Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain, but not limb development.

The Engrailed-1 gene, En1, a murine homologue of the Drosophila homeobox gene engrailed (en), is required for midbrain and cerebellum development and dorsal/ventral patterning of the limbs. In Drosophila, en is involved in regulating a number of key patterning processes including segmentation of the epidermis. An important question is whether, during evolution, the biochemical properties of En proteins have been conserved, revealing a common underlying molecular mechanism to their diverse developmental activities. To address this question, we have replaced the coding sequences of En1 with Drosophila en. Mice expressing Drosophila en in place of En1 have a near complete rescue of the lethal En1 mutant brain defect and most skeletal abnormalities. In contrast, expression of Drosophila en in the embryonic limbs of En1 mutants does not lead to repression of Wnt7a in the embryonic ventral ectoderm or full rescue of the embryonic dorsal/ventral patterning defects. Furthermore, neither En2 nor en rescue the postnatal limb abnormalities that develop in rare En1 null mutants that survive. These studies demonstrate that the biochemical activity utilized in mouse to mediate brain development has been retained by Engrailed proteins across the phyla, and indicate that during evolution vertebrate En proteins have acquired two unique functions during embryonic and postnatal limb development and that only En1 can function postnatally.

Analysis of the genetic pathway leading to formation of ectopic apical ectodermal ridges in mouse Engrailed-1 mutant limbs.

The apical ectodermal ridge (AER), a rim of thickened ectodermal cells at the interface between the dorsal and ventral domains of the limb bud, is required for limb outgrowth and patterning. We have previously shown that the limbs of En1 mutant mice display dorsal-ventral and proximal-distal abnormalities, the latter being reflected in the appearance of a broadened AER and formation of ectopic ventral digits. A detailed genetic analysis of wild-type, En1 and Wnt7a mutant limb buds during AER development has delineated a role for En1 in normal AER formation. Our studies support previous suggestions that AER maturation involves the compression of an early broad ventral domain of limb ectoderm into a narrow rim at the tip and further show that En1 plays a critical role in the compaction phase. Loss of En1 leads to a delay in the distal shift and stratification of cells in the ventral half of the AER. At later stages, this often leads to development of a secondary ventral AER, which can promote formation of an ectopic digit. The second AER forms at the juxtaposition of the ventral border of the broadened mutant AER and the distal border of an ectopic Lmx1b expression domain. Analysis of En1/Wnt7a double mutants demonstrates that the dorsalizing gene Wnt7a is required for the formation of the ectopic AERs in En1 mutants and for ectopic expression of Lmx1b in the ventral mesenchyme. We suggest a model whereby, in En1 mutants, ectopic ventral Wnt7a and/or Lmx1b expression leads to the transformation of ventral cells in the broadened AER to a more dorsal phenotype. This leads to induction of a second zone of compaction ventrally, which in some cases goes on to form an autonomous secondary AER.

Monoclonal antibodies specific for ohmefentanyl.

Ohmefentanyl (Ohm, N[1-(beta-hydroxy-beta-phenylethyl)-3-methyl-4- piperidyl]-N-phenylpro-pronamide), designed and synthesized by our laboratory, is a highly selective mu receptor agonist. After somatic cell fusion between splenocytes of BALB c mice, immunized by Ohm-BSA conjugate and NS-1 myeloma cells, 2 lines of hybridoma (D2 and F4) secreting monoclonal anti-Ohm antibodies (MAb) were obtained. Saturation and competition experiments showed that MAb-D2 and MAb-F4 bound to Ohm-BSA with high affinity and high specificity. Using radioligand binding assay and bioassay, we also found that MAb-D2 and MAb-F4 inhibited [3H] Ohm binding to rat brain opioid receptors in a dose-dependent manner and antagonized the effect of Ohm on the contraction of guinea pig ileum induced by electric field stimulation. These results suggested that MAb-D2 and MAb-F4 were 2 monoclonal antibodies specific for Ohm and could be useful as functional antagonists of Ohm.

[Study of characteristics of monoclonal antibody against substance P].

Cross-reaction of a rat monoclonal antibody (BTP-1) against seventeen substance P analogues was studied. The antibody was of IgG type and related to the carboxyl terminal of substance P, especially methionyl in the terminal, but did not depend on the strength of antagonistic effects of these analogues. It did not show cross-reaction with the following nine peptides: glucagon, endorphin, angiotensin I, II, leucine-enkephalin, methionine-enkephalin, bradykinin, oxytocin and dernorthin, indicating its high specificity to substance P. By means of immuno-enzyme histochemical method, it was shown that stained nerve fibers were located in the gelaliternous substance of Rolando, interpeduncular nucleus, substantia nigra and nerve cell bodies in the vestibular nucleus, lateral tegmental nucleus of mesencephalon and ventral region of third ventricle.