Retinoids and Spinal Cord DevelopmentMalcolm MadenMRC Centre for Developmental Neurobiology, King’s College London, London, United KingdomReceived 23 October 2005; accepted 22 November 2005ABSTRACT: The role that RA plays in the devel-opment and patterning of the spinal cord is discussed.The morphogenetic process of neurulation is describedin which RA plays a role because in the absence of RAsignaling spina bifida results. Following neural induc-tion, RA is involved in several patterning events in thespinal cord. It is one of the posteriorizing factors alongwith FGFs and Wnts and as such patterns the cervicalspinal cord acting via the Hoxc transcription factors. Itis involved in the induction of neural differentiation viagenes such as NeuroM. It plays a part in patterning thedorsoventral axis of the anterior spinal cord where itinteracts with FGF, Shh, and BMPs and induces aninterneuronal population of neurons called V0 and V1and a subset of motor neurons known as LMCs. To per-form these actions RA is synthesized in the adjacent par-axial mesoderm by the enzyme RALDH2 and acts in aparacrine fashion on the developing neural tube. In thefinal action of RA, it begins to be synthesized within theneural tube at brachial and lumbar levels in the LMCs.Later-born neurons migrate through this RALDH2-expressing region and induce differentiation in thesemigrating neurons, which become a subset of LMC neu-rons known as LMCls. Thus RA acts several times overin the development of the spinal cord and not on thecells in which it is synthesized, but in adjacent cells in aparacrine manner.' 2006 Wiley Periodicals, Inc. J Neurobiol 66:726–738, 2006Keywords: retinoic acid; motor neurons; FGF; Wnt;Raldh2; neural plate patterning; spinal cord patterningNEURULATIONThe flat neural plate, which lies on the upper surfaceof the embryo and in which patterning into the fore-brain, midbrain, hindbrain and spinal cord takes place[Fig. 1(A,B)] undergoes a complex series of morpho-genetic movements known as neurulation to producea neural tube that eventually lies just beneath the sur-face of the embryo. During neurulation the open neu-ral plate undergoes a mediolateral narrowing, a ros-trocaudal lengthening, and the neural folds elevateand fuse in the midline, while the epiblast at the lat-eral edges, outside the neural plate, comes to coverthe newly invaginated neural tube. This tube is ini-tially a single layer of pseudostratified epithelium,which then proliferates rapidly, and differentiation ofneurons takes place at the periphery of the expand ingneural tube.Failure to complete closure of the neural tube atthe rostral end leads to anencephaly (no brain) or var-ious degrees of cephalic tissue protruding outside theskull (exencephaly), and failure to comple te closureof the neural tube at the caudal end, within thespinal cord, leads to various degrees of spina bifida.Together these defects are known as neural tube de-fects (NTDs) and they are very important in humansas they are the second-most common congenitaldefect.It is reasonable to ask whether retinoic acid (RA)plays any role in the etiology of NTDs because theoccurrence of spina bifida, anencephaly, and exenc e-phaly is one of the well-established teratogenic ef-fects of excess RA on a wide variety of species (Kal-ter and Warkany, 1959; Shenfelt, 1972; Tibbles andWiley, 1988; Yasuda et al., 1986). The cause of thiseffect has been attributed to vascular damage, malfor-Correspondence to: M. Maden ([email protected]).' 2006 Wiley Periodicals, Inc.Published online in Wiley In terScience (www.in terscience.wiley.com).DOI 10.1002/neu.20 248726Figure 1 (A,B) Drawings of the neural plate stages of frog (A) and chick (B) embryos to showthe presumptive regions. The neural plate is the colored area. In (A) the neural plate is surroundedby the neural folds and in (B) the node (black dot) is still regressing posteriorly through the pre-sumptive spinal cord region. fb ¼ forebrain ¼ red; mb ¼ midbrain ¼ yellow; hb ¼ hindbrain ¼blue; sc ¼ spinal cord ¼ purple. (C) The gene probes and their expression domains marked on achick embryo that are used to identify regions of the neural plate that differentiate after varioustreatments. Red ¼ Otx2 ¼ forebrain marker; yellow ¼ En1 ¼ midbrain/hindbrain border; blue ¼Krox20 ¼ rhombomeres 3 and 5 of the hindbrain; purple ¼ Hoxb9 ¼ spinal cord. (D) The regionsof the nervous system that Wnts, RA, and Fgf are responsible for generating.Retinoids and Spinal Cord Development 727Journal of Neurobiology. DOI 10.1002/neumation of the notochord, distortion of the neural tube,cell death, delayed posterior neuropore closure, orcell death in the tail bud (Alles and Sulik, 1990; Kap-ron-Bras and Trasler, 1988; Tibbles and Wiley, 1988;Padmadabhan, 1998).However, because the levels of RA that are admin-istered during a teratogenic dose are at least 1000-fold higher than physiological levels (Horton andMaden, 1995), it is highly likely that this type ofresult is not telling us anything about the role of endo-genous RA in neurulation. To reveal this role we needto ask whether in the absence of RA signaling NTDsare generated. This has been performed in several ways.Firstly, the generation of null mutant embryos forvarious RARs and combinations thereof has gener-ated embryos with exencephaly. Thus there are highlevels of exencephaly in RAR/ double-null mousemutant embryos (Lohnes et al., 1994), and RAR/mutant embryos are resistant to the spina bifida, exen-cephaly, and anencephaly caused by teratogenic dosesof RA (Iulianella and Lohnes, 1997; Lohnes et al.,1993). The Cyp26A1 / mutant embryos mimic theeffects of excess RA by exhibiting exencephal y andspina bifida (Abu-Abed et al., 2001; Sakai et al., 2001),and this phenotype is rescued in the Cyp26A1/RARdouble-null mutant (Abu-Abed et al., 2003). Theseresults suggest that endogenous RA signaling via theRAR receptor plays a role in proper closure of theneural tube during neurulation.Secondly, the Raldh2 null mutant embryos thathave lost the major RA synthesizing enzyme in theembryo also exhibit spina bifida (Niederreither et al.,1999).Thirdly, in the curly tail mouse mutant, which isused as a model for human NTDs, the incidence ofspina bifida is reduced by the administration of lowdoses of RA at a specific time in development (Sellerand Perkins, 1982). This effect is thought to involvethe straightening of the tail bud, thereby