The homeobox gene Hb9, like its close relative MNR2, is expressed selectively by motor neurons (MNs) in the developing vertebrate CNS. In embryonic chick spinal cord, the ectopic expression of MNR2 or Hb9 is sufficient to trigger MN differentiation and to repress the differentiation of an adjacent population of V2 interneurons. Here, we provide genetic evidence that Hb9 has an essential role in MN differentiation. In mice lacking Hb9 function, MNs are generated on schedule and in normal numbers but transiently acquire molecular features of V2 interneurons. The aberrant specification of MN identity is associated with defects in the migration of MNs, the emergence of the subtype identities of MNs, and the projection of motor axons. These findings show that HB9 has an essential function in consolidating the identity of postmitotic MNs.

The specification of neuronal fates in the ventral spinal cord depends on the regulation of homeodomain (HD) and basic-helix-loop-helix (bHLH) proteins by Sonic hedgehog (Shh). Most of these transcription factors function as repressors, leaving unresolved the link between inductive signaling pathways and transcriptional activators involved in ventral neuronal specification. We show here that retinoid signaling and the activator functions of retinoid receptors are required to pattern the expression of HD and bHLH proteins and to specify motor neuron identity. We also show that fibroblast growth factors (FGFs) repress progenitor HD protein expression, implying that evasion of FGF signaling and exposure to retinoid and Shh signals are obligate steps in the emergence of ventral neural pattern. Moreover, joint exposure of neural progenitors to retinoids and FGFs suffices to induce motor neuron differentiation in a Shh-independent manner.

The diversification of neuronal cell types in the vertebrate central nervous system depends on inductive signals provided by local organizing cell groups of both neural and nonneural origin. The influence of signals provided by postmitotic neurons on the fate of neurons born at subsequent development stages, however, remains unclear. We provide evidence that a retinoid-mediated signal provided by one subset of early-born spinal motor neurons imposes a local variation in the number of motor neurons generated at different axial levels and also specifies the identity of a later-born subset of motor neurons. Thus, in the vertebrate central nervous system the distinct fates of late-born neurons may be acquired in response to signals provided by early-born neurons.

The protein encoded by the human testis determining gene, SRY, contains a high mobility group (HMG) box related to that present in the T cell-specific, DNA-binding protein TCF-1. Recombinant SRY protein was able to bind to the same core sequence AACAAAG recognized by TCF-1 in a sequence dependent manner. In five XY females point mutations were found in the region encoding the HMG box. In four cases DNA binding activity of mutant SRY protein was negligible; in the fifth case DNA binding was reduced. These results imply that the DNA binding activity of SRY is required for sex determination.

Excess retinoids as well as retinoid deprivation cause abnormal development, suggesting that retinoid homeostasis is critical for proper morphogenesis. RALDH-2 and CYP26, two key enzymes that carry out retinoic acid (RA) synthesis and degradation, respectively, were cloned from the chick and show significant homology with their orthologs in other vertebrates. Expression patterns of RALDH-2 and CYP26 genes were determined in the early chick embryo by in situ hybridization. During gastrulation and neurulation RALDH-2 and CYP26 were expressed in nonoverlapping regions, with RALDH-2 transcripts localized to the presumptive presomitic and lateral plate mesoderm and CYP26 mRNA to the presumptive mid- and forebrain. The two domains of expression were separated by an approximately 300-micrometer-wide gap, encompassing the presumptive hindbrain. In the limb region, a similar spatial segregation of RALDH-2 and CYP26 expression was found at stages 14 and 15. Limb region mesoderm expressed RALDH-2, whereas the overlying limb ectoderm expressed CYP26. RA-synthesizing and -degrading enzymatic activities were measured biochemically in regions expressing RALDH-2 or CYP26. Regions expressing RALDH-2 generated RA efficiently from precursor retinal but degraded RA only inefficiently. Conversely, tissue expressing CYP26 efficiently degraded but did not synthesize RA. Localized regions of RA synthesis and degradation mediated by these two enzymes may therefore provide a mechanism to regulate RA homeostasis spatially in vertebrate embryos.

We have studied the mechanism of delta 1‐crystallin gene activation, which occurs early in lens cell differentiation, and have previously shown that an essential element of the delta 1‐crystallin enhancer is bound by a group of nuclear factors, delta EF2, among which delta EF2a is highly enriched in lens cells. In this report we show that the cDNA of delta EF2a codes for the chicken SOX‐2 protein (cSOX‐2), which is structurally related to the sex‐determining factor SRY. Sox‐2 is expressed at high levels in the early developing lens in both chicken and mouse embryos. Overexpression of delta EF2a/cSOX‐2 increased delta 1‐crystallin enhancer activity to a plateau in lens cells, but not in fibroblasts, consistent with the previously drawn conclusion that delta EF2a activates transcription only in concert with another factor present in the lens. This result supports the model that SOX proteins act as architectural components in the activating complex formed on an enhancer, as indicated for another HMG domain protein, lymphoid enhancer binding factor 1 (LEF‐1). We also show that SOX protein binding is essential for lens‐specific promoter activity of the mouse gamma F‐crystallin gene. This work is the first to show delta‐ and gamma‐crystallin genes as examples of direct regulatory targets of SOX proteins and provides evidence that diversified crystallin genes are regulated, at least partly, by a common mechanism.

The six-transmembrane protein glycerophosphodiester phosphodiesterase 2 (GDE2) induces spinal motor neuron differentiation by inhibiting Notch signaling in adjacent motor neuron progenitors. GDE2 function requires activity of its extracellular domain that shares homology with glycerophosphodiester phosphodiesterases (GDPD). GDPDs metabolize glycerophosphodiesters into glycerol-3-phosphate and corresponding alcohols but whether GDE2 inhibits Notch signaling by this mechanism is unclear. Here, we show that GDE2, unlike classical GDPDs, cleaves glycosylphosphatidylinositol (GPI)-anchors. GDE2 GDPD activity inactivates the Notch activator RECK by releasing it from the membrane by GPI-anchor cleavage. RECK release disinhibits ADAM protease-dependent shedding of the Notch ligand Delta-like 1 (Dll1) leading to Notch inactivation. This study identifies a previously unrecognized mechanism to initiate neurogenesis that involves GDE2 mediated surface cleavage of GPI-anchored targets to inhibit Dll1-Notch signaling.

Entry of yeast cells into the mitotic cell cycle (Start) involves a form of the CDC28 kinase that associates with G1-specific cyclins encoded by CLN1 and CLN2 (ref. 1). The onset of Start may be triggered by the activation of CLN1 and CLN2 transcription in late G1 (ref. 2). SWI4 and SWI6 are components of a factor (SBF) that binds the CACGAAAA (SCB) promoter elements responsible for activation in late G1 of the HO endonuclease, CLN1 and CLN2 genes. A related factor (MBF) containing SWI6 and a 120K protein binds to the ACGCGTNA (MCB) promoter elements responsible for late G1-specific transcription of DNA replication genes. Nothing is known about how these heteromeric proteins bind DNA. We show here that SWI4 contains a novel DNA-binding domain at its N terminus that alone binds specifically to SCBs and a C-terminal domain that binds to SWI6. SWI4's DNA-binding domain is similar to an N-terminal domain of the cdc10 protein that is a component of an MBF-like factor from Schizosaccharomyces pombe and is required for Start. An involvement of this kind of DNA-binding domain in transcriptional controls at Start may therefore be a conserved feature of eukaryotic cells.