Conserved regulatory module specifies lateral neural borders across bilaterians

Yongbin Lia,1, Di Zhaoa,1, Takeo Horieb,c,1, Geng Chena,1, Hongcun Baod, Siyu Chena, Weihong Liua, Ryoko Horiec, Tao Lianga, Biyu Donga, Qianqian Fenge, Qinghua Taoa, and Xiao Liua,2

aMinistry of Education Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China; bShimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan; cLewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; dCollege of Life Sciences, Zhejiang University, Hangzhou 310058, China; and eCenter of Biomedical Analysis Platform, Tsinghua University, Beijing 100084, China

Edited by Michael S. Levine, Princeton University, Princeton, NJ, and approved June 21, 2017 (received for review March 13, 2017) The lateral neural plate border (NPB), the neural part of the to PNS neurons; that is, V5 neuroblasts give rise to the mecha- vertebrate neural border, is composed of central nervous system nosensory neuron PVD, whereas QL and QR neuroblasts give rise (CNS) progenitors and peripheral nervous system (PNS) progenitors. to the migratory mechanosensory neurons AVM and PVM, re- In invertebrates, PNS progenitors are also juxtaposed to the lateral spectively (10). Furthermore, Q neuroblasts undergo delamination boundary of the CNS. Whether there are conserved molecular and migrate along prototypical paths while dividing and differen- mechanisms determining vertebrate and invertebrate lateral neural tiating (10), similar to the neural crest in vertebrates and mecha- borders remains unclear. Using single-cell-resolution gene-expression nosensory bipolar tail neurons (BTNs) in tunicate (3, 9). At the profiling and genetic analysis, we present evidence that orthologs molecular level, Msx/vab-15 establishes the identity of the lateral of the NPB specification module specify the invertebrate lateral boundary of the neuroectoderm in fly, annelids, amphioxus, lam- neural border, which is composed of CNS and PNS progenitors. First, prey, and higher vertebrates. Also, their Msx/vab-15–expressing like in vertebrates, the conserved neuroectoderm lateral border domains include both CNS and PNS progenitors (8, 11–14). Worm specifier Msx/vab-15 specifies lateral neuroblasts in Caenorhabditis Msx/vab-15 expression was observed in lateral P neuroblasts, pro- elegans. Second, orthologs of the vertebrate NPB specification genitors of larval CNS motor neurons. Furthermore, development module (Msx/vab-15, Pax3/7/pax-3, and Zic/ref-2) are significantly of mechanosensory neurons is defective in Msx/vab-15 mutants enriched in worm lateral neuroblasts. In addition, like in other bilat- erians, the expression domain of Msx/vab-15 is more lateral than (15). These evolutionary echoes suggest that studies in C. elegans those of Pax3/7/pax-3 and Zic/ref-2inC. elegans. Third, we show that may reveal new clues regarding whether there is a conserved mo- Msx/vab-15 regulates the development of mechanosensory neurons lecular mechanism underlying development of worm lateral derived from lateral neural progenitors in multiple invertebrate spe- neuroblasts and vertebrate NPB. cies, including C. elegans, ,andCiona intes- Using genetic analysis, we show here that worm Msx/vab-15 tinalis. We also identify a novel lateral neural border specifier, expresses in and specifies all lateral neuroblasts. We also did ZNF703/tlp-1, which functions synergistically with Msx/vab-15 in comparative analysis in multiple species, including C. elegans, both C. elegans and Xenopus laevis. These data suggest a common Drosophila melanogaster, and Ciona intestinalis, to show that Msx/ origin of the molecular mechanism specifying lateral neural borders vab-15 regulates the development of mechanosensory neurons across bilaterians. derived from their lateral neural borders, similar to its role in the vertebrate NPB. We profiled the expression of 90% conserved C. elegans | neural plate border | neural border | Msx/vab-15 | ZNF703/tlp-1 transcription factors in worm trunk ectoderm at the resolution of single cells and found that orthologs of the vertebrate NPB he vertebrate neural border is a transient embryonic domain specification module are significantly enriched in worm lateral Tlocated between the neural plate and nonneurogenic ecto- derm from late gastrulation to early neurulation. The neural Significance border is composed of the lateral neural plate border (NPB) and preplacode ectoderm (PPE) subdomains (1, 2). The NPB and PPE The lateral neural plate border (NPB) gives rise to the neural give rise to the neural crest and placode, respectively, both of crest, one of the precursors of the peripheral nervous system which undergo epithelial-to-mesenchymal transition/delamination, (PNS) and generally considered an evolutionary innovation of migrate in prototypical paths, and give rise to the peripheral the vertebrate lineage. Recently, it has been reported that a nervous system (PNS) and many other cell types (3, 4). However, rudimentary neural crest exists in protovertebrate Ciona, but the NPB and PPE also have many different features (5). For ex- whether this is true in other invertebrates and there is con- ample, the PPE is confined to the anterior half of embryos and served molecular machinery specifying the NPB lineage are unknown. We present evidence that orthologs of the NPB does not contribute to the central nervous system (CNS), whereas specification module specify lateral neural progenitor cells in the NPB is the lateral border of the whole neural plate and consists several invertebrates, including worm, fly, and tunicate. We not only of progenitors for the PNS but also those for the CNS in propose that an ancient lateral neuroblast gene regulatory the dorsal neural tube. The juxtaposed localization of the CNS module was coopted by chordates during the evolution of neuroectoderm and PNS progenitors also occurs in the trunk of PNS progenitors. invertebrate embryos such as nematodes, arthropods, annelids, and – urochordates (6 9), reminiscent of vertebrate NPB. In Caeno- Author contributions: Y.L., D.Z., T.H., and G.C. designed research; S.C., W.L., R.H., and B.D. rhabditis elegans, lateral neuroblasts (P, Q, and V5 cells) are located performed research; H.B., T.L., and Q.F. analyzed data; and Q.T. and X.L. wrote the paper. between the embryonic CNS and skin from the birth of these cells The authors declare no conflict of interest. (10). In addition, worm lateral neuroblasts possess several key This article is a PNAS Direct Submission. cellular and developmental features of vertebrate NPB. For ex- 1Y.L., D.Z., T.H., and G.C. contributed equally to this work. ample, the medially located P neuroblasts display nuclear migra- 2To whom correspondence should be addressed. Email: [email protected]. tion and give rise to multiple cell types, including CNS motor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. neurons (10). The laterally located V5 and Q neuroblasts give rise 1073/pnas.1704194114/-/DCSupplemental.

E6352–E6360 | PNAS | Published online July 17, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1704194114 Downloaded by guest on October 1, 2021 neuroblasts. More interestingly, a novel NPB specifier, ZNF703/ V5 neuroblasts but is maintained in Q cells until they divide (Fig. 1 PNAS PLUS tlp-1, was identified and shown to be synergistic with Msx/vab-15 B and C). P-neuroblast expression of Msx/vab-15 starts before in regulating lateral neural progenitors in both C. elegans and hatching, is maintained while P nuclei migrate into the ventral Xenopus laevis, providing another molecular clue to trace the midline in early L1-stage larvae, and shuts down after P neuroblasts origin and evolution of the lateral neural border. divide (Fig. 1 B and D). To investigate the function of Msx/vab-15 during worm de- Results velopment, we phenotyped Msx/vab-15 mutants. These mutants Msx/vab-15 Specifies Lateral Neuroblasts in C. elegans. A previous appear to have wild-type embryonic cell lineage and produce study in C. elegans did not detect Msx/vab-15 reporter expression in lateral neuroblasts that can even divide in larvae (Figs. 2 and 3B V5 and Q neuroblasts (15). To reveal the expression pattern of and Fig. S1), but their P neuroblasts are significantly defective in endogenous Msx/vab-15, we generated a knockin worm strain in nuclear migration, and those that migrate into the midline fre- whichGFPwasintegratedintothe3′ end of the Msx/vab-15–coding quently fail to give rise to hypodermal daughter cells or neurons sequence in the genome. Worms of the knockin strain do not show (Fig. 2 and Table 1). Q neuroblasts in mutants show a defect in any detectable phenotype, suggesting the GFP knockin does not migration too (Fig. 3B), and fail to give rise to mechanosensory disturb the expression of vab-15. The knockin strain shows signif- neurons with nearly full penetrance (Fig. 3 D and F and Table 1). icant Msx/vab-15 expression in embryonic AB.p(lr)apapaa, which is Similarly, V5 neuroblasts in mutants also fail to give rise to the mother cell of Q and V5 neuroblasts (Fig. 1A). After the di- neurons (Fig. 3H). vision of this mother cell, Msx/vab-15 expression turns off in V5 neuroblasts show a strong phenotype in Msx/vab-15 mu- tants even though Msx/vab-15 expression shuts down right after the separation from its sister cell Q in late embryos, indicating Msx/vab-15 specifies the cell fate of these neuroblasts at the embryo stage, instead of functioning at the larval stage. To dis- sect the temporal role of Msx/vab-15,wegeneratedanMsx/vab-15 somatic knockout strain in which CAS9 is driven by a heat- shock promoter. Disrupting Msx/vab-15 at an early stage by heat shocking embryos results in a significant migration and differ- entiation defect of the lateral neuroblasts. On the contrary, disrupting Msx/vab-15 in newly hatched L1 larvae does not lead to a significant differentiation phenotype in V5 and Q neuro- blasts (Table 1). Similarly, knocking out Msx/vab-15 at the early L1 stage does not affect the development of P neuroblasts, either (Table 1). In summary, our endogenous gene-expression analysis and temporal knockout assay show that Msx/vab-15 specifies the lateral neuroblasts at their initial formation and regulates the development of both the CNS and PNS.

Msx/vab-15 Regulates Development of PNS Derived from Lateral Neuroblasts in Fly. As in nematodes and vertebrates, Msx/vab-15 in D. melanogaster is also expressed in the lateral neural border encompassing both CNS and PNS progenitors (7, 16). Further- more, fly Msx/vab-15 is a specifier of lateral CNS progenitors (7, 16). To test whether Msx/vab-15 also regulates the PNS in fly, we generated an Msx/vab-15 mutant, msh4-4,inD. melanogaster and focused on the lateral clusters of the mechanosensors, the chor- dotonal organs. In wild-type Drosophila embryos, each chordotonal organ is made of four cells derived from a single sense organ precursor (SOP) cell, including a neuron, glial cell, and two sup- porting cells. During PNS development, five SOPs for the chor- dotonal organs are formed in the Msx/vab-15–expressing domain lateral to the neuroectoderm in each segment (7). The progeny cells of these five SOPs migrate dorsally and give rise to a stretch of BIOLOGY Fig. 1. Expression of Msx/vab-15 in the trunk nervous systems of bilaterians. five characteristically arranged chordotonal organs (17). However, DEVELOPMENTAL – (A D) Lateral view of Msx/vab-15::GFP knockin comma-stage embryo (A), in msh4-4 mutants, the chordotonal organs contained fewer neu- newly hatched L1 larvae (B), middle L1 (C), and late L1 (D). The red channel rons and glial cells (Fig. 4A). Furthermore, the migration of the represents Histone1::mCherry knockin to label nuclei universally. Arrows 4-4 indicate the mother cell of Q and V5 (A), Q neuroblast (B), or Q daughter progeny cells derived from the SOPs in msh embryos appeared cells (C). Arrowheads indicate P neuroblasts (B and C) and their daughter to be significantly desynchronized among different segments, as and granddaughter cells (D). In middle L1 stage, P nuclei have arrived at the indicated by the position of the glial cells, whereas in the wild-type ventral midline (C). Anterior end of larvae is to the right, and ventral side is embryos these cells migrated to a similar position in all segments up. (Scale bars, 10 μm.) (E) The schematic drawing of neurulae represents the (Fig. 4B). So, like its role in nematodes and vertebrates, Msx/vab-15 dorsal view of vertebrates and the ventral view of protostomes. The head in fly regulates PNS development in the lateral neural border. region in each animal lies above the dotted arc. Only in the ectoderm of C. elegans are neuroblasts drawn according to their precise numbers: a pair Systematic Prediction of Lateral Neuroblast Regulators in C. elegans. of PNS progenitors [AB.p(lr)apapaa, the mother cell of V5 and Q neuro- To comprehensively reveal regulators that distinguish lateral blasts]; six pairs of P neuroblasts; and nine progenitors of ventral cord motor neuroblasts from trunk CNS and the skin cells flanking them, we neurons in the midline. The Msx/vab-15–expressing domain in the annelid is based on Platynereis dumerilii (8). The Msx/vab-15–expressing domain in fly generated reporter transgenic lines for 339 out of all 377 worm is based on D. melanogaster (7). The vertebrate Msx/vab-15–expressing do- transcription factors that have human orthologs (18) (Dataset main represents the merged expression patterns of Msx1, Msx2,andMsx3 in S1). Seventy-five percent of our cloned promoters are more than mouse (34, 35). 2-kb-long upstream regions. Other promoters are less than 2 kb,

Li et al. PNAS | Published online July 17, 2017 | E6353 Downloaded by guest on October 1, 2021 Fig. 2. Msx/vab-15, Pax3/7/pax-3, and Zic/ref-2 are required for migration and differentiation of P neuroblasts. Somatic knockout was induced by heat shocking embryos carrying the Phsp::CAS9 transgene. The Pref-2::H1::mCherry reporter was used as a marker to label P-neuroblast progeny. The Punc-25::GFP reporter was used to label D-type ventral cord motor neurons. VDs are P-derived D-type neurons. (A, E, I, and M) Wild-type L2 larvae, when all P progenies arrive at the ventral midline (A) and give rise to hypodermal Pn.p (E) and neurons (I and M) distributed with stereotypical patterns. Arrowheads indicate progenies of P neuroblasts that are defective in migration, as shown retained in the lateral body. In mutant worms, some P neuroblasts cannot migrate tothe ventral cord and retain in the middle body of worm (B–D). Among P neuroblasts that successfully migrated into the ventral cord, some failed to give rise to hypodermal Pn.p (F–H), neurons (J–L), such as VD neurons (N–P). Dotted circles indicate missing nuclei. Each worm was arranged such that its anterior end was to the right and its ventral midline was at the top. Green fluorescence in pharynx represents the Pmyo-2::GFP coinjection marker (K, L, O, and P). All of the worms detected were at L2 stage. (Scale bar, 10 μm.)

mostly because their upstream intergenic regions are short. It has To predict neuroblast regulators, we defined a neuroblast- been demonstrated that 2 kb upstream of a worm gene accu- enrichment score as the logarithmic ratio of expression in neu- rately drives in most cases (19, 20). Therefore, roblasts over expression in skin or ventral cord motor neurons the majority of our reporters should largely recapitulate endog- (see Materials and Methods for details). To test the prediction enous gene expression. We profiled their expression in trunk power of our neuroblast-enrichment score, we functionally vali- ectoderm of newly hatched L1 larvae at the resolution of single dated seven top-scored : ZNF703/tlp-1, Msx/vab-15, Atonal/ cells using an imaging analysis pipeline (19) (Fig. 5). The an- lin-32, Zic/ref-2, Znf/ztf-6, Ash/hlh-3, and Pax3/7/pax-3 (Fig. 5). In notation of L1 trunk ectodermal cells is highly reliable because addition to Msx/vab-15 described in this study, Atonal/lin-32 is a of their well-separated and invariable position and characteristic well-known master regulator of Q and V5 neuroblast develop- nuclear morphology (10). All cell annotations were manually ment (21, 22). Our genetic analysis showed that Pax3/7/pax-3 and verified to be precise. Zic/ref-2 are required for both migration and differentiation of P

E6354 | www.pnas.org/cgi/doi/10.1073/pnas.1704194114 Li et al. Downloaded by guest on October 1, 2021 (Msx/vab-15, Pax3/7/pax-3, Zic/ref-2,andAP2/aptf-1)estab- PNAS PLUS lishes the identity of the NPB from jawless fish lamprey to mouse (11, 23). This gene set has neuroblast-enrichment scores significantly higher than those of other genes (Mann–Whitney test − based on neuroblast-enrichment score, P value < 1.8 × 10 4). Actually, three out of these four genes are among the top-seven neuroblast-enriched genes (Fig. 5). Although the expression pat- terns of these three NPB specifiers (Msx/vab-15, Pax3/7/pax-3,and Zic/ref-2) are overlapping, the expression domain of Msx/vab-15 in many bilaterians expands more laterally than those of Pax3/7/pax-3 and Zic/ref-2 (8, 24). Consistently, only Msx/vab-15, but not Pax3/7/ pax-3 and Zic/ref-2, is expressed and plays a critical role in the most lateral V5 and Q neuroblasts, which are PNS progenitors in worm (Fig. 1 and Table 1). Another example of more lateral Msx/ vab-15 expression exists in the urochordate C. intestinalis.Two NPB cells (b8.18 and b8.21) give rise to BTNs, which are likely the counterparts to vertebrate dorsal root ganglion (DRG) mecha- nosensory neurons (9). These two NPB cells express Msx/vab-15 and Pax3/7/pax-3 but not Zic/ref-2 (9, 25). Nevertheless, our gene- knockdown experiments showed that Msx/vab-15 is required for BTN differentiation (Fig. 4C). Therefore, Msx/vab-15 regulates the development of the PNS from the lateral neural border in both Protostomia and Deuterostomia, regardless of the coexistence of other NPB specifiers such as Zic/ref-2.

ZNF703/tlp-1 Is a Novel NPB Specifier in X. laevis. We have identified ZNF703/tlp-1 as a novel regulator of lateral neuroblasts cooper- Fig. 3. Msx/vab-15 is required for the development of worm lateral PNS ating with Msx/vab-15 in C. elegans. To investigate whether progenitors. The boundary of the worm body is delineated by dashed lines. ZNF703/tlp-1 has conserved function in vertebrates, we examined (A and B) Migration of Q neuroblast progeny cells. Dotted circles indicate Q its expression and function in X. laevis. In situ RNA hybridization daughter or granddaughter cells. Examined worms were at late L1 stage. Q daughters were separating due to their different migration rate in wild type showed that ZNF703/tlp-1 is expressed at the posterior ectoderm (A), whereas they failed in migration in the vab-15 mutant so that even their except for the dorsal midline in frog gastrulae. During neurulation, granddaughters are clustered together (B). (C–F) Q-derived mechanosensory the expression of ZNF703/tlp-1 in nonneurogenic lateral ectoderm neurons AVM (arrow in C) and PVM (arrow in E) were missed in the vab-15 diminishes so as to become enriched in the NPB and neural crest mutant (D and F). Examined were L2 larvae. Arrowheads indicate ALM (Fig. 6F). As in C. elegans,disturbingZNF703/tlp-1 alone has no (C and D) or PLM (E and F) neurons as a landmark. (G and H) V5-derived detectable phenotype. But moderate knockdown of both ZNF703/ mechanosensory neuron PVD (arrow). egl-17: markers of Q neuroblasts and tlp-1 and Msx/vab-15 significantly down-regulates the trunk ex- their progeny (47). mec-4: markers of AVM/PVM (58). mec-10: markers of pression of the neural crest specifier FoxD3, migratory DRG progen- AVM/PVM and PVD (59). Arrowheads indicate the PLM neuron as a land- itor marker islet1, and nonmigratory Rohon–Beard mechanosensory mark. Examined were L3 larvae. (Scale bars, 10 μm.) neuron progenitor marker Runx1 (Fig. 6G), with no detectable effect on cell proliferation or apoptosis (Fig. S2). High dosage nuclei (Fig. 2 and Table 1). However, mutants of the other three Msx/vab-15 morpholino blocks the differentiation of NPB in candidates (ZNF703/tlp-1, Ash/hlh-3, and Znf/ztf-6) show a weak Xenopus, while low dosage does not (Fig. 6G and Fig. S3). to marginal phenotype (Table 1). ZNF703/tlp-1 is expressed both Hence, ZNF703/tlp-1 acts as a modulator of Msx/vab-15 in both in P neuroblasts and the mother cells of V5 and Q, similar to worm lateral neuroblasts and the vertebrate NPB, supporting our Msx/vab-15 (Figs. 5 and 6A), so we further investigated the ge- hypothesis on the conservation of the gene regulatory module netic interaction between ZNF703/tlp-1 and Msx/vab-15. Al- specifying the lateral neural border across bilaterians. though P neuroblasts appear, there are no phenotypes in Discussion ZNF703/tlp-1 single mutants. But P neuroblasts in ZNF703/tlp-1; An important feature of Bilateria is the centralized nervous Msx/vab-15 BIOLOGY double mutants show a stronger defect in migration system, in which the CNS in the head and trunk midline inte- and differentiation than those in Msx/vab-15 single mutants (Fig. grates and processes sensory information from the PNS. De- DEVELOPMENTAL 6 B and C). Q neuroblasts display little migration defect in velopmental processes to generate centralized nervous systems ZNF703/tlp-1 single mutants, whereas ZNF703/tlp-1;Msx/vab-15 differ dramatically among different bilaterian phyla. In nema- double mutants showed a stronger phenotype than Msx/vab-15 todes such as C. elegans, cell fate is coupled with cell lineage and single mutants (Fig. 6D). Significant synergistic effect was also neurogenesis is multiclonal; that is, many independent pro- observed upon V5 differentiation (Fig. 6E). Therefore, ZNF703/ genitor cells give rise to neurons (6, 10). However, in vertebrate tlp-1 is a novel neuroblast regulator that functions as a modulator gastrulae, accompanying the determination of the dorsoventral of Msx/vab-15 activity in worms. These data demonstrated that axis by BMP signaling, ectoderm in the dorsal midline thickens to the neuroblast-enrichment score is strongly related to the mo- form a neural plate, from which their CNS is derived. Between lecular mechanisms specifying worm lateral neural precursor the neural plate and nonneurogenic ectoderm are the NPB and cells. Then, examining the correlation between the worm PPE, from which the majority of the vertebrate PNS is derived neuroblast-enrichment score and orthologs of the NPB specifi- (24). How nervous system centralization patterns come into be- cation module can suggest the conservation between the worm ing is a long-standing question in neural development and evo- lateral neuroblasts and the vertebrate NPB. lution (3, 26). Regarding the molecular mechanisms determining CNS de- The NPB Specification Module Is Conserved Between Worm and velopment, comparative studies have provided evidence that the Chordates. A conserved four-gene NPB specification module CNS of vertebrates, annelids, and arthropods originated from a

Li et al. PNAS | Published online July 17, 2017 | E6355 Downloaded by guest on October 1, 2021 Table 1. Genetic analysis of migration and differentiation of lateral neuroblasts Frequency of worms with neuroblast migration defect, % (n) Frequency of worms with neuroblast differentiation defect, % (n)

† † Missing PDE§, Missing AVM , Missing PVM , Not 13 VDs¶, † ‡ Gene Allele Q*, P (V5.paaa#) (QR.paa#) (QL.paa#) (Pn.app#)

Wild type 0 (100) 0 (30) 1 (105) 0 (100) 0 (100) 0.5 (200) Msh/vab-15 tm260 34 (44) 81 (101) 58 (112) 96 (104) 96 (101) 100 (44) Knockout in embryojj 19 (32) 46.46 (99) 30 (50) 28 (50) 16 (50) 46.46 (99) jj Knockout in L1 13 (23) 0 (27) 6 (50) 4 (25) 8 (50) 0 (27) NLZ/tlp-1 bx85 0 (50) 0 (55) 2 (100) 13 (101) 4 (104) 0 (101) ZNF/ztf-6 tm1803 0 (100) 0 (100) 2 (42) 0 (100) 0 (100) 0 (100) Ash/hlh-3 tm1688 0 (100) 0 (100) 0 (100) 0 (100) 0 (100) 3.6 (280) jj Zic/ref-2 Knockout in embryo 0 (42) 54 (63) 0 (50) 0 (42) 0 (42) 54 (63) jj /7/pax-3 Knockout in embryo 0 (22) 41.7 (60) 4 (50) 0 (22) 0 (22) 45 (60)

The number of scored animals in each experiment is in parentheses. *A late-stage larva is considered abnormal if either the AVM or PVM is mislocated. †mec-4 reporter as an AVM/PVM neuron marker. ‡ ref-2 reporter as a marker of P neuroblasts and their progeny. §dat-1 reporter as a PDE marker. ¶unc-25 reporter as a VD neuron marker. #Cell lineage of the specified neuron. jj Somatic knockout is induced by heat shock activating Phsp::CAS9 in a specified stage.

common bilaterian ancestor (8, 27). For example, both the lateral neural borders in nematodes, fly, annelids, and urochor- neuroectoderm of annelids and neural plate of vertebrates are dates show conserved neural patterning where the medial por- subdivided into a sim/hlh-34–expressing midline and longitudinal tion of the Msx/vab-15–expressing domain gives rise to the CNS Nk2.2/ceh-22–, Nkx6/cog-1–, Pax6/vab-3–, Pax3/7/pax-3–,andMsx/ whereas the lateral portion generates the PNS. Second, their vab-15–expressing domains from medial to lateral, likely repre- lateral PNS progenitors exhibit several key features of vertebrate senting the molecular architecture of trunk CNS in their last PNS progenitors, such as migration and differentiation into common ancestor, Urbilateria (8). However, there has been de- mechanosensory neurons (7, 8, 10, 13, 34). Our genetic analysis bate on the common origin of CNS centralization because some also shows that Msx/vab-15 specifies these PNS progenitors in sister or outgroup species of annelids, arthropods, and chordates nematodes, fly (Fig. S4), and urochordates, similar to its role in lack a centralized ventral cord (26, 28). For example, hemichor- the vertebrate NPB and PPE. The PPE is the neurogenic ecto- dates differ from major chordates by possessing a diffuse nervous derm anterolateral to the NPB, and is considered the epidermal system and do not have mediolateral neural patterning (29). subdomain of the vertebrate neural border that mostly contrib- Characterization of the molecular mechanisms of neural pattern- utes to cranial sensory systems (1, 37). The PPE also gives rise to ing in distantly related bilaterians should provide a broader view of mechanosensing hair cells of lateral lines in the trunk of anam- the evolution of nervous system centralization (28). niote vertebrates and those in vertebrate inner ears (1). The Nematodes diverged from arthropods about 550 Mya, close to development of the mechanosensing hair cells in vertebrates and the origin of Ecdysozoa (30). Unlike annelids and arthropods, that of the mechanosensory neurons in worm and fly both de- nematodes have an unsegmented body plan and no appendages. pend on Atoh1/lin-32 (22, 38). On the contrary, Ciona BTNs and C. elegans burrows in rotten matter and has a feeding organ vertebrate DRG neurons require Neurogenin/ngn-1 (9). Hence, isolated from the rest of the animal (31, 32). Correspondingly, its more molecular and comparative studies are needed to un- nervous system is more modestly organized than those in anne- derstand the evolutionary relationship of these mechanosensing lids and arthropods. The complex mediolateral patterning of cell types. Nevertheless, our study on the neural borders suggests trunk CNS is largely absent in worm, where there are only motor the conservation of the molecular mechanism underlying the neurons in its ventral cord. Nevertheless, Pax3/7/pax-3 and Msx/ specification of PNS progenitors across bilaterians (39). vab-15 still express in the lateral border of the CNS neuro- In theory, the similarity between the invertebrate and verte- ectoderm in worm (15, 33), consistent with the conserved ex- brate lateral neural border can be explained by either common pression patterns of these two lateral genes in arthropods, origin or convergence of independent evolution. To distinguish annelids, and chordates (8). Our genetic analysis shows that both between these two hypotheses, it is necessary to compare mo- Msx/vab-15 and Pax3/7/pax-3 specify P neuroblasts, which are lecular mechanisms specifying lateral neural borders in various worm CNS progenitors, similar to their roles in the lateral CNS bilaterians. Unlike the vertebrate NPB and neural crest, the progenitors in fly and vertebrates (11, 16, 34–36). Combining our molecular mechanism underlying the specification of worm data with the existing evidence of the common origin of trunk lateral neuroblasts still remains largely unknown after years of CNS from fly to vertebrates (8, 27), it is more likely that nem- genetic study (33, 40, 41). Our systematic expression profiling atodes inherited the Msx/vab-15 specification of P neuroblasts of 90% conserved transcription factors in C. elegans revealed from Urbilateria than the nematode branch lost the conserved that three NPB specifiers (Msx/vab-15, Pax3/7/pax-3,andZic/ lateral domain after diverging from fly and then regenerated ref-2) are coexpressed in worm lateral neuroblasts and that Msx/ it independently. vab-15 expression is more lateral than that of Pax3/7/pax-3 and In addition to the conserved CNS molecular module shown in Zic/ref-2, like their expression patterns in neural borders of C. elegans, our data suggest that the molecular mechanism de- vertebrates, urochordates, amphioxus, and likely annelids (7, 8, fining the lateral neural border, from which the majority of the 11, 13, 24). Furthermore, we showed that Msx/vab-15 regulates PNS is derived, is also conserved across bilaterians. First, the the differentiation of PNS progenitors in worm and fly, similar

E6356 | www.pnas.org/cgi/doi/10.1073/pnas.1704194114 Li et al. Downloaded by guest on October 1, 2021 and demonstrated that it functions synergistically with Msx/vab-15 PNAS PLUS in regulating the migration and differentiation of lateral neural progenitor cells in both C. elegans and Xenopus. Altogether, our data revealed deep conservation of vertebrate NPB specifiers’ module composition, expression pattern, and regulatory role in neural border development among distantly related species with drastically different neural anatomy, favoring the common ori- gin hypothesis in the molecular mechanism underlying neural border specification. Materials and Methods C. elegans-Related Procedures. Orthologous gene pairs. Orthologous gene pairs were obtained from a previous publication (18) or annotation in WormBase (www.wormbase.org)orTreeFam (www.treefam.org). A 1:N ortholog relationship is allowed. C. elegans strains and reporter transgenes. Mutant strains were obtained from the Caenorhabditis Genetics Center or National BioResource Project of Japan. vab-15 mutants: TU2362(u781), FX0260(tm260); tlp-1 mutant: EM347(bx85); lin-32 mutant: FXO1446(tm1446); dat-1 reporter: BZ555(Pdat-1::GFP). Promoter reporter constructs and their transgenic strains were generated as described (19, 44), and their description can be found in Dataset S1. vab-15::GFP knockin strain was generated using the CRISPR/Cas9 system as described (45). mec-10 reporter (Pmec-10::H1::mCherry) was generated through mosSCI insertion (46). Primer sequences of mec-10 promoter: 5′-accgaaagatgcacgtccta-3′ and 5′-ATATTGTGGTCCAGCTCTCACACTG-3′. Somatic knockout. Somatic KO was conducted as described (47). Guide RNA se- quences: vab-15,5′-GTGAATGTTGCATAGACAG-3′; pax-3,5′-GTCGTGTTAAC- CAACTCGG-3′; ref-2,5′-CTTGGGCTGGAAGAAAGTG-3′. These sequences were cloned into the vector pOG2306 (47). Each vector was injected into worms mixedwiththePmyo-2::GFP coinjection marker. Synchronized embryos of transgenic strain were transferred to OP50-seeded nematode growth medium (NGM) plates and heat shocked as described, at 33 °C for 1 h (47). The embryos then grew at 20 °C until L2 stage. Fig. 4. Msx/vab-15 is required for the development of worm lateral PNS Imaging protocol. Larvae that grew for no more than 4 h after hatching were progenitors in other invertebrates. (A and B) Disrupted chordotonal organs in considered to be at early L1 stage. Larvae whose neuronal lineage P neu- Msx/vab-15/msh mutant Drosophila embryos. Shown are lateral views of wild- roblasts finished cell division and whose gonad was composed of about type and msh4-4 embryos at early stage 12 with the anterior side at the left 50 cells were considered to be at L2 stage. For live imaging, worms were and ventral side at the bottom. (A and B) Neurons in the chordotonal organs placed on a 2% agarose pad and immobilized using levamisole (2.5 mg/mL). are revealed by 22C10 immunostaining (A) and glial cells by Repo staining (B). Images were captured using a Zeiss Imager A2 epifluorescence microscope. Pictures of whole embryos are in Fig. S4. Arrowheads indicate a five-neuron For 3D imaging, collected worms were frozen in liquid nitrogen for a night. array of chordotonal organs in wild type but disorganized with only four Frozen worms were thawed in 200 μL acetone. Then, the acetone was dis- neurons in msh4-4. Arrows indicate glial cells in the chordotonal organs. The carded and the worms were fixed with a solution composed of 1× modified migration of five glial cells of chordotonal organs is significantly Ruvkun’s witches brew and 20% formalin for 1.5 h, and then proceeded to desynchronized, and the glial cell numbers vary in different segments com- DAPI staining and 60% glycerol mounting as described (19). Three- pared with the WT embryos. (Scale bar, 100 μm.) (C) Defective BTN differ- dimensional images were obtained with a Zeiss LSM 780 confocal micro- entiation in Msx/vab-15 knockdown Ciona. Examined were larvae whose scope. The pixel size was 0.132 μminthex-y plane and 0.300 μminthe dorsal sides were up. Arrows point to BTNs. (C, Insets) Enlarged areas in the z direction. morpholino-injected larva represent BTNs with the down-regulated Asicb Cell annotation of 3D image stacks of L1 larvae. The 3D image stacks of worms reporter, a marker of BTN mechanosensory neurons. Fractions of scored larvae were straightened computationally along the anterior–posterior axis. Their with down-regulated reporter activity are shown. (Scale bars, 100 μm.) cell nuclei were segmented and annotated, and then manually edited with the VANO interactive interface as described (19). The identities of hyp7 hypodermal nuclei, seam cells, ventral cord motor neurons, neuroblasts, and to its critical role in specifying PNS lineage in urochordates and their progeny’s nuclei were manually confirmed according to WormAtlas vertebrates. We noticed that in PNS specification, only the role (www.wormatlas.org) (10). of Msx/vab-15 is conserved in worm, but not the other two NPB Gene-expression measurement. The gene-expression level for every cell was BIOLOGY

specifiers, Pax3/7/pax-3 and Zic/ref-2. This is not totally sur- measured as described (19). Briefly, background fluorescence was estimated DEVELOPMENTAL prising because there have been indications that the expression using 10 pseudonuclei. After subtracting background fluorescence, mCherry andfunctionofPax3/7/pax-3 and Zic/ref-2 are not strictly con- fluorescence was normalized by DAPI fluorescence to account for spherical served in the evolution of PNS development. For example, aberration. A nucleus with a normalized mCherry fluorescence of 500 was whereas Pax3/7/pax-3 isexpressedinthelateraldomainin often barely distinguishable from background red fluorescence according to visual inspection. To become robust to background, gene expression was both annelids and nematodes (8, 33), it is active in transverse calculated as (normalized mCherry fluorescence + 500)/500. stripes in fly (27). In addition, Pax3/7/pax-3 is only marginally Neuroblast-enrichment score. For each gene, we calculated its median log2 expressedintheputativePNSprogenitorregioninannelids(8). expression level for hypodermal cell group (hyp7, V1 to V4, plus V6), lateral Their expression even varies within vertebrates. Pax3/7/pax-3 neuroblast group (Q, P, V5), and ventral cord motor neuron (VMN) (Dataset − and Zic/ref-2 expression domains are significantly more medial S2). Mean (MedianP, MedianQ) Max (Medianhyp7, MedianV, MedianVMN) in lamprey (11) than those in zebrafish (42). Also, in Ciona,the represents the neuroblast-enrichment score of a gene. NPB cells that give rise to mechanosensory BTNs do not ex- Embryo-lineage tracing. vab-15 mutant strain TU2362(u781)wascrossedwith press Zic/ref-2 (25). Therefore, our data, consistent with other RW10029 (zuIs178; stIs10024) (48), which carries the his-72::GFP transgene so that all somatic nuclei become detectable during embryogenesis. studies, showed that only Msx/vab-15, but not other NPB Then, one- to four-cell-stage embryos were transferred onto an agarose pad specifiers, exhibits conserved expression and a functional role for imaging. We performed confocal imaging with an inverted Leica SP5 in PNS progenitors across bilaterians (3, 43). Finally, we have confocal microscope. Embryo-lineage tracing and imaging were processed as identified a novel lateral neural border specifier, ZNF703/tlp-1, described (49). The u781 mutant is edited up to 200 time points with 398 cells.

Li et al. PNAS | Published online July 17, 2017 | E6357 Downloaded by guest on October 1, 2021 Fig. 5. Expression profile of conserved transcription factors in the trunk ectoderm of L1 larvae at single-cell resolution. The data are in Dataset S2. Cells were manually arranged according to their cell types. Genes are ranked according to their neuroblast-enrichment score (see Materials and Methods) in descending order. (Upper) Expression profile of the top-seven neuroblast-enriched genes, which are barely expressed in skin cells and ventral cord motor neurons. Orthologs of vertebrate NPB specifiers are underlined.

D. melanogaster-Related Procedures. The MO sequences were as follows: Msx1:5′-GCCATACAGAGAGATCCG- Generation of Drosophila mutant. Msh mutant was generated using the CRISPR/Cas9 AGCTGAG-3′; ZNF703a:5′-ACCAGGGCGCAAAGATTCCCCTTGT-3′;andZNF703b: method (50) and guide sequence 5′-GGCTAGGACTCAGGTTGGTC-3′ target- 5′-ACCAGGGCGCGGAGATTCCCCTTAT-3′. 4-4 ing the N-terminal msh coding region. Msh contains a single-nucleotide All MOs are purchased from Gene Tools. ZNF703 MO was a mixture of 5 ng insertion at position 185 of the ORF so that it is a frameshift allele and pro- ZNF703a MO and 5 ng ZNF703b MO per embryo. Msx1 MO and/or ZNF703 MO duces a truncated MSH of only 67 amino acids. mixed with 2 ng RLDx as lineage tracer were injected into the animal-pole two Immunocytochemistry and microscopy. Embryos were fixed and stained as pre- left-side cells at the eight-cell stage. For rescue experiments, after MO injection, viously described (51). Rat anti-Repo was from A. Tomlinson (Columbia University, ZNF703 mRNA was immediately injected into the same two cells before the New York). Embryos were observed using differential interference contrast op- needle hole cured. Beta-gal mRNA was coinjected as a lineage tracer in the tics with a Nikon Eclipse 80i microscope. ZNF703 mRNA-alone group. The right-injected embryos were sorted out under a fluorescence dissecting microscope (Olympus; SZX16) and cultured to stages

X. laevis-Related Procedures. Frogs were used with ethical approval from the 16 to 17. Embryos were fixed in Mops EGTA MgSO4 formaldehyde (MEMFA) Animal Care and Use Committee of Tsinghua University. solutionin for 4 h at room temperature followed by whole-mount in situ hy- Xenopus whole-mount in situ hybridization. Whole-mount in situ hybridization bridization. ZNF703 mRNA (mixed with beta-gal)-injected embryos were stained was performed based on standard protocols (52, 53). cDNA sequences for by red-gal before in situ hybridization. ZNF703, Runx1, Islet1,andSox2 were cloned into the vector pCS107 (www. Western blotting. Western blotting was performed as described previously (55). xenbase.org). Primer sequences were as follows: ZNF703:5′-TCCatcga- Briefly, Xenopus embryos were lysed in TNE buffer (10 mM NaCl, 10 mM tATGAACTGTTCTCCCCCTGGATCT-3′ and 5′-cacGTCGACctggtatcctagcgc- Tris·HCl, 1 mM EDTA, pH 7.4) plus protease inhibitors, and pipetted several tgaagctg-3′; Runx1:5′-tccATCGATATGGCCTCACATAGTGCCTTCCA-3′ and times on ice. Lysates were then cleared by centrifugation at 1,000 × g for 5′-gagcCTCGAGtcagtatggccgccatacggctt-3′; Islet1:5′-cgcGAATTCatggga- 5 min. Supernatant was mixed with loading buffer and boiled for 15 min gatatgggagaccca-3′ and 5′-gagcCTCGAGtgcctctatagggctggctaccatg-3′;and followed by SDS/PAGE separation. Primary antibody of HA (Roche; 1:2,000 di- :5′-gagaGAATTCatgtacagcatgatggagaccgag-3′ and 5′-gcgcctcgagt- lution) and primary antibody of β-actin (1:5,000 dilution) were used. cacatgtgcgacagaggcagcgtgcc-3′. In situ probes were labeled with Dig-UTP using a Promega T7 polymerase kit. C. intestinalis-Related Procedures. C. intestinalis adults were obtained from M- Xenopus embryo manipulation and microinjection. Xenopus eggs and embryos Rep. Sperm and eggs were collected by dissecting the sperm and gonad ducts. were obtained through standard procedures (54). Briefly, eggs harvested Construct. The Asicb reporter construct was designed based on the previously from HCG-injected female frogs were fertilized through artificial in- published enhancer sequence (56). semination techniques. Fertilized eggs were dejellied using 2% cysteine Microinjection of antisense morpholino oligonucleotide. MOs were obtained from prepared in 0.1× Marc’s modified Ringer’s (MMR) (pH 7.9), followed by Gene Tools. The antisense oligonucleotide sequence of the Msx MO is 5′-ATT a thorough wash with 0.1× MMR. The dejellied embryos were then transferred CGTTTACTGTCATTTTTAATTT-3′. The MO was dissolved at a concentration of into 2% Ficoll in 0.5× MMR for morpholino (MO) and/or mRNA injection. 0.5 mM in 1 mM Tris·HCl (pH 8.0), 0.1 mM EDTA, 1 mg/mL tetramethylrhodamine

E6358 | www.pnas.org/cgi/doi/10.1073/pnas.1704194114 Li et al. Downloaded by guest on October 1, 2021 PNAS PLUS

Fig. 6. ZNF703/tlp-1 is a novel regulator functioning as an Msx/vab-15 modulator both in worm lateral neuroblasts and vertebrate NPB. (A) Embryonic expression of worm ZNF703/tlp-1. A comma-stage embryo carrying the Ptlp-1::H1::mCherry reporter trangene is shown. Arrowheads indicate P neuroblasts. μ –

Arrow indicates the mother cell of V5 and Q neuroblasts. (Scale bar, 10 m.) (B E) Phenotypes of ZNF703/tlp-1 and Msx/vab-15 mutant worms of migration BIOLOGY and differentiation of lateral neuroblasts. The y axis represents the number of VD neurons recognized by the Punc-25::GFP marker (B) or the fraction of worms showing the specified defect (C–E). The number of scored worms is shown. (F) The expression of ZNF703 in Xenopus embryos from stage (st)12 to st19. DEVELOPMENTAL (Scale bar, 0.5 mm.) The red dot lines represent the location of cross section. (G) Whole-mount in situ hybridization assay in Xenopus embryos (st16 to st17) to examine the effect of MO knockdown of Msx/vab-15 and/or ZNF703/tlp-1 on the expression of the neural plate marker Sox2, neural crest specifier FoxD3,and Rohon–Beard sensory neuron progenitor marker Runx1 and DRG progenitor marker Islet1. Asterisks indicate the injected side of an embryo. The red lines and green lines indicate the length of left and right parts of neural tubes, respectively. # indicates mRNA resistant to the ZNF703 MO. Fractions of scored embryos with phenotype are shown. (Scale bar, 0.5 mm.) Error bars represent standard errors.

dextran (D-1817; Invitrogen). Microinjection of MO was performed as described the assumption that the fraction of animals with a phenotype followed previously (57). binomial distribution, and a t test was used when a quantitative pheno- Image acquisition. Images of larvae were captured using a Zeiss AX10 epifluorescence type was scored under the assumption that the quantitative phenotype microscope. followed normal distribution. Variation was calculated based on binomial or normal distribution, and the similarity of variations was not assumed to – Statistics. Wilcoxon and Mann Whitney tests, robust statistics tests with no decrease the false positive rate. The minimum number of scored animals assumption on the distribution of samples, were used to examine whether was 22 in one batch analyzed, whereas the maximum was 300. a set of worm genes orthologous to a vertebrate gene module had sig- nificantly higher neuroblast-enrichment or mechano-enrichment scores. In ACKNOWLEDGMENTS. We thank Haidong Li from the Center of Biomedi- genetics and gene-knockdown analyses, Fisher’s exact test was used under cal Analysis, Tsinghua University for confocal imaging, and Lei Qu and

Li et al. PNAS | Published online July 17, 2017 | E6359 Downloaded by guest on October 1, 2021 Hanchuan Peng for processing imaging data. Drs. Cecilia Mello and Robert script. This work was supported by Ministry of Science and Technology Waterston edited the manuscript. Dr. Dionne Vafeados provided transgenic of China Grant 20131970194; National Natural Science Foundation of worms. We are very grateful to Drs. Martin Chalfie, Andy Fire, David Kingsley, China Grants 20141300429 and 91519326; Tsinghua 985 funding; Grant-in- Chris Amemiya, Xiaohang Yang, Ding Xue, Zhirong Bao, Margaret Fuller, Aid for Scientific Research from JSPS Grant 24687008; and Pre-Strategic Ini- and Joanna Wysocka for helpful discussions and comments on the manu- tiatives, University of Tsukuba.

1. Schlosser G, Patthey C, Shimeld SM (2014) The evolutionary history of vertebrate importance of substrate type, abiotic parameters, and Caenorhabditis competitors. cranial placodes II. Evolution of ectodermal patterning. Dev Biol 389:98–119. BMC Ecol 14:4. 2. Medeiros DM (2013) The evolution of the neural crest: New perspectives from lam- 32. Albertson DG, Thomson JN (1976) The pharynx of Caenorhabditis elegans. Philos prey and invertebrate neural crest-like cells. Wiley Interdiscip Rev Dev Biol 2:1–15. Trans R Soc Lond B Biol Sci 275:299–325. 3. Green SA, Simoes-Costa M, Bronner ME (2015) Evolution of vertebrates as viewed 33. Thompson KW, et al. (2016) The paired-box protein PAX-3 regulates the choice be- from the crest. Nature 520:474–482. tween lateral and ventral epidermal cell fates in C. elegans. Dev Biol 412:191–207. 4. Milet C, Monsoro-Burq AH (2012) Neural crest induction at the neural plate border in 34. Wang W, Chen X, Xu H, Lufkin T (1996) Msx3: A novel murine homologue of the vertebrates. Dev Biol 366:22–33. Drosophila msh gene restricted to the dorsal embryonic central nervous 5. Schlosser G (2008) Do vertebrate neural crest and cranial placodes have a common system. Mech Dev 58:203–215. evolutionary origin? BioEssays 30:659–672. 35. Duval N, et al. (2014) Msx1 and Msx2 act as essential activators of Atoh1 expression in 6. Sulston JE, Schierenberg E, White JG, Thomson JN (1983) The embryonic cell lineage the murine spinal cord. Development 141:1726–1736. of the nematode Caenorhabditis elegans. Dev Biol 100:64–119. 36. Lin J, Wang C, Yang C, Fu S, Redies C (2016) Pax3 and Pax7 interact reciprocally and 7. D’Alessio M, Frasch M (1996) msh may play a conserved role in dorsoventral pat- regulate the expression of cadherin-7 through inducing neuron differentiation in the terning of the neuroectoderm and mesoderm. Mech Dev 58:217–231. developing chicken spinal cord. J Comp Neurol 524:940–962. 8. Denes AS, et al. (2007) Molecular architecture of annelid nerve cord supports common 37. Abitua PB, et al. (2015) The pre-vertebrate origins of neurogenic placodes. Nature origin of nervous system centralization in bilateria. Cell 129:277–288. 524:462–465. 9. Stolfi A, Ryan K, Meinertzhagen IA, Christiaen L (2015) Migratory neuronal progen- 38. Arendt D, et al. (2016) The origin and evolution of cell types. Nat Rev Genet 17:744–757. itors arise from the neural plate borders in tunicates. Nature 527:371–374. 39. Fritzsch B, Beisel KW, Pauley S, Soukup G (2007) Molecular evolution of the vertebrate 10. Sulston JE, Horvitz HR (1977) Post-embryonic cell lineages of the nematode, Caenorhabditis mechanosensory cell and ear. Int J Dev Biol 51:663–678. elegans. Dev Biol 56:110–156. 40. Feng G, et al. (2013) Developmental stage-dependent transcriptional regulatory 11. Sauka-Spengler T, Meulemans D, Jones M, Bronner-Fraser M (2007) Ancient evolu- pathways control neuroblast lineage progression. Development 140:3838–3847. tionary origin of the neural crest gene regulatory network. Dev Cell 13:405–420. 41. Wrischnik LA, Kenyon CJ (1997) The role of lin-22, a hairy/enhancer of split homolog, in 12. Ramos C, Robert B (2005) msh/Msx gene family in neural development. Trends Genet patterning the peripheral nervous system of C. elegans. Development 124:2875–2888. 21:624–632. 42. Garnett AT, Square TA, Medeiros DM (2012) BMP, Wnt and FGF signals are integrated 13. Abitua PB, Wagner E, Navarrete IA, Levine M (2012) Identification of a rudimentary through evolutionarily conserved enhancers to achieve robust expression of Pax3 and neural crest in a non-vertebrate chordate. Nature 492:104–107. Zic genes at the zebrafish neural plate border. Development 139:4220–4231. 14. Sharman AC, Shimeld SM, Holland PW (1999) An amphioxus Msx gene expressed 43. Waki K, Imai KS, Satou Y (2015) Genetic pathways for differentiation of the periph- predominantly in the dorsal neural tube. Dev Genes Evol 209:260–263. eral nervous system in ascidians. Nat Commun 6:8719. 15. Du H, Chalfie M (2001) Genes regulating touch cell development in Caenorhabditis 44. Murray JI, et al. (2012) Multidimensional regulation of gene expression in the elegans. Genetics 158:197–207. C. elegans embryo. Genome Res 22:1282–1294. 16. Isshiki T, Takeichi M, Nose A (1997) The role of the msh homeobox gene during 45. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B (2013) Engineering the Caenorhabditis Drosophila neurogenesis: Implication for the dorsoventral specification of the neu- elegans genome using Cas9-triggered homologous recombination. Nat Methods 10: roectoderm. Development 124:3099–3109. 1028–1034. 17. Orgogozo V, Grueber WB (2005) FlyPNS, a database of the Drosophila embryonic and 46. Frøkjær-Jensen C, et al. (2014) Random and targeted transgene insertion in larval peripheral nervous system. BMC Dev Biol 5:4. Caenorhabditis elegans using a modified Mos1 transposon. Nat Methods 11:529–534. 18. Shaye DD, Greenwald I (2011) OrthoList: A compendium of C. elegans genes with 47. Shen Z, et al. (2014) Conditional knockouts generated by engineered CRISPR- human orthologs. PLoS One 6:e20085. Cas9 endonuclease reveal the roles of coronin in C. elegans neural development. Dev 19. Liu X, et al. (2009) Analysis of cell fate from single-cell gene expression profiles in Cell 30:625–636. C. elegans. Cell 139:623–633. 48. Murray JI, et al. (2008) Automated analysis of embryonic gene expression with cellular 20. Reece-Hoyes JS, et al. (2007) Insight into gene duplication from resolution in C. elegans. Nat Methods 5:703–709. Caenorhabditis elegans Promoterome-driven expression patterns. BMC Genomics 49. Ho VWS, et al. (2015) Systems-level quantification of division timing reveals a com- 8:27. mon genetic architecture controlling asynchrony and fate asymmetry. Mol Syst Biol 21. Zhu Z, et al. (2014) A proneural gene controls C. elegans neuroblast asymmetric di- 11:814. vision and migration. FEBS Lett 588:1136–1143. 50. Gratz SJ, et al. (2013) Genome engineering of Drosophila with the CRISPR RNA- 22. Zhao C, Emmons SW (1995) A transcription factor controlling development of pe- guided Cas9 nuclease. Genetics 194:1029–1035. ripheral sense organs in C. elegans. Nature 373:74–78. 51. Yang X, Yeo S, Dick T, Chia W (1993) The role of a Drosophila POU homeo domain 23. Nikitina N, Sauka-Spengler T, Bronner-Fraser M (2008) Dissecting early regulatory gene in the specification of neural precursor cell identity in the developing embryonic relationships in the lamprey neural crest gene network. Proc Natl Acad Sci USA 105: central nervous system. Genes Dev 7:504–516. 20083–20088. 52. Lander R, et al. (2013) Interactions between Twist and other core epithelial- 24. Holland LZ (2009) Chordate roots of the vertebrate nervous system: Expanding the mesenchymal transition factors are controlled by GSK3-mediated phosphorylation. molecular toolkit. Nat Rev Neurosci 10:736–746. Nat Commun 4:1542. 25. Imai KS, Levine M, Satoh N, Satou Y (2006) Regulatory blueprint for a chordate em- 53. Zhang Y, Ding Y, Chen YG, Tao Q (2014) NEDD4L regulates convergent extension bryo. Science 312:1183–1187. movements in Xenopus embryos via Disheveled-mediated non-canonical Wnt sig- 26. Strausfeld NJ, Hirth F (2016) Introduction to ‘Homology and convergence in nervous naling. Dev Biol 392:15–25. system evolution.’ Philos Trans R Soc Lond B Biol Sci 371:20150034. 54. Sive HL, Grainger RM, Harland RM (2000) Early Development of Xenopus laevis: A 27. Arendt D, Nübler-Jung K (1999) Comparison of early nerve cord development in in- Laboratory Manual (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY). sects and vertebrates. Development 126:2309–2325. 55. Gao L, et al. (2016) A novel role for Ascl1 in the regulation of mesendoderm for- 28. Hejnol A, Lowe CJ (2015) Embracing the comparative approach: How robust phy- mation via HDAC-dependent antagonism of VegT. Development 143:492–503. logenies and broader developmental sampling impacts the understanding of nervous 56. Coric T, Passamaneck YJ, Zhang P, Di Gregorio A, Canessa CM (2008) Simple chordates system evolution. Philos Trans R Soc Lond B Biol Sci 370:20150045. exhibit a proton-independent function of acid-sensing ion channels. FASEB J 22: 29. Lowe CJ, et al. (2006) Dorsoventral patterning in hemichordates: Insights into early 1914–1923. chordate evolution. PLoS Biol 4:e291. 57. Satou Y, Imai KS, Satoh N (2001) Action of morpholinos in Ciona embryos. Genesis 30: 30. Rota-Stabelli O, Daley AC, Pisani D (2013) Molecular timetrees reveal a Cambrian 103–106. colonization of land and a new scenario for ecdysozoan evolution. Curr Biol 23: 58. Duggan A, Ma C, Chalfie M (1998) Regulation of touch differentiation by the 392–398. Caenorhabditis elegans mec-3 and unc-86 genes. Development 125:4107–4119. 31. Petersen C, Dirksen P, Prahl S, Strathmann EA, Schulenburg H (2014) The prevalence 59. Huang M, Chalfie M (1994) Gene interactions affecting mechanosensory transduction of Caenorhabditis elegans across 1.5 years in selected north German locations: The in Caenorhabditis elegans. Nature 367:467–470.

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