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During vertebrate limb development, the ectoderm directs the dorsoventral patterning of the underlying mesoderm. To define the molecular events involved in this process, we have analyzed the function of WNT7a, a secreted factor expressed in the dorsal ectoderm, and LMX1, a LIM homeodomain transcription factor expressed in the dorsal mesenchyme. Ectopic expression of Wnt7a is sufficient to induce and maintain Lmx1 expression in limb mesenchyme, both in vivo and in vitro. Ectopic expression of Lmx1 in the ventral mesenchyme is sufficient to generate double-dorsal limbs. Thus, the dorsalization of limb mesoderm appears to involve the WNT7a-mediated induction of Lmx1 in limb mesenchymal cells.

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A controversy has existed in the literature for the past several years regarding the number of vertebrate genes encoding the mitochondrial protein that initiates the first step in heme biosynthesis, 8-aminolevulinate synthase [ALAS; succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37]. By analysis of chicken ALAS cDNA clones isolated from both liver and erythroid cells, we show that at least two separate genes encode ALAS mRNAs. These experiments show that (i) two different genes encode the ALAS isozymes found in erythroid and in liver tissues, and (i) while the product of the erythroid gene (ALASE) is expressed exclusively in erythroid cells, the hepatic form of the enzyme is expressed ubiquitously, suggesting that this is the nonspecific form (ALASN) found in all chicken tissues.S-Aminolevulinate synthase [ALAS; succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] is the first enzyme in the heme biosynthetic pathway (1). The mRNA for this mitochondrial enzyme is encoded by one or more nuclear genes, translated into an enzymatically active precursor form, and proteolytically cleaved as it traverses the mitochondrial membrane. The functional form ofthe enzyme is a homodimer, found in the intracellular compartment ofthe mitochondrial matrix (2, 3). Although expressed in all tissues, the highest levels of ALAS are found in erythroid and liver cells, where high concentrations of heme are required for hemoglobin or cytochrome P-450 biosynthesis, respectively.Numerous studies have shown that both the ALAS gene and enzyme are subject to a broad array of regulatory influences in the liver. For example, the enzyme is negatively regulated by hemin (4-11) and can be induced by chemical effectors such as 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) and allylisopropylacetamide (AIA;. In contrast, ALAS as isolated from reticulocytes appears to be distinct from the hepatic form of the enzyme. The erythroid enzyme is neither induced by the poryphyrinogenic effectors of hepatic ALAS nor significantly affected by the addition of hemin (15-18). Furthermore, Bishop et al. (19) have partially purified both erythroid and nonerythroid ALAS from guinea pigs and have shown that they differ widely in biochemical properties. Of most direct significance to the studies presented here, Watanabe et al. (20) showed immunochemically that ALAS preparations from chicken erythroid and hepatic tissues differ in size, both as precursor and mature proteins. These and other studies (21) have led to the conclusion that the two-tissues contain different isoforms of ALAS.In earlier investigations of the molecular basis of these (24). Finally, we compare the relative abundance of erythroid and liver ALAS transcripts in both erythroid and hepatic tissue and show that, although hepatic ALAS mRNA is present in erythroid cells, that mRNA is substantially less abundant than the erythroidspecific ALAS mRNA. We conclude, in agreement with our earlier work (22), that at least two different ALAS genes exist in...

The mesencephalic and metencephalic region (MMR) of the vertebrate central nervous system develops in response to signals produced by the isthmic organizer (IsO). We have previously reported that the LIM homeobox transcription factor Lmx1b is expressed within the chick IsO, where it is sufficient to maintain expression of the secreted factor wnt1. In this paper, we show that zebrafish express two Lmx1b orthologs, lmx1b.1 and lmx1b.2, in the rostral IsO, and demonstrate that these genes are necessary for key aspects of MMR development. Simultaneous knockdown of Lmx1b.1 and Lmx1b.2 using morpholino antisense oligos results in a loss of wnt1, wnt3a, wnt10b, pax8 and fgf8 expression at the IsO, leading ultimately to programmed cell death and the loss of the isthmic constriction and cerebellum. Single morpholino knockdown of either Lmx1b.1 or Lmx1b.2 has no discernible effect on MMR development. Maintenance of lmx1b.1 and lmx1b.2 expression at the isthmus requires the function of no isthmus/pax2.1, as well as Fgf signaling. Transient misexpression of Lmx1b.1 or Lmx1b.2 during early MMR development induces ectopic wnt1 and fgf8 expression in the MMR, as well as throughout much of the embryo. We propose that Lmx1b.1- and Lmx1b.2-mediated regulation of wnt1, wnt3a, wnt10b, pax8and fgf8 maintains cell survival in the isthmocerebellar region.