Zinc Finger Gene Nolz1 Regulates the Formation of
Retinal Progenitor Cells and Suppresses the Lim3/Lhx3
Phenotype of Retinal Bipolar Cells in Chicken Retina
Maria K.E. Blixt, Dardan Konjusha, Henrik Ring, and Finn Hallb
Department of Neuroscience, Uppsala University, Uppsala, Sweden
Background: The zinc-ﬁnger transcription factor Nolz1 regulates spinal cord neuron development by interacting with the tran-
scription factors Isl1, Lim1, and Lim3, which are also important for photoreceptors, horizontal and bipolar cells during retinal
development. We, therefore, studied Nolz1 during retinal development.
Results: Nolz1 expression was seen in two waves dur-
ing development: one early (peak at embryonic day 3–4.5) in retinal progenitors and one late (embryonic day 8) in newly dif-
ferentiated cells in the inner nuclear layer. Overexpression and knockdown showed that Nolz1 decreases proliferation and
stimulates cell cycle withdrawal in retinal progenitors with effects on the generation of retinal ganglion cells, photoreceptors,
and horizontal cells without triggering apoptosis. Overexpression of Nolz1 gave more p27 positive cells. Sustained overexpres-
sion of Nolz1 in the retina gave fewer Lim3/Lhx3 bipolar cells.
Conclusions: We conclude that Nolz1 has multiple functions
during development and suggest a mechanism in which Nolz1 initially regulates the proliferation state of the retinal progeni-
tor cells and then acts as a repressor that suppresses the Lim3/Lhx3 bipolar cell phenotype at the time of bipolar cell differen-
tiation. Developmental Dynamics 247:630–641, 2018.
2017 Wiley Periodicals, Inc.
Key words: chicken embryo; differentiation; horizontal cells; in ovo electroporation; Isl1; Lim1; morpholino; p27; piggyback; pho-
Submitted 11 January 2017; First Decision 29 September 2017; Accepted 17 October 2017; Published online 15 November 2017
The transcription factor Nolz1, also known as zinc ﬁnger protein
503 (ZNF503), is encoded by the ZNF503 gene and forms,
together with its paralogue ZNF703, the atypical zinc ﬁnger sub-
family NET (Noc/Nolz, Elbow, Tlp-1). This subfamily is character-
ized by a single zinc ﬁnger domain (Nakamura et al., 2004), yet it
is sufﬁcient for speciﬁc DNA binding (Pedone et al., 1996; Brown
et al., 2009). Both Nolz1/ZNF503 and ZNF703 repress transcrip-
tion (Nakamura et al., 2004, 2008; Runko and Sagerstrom, 2004).
The NET family paralogues are found in genomes from species
throughout the vertebrate subphylum and orthologues to ZNF503
have been isolated from several vertebrate species including
zebraﬁsh, chicken, mouse, rat and human (Chang et al., 2004;
Runko and Sagerstrom, 2004; McGlinn et al., 2008; Ji et al.,
Neuronal progenitors and early postmitotic immature neurons
express Nolz1, and the expression is later downregulated in dif-
ferentiated neurons (Chang et al., 2004; Urban et al., 2010; Danjo
et al., 2011). In a recent study, Nolz1 immunoreactivity was
exclusively seen in early differentiating neurons and not in the
proliferating progenitors (Ko et al., 2013), suggesting that Nolz1
has a function during the process of cell differentiation. A study
using overexpression and knockdown of Nolz1 in mouse neuro-
spheres suggests that Nolz1 promotes cell cycle withdrawal and
exit (Urban et al., 2010). This is further supported by a study,
which demonstrated that ectopic Nolz1 expression in the devel-
oping mouse telencephalon promoted cell cycle exit and neuronal
differentiation, as well as induced apoptosis (Chang et al., 2013).
Nolz1 is expressed in a subset of developing spinal motoneur-
ons. It controls the initial formation of the prospective Isl1-
positive (þ) forelimb and thoracic Lim3-negative motor columns
by downregulating Lim3 expression and maintaining the expres-
sion of other key differentiation transcription factors. Isl1, Lim1
(Lhx1), and Lim3 (Lhx3) are transcription factors that are neces-
sary for establishing motoneuron identity and promoting neuro-
nal survival (Ji et al., 2009). Coexpression of Lim3 and Isl1
promotes transformation of developing spinal cord neurons from
an interneuron to a motor neuron-phenotype (Thaler et al., 2002).
Additional supporting information may be found in the online ver-
sion of this article.
Grant sponsor: the Swedish Research Council; Grant number:
MH521.2013.3346; Grant sponsor: Barncancerfonden; Grant number:
PR20150122; Grant sponsor:
Ogonfonden, and Stiftelsen Kronprin-
sessan Margaretas Arbetsn
or synskadade; Grant number:
*Correspondence to: Finn Hallb
ok, Department of Neuroscience, Uppsala
University, Box 593, 75124 Uppsala, Sweden. E-mail: ﬁnn.hallbook@
Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.
2017 Wiley Periodicals, Inc.
DEVELOPMENTAL DYNAMICS 247:630–641, 2018