Ectopic expression of Nkx2.5 suppresses the formation of the sinoatrial node in mice

The sinoatrial node (SAN), functionally known as the pacemaker, regulates the cardiac rhythm or heartbeat. Several genes are expressed in the developing SAN and form a genetic network regulating the fate of the SAN cells. The short stature homeobox gene Shox2 is an important player in the SAN genetic network by regulating the expression of different cardiac conduction molecular markers including the early cardiac differentiation marker Nkx2.5. Here we report that the expression patterns of Shox2 and Nkx2.5 are mutually exclusive from the earliest stages of the venous pole and the SAN formation. We show that tissue specific ectopic expression of Shox2 in the developing mouse heart downregulates the expression of Nkx2.5 and causes cardiac malformations; however, it is not sufficient to induce a SAN cell fate switch in the working myocardium. On the other hand, tissue specific overexpression of Nkx2.5 in the heart leads to severe hypoplasia of the SAN and the venous valves, dis-regulation of the SAN genetic network, and change of the SAN cell fate into working myocardium, and causes embryonic lethality, recapitulating the phenotypes including bradycardia observed in Shox2^−/− mutants. These results indicate that Nkx2.5 activity is detrimental to the normal formation of the SAN. Taken together, our results demonstrate that Shox2 downregulation of Nkx2.5 is essential for the proper development of the SAN and that Shox2 functions to shield the SAN from becoming working myocardium by acting upstream of Nkx2.5.     

The homeobox leucine zipper gene Homez plays a role in Xenopus laevis neurogenesis

The Homez gene encodes a protein with three atypical homeodomains and two leucine zipper motifs of unknown function. Here we show that during neurula stages, Xenopus Homez is broadly expressed throughout the neural plate, the strongest expression being detected in the domains where primary neurons arise. At later stages, Homez is maintained throughout the central nervous system in differentiating progenitors. In accordance with this expression, Homez is positively regulated by neural inducers and by Ngnr1 and negatively by Notch signaling. Interference with Homez function in embryos by injection of an antisense morpholino oligonucleotide results in the specific disruption of the expression of late neuronal markers, without affecting the expression of earlier neuronal and early neurectodermal markers. Consistent with this finding, Homez inhibition also interferes with the expression of late neuronal markers in Ngnr1 overexpressing animal cap explants and in Notch inhibited embryos. In gain of function experiments, Homez inhibits the expression of late neuronal markers but has no effect on earlier ones. These data suggest a role for Homez in neuronal development downstream of proneural/neurogenic genes.     

Two alloalleles of <Emphasis Type="Italic">Xenopus laevis hairy2</Emphasis> gene—evolution of duplicated gene function from a developmental perspective

Gene duplication is a fundamental source of a new gene in the process of evolution. A duplicated gene is able to accept many kinds of mutations that could lead to loss of function or novel phenotypic diversity. Alternatively, the duplicated genes complementarily lose part of their functions to play original roles as a set of genes, a process called subfunctionalization. Pseudotetraploid frog Xenopus laevis has four sets of genes, and it is generally thought that the alloalleles in X. laevis have mutually indistinguishable functions. In this paper, we report differences and similarities between Xhairy2a and Xhairy2b in the neural crest, floor plate, and prechordal plate. Knockdown studies showed that Xhairy2a seems not to function in the neural crest, although both of them are required in the floor plate and the prechordal plate. Temporal expression pattern analysis revealed that Xhairy2a is a maternal factor having lower zygotic expression than Xhairy2b, while Xhairy2b is not loaded in the egg but has high zygotic expression. Spatial expression pattern analysis demonstrated that future floor plate expression is shared by both alloalleles, but Xhairy2b expression in the neural crest is much higher than Xhairy2a expression, consistent with the results of individual knockdown experiments. Therefore, our data suggest that subfunctionalization occurs in Xhairy2. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The words of the regulatory code are arranged in a variable manner in highly conserved enhancers

The mammillary body, a ventral specialization of the caudal hypothalamus, lies close to the transition between epichordal and prechordal parts of the forebrain (Puelles and Rubenstein, 2003). This report examines its presumed causal connection with either prechordal or notochordal mesodermal induction, as well as the timing of its specification, in the context of early ventral forebrain patterning. It was recently found that the ephrin receptor gene EphA7 is selectively expressed in the mammillary pouch from early stages of development (HH14: García-Calero et al., 2006). We used mammillary EphA7 expression as well as ventral hypothalamic expression of the gene markers Nkx2.1 and Shh to analyze experimental effects on mammillary specification and morphogenesis after axial mesoderm ablation at stages HH4+ to HH6. Progressively delayed ablation of the prechordal plate revealed its sequential implication in molecular specification of the entire ventral forebrain, including the mammillary and tuberal regions of the hypothalamus. We observed differential contact requirements for induction by the prechordal plate of all the forebrain regions expressing Shh and Nkx2.1, including distant subpallial ones. In contrast, ablation of the anterior notochordal tip at these stages did not elicit significant patterning changes, particularly no effects on mammillary EphA7 expression or mammillary pouch development.     

Endodermal expression of Nkx6 genes depends differentially on Pdx1

Nkx family members are essential for normal development of many different tissues such as the heart, lungs, thyroid, prostate, and CNS. Here, we describe the endodermal expression pattern of three Nkx6 family genes of which two shows conserved expression in the early pancreatic epithelium. In chicken, Nkx6.1 expression is not restricted to the presumptive pancreatic area but is more broadly expressed in the endoderm. In mice, expression of Nkx6.1 is restricted to the pancreatic epithelium. In both mice and chicken, Nkx6.2 and Pdx1 are expressed in very similar domains, identifying Nkx6.2 as a novel marker of pancreas endoderm. Additionally, our results show that Nkx6.3 is expressed transiently in pancreatic endoderm in chicken but not mouse embryos. At later stages, Nkx6.3 is found in the caudal stomach and rostral duodenum in both species. Finally, we demonstrate that Pdx1 is required for Nkx6.1 but not Nkx6.2 expression in mice and that ectopic Pdx1 can induce Nkx6.1 but not Nkx6.2 or Nkx6.3 expression in anterior chicken endoderm. These results demonstrate that Nkx6.1 lies downstream of Pdx1 in a genetic pathway and that Pdx1 is required and sufficient for Nkx6.1 expression in the early foregut endoderm.     

Analysis of Gene Expressions during Xenopus Forelimb Regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.     

Nkx6.1 and nkx6.2 regulate α- and β-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells

In mice, the Nkx6 genes are crucial to α- and β-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in β-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of α-cells but has no effect on β-, δ-, or ε-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of β-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both β- and α-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for α-cell than for β-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control α- and β-cell differentiation.     

Mice lacking the Nkx6.2 (Gtx) homeodomain transcription factor develop and reproduce normally

The Nkx homeobox genes are expressed in a variety of developing tissues and have been implicated in controlling tissue patterning and cell differentiation. Expression of Nkx6.2 (Gtx) was previously observed in the embryonic neural tube, testis, and differentiating oligodendrocytes. To investigate the role of Nkx6.2 in the control of cell specification and differentiation, we generated mice with null mutations in Nkx6.2 using the standard gene targeting approach. Null mutant mice were viable and fertile without apparent histological and immunohistochemical changes in the central nervous systems and testis. The absence of detectable phenotypes suggests a redundant function of Nkx6.2 in mouse development.     

Neural crests are actively precluded from the anterior neural fold by a novel inhibitory mechanism dependent on Dickkopf1 secreted by the prechordal mesoderm

It is known the interactions between the neural plate and epidermis generate neural crest (NC), but it is unknown why the NC develops only at the lateral border of the neural plate and not in the anterior fold. Using grafting experiments we show that there is a previously unidentified mechanism that precludes NC from the anterior region. We identify prechordal mesoderm as the tissue that inhibits NC in the anterior territory and show that the Wnt/beta-catenin antagonist Dkk1, secreted by this tissue, is sufficient to mimic this NC inhibition. We show that Dkk1 is required for preventing the formation of NC in the anterior neural folds as loss-of-function experiments using a Dkk1 blocking antibody in Xenopus as well as the analysis of Dkk1-null mouse embryos transform the anterior neural fold into NC. This can be mimicked by Wnt/beta-catenin signaling activation without affecting the anterior posterior patterning of the neural plate, or placodal specification. Finally, we show that the NC cells induced at the anterior neural fold are able to migrate and differentiate as normal NC. These results demonstrate that anterior regions of the embryo lack NC because of a mechanism, conserved from fish to mammals, that suppresses Wnt/beta-catenin signaling via Dkk1.     

Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain

The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. We have examined the role of Nkx6 and Nkx2 class homeodomain proteins in this process, and provide evidence that these proteins mediate complementary roles in the specification of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor cells is sufficient to mediate the activation of Phox2b, a homeodomain protein required for the generation of hindbrain visceral motoneurons. The redundant activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral motoneuron generation but are necessary to prevent these cells from adopting a parallel program of interneuron differentiation. The expression of Nkx6.1 and Nkx6.2 is further maintained in differentiating visceral motoneurons, and consistent with this the migration and axonal projection properties of visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2 function. Our analysis provides insight also into the role of Nkx6 proteins in the generation of somatic motoneurons. Studies in the spinal cord have shown that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons, and that the loss of motoneurons at this level correlates with the extinguished expression of the motoneuron determinant Olig2. Unexpectedly, we find that the initial expression of Olig2 is left intact in the caudal hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all somatic motoneurons are missing. These data argue against models in which Nkx6 proteins and Olig2 operate in a linear pathway, and instead indicate a parallel requirement for these proteins in the progression of somatic motoneuron differentiation. Thus, both visceral and somatic motoneuron differentiation appear to rely on the combined activity of cell intrinsic determinants, rather than on a single key determinant of neuronal cell fate.