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In this study, the initial specification of foregut endoderm in the chick embryo was analyzed. A fate map constructed for the area pellucida endoderm at definitive streak-stage showed centrally-located presumptive cells of foregut-derived organs around Hensen's node. Intracoelomic cultivation of the area pellucida endoderm at this stage combined with somatic mesoderm resulted in the differentiation predominantly into intestinal epithelium, suggesting that this endoderm may not yet be regionally specified. In vitro cultivation of this endoderm for 1-1.5 day combined with Hensen's node or its derivatives but not with other embryonic structures/tissues elicited endodermal expression of cSox2 but not of cHoxb9, which is characteristic of specified foregut endoderm. When the anteriormost or posteriormost part of the area pellucida endoderm at this stage, whose fate is extraembryonic, was combined with Hensen's node or its derivatives for 1 day, then enwrapped with somatic mesoderm and cultivated for a long period intracoelomically, differentiation of various foregut organ epithelia was observed. Such epithelia never appeared in the endoderm associated with other embryonic structures/tissues and cultured similarly. Thus, Hensen's node and its derivatives that lie centrally in the presumptive endodermal area of the foregut are likely to play an important role in the initial specification of the foregut. Chordin-expressing COS cells or noggin-producing CHO cells transplanted into the anteriormost area pellucida of the definitve streak-stage embryo could induce endodermal expression of cSox2 but not of cHoxb9, suggesting that chordin and noggin that emanate from Hensen's node and its derivatives, may be involved in this process.

Caspase-3-deficient 129/Sv mice show hyperplasia of the forebrain at embryonic day (E) 10.5, which suggests that caspase-3-dependent programmed cell death (PCD) plays an essential role in brain morphogenesis prior to neurogenesis. However, little is known about region-specific caspase-3-dependent PCD in the developing forebrain. We examined the PCD region in the early developmental brain at E9.5 by whole mount terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL). In addition to hindbrain, TUNEL-reactivity was detected in the ventral forebrain and in the caudal portion of the front nasal region, just behind the regions expressing fgf-8 and otx-2. It has been shown recently that brain hyperplasia induced by caspase-3-deficiency is mouse strain-dependent; such that brain abnormalities were observed in caspase-3-deficient 129/Sv mice but not in caspase-3-deficient C57BL/6 mice. We examined the caspase-3-dependent PCD in the ventral forebrain of 129/Sv and C57BL/6 mouse embryos (E8.5-9 and E9.5) by double staining of TUNEL and antiserum against the active form of caspase-3 (anti-m3D175). TUNEL/anti-m3D175 reactivity in the ventral forebrain was mouse strain-dependent, such that many TUNEL/anti-m3D175-positive cells were detected in the ventral forebrains of 129/Sv mice, but were not observed in C57BL/6 mice. Thus, it is likely that this region is the site of the strain-specific caspase-3-dependent PCD. A strain-dependent 'modulator' that regulates both caspase-3-dependent and -independent cell death pathways may control PCD in the ventral forebrain at E8.5-9.5.

Src kinase-associated phosphoprotein 2 (Ra70/scap2), which was originally isolated as a retinoic acid (RA)-induced gene, associates with molecules that modulate integrin-survival signals. Although RA is essential for vertebrate organogenesis in the posterior region, little is known about the biological role of RA70/Scap2 during development. In the present study, we demonstrate that Ra70/scap2 mRNA is temporally expressed during the RA-induced neuronal differentiation of P19 embryonic carcinoma cells. Homozygous knockout mice in which the Ra70/scap2 gene was replaced with LacZ exhibited embryonic lethality, while heterozygous mice displayed preferential expression of LacZ in posterior neural tissues, including the neural tube and hindbrain during development (E7.5–11.5), but not the forebrain. Ra70/scap2 was expressed in the ependymal layer and ventricular zone in the neural tube, where neuroepithelial cells and neuroblasts with proliferation capacity are localized, respectively. Thus, RA70/Scap2 may be necessary for RA-induced neuronal differentiation from the posterior neuroectoderm.

RA175, a new member of the immunoglobulin superfamily, is highly expressed during neuronal differentiation of P19 embryonal carcinoma cells. In situ hybridization showed that RA175 mRNA was detected in the developing nervous system, as well as the epithelium of the various non-neuronal tissues of mouse embryo. In contrast with the epithelia of the non-neuronal tissues, RA175 mRNA was not co-expressed with Sonic hedgehog mRNA and Patched mRNA during brain morphogenesis. RA175 mRNA was highly expressed in the anterior horn cells and the peripheral nervous system at embryonic day (E) 11.5 and in the central nervous system at E14.5-E18.5, but its expression decreased after birth and was undetectable in the adult mouse brain.

Caspase-3 is one of the cystein proteases that play essential roles in programmed cell death. As such, brain development is profoundly affected by caspase-3-deficiency, resulting in hyperplasia and abnormal cell organization (Kuida et al., Nature 1996;384:368-372). In the present study, we used caspase-3 (-/-) mice to show that caspase-3 deficiency results in severe hearing loss, hyperplasia of supporting cells and degeneration of sensory hair cells. The greater epithelial ridge, a remnant of the primordial organ of Corti, persists throughout all of the turns of cochlea in 2-week-old caspase-3 (-/-) mice, which indicates that the morphology of the cochlea is immature. The number of border cells, that develop from the greater epithelial ridge and are one of the supporting cells of the inner hair cell, increase significantly in both 2- and 5-week-old caspase-3 (-/-) mice. On the other hand, abnormal fused stereocilia can be seen in both 2- and 5-week-old caspase-3 (-/-) mice, and disarrangement and loss of sensory hair cells are observed in 5-week-old caspase-3 (-/-) mice. Taken together, both hyperplasia and degeneration occur simultaneously in the inner ear of the caspase-3 (-/-) mice, suggesting that caspase-3-dependent apoptosis is necessary for the development and formation of a properly functioning auditory system in mammals.

Truncated polypeptides containing expanded polyglutamine (polyQ) stretches tend to form cytoplasmic or nuclear aggregates in cultured cells, leading to cell death. Although it has been shown recently that caspase-8 coaggregates with polyQ and is activated during polyQ-mediated cell death, little is known of the location and timing of caspase-8 activation by nuclear polyQ aggregates. Also, the relationship between nuclear polyQ aggregate-mediated cell death and activation of other caspases is unclear. In P19 embryonal carcinoma (EC) cells, which can be made to differentiate into neuronal cells, polyQ72 repeats preferentially aggregate in the nucleus. Nuclear aggregates of polyQ72 induced P19 EC cell death, with a high frequency of cells exhibiting morphology characteristic of apoptosis (i.e., roundness, cell shrinkage, chromatin condensation) and DNA fragmentation. In the present study, we used antisera that specifically recognized the active forms of caspase-8, -3, and -9 but not their proforms, and showed that only caspase-8 and -3 were activated during the generation of polyQ72 aggregates in P19 EC cell nuclei. Furthermore, we showed that the caspase inhibitor z-VAD-fmk inhibited DNA fragmentation, but only partially inhibited the appearance of apoptotic morphology. Thus, caspase activation, including caspase-8 and -3, is necessary for polyQ-mediated DNA fragmentation but not sufficient for polyQ-mediated cell death in P19 EC cells.

Caspases and Bcl-xL, the mammalian homologues of the Caenorhabditis elegans (C. elegans) ced-3 and ced-9 genes, respectively, regulate apoptosis of various cells. Caspase-3 is processed into an active form (p20 or p17 and p12) during apoptosis. We investigated the relation between caspase-3 and Bcl-xL during development by examining activation of caspase-3 and apoptotic cells in Bcl-x-deficient (bcl-x−/−) mice at embryonic (E) day 11.5. We used a double-staining technique with a cleavage site-directed antibody against caspase-3 (anti-p20/17) and terminal-deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). Bcl-xL-deficiency increased both numbers of p20/17-positive and -negative apoptotic cells in dorsal root ganglia (DRG); the numbers of p20/17-positive apoptotic cells in the caudal parts of the ventral hindbrain and ventral spinal cord; and the numbers of p20/17-negative apoptotic cells in the dorsal midbrain, dorsal hindbrain, and dorsal spinal cord. Thus, Bcl-xL blocks the caspase-3-dependent apoptotic pathway in the restricted regions of the nervous system during development. Furthermore, these observations suggest that Bcl-xL protects against activation of the caspase-3-independent apoptotic pathway. Other caspases or apoptotic mechanisms may also be activated in the nervous systems of bcl-x−/− mice.

PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.

P19 embryonal carcinoma (EC) cells undergo apoptosis during neuronal differentiation induced by all-trans retinoic acid (RA). Caspase-3-like proteases are activated and involved in the apoptosis of P19 EC cells during neuronal differentiation.1 Recently it has been shown that growth factor signals protect against apoptosis by phosphorylation of Bad. Phosphorylated Bad, an apoptotic member of the Bcl-2 family, cannot bind to Bcl-xL and results in Bcl-xL homodimer formation and subsequent antiapoptotic activity. In the present study, we demonstrate that this system is used generally to protect against apoptosis during neuronal differentiation. Bcl-xL inhibited the activation of caspase-3-like proteases. Basic fibroblast growth factor (bFGF) inhibited more than 90% of the caspase-3-like activity, inhibited processing of caspase-3 into its active form, and inhibited DNA fragmentation. bFGF activated phosphatidyl-inositol-3-kinase (PI3K) and stimulated the phosphorylation of Bad. Phosphorylation was inhibited by wortmannin, an inhibitor of PI3K and its downstream target Akt. Thus, Bad is a target of the FGF receptor-mediated signals involved in the protection against activation of caspase-3.

We previously demonstrated that Caspase-3 is highly expressed in dorsal root ganglia and trigeminal ganglia of mouse embryos [T. Mukasa, K. Urase, Y.M. Momoi, I. Kimura, T. Momoi, Specific expression of CPP32 in sensory neurons of mouse embryos and activation of CPP32 in the apoptosis induced by a withdrawal of NGF, Biochem. Biophys. Res. Commun., 231 (1997) 770-774.]. Since, however, Caspases are processed into active form during apoptosis, it is difficult to examine the involvement of activated Caspases in naturally occurring cell death during development by immunohistochemical staining or in situ hybridization method. We prepared a cleavage site-directed antiserum against Caspase-3 (anti-p20/17). This antiserum reacted with fragment (p20/17) of Caspase-3, but not proCaspase-3 (p32), proCaspase-7 (p34) and its cleaved fragment (p24). We examined the relationship between the activation of Caspase-3 and the appearance of the naturally occurring apoptotic cells in the nervous system during development. In the trigeminal ganglia and dorsal root ganglia, the expression of Caspase-3 mRNA was maximal before the appearance of p20/17-positive cells and apoptotic cells. In the mouse brain, many p20/17-positive cells and apoptotic cells were observed in the neuroepithelium in the early developmental stages, but very few p20/17-positive cells were detected in postmitotic neurons in the cerebral cortex although Caspase-3 mRNA was expressed highly. Caspase-3 is activated mainly during apoptosis of neuroepithelial cells in the early developmental stages but not of mature neurons at postnatal stages.

We prepared a cleavage site-directed antiserum against Caspase-3 (anti-p20/17), which reacts with the p20/17 fragment (p20/17) activated by cleavage but not proCaspase-3 (p32), and examined the relationship between the activation of Caspase-3 and apoptosis. We identified p20/17-positive cells where cell death occurs naturally: interdigits of the forelimbs, small intestine epithelium, thymus, trigeminal ganglia, and dorsal root ganglia of mouse embryos. Withdrawal of nerve growth factor induced the appearance of p20/17-positive cells with DNA fragmentation in the culture of dorsal root ganglia neurons, while DNA fragmentation was detected in both p20/17-positive and -negative neurons in dorsal root ganglia of mouse embryos. These results suggest that not only activation of Caspase-3 but also other molecular mechanism play a role in the naturally occurring dorsal root ganglia apoptosis. Cleavage site-directed antisera against Caspases will be useful for the analysis of the molecular mechanism of naturally occurring apoptosis during development.

Mutation of the Patched gene has been detected in human inherited basal cell nevus syndrome (BCNS) and sporadic basal cell carcinomas (BCC), suggesting a strong relation between a Sonic hedgehog-Patched signal and cell proliferation. In the present study, we demonstrate that Sonic hedgehog is expressed in human lung squamous carcinoma (LK-2 and EBC-1) and some adenocarcinoma cell lines. The expression of Sonic hedgehog is also detected in the human lung squamous carcinoma tissues, but not in the normal lung tissue of the same patient. The N-terminal region of Sonic hedgehog stimulates the incorporation of BrdU into LK-2 cells and stimulates their cell growth, while anti-Shh-N inhibits their cell growth. These results suggest that a Sonic hedgehog signal is involved in the cell growth of LK-2 cells.

The embryonic chicken digestive tract consists of endodermal epithelium and mesenchyme derived from splanchnic mesoderm. Interactions between these two tissues are important for the establishment of regionality and the subsequent differentiation of digestive organs. In the present study we obtained a monoclonal antibody that reacted with mucus-associated antigen and named it the MA antibody. From 6 days of incubation, this antibody reacted with the esophageal, proventricular and gizzard epithelia. In the proventriculus, the MA antigen was expressed in luminal epithelial cells, while pepsinogen-producing gland cells became MA antigen-negative. The intestinal goblet cells, which secrete mucus, became positive to the antibody from day 13 of incubation. When the esophageal, proventricular or gizzard epithelium of a 6 day embryo was associated and cultivated with the proventricular mesenchyme, the luminal epithelial cells remained reactive to the MA antibody while gland cells were negative or only weakly positive. If the small-intestinal epithelium was cultivated with the proventricular or gizzard mesenchyme, the antigen was detected on the apical surface of the epithelium, suggesting that the expression of the MA antigen was induced by mesenchymal influences in the small-intestinal epithelium. These results suggest that spatio-temporally regulated expression of the MA antigen is controlled by the epithelial-mesenchymal interactions.

Epithelial-mesenchymal interaction plays an important role in the differentiation of digestive tract. However, the factors of these mesenchymes involved in induction of the epithelial differentiation of each organs are still unknown. In the present study, we made reconstituted mesenchymal cell aggregates by mixing proventricular mesenchymal cells with other mesenchymal cells, recombined the reconstituted mesenchyme with gizzard epithelium, and observed the differentiation of the gizzard epithelium in the explants with special attention to the appearance of embryonic chicken pepsinogen, one of the molecular marker of the proventricular epithelial cells, in the gizzard epithelium. The results showed that the proventricular mesenchymal cells induce gland formation and pepsinogen in the gizzard epithelium and that the esophageal and gizzard mesenchymal cells have the inhibitory influence on the differentiation of epithelia toward proventricular epithelium. The cells from small-intestinal, lung and dorsal dermal mesenchyme have no such effect. Based on the results obtained so far, a hypothesis was presented to explain the mechanism regulating the differentiation of the epithelium in the digestive tract in the chicken embryo.

RA175 is a new member of the immunoglobulin superfamily with trans interaction activity, and it plays a role as a tumor suppressor in lung carcinoma (TSLC1) and as a cell adhesion molecule promoting the formation of functional synapses (SynCAM). Little is known about the biological function of RA175/TSLC1/SynCAM neural network formation during neurogenesis. We examined the distribution and colocalization of the RA175/TSLC1/SynCAM protein with other members of the immunoglobulin superfamily such as NCAM, L1, and TAG-1 in the mouse developing nervous system. Consistent with the expression of RA175/TSLC1/SynCAM mRNA, the protein was localized in the brain neuroepithelium at embryonic day (E) 9.5, neural crest at E10.5, motor neurons at E10.5, and olfactory epithelium at E16.5. In contrast with its mRNA, the protein was intensely detected on the fasciculated axons in the floor plates, ventral root, and dorsal funiculus in the E10.5-11.5 spinal cord and colocalized with NCAM and L1 on the ventral root and dorsal funiculus and partly colocalized with TAG-1 on the commissural axons and dorsal funiculus. In the E13.5-15.5 brain, RA175/TSLC1/SynCAM colocalized with NCAM and L1 on the developing thalamocortical fibers from the internal capsule (IC) and partly colocalized with TAG-1 on the cortical efferent axons in the intermediate zone (IZ). RA175/TSLC1/SynCAM was localized on the axons of some of the cortical neurons cultured in vitro. Thus, in addition to cell adhesion activity in the neuroepithelium and the synapses, RA175/TSLC1/SynCAM may be involved in neuronal migration, axon growth, pathfinding, and fasciculation on the axons of differentiating neurons.

Caspase-3-deficient 129/Sv mice show hyperplasia of the brain at embryonic (E) day 10.5-12.5, but caspase-3-deficient C57L/B6 mice do not. We examined the relationship between activation of caspase-3 and programmed cell death (PCD) during forebrain development of various mouse strains (129/Sv, ICR, C57L/B6, and CBA) using terminal deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and immunostaining with antiserum against the caspase-3 (anti-m3D175) cleavage site. A number of anti-m3D175 positive cells and TUNEL positive cells were detected in the ventral side of the forebrain of 129/Sv and ICR mice at E8.5-9 but not in C57L/B6 and CBA mice. Ac-DEVD-MCA cleavage activity, a caspase-3-like activity, also suggests the preferential activation of caspase-3 in the ventral forebrain of ICR mice but not in C57L/B6 mice. Developmental changes of TUNEL and anti-m3D175 reactivities were essentially similar during brain morphogenesis of ICR and 129/Sv mice. The number of TUNEL/anti-m3D175 positive cells decreased in the neuroepithelium of the ventral forebrain at E9.5 before generation of the medial ganglionic eminence (MGE). TUNEL and/or anti-m3D175 reactivity was slightly detectable in the MGE at E10.5, from which neuroprogenitor cells follow a tangential migratory route to the cortex. Activation of caspase-9 was also immunohistochemically detected in the ventral forebrain at E8.5-9, suggesting that activation of caspase-3 and caspase-9 occurs in the PCD of this region. Thus, it is likely that decreased cell death in the ventral forebrain of caspase-3- and caspase-9-deficient 129/Sv mice increases the number of neuroprogenitor cells in the MGE, leading to hyperplasia of the forebrain.

Embryonic induction of hair follicles is a fascinating model of localized morphogenesis from a simple homogeneous epithelial cell sheet. Accumulating evidence indicates that Sonic hedgehog (Shh) signaling plays a central role in hair follicle formation. We quantitated the expression levels of Shh and its receptor genes, Patched (Ptc) and Patched-2 (Ptch2), in two distinct experimental systems in which the development of hair follicles was suppressed. Shh, Ptc, and Ptch2 were induced about six- to tenfold in normal embryonic hair germs in vivo as well as in developing skin tissue maintained in organ culture. This induction was almost completely inhibited both in the developing skin tissue of ICR mice cultured with 30 ng/ml epidermal growth factor and in embryos of Tabby mutant mice (a model of hypohidrotic ectodermal dysplasia) at 14.5-15.5 days postcoitus. Expression of Shh, Ptc and Ptch2 was induced in the Tabby embryos at 16.5 days postcoitus, indicating that Shh signaling may be involved in the formation not only of the well-studied guard hair but also of the awl hair. The potential of the two biological systems for studying molecular mechanisms in hair follicle formation, particularly at an early phase including Shh signaling, is discussed.

Embryonic mouse tracheal epithelium, which branches in an epithelial–mesenchymal recombination culture with bronchial mesenchyme, was cultured under mesenchyme-free conditions. When embedded in a basement-membrane–like matrix and cultured in a serum-free medium supplemented with fibroblast growth factor 1 (FGF1), the tracheal epithelium did not branch, whereas the bronchial epithelium underwent branching morphogenesis. When the medium was enriched with transferrin (Tf), bud formation was induced in the tracheal epithelium and some buds branched secondarily. FGF7 and FGF10, in cooperation with Tf, induced tracheal bud formation to the same extent as FGF1, although the optimum concentrations differed. A bromodeoxyuridine-labeling study comparing cultures with and without Tf showed no Tf-specific amplification of cell proliferation. A whole-mount in situ hybridization study of the expression of Bmp4 and Shh genes in explants of mesenchyme-free culture revealed that both genes were ubiquitously expressed and that expression did not correlate with bud formation. This expression pattern was different from the distally localized expression pattern observed in normal lung rudiments and in extratracheal buds induced by the recombined bronchial mesenchyme. These results suggest that both bronchial and tracheal bud formations were initiated without localized exposure of the epithelium to FGFs and were not accompanied by localized expression of Bmp4 and Shh in the epithelium. © 2001 Wiley-Liss, Inc.

Six family genes are homologues of Drosophila melanogaster sine oculis, one of the homeobox genes essential for compound eye formation (5). They are characterized by the presence of the Six domain and Six-type homeodomain in the encoded proteins, which confer specific DNA binding activity and function as transcription factors (12, 28). In mammals, six members of the family have so far been identified (2–4, 7, 8, 10, 12, 13, 31, 32, 34, 41; for a review, see reference 14). Each member of the family is expressed in a spatiotemporally regulated manner during embryogenesis. In mice, Six3 and Six6 are exclusively expressed in the developing forebrain and eyes (10, 22, 31, 40). In contrast, Six1, Six2, and Six5 show relatively broader expression patterns (15, 32). Six1 is expressed in the cranial and dorsal root ganglia, somites, otic and nasal placodes, branchial arches, limbs, Rathke's pouch, and nephrogenic cords (32). Six2 is expressed in head mesenchyme, branchial arches, limbs, and some mesenchymal regions surrounding the gastrointestinal tract (32). Six5 shows a broad expression in branchial arches, limb buds, telencephalon, eye, sclerotomes, and cartilages (15). Such distribution suggests that these genes play specific roles in embryogenesis. Overexpression and misexpression experiments showed that Six3 and Six6 genes are involved in forebrain and eye organogenesis (18, 21, 30, 48). Consistently, SIX3 mutations cause holoprosencephaly, a severe malformation of the brain in humans (42). SIX5 is located immediately downstream of the CTG trinucleotide repeats whose expansion causes myotonic dystrophy (3). Downregulation of the gene was observed in myotonic dystrophy patients and is thought to be responsible for some of the symptoms of the disease such as cataracts (16, 44). In agreement with this, Six5-heterozygous and -homozygous mutant mice develop cataracts (15, 35). Six4 was originally identified as a binding factor to the positive regulatory element of the Na+,K+-ATPase α1 subunit gene (Atp1a1) (12, 38). Immunostaining showed the presence of Six4 protein in some populations of neuronal cells in developing mouse embryos and developing retina (27, 29). In adult mice, Six4 protein is localized in skeletal muscles, as demonstrated by gel retardation assay (37). These observations suggest that this gene is involved in neurogenesis, myogenesis, and probably the development of other organs. In myogenesis, myogenin plays an important role along with other myogenic genes such as MyoD, Myf5, and MRF4 (33). Promoter analysis with transgenic mice demonstrated that the MEF3 site in the promoter region is essential for myogenin expression (37). Mouse Six1 and Six4 proteins are present in the developing somites in which myogenin expression is activated and bind to the MEF3 site in the myogenin promoter, as shown by gel retardation assay (37). Furthermore, Six4 can activate the myogenin gene promoter alone or in synergy with the specific cofactor, Eya, through direct binding to the MEF3 site in cultured cells (28). These results support the notion that Six4 is one of the genes that control myogenesis through activation of myogenin. In addition, Eya1, one of the specific cofactors of Six4, is implicated in branchio-oto-renal syndrome, a dominantly inherited disorder characterized by hearing loss and branchial arch and renal anomalies in humans (1). Eya1-deficient mice lack ears and kidneys, and heterozygous mutant mice show hearing loss and renal anomalies, as seen in human branchio-oto-renal syndrome (46). Because immunostaining showed the presence of Six4 protein in acoustic ganglia and otic vesicles (29), it is possible that Six4 is involved in the development of the ear in association with Eya1. Nevertheless, the biological function of Six4 in development is not clear, due to the lack of natural mutants, knockout models, and overexpression experiments. To access the biological function of Six4, we generated Six4-deficient mice and analyzed phenotypic changes both in adults and in embryos. We found no apparent changes in morphology and expression patterns of some marker genes in Six4-deficient mice. Functionally, hearing ability was normal. The reason for the lack of phenotype in Six4-deficient mice and the possible compensation among Six family genes is discussed.

Caspases, which play crucial roles during apoptosis, are activated from their inactive proforms in a sequential cascade of cleavage by other members of the caspase family. Caspase-9 is autoprocessed by the Apaf-1/cytochrome c pathway and acts at an early point in this cascade, whereas Bcl-xL, an antiapoptotic member of the Bcl-2 family, prevents activation of caspases in vitro. Little is known, however, about the relation between caspase-9 and Bcl-xL during development of the mammalian nervous system. We used antisera against two cleavage sites in mouse caspase-9 that recognize only the activated form of mouse caspase-9, and we examined immunohistochemically the activation of mouse caspase-9 in the nervous system of Bcl-x-deficient mouse embryos. Mouse caspase-9 is processed at both D353 and D368, but it is processed preferentially at D368 during apoptosis of cultured cells induced by various stimuli and in the nervous system of Bcl-x-deficient mouse embryos. We show that Bcl-xL protects against caspase-9- and/or caspase-3-dependent apoptosis in the caudal portion of the ventral hindbrain, anterior horn cells, and dorsal root ganglia neurons of the normal mouse embryos and against caspase-9/caspase-3-independent apoptosis in the dorsal region of the nervous system including the dorsal spinal cord. Furthermore, we demonstrate that Bcl-xL blocks cytochrome c release from mitochondria, causing activation of caspase-9 in anterior horn cells and dorsal root ganglia neurons in mouse embryos at embryonic day 11.5.

In contrast to the autoprocessing of caspase-9, little is known about the biological significance of caspase-9 processing by caspase-3 via a feedback loop in vivo. We prepared antisera against mouse caspase-9 cleavage sites so that only the activated form of mouse caspase-9 was recognized. Using these antisera and caspase-9- and caspase-3-deficient mouse embryonic fibroblasts, we demonstrated that mouse caspase-9 is initially autoprocessed at D(353) and D(368) at low levels during staurosporine-induced apoptosis, whereupon the D(368) and D(168) sites are preferentially processed over D(353) by activated caspase-3 as part of a feedback amplification loop. Ac-DEVD-MCA (caspase-3-like) and Ac-LEHD-MCA (caspase-9-like) cleavage activities clearly showed that caspase-9 autoprocessing was necessary for the activation of caspase-3, whereas full activation of caspase-3 and caspase-9 was achieved only through the feedback amplification loop. This feedback amplification loop also played a predominant role during programmed cell death of dorsal root ganglia neurons at mouse embryonic day 11.5.

Sonic hedgehog (Shh), a homologue of Drosophila hedgehog, was specifically expressed in lung epithelium during branching morphogenesis, but was not uniformly expressed in lung epithelium. Shh was intensely expressed in the distal tips of the bronchial tubes during branching morphogenesis, and Shh was localized on the apical side of the epithelium. On the other hand, Bmp-4, one of the target genes of Shh, was also specifically expressed in the epithelium at the branching point. These results suggest that Shh and Bmp-4 are involved in the branching morphogenesis of lung epithelium.