PATTERNS & PHENOTYPES
Zebraﬁsh Rfx4 Controls Dorsal and Ventral Midline
Formation in the Neural Tube
Abigail N. Keller,
Oleg V. Moskvin,
and Yevgenya Grinblat
Department of Integrative Biology, University of Wisconsin, Madison, Wisconsin
Department of Neuroscience, University of Wisconsin, Madison, Wisconsin
Zoology Ph.D. Program, University of Wisconsin, Madison, Wisconsin
Genetics Ph.D. Training Program, University of Wisconsin, Madison, Wisconsin
Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin
McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin
Background: Rfx winged-helix transcription factors, best known as key regulators of core ciliogenesis, also play ciliogenesis-
independent roles during neural development. Mammalian Rfx4 controls neural tube morphogenesis via both mechanisms.
Results: We set out to identify conserved aspects of rfx4 gene function during vertebrate development and to establish a new
genetic model in which to analyze these mechanisms further. To this end, we have generated frame-shift alleles in the zebra-
ﬁsh rfx4 locus using CRISPR/Cas9 mutagenesis. Using RNAseq-based transcriptome analysis, in situ hybridization and immu-
nostaining we identiﬁed a requirement for zebraﬁsh rfx4 in the forming midlines of the caudal neural tube. These functions
are mediated, least in part, through transcriptional regulation of several zic genes in the dorsal hindbrain and of foxa2 in the
ventral hindbrain and spinal cord (ﬂoor plate).
Conclusions: The midline patterning functions of rfx4 are conserved, because
rfx4 regulates transcription of foxa2 and zic2 in zebraﬁsh and in mouse. In contrast, zebraﬁsh rfx4 function is dispensable for
forebrain morphogenesis, while mouse rfx4 is required for normal formation of forebrain ventricles in a ciliogenesis-
dependent manner. Collectively, this report identiﬁes conserved aspects of rfx4 function and establishes a robust new genetic
model for in-depth dissection of these mechanisms. Developmental Dynamics 247:650–659, 2018.
2017 Wiley Periodicals, Inc.
Key words: Rfx4; Zic; foxa2; Hh signaling; zebraﬁsh; neural tube
Submitted 12 August 2017; First Decision 13 October 2017; Accepted 6 December 2017; Published online 15 December 2017
Neural development begins with neural induction and patterning;
the resulting ﬂat neural plate is converted to a tube via a mor-
phogenetic process known as neurulation (Smith and Schoen-
wolf, 1997; Lowery and Sive, 2004; Harrington et al., 2009). As a
result, cells near the lateral edges of the neural plate form the
dorsal midline of the neural tube, the roof plate, while the medial
cells of the neural plate become the ventral midline of the neural
tube, the ﬂoor plate. Despite their distinct origins, the roof plate
and ﬂoor plate share a key property: each serves as a source of
global coordinates for subsequent dorsoventral patterning of the
neural tube, which in turn is crucial for neuronal subtype speciﬁ-
cation. At the molecular level, these coordinates are represented
by gradients of bone morphogenetic proteins (BMPs) produced by
the roof plate and Hedgehogs (Hhs) produced by the ﬂoor plate;
these gradients specify the precise pattern of neuronal subtypes
along the DV axis (Lee et al., 2000; Chizhikov and Millen, 2005;
Wilson and Maden; 2005; Lupo et al., 2006).
The two midlines are controlled by distinct and largely inde-
pendent genetic programs. In the ventral midline, prospective
ﬂoor plate, Sonic hedgehog (Shh) produced by the adjacent noto-
chord induces speciﬁcation of ﬂoor plate cells, which also begin
to express Shh (Dodd et al., 1998). Foxa2 is the key component of
this process, as it is induced in the ﬂoor plate by Hh signaling
from the notochord, activates shh transcription in the ﬂoor plate
cells and is required subsequently for ﬂoor plate differentiation
(Sasaki and Hogan, 1994; Chang et al., 1997; Lek et al., 2010;
Ribes et al., 2010; Balaskas et al., 2012).
In the dorsal neural tube, roof plate speciﬁcation is triggered
by inductive signals (BMPs) secreted by the adjacent nonneural
ectoderm (Lee and Jessell, 1999; Lupo et al., 2006). The newly
formed roof plate, in turn, becomes an important source of BMPs
as well as Wnts, which organize DV pattern and are essential for
dorsal interneuron speciﬁcation (reviewed in Chizhikov and Mil-
len, 2004). Earlier in development, the roof plate lineage shares a
common progenitor with the neural crest lineage (Krispin et al.,
Author Keller’s current address Molecular and Cellular Biology Pro-
gram and Department of Genome Sciences, University of Washing-
ton, Seattle, WA, 98195-5065
Grant sponsor: NIH; Grant number: EY022098-01; Grant sponsor:
AHA; Grant number: 11GRNT7770002.
*Correspondence to: Yevgenya Grinblat, McPherson Eye Research Insti-
tute, University of Wisconsin, Madison, WI, 53706. E-mail: ygrinblat@
Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.
2017 Wiley Periodicals, Inc.
DEVELOPMENTAL DYNAMICS 247:650–659, 2018