18 embryos. We

detected low levels of HIF-2

protein in neural crest cells

within the neural tube of HH11 and HH13 embryos

(Figure 1A,B, respectively), as well as other sites in the

embryo. This contrasts with previous reports on HIF-2

reporting expression exclusively in extraembryonic tissue

at these stages.

Indeed, we also detect HIF-2

staining

in extraembryonic tissue of HH11 and HH13 embryos

(Figure 1A,B). Differences in results may be due to differ-

ent detection methods (eg, in situ hybridization in previ-

ous reports vs antibody staining here), staging, or species

differences. At these stages, trunk neural crest cells are

still premigratory, and although not all cells within

the neural tube will emigrate, a large fraction of these

cells will generate progeny that become

bona fide

neural

crest cells. We further detected HIF-2

in cells that

had delaminated from the neural tube and initiated

migration in older embryos (HH18; Figure 1C), in line

with data from Nanka et al.

To identify these cells as

trunk neural crest cells, we co-stained with HNK-1 anti-

body (Figure 1D). Secondary antibody alone confirmed

192

NIKLASSON

ET AL

that there was no nonspecific binding (Figure 1E), and

we ruled out that the primary antibody (ab199, rabbit

anti-HIF-2

; Abcam) also detected related protein HIF-

by knocking down both HIF-

isoforms and blotting

for HIF-2

. The antibody did not detect any protein in

the HIF-2

siRNA lane, ensuring specificity (Figure 1F).

Along the same line, we used immunohistochemistry to

stain cells cultured at normoxia (21%) or hypoxia (1% O

)

with the NB100-132 primary antibody (mouse anti-HIF-

; Novus Biologicals) and as expected only observed

FIGURE 1

Hypoxia inducible factor (HIF)-2

expressed in trunk neural crest cells. A,C,

Immunostaining of HIF-2

in sections from trunk

axial level of wild-type chick embryos at HH11, A,

HH13, B, and HH18, C. Arrow denotes ventrally

migrating HIF-2

positive cells. D, Co-

immunostaining of HIF-2

and HNK1 (marker of

migrating neural crest) in sections from trunk axial

level of wild-type HH18 chick embryos. Arrows

denote migrating cells double positive for the two

proteins. E, Sections of HH13 wild-type embryo

immunostained with DAPI for visualization of nuclei

and secondary antibody only (donkey anti-rabbit

Alexa Fluor-546). F, Western blot analysis for

detection of HIF-2

protein at 21% and 1% O

following siRNA mediated knockdown of HIF-1

HIF-2

. DIP treated cells were used as a positive

control and SDHA as loading control. Lanes between

21% and 1% siCTRL were removed from this figure,

indicated by the black line. G, Immunohistochemical

staining for HIF-2

in sections of SK-N-BE(2)c

neuroblastoma cells cultured at 21% or 1% O

.H,

Schematic of where oxygen measurements were

performed. I, Oxygen saturation in the trunk of chick

embryos during development measured ex ovo using

microsensor technique. Error bars represent SEM,

≥

3 biologically independent replicates for each

time point

NIKLASSON

ET AL

193

HIF-2

expression at lowered oxygen concentrations

(Figure1G).Togetherwithpreviousdataonthese

antibodies,

6,9,15,16

these results ensure antibody specificity.

2.2

Development from environmental

to physiological oxygen

In adult vertebrate animals, HIF-2

is canonically induced

at low oxygen levels. To unde

rstand variations in oxygen

consumption during the developmental stages of interest,

we measured O

saturation in real time in the developing

chick embryo utilizing STOX microsensors. Oxygen avail-

ability is referred to as changes of full saturation, meaning

that anything below 100% sat

uration reflects a reduction

from what liquid would hold if in equilibrium with air,

which is to be expected when organisms develop into 3D

structures. Embryos were removed from the egg at desired

developmental time points (minimum three embryos per

time point) and oxygen saturat

ion was measured specifically

within the neural tube at the trunk axial level (Figure 1H).

The handling of embryos outside the egg did not change

intratissue oxygen saturatio

n over the first 4 hours. Since

our measurements were performed within 30 minutes, we

believe that these numbers reflect near-endogenous levels.

Within the trunk neural tube, oxygen saturation starts out

high (up to 85% ± 5 SEM O

saturation) at trunk specific

premigratory to migratory st

ages of neural crest develop-

ment (HH10-HH16) and gradually decreases (Figure 1I). At

the time when the majority of trunk neural crest cells have

delaminated from the tube (HH18), oxygen saturation is

low (23% ± 10 SEM O

saturation), only to rise and fall

again at later time points (Figure 1I).

2.3

HIF-2

α

is expressed in sympathetic

neuroblasts in human and mouse embryos

EPAS1

knockout mice have severe abnormalities in the

sympathetic nervous system (SNS)

; consistent with this,

there is some, albeit limited, data suggesting that HIF-2

is expressed in sympathetic chain ganglia up to murine

day E11.5 (corresponding to human embryonic week 5).

Moreover, mice lacking

PHD3

(HIF prolyl hydroxylase),

a gene critical for regulation of HIF-2

, display reduced

SNS function that is rescued by crossing these mutants

with EPAS1

−

mice.

We have previously shown that HIF-2

is expressed

in sympathetic ganglia of human embryos at embryonic

week 6.5 (

E12.5 in mice) but that expression is lost

in these cells at later stages (fetal week 8).

Here, we

detected expression of HIF-2

positive cells in the dorsal

neural tube, as well as in migrating cells in sections

through the trunk region of a human embryo of embry-

onic week ew5 (Carnegie stage 13; Figure 2A). In con-

trast, there were virtually no HIF-2

positive cells left

within the neural tube at embryonic week ew6 (Carnegie

stage 16; Figure 2B). Rather, positive cells could be

detected migrating along the ventral pathway followed by

sympathoadrenal precursors (Figure 2B). To confirm that

that these HIF-2

positive cells were trunk neural crest

cells in human embryos, we co-stained with HNK-1 anti-

body, which is expressed on migrating neural crest cells

of human embryos similar to expression in the chick

(Figure 2C, cf Figure 1D). This resembled the staining

pattern found in chick embryos, but also highlights some

differences in the number of positive cells as well as tis-

sues positive for HIF-2

(compare Figures 1 and 2). These

differences likely reflect variation between species as well

as the fact that it is difficult to assess exact corresponding

developmental stages between them. We further detected

HIF-2

in sympathetic ganglia in mouse embryos at

E12.5 by staining adjacent sections for HIF-2

and TH

antibodies, with the latter indicating the location of sym-

pathetic ganglia (Figure 2D). HIF-2

is a transcription

factor that localizes to the nucleus but it has lately also

been shown to be expressed in the cytoplasm,

6,9,19

though

its role in the cytoplasm remains unknown. Consistent

with this dual localization, we noted HIF-2

expression

in both the nucleus and cytoplasm (Figure 2E), similar to

what has been observed in perivascular oxygenated neu-

roblastoma and glioblastoma cells.

6,20

2.4

Knockdown of HIF-2

α

delays

embryogenesis and alters gene expression

To examine the role of HIF-2

in vivo, we performed

loss-of-function experiments in chick embryos using a

morpholino-mediated knockdown approach. Functioning

as a surrogate marker, successful electroporation was

confirmed by

EGFP

expression (Figure 3A). Experimen-

tally, to ensure that we specifically affected the neural

crest and not surrounding tissue such as mesoderm, we

injected from the posterior end of the embryo and elec-

troporated the constructs into the lumen of the neural

tube. We then let the embryos develop for an additional

24 or 44 to 48 hours (for gene expression and staging/

migration assessment, respectively) and analyzed several

potentially affected biological processes. Surprisingly, we

noticed that HIF-2

knockdown embryos were develop-

mentally delayed compared with their control counter-

parts (Figure 3B,C). The stages of embryos following

loss of HIF-2

were determined by their Hamburger

and Hamilton developmental stage in ovo (Figure 3B)

and by counting somites ex ovo (Figure 3C) 44 hours

194

NIKLASSON

ET AL

postinjection. The number of somites was equal on both

sides and effects observed were embryo wide.

Knockdown of HIF-2

further led to decreased

expression levels of genes representative of early and

migrating neural crest as well as trunk neural crest cells

in particular

21,22

(Figure 3D). The cranial neural crest

associated gene

HOXA2

was also slightly downregulated

(Figure 3E), though not consistently.

FIGURE 2

Hypoxia inducible factor

(HIF)-2

is expressed in human and

mouse trunk neural crest cells. A,B,

Immunostaining of HIF-2

in sections

from trunk axial level of human embryos

at embryonic week 5, A, and embryonic

week 6, B. Asterisks denote magnified

area in the two right panels. ew,

embryonic week. DAPI was used to

counterstain nuclei. C, Co-

immunostaining of HIF-2

and HNK1

(marker of migrating neural crest) in

sections from trunk axial level of human

embryos at embryonic week 6. Arrows

denote areas staining positive for both

proteins. Right panel: open arrowheads

denote double positive individual cells;

closed arrowheads denote cells positive for

HNK1 alone. D, Immunohistochemical

staining of HIF-2

and TH in adjacent

sections from a mouse embryo at

embryonic day E12.5. TH is used to locate

sympathetic ganglia. Asterisks in left

panels indicate magnified area in middle

panels and dashed square indicates

magnification area in right panels. E,

Magnification of an embryo

immunostained for HIF-2

in a

section from trunk axial level of a human

embryo at embryonic week 6. Closed

arrowheads denote nuclear HIF-2

staining; open arrowheads denote

cytoplasmic HIF-2

staining

NIKLASSON

ET AL

195

2.5

CRISPR/Cas9 mediated knockout

of HIF-2

α

recapitulates the morpholino

phenotype

Our

EPAS1

morpholino is a splice targeting morpholino,

predicted to confer either nonsense-mediated decay of

mRNA or a mutant dysfunctional protein. We could not

convincingly detect any changes in HIF-2

protein expres-

sion following morpholino treatment, nor a shift in protein

size. This could be explained b

y other mechanisms-of-action

for decrease in protein activity or the mosaicism that arises

with morpholino treatments in chick embryos. To ensure

that the observed biological phenotypes were not due to off-

target effects of our morpholino, we used CRISPR/Cas9 as a

second approach to knock out HIF-2

by designing three

different gRNAs targeting

EPAS1

at three different sites.

Functional CRISPR mediated knockout of the HIF-2

pro-

tein was demonstrated by immunofluorescence (Figure 4A).

The fact that both morpholino and several CRISPR/Cas9

constructs with in total four different target sites within the

gene produced the same biological phenotype nicely vali-

dates our results and serves as important controls.

After ensuring electroporation efficiency by

EGFP

expression (Figure 4B), we determined the age of the

embryos following CRISPR/C

as9 mediated knockout of the

protein using head- and tail morphology (converted into

HH stage; Figure 4C) or by counting somites (Figure 4D)

36 hours postinjection.

FIGURE 3

Morpholino mediated knockdown of hypoxia inducible factor (HIF)-2

delays embryogenesis. A, Relative mRNA

expression as measured by qRT-PCR. WT, wild-type HH18 embryos. Error bars represent SEM, n = 2 biologically independent replicates.

B,C, Determination of developmental age 44 hours postelectroporation with 5

-mispair or

EPAS1

targeting morpholinos as assessed by head-

and tail morphology, B, (converted to Hamburger Hamilton (HH) stages. Number of embryos analyzed were n = 20 [5

-mispair], n = 16

[EPAS1]) or counting somites ex ovo, C, (number of embryos analyzed were n = 17 (5

-mispair), n = 15 [EPAS1]). Statistical significance was

determined by one-way analysis of variance (ANOVA). D,E, Relative mRNA expression of trunk, D, and cranial, E, neural crest associated

genes in dissected neural tube tissue derived from the trunk axial level of embryos electroporated with 5

-mispair or

EPAS1

morpholinos,

measured by qRT-PCR 24 hours postelectroporation. Data presented as mean of n = 2 biologically independent repeats, error bars denote

SEM. Statistical significance was determined by two-sided student's

test

196

NIKLASSON

ET AL

FIGURE 4

Legend on next page.

NIKLASSON

ET AL

197

2.6

Knockdown of HIF-2

α

affects cell

numbers along the ventral neural crest

migratory pathway

One of the most important features of neural crest cells

is their migratory ability. Trunk neural crest cells des-

tined to form the sympathetic chain ganglia migrate ven-

trally. Following HIF-2

loss of function using either

morpholino or CRISPR/Cas9, HNK1 positive migratory

trunk neural crest cells were detected on the control side

in all embryos (right panel, left side; Figure 5A-E) as well

as on the side electroporated with nontargeting gRNA

CTRL and control 5

-mismatch morpholino (right panel,

left side; Figure 5A,D, respectively). In contrast, loss

of HIF-2

profoundly reduced the number of HNK1 posi-

tive cells migrating to ventral regions of the embryo

(CRISPR/Cas9, Figure 5B,C; morpholino, Figure 5E,F).

2.7

Overexpression of HIF-2

α

presents

similar effects as loss-of-function

Similar to the loss-of-function experiments, over-

expression of HIF-2

led to delayed embryonic develop-

ment (Figure 6A) and perturbed migration as visualized

by HNK1 staining (Figure 6B,C). To investigate spatially

whether affected genes (Figures 3D and 4E,F) were

indeed downregulated in neural crest cells (as indicated

by qPCR analyses of gene expression in dissected neural

tubes of electroporated embryos), we performed

in situ

hybridization for

TFAP2B

on whole HIF-2

wild-type

and overexpression embryos. We could detect down-

regulated levels of

TFAP2B

in delaminated cells on the

electroporated side of embryos after overexpression of

HIF-2

, visualized by whole embryo imaging (Figure 6D)

and transverse sections (Figure 6E) at trunk axial level.

We also performed qPCR to extend our panel of investi-

gated genes and observed slightly suppressed expression

of neural crest- and trunk specific genes (Figure 7A,B)

whereas expression of cranial neural crest gene

HOXA2

was instead slightly induced (Figure 7C). The less profound

effects on neural crest genes from overexpression as com-

pared to knockdown may be attributed HIF-2

expression

level dependent efficiency of the constructs. Overexpression

EPAS1

was confirmed by qRT-PCR (Figure 7D).

2.8

HIF-2

α

knockout does not affect

SOX9 distribution

SOX9, a member of the SoxE family of transcription fac-

tors, is important for neural crest fate. It is expressed in

premigratory neural crest cells at all axial levels and pro-

motes their lineage progression. Importantly, transverse

sections through the trunk of embryos electroporated

with control (Figure 8A) or two different

EPAS1

targeting

gRNA constructs (EPAS1.1 and EPAS1.3, Figure 8B,C,

respectively) showed no differences in SOX9 expression.

These results suggest that neural crest lineage specifica-

tion, at least as assessed by SOX9, was unaffected by loss

of HIF-2

2.9

Trunk neural crest cells proliferate

extensively in response to dysregulated

HIF-2

α

We next examined cell prolif

eration in premigratory and

early migrating trunk neural crest cells after loss of HIF-2

using real-time EdU pulse ch

ase labeling optimized for

avian embryos.

Quantifying the proportion of electro-

porated premigratory and early migrating trunk neural crest

cells that had incorporated EdU (by counting RFP

only

and RFP

/GFP

cells above and outside of the dotted line;

Figure 9A) demonstrated a signif

icant increase in proliferat-

ing cells with an average propor

tion of double positive cells

of 22% and 70% in the 5

-mismatch vs EPAS1 morpholino

targeted embryos, respectively (

.029; Figure 9A,B).

FIGURE 4

CRISPR/Cas9 mediated knockout of hypoxia inducible factor (HIF)-2

delays embryogenesis. A, Immunofluorescent

staining for HIF-2

in embryos electroporated with control (CTRL) or HIF-2

(EPAS1.2) targeting gRNAs. Arrowheads denote GFP+ cells

lacking HIF-2

in knockout embryos. Sections from trunk. B, Relative mRNA expression measured by qRT-PCR. WT, wild-type HH18

embryos. C,D, Determination of developmental age 36 hours postelectroporation with a nontargeting (CTRL) gRNA compared to three

different gRNAs targeting

EPAS1

(EPAS1.1, EPAS1.2, EPAS1.3) as assessed by head- and tail morphology (converted to Hamburger

Hamilton [HH] stages, C. Number of embryos analyzed were n = 14 [CTRL], n = 10 [EPAS1.1], n = 14 [EPAS1.2], and n = 14 [EPAS1.3]) or

by counting somites ex ovo. (D, Number of embryos analyzed were n = 8 [CTRL], n = 13 [EPAS1.1], and n = 14 [EPAS1.3].) Statistical

significance was determined by one-way analysis of variance (ANOVA), comparing nontargeting CTRL to each individual

EPAS1

gRNA.

E-G, Relative mRNA expression of trunk neural crest, E, neural crest, F, and cranial neural crest, G, associated genes in dissected trunk axial

level derived neural tube tissue, measured by qRT-PCR 36 hours postelectroporation. Data presented as mean of n = 2 biologically

independent repeats, error bars denote SEM, B,E-G. Statistical significance was determined by two-sided student's

test, comparing

nontargeting CTRL with each individual

EPAS1

gRNA

198

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ET AL

After overexpression of HIF-2

, real-time EdU incor-

poration demonstrated that cells with increased expres-

sion of HIF-2

, similar to HIF-2

knockdown cells,

became highly proliferative with an average proportion

of double positive cells of 11% and 52% in the control

and HIF-2

overexpressing embryos, respectively (

.011;

Figure 9C,D). We conclude that neural crest prolifera-

tion, embryonic development and migration is highly

sensitive to dysregulated expression of HIF-2

suggesting

that levels must be strictly controlled for proper develop-

ment (Figures 3B-E, 4C-G, 5A-F, 6A-E, and 7A-C).

2.10

HIF-2

α

downregulation enhances

self-renewal capacity of trunk NC cells

Neural crest-derived crestosphere cultures

24,25

enable

studies on stemness properties of neural crest cells

in vitro. Therefore, we examined

EPAS1

expression in

crestosphere cultures, in which multipotent neural crest

cells can be maintained in a stem cell-like state

in vitro.

25,26

Comparing crestosphere cultures derived

from trunk vs cranial axial levels (respective axial identi-

ties have been extensively characterized in References 25

FIGURE 5

Dysregulation of hypoxia inducible factor (HIF)-2

expression affects migration of trunk neural crest cells. A-E,

Immunostaining of HNK1 (red) marking migrating crest cells in one-sided electroporated embryos (right side). Electroporated cells

(nontargeting CTRL gRNA, A, gRNA #2 targeting

EPAS1

(EPAS1.2; B), 5

-mispair morpholino, D, or

EPAS1

morpholino, E) are seen in

green. DAPI was used to counterstain nuclei. Embryo sections from trunk axial level are from 36 hours, A,B, or 44 hours, D,E,

postelectroporation. Arrowheads highlight the difference in HNK1+ area in control vs electroporated side. C,F, Quantification of area

positive for HNK1. Area on electroporated side in EPAS1.2, B, or EPAS1 morpholino, E, embryos was normalized to that of respective

control side. Data are presented as mean ± SEM. Statistical significance was calculated using one-way analysis of variance (ANOVA)

NIKLASSON

ET AL

199

and 26), showed that

EPAS1

was enriched in trunk

crestospheres (Figure 9E).

We further established trunk crestospheres from

embryos previously electroporated with a control gRNA

construct or two different gRNAs targeting

EPAS1

(EPAS1.1 and EPAS1.2). Primary sphere assays demon-

strated that cells with dysregulated HIF-2

levels had an

increased ability to form new spheres when seeded as single

cells (1 cell/well; Figure 9F-G). In addition, crestosphere

cultures derived from embryos electroporated with the

EPAS1.2 construct formed larger spheres compared to their

control counterparts (Figure 9H).

2.11

RNA sequencing after loss of HIF-

α

identifies downstream genes associated

with invasion, growth arrest, and

developmental regulation

To investigate global gene expression changes in cells

with dysregulated levels of HIF-2

, we performed loss

of function experiments at premigratory stages of

trunk neural crest development (HH10

/HH11 in avian

embryos) using the splice targeting morpholino as

above. Neural tubes from trunk region were dissected

24 hours postelectroporation (at stage

HH16, when

trunk neural crest cells are in the premigratory to early

delaminating phase) and subsequently analyzed these

by RNA sequencing. Correlation plot of all genes

from the dataset demonstrated that HIF-2

knockdown

cells indeed differ from those injected with control scram-

bled morpholino (spearman

> .96; Figure 10A).

Setting a cut-off at

< .005 and removing all hits that

were not annotated (NA), identified 97 genes of interest

(Figure 10B). The top 10 genes downregulated and

upregulated (assessed by log2 fold differences in expres-

sion) by knockdown of HIF-2

are summarized in

Figure 10C, while the complete list of these 97 genes can

be found in Table 1. RNA sequencing results were vali-

dated by analyzing selected genes from the top list by

qPCR using the samples assessed for neural crest specific

gene expression (Figure 3D,E). Genes analyzed by qPCR

FIGURE 6

Overexpression of hypoxia inducible factor (HIF)-2

reflects the knockdown phenotype. A, Hamburger Hamilton

(HH) staging of embryos 24 hours postelectroporation with a control (pCI-CTRL) or

EPAS1

overexpression construct (pCI-EPAS1),

determined by head- and tail morphology. Number of embryos analyzed were n = 16 (CTRL), n = 20 (EPAS1). Statistical significance was

determined by one-way analysis of variance (ANOVA). B, Immunostaining of HNK1 (green) marking migrating crest cells in one-sided

electroporated embryos (right side). Electroporated cells (CTRL or EPAS1) are seen in red. DAPI was used to counterstain nuclei. Embryo

sections from trunk axial level are taken 48 hours postelectroporation. C, Quantification of area positive for HNK1. Area on electroporated

side in pCI-EPAS1 embryos was normalized to that of respective control side. Data are presented as mean ± SEM. Statistical significance was

calculated using one-way ANOVA. D, In situ hybridization for

TFAP2B

in whole embryos postelectroporation with pCI-CTRL vs pCI-EPAS1

constructs. E, Sections at trunk axial level of embryos in, D

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