The role of chick Ebf genes in the mediolateral patterning of the somites

This study was conducted to check whether the three chick Early B‐cell Factor (Ebf) genes, particularly cEbf1, would be targets for Shh and Bmp signals during somites mediolateral (ML) patterning. Tissue manipulations and gain and loss of function experiments for Shh and Bmp4 were performed and the results revealed that cEbf1 expression was initiated in the cranial presomitic mesoderm by low dose of Bmp4 from the lateral mesoderm and maintained in the ventromedial part of the epithelial somite and the medial sclerotome by Shh from the notochord; while cEbf2/3 expression was induced and maintained by Bmp4 and inhibited by high dose of Shh. To determine whether Ebf1 plays a role in somite patterning, transfection of a dominant‐negative construct was carried out; this showed suppression of cPax1 expression in the medial sclerotome and upregulation and medial expansion of cEbf3 and cPax3 expression in sclerotome and dermomyotome, respectively, suggesting that Ebf1 is important for ML patterning. Thus, it is possible that low doses of Bmp4 set up Ebf1 expression which, together with Shh from the notochord, leads to establishment of the medial sclerotome and suppression of lateral identities. These data also conclude that Bmp4 is required in both the medial and lateral domain of the somitic mesoderm to keep the ML identity of the sclerotome through maintenance of cEbf gene expression. These striking findings are novel and give a new insight on the role of Bmp4 on mediolateral patterning of somites.     

The effect of RA on the chick Ebf1-3 genes expression in somites and pharyngeal arches

Expression of chick early B cell factor 1-3 (cEbf1-3) genes in regions of high retinoic acid (RA) activity, such as somites and pharyngeal arches (PAs), and regulation of other EBF members by RA raise the possibility that the internal cue RA may regulate cEbf1-3 expression in these tissues. To check this possibility, RA gain and loss of function experiments were conducted. Ectopic expression of RA led to up-regulation of cEbf2, 3 but did not change cEbf1 expression in somites. Expectedly, inhibition of RA by disulfiram resulted in downregulation of cEbf2, 3, but did not change cEbf1 expression in somites. The same RA gain and loss of function experiments did not change cEbf1-3 expression in PAs. However, ectopic expression of RA in the cranial neural tube before migration of neural crest cells downregulated cEbf1, 3 and up-regulated cEbf2 expression in the PAs. The same experiment, but with application of disulfiram, resulted in downregulation of cEbf2, but did not alter the expression of the other two genes. We conclude that the three cEbf genes act differently in response to RA signals in somitic mesoderm. cEbf1 may be not RA dependant in somites; however, the other two cEbf genes positively respond to RA signalling in somites. Additionally, only the migratory cEbf-expressing cells into the PAs are affected by RA signals.     

EBF proteins participate in transcriptional regulation of Xenopus muscle development

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells.     

Conserved origin of the ventral pancreas in chicken

To determine the origin of the ventral pancreas, a fate map of the ventral pancreas was constructed using DiI crystal or CM-DiI to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. First, the region lateral to the 7- to 9-somite level, which has been reported to contribute to the ventral pancreas, was shown to contribute mainly to the intestine or the dorsal pancreas. At the 10 somite stage (ss), the ventral pre-pancreatic cells reside laterally at the 2-somite level, at the lateral boarder of the somite. At this stage, however, the fate of these cells has not yet segregated and they contribute to the ventral pancreas and to the intestine or bile duct. The ventral pancreas fate segregated at the 17 ss; the cells residing at the somite boarder at the 4-somite level at the 17 ss were revealed to contribute to the ventral pancreas. Interestingly, the dorsal and the ventral pancreatic buds are different in both origin and function. These two pancreatic buds begin to fuse at day 7 (HH 30) of embryonic development. However, whereas the dorsal pancreas gives rise to both Insulin-expressing endocrine and Amylase-expressing exocrine cells, the ventral pancreas gives rise to Amylase-expressing exocrine cells, but not insulin-expressing endocrine cells before day 7 (HH 30) of embryonic development.     

Shh signalling restricts the expression of Gcm2 and controls the position of the developing parathyroids

The parathyroid glands originate from the endoderm of the caudal pharyngeal pouches. How these parathyroids are restricted to developing in the caudal pouches is unclear. In this paper we investigate the role of Shh signalling in patterning the vertebrate pharyngeal pouches, and show that Hh signalling may be involved in restricting the expression of the parathyroid marker Gcm2 in the pharyngeal epithelium. In the chick and mouse, Shh signalling is excluded or highly reduced in the posterior/caudal pouches, where the parathyroid marker Gcm2 is expressed, while remaining at high levels in the more anterior pouches. Moreover, though the block of Shh signalling at early developmental stages results in the loss of chick Gcm2 expression, at later stages, it induces ectopic Gcm2 expression domains in the second and first pharyngeal epithelium, suggesting that HH signalling prevents Gcm2 in those tissues. These ectopic domains go on to express other parathyroid markers but do not migrate and develop into ectopic parathyroids. Differences in the expression of Gcm2 in the chick, mouse and zebrafish, correlate with changing patterns of Shh signalling, indicating a conserved regulatory mechanism that acts to define pouch derivatives.     

BMP is an important regulator of proepicardial identity in the chick embryo

The proepicardium (PE) is a transient structure formed by pericardial coelomic mesothelium at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. In order to study PE development in chick embryos, we have analyzed the expression pattern of the marker genes Tbx18, Wt1, and Cfc. During PE induction, the three marker genes displayed a left-right asymmetric expression pattern. In each case, expression on the right side was stronger than on the left side. The left-right asymmetric gene expression observed here is in accord with the asymmetric formation of the proepicardium in the chick embryo. While initially the marker genes were expressed in the primitive sinus horn, subsequently, expression became confined to the PE mesothelium. In order to search for signaling factors involved in PE development, we studied Bmp2 and Bmp4 expression. Bmp2 was bilaterally expressed in the sinus venosus. In contrast, Bmp4 expression was initially expressed unilaterally in the right sinus horn and subsequently in the PE. In order to assess its functional role, BMP signaling was experimentally modulated by supplying exogenous BMP2 and by inhibiting endogenous BMP signaling through the addition of Noggin. Both supplying BMP and blocking BMP signaling resulted in a loss of PE marker gene expression. Surprisingly, both experimental situations lead to cardiac myocyte formation in the PE cultures. Careful titration experiments with exogenously added BMP2 or Noggin revealed that PE-specific marker gene expression depends on a low level of BMP signaling. Implantation of BMP2-secreting cells or beads filled with Noggin protein into the right sinus horn of HH stage 11 embryos resulted in downregulation of Tbx18 expression, corresponding to the results of the explant assay. Thus, a distinct level of BMP signaling is required for PE formation in the chick embryo.     

The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup

Background  

Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4) is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp (homologue of vertebrate Bmp4) acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse.     

Signals from trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm

We used Pax-2 mRNA expression and Lim 1/2 antibody staining as markers for the conversion of chick intermediate mesoderm (IM) to pronephric tissue and Lmx-1 mRNA expression as a marker for mesonephros. Pronephric markers were strongly expressed caudal to the fifth somite by stage 9. To determine whether the pronephros was induced by adjacent tissues and, if so, to identify the inducing tissues and the timing of induction, we microsurgically dissected one side of chick embryos developing in culture and then incubated them for up to 3 days. The undisturbed contralateral side served as a control. Most embryos cut parallel to the rostrocaudal axis between the trunk paraxial mesoderm and IM before stage 8 developed a pronephros on the control side only. Embryos manipulated after stage 9 developed pronephric structures on both sides, but the caudal pronephric extension was attenuated on the cut side. These results suggest that a medial signal is required for pronephric development and show that the signal is propagated in a rostral to caudal sequence. In manipulated embryos cultured for 3 days in ovo, the mesonephros as well as the pronephros failed to develop on the experimental side. In contrast, embryos cut between the notochord and the trunk paraxial mesoderm formed pronephric structures on both sides, regardless of the stage at which the operation was performed, indicating that the signal arises from the paraxial mesoderm (PM) and not from axial mesoderm. This cut also served as a control for cuts between the PM and the IM and showed that signaling itself was blocked in the former experiments, not the migration of pronephric or mesonephric precursor cells from the primitive streak. Additional control experiments ruled out the need for signals from lateral plate mesoderm, ectoderm, or endoderm. To determine whether the trunk paraxial mesoderm caudal to the fifth somite maintains its inductive capacity in the absence of contact with more rostral tissue, embryos were transected. Those transected below the prospective level of the fifth somite expressed Pax-2 in both the rostral and the caudal isolates, whereas embryos transected rostral to this level expressed Pax-2 in the caudal isolate only. Thus, a rostral signal is not required to establish the normal pattern of Pax-2 expression and pronephros formation. To determine whether paraxial mesoderm is sufficient for pronephros induction, stage 7 or earlier chick lateral plate mesoderm was cocultured with caudal stage 8 or 9 quail somites in collagen gels. Pax-2 was expressed in chick tissues in 21 of 25 embryos. Isochronic transplantation of stage 4 or 5 quail node into caudal chick primitive streak resulted in the generation of ectopic somites. These somites induced ectopic pronephroi in lateral plate mesoderm, and the IM that received signals from both native and ectopic somites formed enlarged pronephroi with increased Pax-2 expression. We conclude that signals from a localized region of the trunk paraxial mesoderm are both required and sufficient for the induction of the pronephros from the chick IM. Studies to identify the molecular nature of the induction are in progress.     

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung‐to‐gas bladder evolutionary transition in ray‐finned fishes

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray‐finned fishes is the gas bladder, an air‐filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe‐finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral‐to‐dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray‐finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.     

Ontogeny of angiopoietin‐like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development

Angiopoietin‐like proteins (ANGPTLs) are secreted proteins possessing an amino‐terminal coiled‐coil domain and a carboxyl‐terminal fibrinogen‐like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole‐mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite‐derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.     

Maintenance of asymmetric nodal expression in Xenopus laevis

Vertebrate species display consistent left-right asymmetry in the arrangement of their internal organs. This asymmetry reflects the establishment of the left-right axis and the alignment of the organs along this axis during development. Members of the TGF-β family of molecules have been implicated in both the establishment and signaling of left-right axis information. Asymmetric expression of one member, nodal (called Xnr-1 in the frog, Xenopus laevis), is highly conserved among species. The nodal-related genes are normally expressed in the left lateral plate mesoderm prior to the development of morphologic asymmetry. Expression patterns of nodal have been correlated with heart situs in mouse, chick, and frog and our previous work has implicated the dorsal midline structures in the regulation of nodal expression and cardiac laterality. In this study, three approaches were used to address the embryologic and molecular basis of asymmetric Xnr-1 expression. First, notochord and lateral plate recombinants were performed and showed that notochord can repress Xnr-1 expression in lateral plate mesoderm explants derived from either the left or the right side. Second, lateral plate mesoderm grafts indicated that Xnr-1 expression is specified but not determined at neurula stages and can subsequently be repatterned. These experiments suggest that a repressive signal from the notochord is required for maintenance of asymmetric Xnr-1 expression and that Xnr-1 expression is regulated by signals outside of the lateral plate mesoderm. Third, candidate molecules were injected to test for their ability to alter Xnr-1 expression pattern in the lateral plate. Late injection of activin protein on the right side of the embryo induced ectopic Xnr-1 expression and randomized cardiac orientation. This suggests that activin or a related TGF-β molecule is involved in the proximal regulation of asymmetric Xnr-1 expression. Dev. Genet. 23:194–202, 1998. © 1998 Wiley-Liss, Inc.