Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function.

The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern.

Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3.

Specification of distinct neuron types in the ventral spinal cord is thought to be mediated by a graded concentration of Sonic hedgehog (Shh), a secreted signaling protein. Shh is made in the notochord, the most ventral part of the spinal cord, and in mice lacking Shh, ventral cell types are reduced or absent. The response to Shh depends on transcription factors of the Gli family, but the detailed mechanism is not understood. Here we show that Gli3 represses ventral fates in a dose-dependent manner. Whereas Shh -/- mutant mice show reductions in several classes of ventral interneurons and a complete absence of motor neurons, these cell types were rescued in Shh-/-;Gli3 -/- double mutants. This rescue of the Shh null phenotype depended on the level of Gli3 function; a partial rescue was observed in Shh-/-;Gli3 +/- embryos. We propose that Shh is required to antagonize Gli3, which would otherwise repress ventral fates. Differences between rostral and caudal regions suggest that other signaling molecules-in addition to Shh-may be involved in specifying ventral fates, particularly in the caudal region of the spinal cord.

Control of Shh activity and signaling in the neural tube.

The generation of diverse cell types in the neural tube requires inductive signals that are generally derived from tissues adjacent to the neural tube and capable of patterning cell fates at a distance. Shh, a signaling molecule secreted from the notochord and the floor plate, has been shown to induce motor neurons (MN) as well as interneurons, dorsal to the MN, in a concentration-dependent manner. The cellular response to the Shh signal is mediated by receptors, cytoplasmic factors as well as transcription factors, which act both positively and negatively to modulate Shh activity in the patterning of diverse cell types in the ventral neural tube. Additionally, Shh also cooperates with Bmp and Fgf molecules in the control of diverse neuronal cell fates in the brain.

Growth retardation and neonatal lethality in mice with a homozygous deletion in the C-terminal domain of RNA polymerase II.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandem repeats of the consensus heptapeptide YSPTSPS. Deletion studies in tissue culture cells have indicated that the CTD plays an essential role in transcription, although the nature of this essential function remains unclear. About half of the CTD can be deleted without affecting the viability of cells in tissue culture. Paradoxically, the dispensable CTD repeats are precisely conserved among all mammals whose CTD sequences are known. To determine whether the mammalian CTD is important in transcription during mouse development, we developed a gene targeting approach to introduce deletions into the CTD coding region of mouse embryonic stem (ES) cells. To maintain a functional Rpo2-1 gene, the neo marker in the targeting vector was positioned outside of the Rpo2-1 transcribed region, 1.2 kb from the site of the CTD deletion. G418-resistant clones were screened for co-integration of the CTD deletion, and the resulting ES lines were used to create germline chimeric mice. Stable heterozygous lines were established and mated to produce animals homozygous for the CTD deletion. We show here that mice homozygous for a deletion of thirteen of the 52 heptapeptide repeats are smaller than wild-type littermates and have a high rate of neonatal lethality. Surviving adults, although small, appear morphologically normal and are fertile. This result suggests that the CTD plays a role in regulating growth during mammalian development. The gene targeting approach described here should be useful for making further deletions in the CTD and may be of general applicability where it is desirable to engineer specific mutations in the germline of mice.

Essential role for Sonic hedgehog during hair follicle morphogenesis.

The hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control and Shh -/- embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression of Gli1 and Ptc1 was reduced in Shh -/- dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected in Shh -/- skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development.

Sonic hedgehog is essential to foregut development.

Congenital malformation of the foregut is common in humans, with an estimated incidence of 1 in 3000 live births, although its aetiology remains largely unknown. Mice with a targeted deletion of Sonic hedgehog (Shh) have foregut defects that are apparent as early as embryonic day 9.5, when the tracheal diverticulum begins to outgrow. Homozygous Shh-null mutant mice show oesophageal atresia/stenosis, tracheo-oesophageal fistula and tracheal and lung anomalies, features similar to those observed in humans with foregut defects. The lung mesenchyme shows enhanced cell death, decreased cell proliferation and downregulation of Shh target genes. These results indicate that Shh is required for the growth and differentiation of the oesophagus, trachea and lung, and suggest that mutations in SHH and its signalling components may be involved in foregut defects in humans.

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing.

RNA polymerase II C-terminal domain required for enhancer-driven transcription.

The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD may participate in activated transcription through interaction with a high-molecular-weight mediator complex. Such a role would be consistent with observations that some genes are preferentially sensitive to CTD mutations. Here we investigate the function of the mouse RNA polymerase CTD in enhancer-driven transcription. Transcription by alpha-amanitin-resistant CTD-deletion mutants was tested by transient transfection of tissue culture cells in the presence of alpha-amanitin in order to inhibit endogenous RNA polymerase II. Removal of most of the CTD abolishes transcriptional activation by all enhancers tested, whereas transcription from promoters driven by Sp1, a factor that typically activates housekeeping genes from positions proximal to the initiation sites, is not affected. These findings show that the CTD is essential in mediating 'enhancer'-type activation of mammalian transcription.