3693 Introduction Dorsal spinal cord formation begins with neuronal and glial progenitors exiting the cell cycle in the ventricular zone (VZ), and is followed by sequential migration and further differentiation of migrating neurons and glia. Once neurons have reached their appropriate region of the dorsal spinal cord, they differentiate further into distinct layers or laminae that carry out distinct physiological function (Christensen and Perl, 1970; Rexed, 1952). For example, lamina I primarily contains relay and local interneurons, whereas lamina II (substantia gelatinosa) consists of few projection neurons but contains numerous small-size interneurons, many of which make synaptic contacts with lamina I neurons (Molander and Grant, 1995; Willis, 1995). The primary afferents of dorsal root ganglia (DRG) neurons project to distinct laminae of the spinal cord and establish precise neuronal connections with their targets. Small-diameter DRG neurons that express TrkA, a high-affinity neurotrophin receptor for nerve growth factor (NGF), project to laminae I-II and relay noxious and thermal sensory information, whereas large-diameter DRG neurons that express the neurotrophin receptor TrkC make connections with motoneurons in the ventral horn and relay muscle proprioceptive information (Christensen and Perl, 1970; Huang and Reichardt, 2001; Lawson and Biscoe, 1979; Scott, 1992; Snider, 1994). During spinal cord development, the specification of dorsal interneurons is mediated by both extrinsic and intrinsic factors (Caspary and Anderson, 2003; Helms and Johnson, 2003; Lee and Jessell, 1999; Liem et al., 1997). Specifically, the intrinsic factors that control the specification and development of several classes of early-born neurons (dI1-6) have been analyzed in greater detail. In mice lacking transcription factors Lbx1 (Lbx1h – Mouse Genome Informatics) or Rnx (Tlx3 – Mouse Genome Informatics), the specification of dI5 neurons is affected, as indicated by the loss or downregulation of Lmx1b (dI5 marker) (Gross et al., 2002; Muller et al., 2002; Qian et al., 2002). These two genes are also involved in the development of late-born neurons that make up the dorsal horn of the spinal cord (Caspary and Anderson, 2003). In addition, the transcription factors Drg11 (Prxxl1 – Mouse Genome Informatics), Ebf1, Ebf3 and Zic1 have also been shown to be important for the early specification of the dorsal spinal cord neurons (Aruga et al., 1998; Aruga et al., 2002b; Chen et al., 2001; Ebert et al., 2003; Garcia-Dominguez et al., 2003; Garel et al., 1997; Wang et al., 1997). Among them, Zic1 has been shown to be a negative regulator of the differentiation of the dorsal horn neurons in mice (Aruga et al., 2002a; Aruga et al., 1998; Aruga et al., 1996a; Aruga et al., 2002b; Aruga et al., 1994; Aruga et al., 1996b). Despite these studies, our knowledge about the molecular mechanisms that govern the development of early-born neurons and the assembly of the dorsal horn circuits is still rather fragmentary. Lmx1b is an LIM homeobox-containing gene, and was originally isolated as a mouse ortholog of the chicken Lmx1 (Chen et al., 1998a; Riddle et al., 1995; Vogel et al., 1995). In the chicken, Lmx1 is involved in the specification of the dorsal cell The differentiation and migration of superficial dorsal horn neurons and subsequent ingrowth of cutaneous afferents are crucial events in the formation of somatosensory circuitry in the dorsal spinal cord. We report that the differentiation and migration of the superficial dorsal horn neurons are regulated by the LIM homeobox gene Lmx1b, and its downstream targets Rnx and Drg11, two transcription factors implicated in the development of dorsal horn circuitry. An analysis of Lmx1b mutants shows that Lmx1b normally acts to maintain the expression of the Ebf genes and to repress the Zic genes. Lmx1b mutants also exhibit the disruption of the cutaneous afferent ingrowth, suggesting that the dorsal horn cells might provide important cues guiding sensory axons into the dorsal spinal cord. Our results thus indicate that Lmx1b has a pivotal role in genetic cascades that control the assembly of circuitry in the superficial dorsal horn. Supplemental data available online Key words: Lmx1b, Dorsal horn, Migration, Differentiation, Cutaneous afferents, Mouse Summary Lmx1b controls the differentiation and migration of the superficial dorsal horn neurons of the spinal cord Yu-Qiang Ding 1, *, Jun Yin 1 , Artur Kania 2 , Zhong-Qiu Zhao 1 , Randy L. Johnson 3 and Zhou-Feng Chen 1,† 1 Departments of Anesthesiology, Psychiatry, Molecular Biology and Pharmacology, Washington University School of Medicine Pain Center, St. Louis, MO 63110, USA 2 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10027, USA 3 Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 70030, USA *Present address: Laboratory of Neural Development, Institute of Neuroscience, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, 20031, PR China † Author for correspondence (e-mail: chenz@morpheus.wustl.edu) Accepted 4 May 2004 Development 131, 3693-3703 Published by The Company of Biologists 2004 doi:10.1242/dev.01250 Research article