T-box family and limb morphogenesis

Four members of the T-box family of transcription factors (Tbx2-Tbx5) are expressed in developing limb buds, and expression of two of these genes, Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. The role of these genes has been investigated in limb specification and development, using the chick as a model system. The formation of ectopic limbs was induced in the flank of chick embryos to examine the relationship between the identity of the limb-specific T-box genes being expressed and the identity of limb structures that subsequently develop. Whereas bud regions expressing Tbx4 develop characteristic leg structures, regions expressing Tbx5 develop characteristic wing features. In addition, heterotopic grafts of limb mesenchyme (wing bud into leg bud, and vice versa), which are known to retain the identity of the donor tissue after transplantation, retain autonomous expression of the appropriate, limb-specific T-box gene, with no evidence of regulation by the host bud. Thus there is a direct relationship between the identity of the structures that develop in normal, ectopic and recombinant limbs, and the identity of the T-box gene(s) being expressed. To investigate the regulation of T-box gene expression during limb development, several other embryological manipulations were employed. By surgically removing the apical ectodermal ridge (AER) from either wing or leg buds, it was found that, in contrast to all other genes implicated in the patterning of developing appendages, maintenance of T-box gene expression is not dependent on the continued provision of signals from the AER or the zone of polarizing activity (ZPA). By generating an ectopic ZPA (by grafting a sonic hedgehog [SHH]-expressing cell pellet under the anterior AER), it was found that Tbx2 expression can lie downstream of SHH. Finally, by grafting a SHH-expressing cell pellet to the anterior margin of a bud from which the AER had been removed, it was found that Tbx2 may be a direct, short-range target of SHH (Gibson-Brown, 1998).

Tbx-4 and Tbx-5 are first expressed in lateral plate mesoderm within clearly defined territories at the time the prospective limb fields are being specified by Hox genes. Hox genes may therefore be responsible for regulating expression of these T-box genes within the limb fields. Fgf-10 expression is also initiated in lateral plate mesoderm around this time, and FGF10 is a good candidate for the mesodermal factor that initiates limb outgrowth and signals the adjacent ectoderm to express FGF8. This makes Tbx4 and Tbx5 prime candidates to encode transcription factors, directly or indirectly regulated by Hox genes, required for the initiation of bud outgrowth and makes Fgf-10 a possible downstream target in the mesoderm (Gibson-Brown, 1998).

The first vertebrates to develop paired appendages, the osteostracan fishes, appeared in Devonian seas around 400 million years ago. However, these jawless fishes only possessed paired pectoral appendages; no evidence of pelvic fins has ever been discovered in a fossil or extant agnathan. Tbx2 and Tbx3 are expressed in very similar patterns in both the forelimb and hindlimb, and are both derived from a common ancestral locus (the Tbx2/3 locus) that was duplicated at a very early point along the vertebrate lineage. Tbx2 and Tbx3 are linked, respectively, to Tbx4 and Tbx5, which were also derived from a common ancestral locus (the Tbx4/5 locus) in the same duplication event. This raises the possibility that the ancestral Tbx2/3, Tbx4/5 gene pair was involved in development of the paired pectoral fins of ancient agnathans, and that evolution of paired pelvic fins may only have been possible following duplication of these genes and establishment of the two cognate gene pairs (Tbx2/Tbx3 and Tbx4/Tbx5). According to this model Tbx4/5 gene function was conserved by Tbx5 for specification and development of the pectoral appendages, whereas Tbx4 was then available to be recruited (co-opted) into serving an analogous role in specifying novel structures, the paired pelvic fins, at a different level along the primary body axis. Elaboration of the Tbx2-Tbx5 subfamily may therefore have been an important element in the evolution of gnathostome appendages (Gibson-Brown, 1998).

Tbx-2, Tbx-3, Tbx-4 and Tbx-5 chick genes have been isolated and, like their mouse homologs, are expressed in the limb regions. The T-box family has a slightly greater sequence affinity to Drosophila Optomotor blind than to Drosophila Brachyenteron (T-related gene). Tbx-2 and Tbx-3 are expressed in anterior and posterior domains in wings and legs, as well as throughout the flank. Of particular interest, however, are Tbx-5, which is expressed in wing and flank but not leg, and Tbx-4, which is expressed very strongly in leg but not wing. Grafts of leg tissue to wing and wing tissue to leg give rise to toe-like or wing-like digits in wing and leg respectively. Expression of Tbx-4 is stable when leg tissue is grafted to wing, and Tbx-5 expression is stable when wing tissue is grafted to leg. Induction of either extra wings or legs from the flank by applying FGF-2 in different positions alters the expression of Tbx-4 and Tbx-5 in such a way that suggests that the amount of Tbx-4 that is expressed in the limb determines the type that will form. The ectopic limb always displays a limb-like Tbx-3 expression. Thus Tbx-4 and Tbx-5 are strong candidates for encoding 'legness' and 'wingness' respectively (Isaac, 1998).

Much progress has been made in understanding limb development. Most genes are expressed equally and in the same pattern in the forelimbs and hindlimbs, which nevertheless develop into distinct structures. In contrast, the T-box genes Tbx5 and Tbx4 are expressed differently in chick wing (Tbx5) and leg (Tbx4) buds. Molecular analysis of the optomotor blind gene, which belongs to the same family of transcription factors, has revealed that this gene is involved in the transdetermination of Drosophila wing and leg imaginal discs. In addition, expression of Tbx5 and Tbx4 correlates well with the identity of ectopic limb buds induced by fibroblast growth factor. Thus, it is thought that Tbx5 and Tbx4 might be involved in determining limb identity. Another candidate is the Pitx1 gene, which encodes a bicoid-type homeodomain transcription factor that is expressed in leg buds. These factors are determined to be important in establishing limb identity (Takeuchi, 1999).

During embryonic development, initially similar fields can develop into distinct structures, such as the vertebrate forelimbs and hindlimbs. Although considerable progress has been made in understanding of the genetic control underlying the establishment of the different limb axes, the molecular cues that specify the differential development of the forelimbs and hindlimbs are unknown. Possible candidates for genes determining limb identity are Pitx1, a gene whose transcripts are detected in the early hindlimb bud, but not in the forelimb bud, and two members of the T-box (Tbx) gene family, Tbx4 and Tbx5, which are specifically expressed in the hindlimb and forelimb buds, respectively. Tbx4 and Tbx5 are shown to be essential regulators of limb outgrowth whose roles seem to be tightly linked to the activity of three signaling proteins that are required for limb outgrowth and patterning: fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and Wnt. In addition, evidence is provided that Tbx4 and Tbx5 are involved in controlling limb identity. These findings provide insight into how similar developmental fields can evolve into homologous but distinct structures (Rodriguez-Esteban, 1999).

When mesodermal tissue from the leg bud is grafted beneath the apical ridge of the wing bud, toe-like digits will form in the wing. This tissue retains the expression of Tbx-4. When wing mesoderm is grafted beneath the apical ridge of the leg bud, this graft retains Tbx-5 expression. The extent of leg structures that form in the wing relates directly to the amount of mesoderm along the proximodistal axis of the bud that is transferred; a complete leg will form in the wing region if the entire leg bud mesoderm is grafted in place of the wing mesoderm. It is likely therefore that the type of limb structures that form relates directly to the amount of Tbx expression (Isaac, 1998 and references).

In spite of recent breakthroughs in understanding limb patterning, the genetic factors determining the differences between the forelimb and the hindlimb have not been understood. The genes Pitx1 (a Bicoid-related Hox gene) and Tbx4 encode transcription factors that are expressed throughout the developing hindlimb but not forelimb buds. Misexpression of Pitx1 in the chick wing bud induces distal expression of Tbx4, as well as HoxC10 and HoxC11, which are normally restricted to hindlimb expression domains. Wing buds in which Pitx1 is misexpressed develop into limbs with some morphological characteristics of hindlimbs: the flexure is altered to that normally observed in legs; the digits are more toe-like in their relative size and shape, and the muscle pattern is transformed to that of a leg (Logan, 1999).

In certain urodeles, a lost appendage, including hand and foot, can be completely replaced through epimorphic regeneration. The regeneration process involves cellular activities similar to those described for embryogenesis. Working on the assumption that the morphological pattern specific for a forelimb or a hindlimb is controlled by different gene activities in the two limbs, an mRNA differential display screen was employed for the detection of candidate limb identity genes. Using this approach, a newt gene was isolated which in regenerating and developing limbs reveals properties expected of a gene having a role in controlling limb morphology: (1) it is exclusively expressed in the forelimbs, but not hindlimbs, (2) during embryonic development its expression is co-incident with forelimb bud formation, (3) it has an elevated message level throughout the undifferentiated limb bud and the blastema, respectively, and (4) it is expressed only in mesenchymal, but not in epidermal tissues. This novel newt gene shares a conserved DNA-binding domain, the T-box, with putative transcription factors including the Brachyury (T) gene product. The most closely related Drosophila gene is optomotor blind. In a following PCR-based screen, the evolutionarily conserved T-box motif was used to amplify a family of related genes in the newt; their different expression patterns in normal and regenerating forelimbs, hindlimbs and tail suggest, in general, an important role of T-domain proteins in vertebrate pattern formation (Simon, 1997).

To better understand the role of TBX5, a T-box containing transcription factor in forelimb and heart development, the clinical features of Holt-Oram syndrome caused by 10 different TBX5 mutations have been studied. Defects predicted to create null alleles cause substantial abnormalities both in limb and heart. In contrast, missense mutations produce distinct phenotypes: Gly80Arg causes significant cardiac malformations but only minor skeletal abnormalities; and Arg237Gln and Arg237Trp causes extensive upper limb malformations but less significant cardiac abnormalities. Amino acids altered by missense mutations were located on the three-dimensional structure of a related T-box transcription factor, Xbra, bound to DNA. Residue 80 is highly conserved within T-box sequences that interact with the major groove of target DNA; residue 237 is located in the T-box domain that selectively binds to the minor groove of DNA. These structural data, taken together with the predominant cardiac or skeletal phenotype produced by each missense mutation, suggest that organ-specific gene activation by TBX5 is predicated on biophysical interactions with different target DNA sequences (Basson, 1999).

Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structures such as limbs are not well established. Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations along the anteroposterior axis of the embryo is not known. The T-box transcription factor TBX5 is important for normal heart and limb formation, but its role in early limb development is not well defined. Mouse embryos lacking Tbx5 do not form forelimb buds, although the patterning of the lateral plate mesoderm into the limb field is intact. Tbx5 is not essential for an early establishment of forelimb versus hindlimb identity. In the absence of Tbx5, the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation. Lef1/Tcf1-dependent Wnt signaling is not essential for initiation of Tbx5 or Fgf10 transcription, but is required in concert with Tbx5 for maintenance of normal levels of Fgf10 expression. It is conclude that Tbx5 is not essential for the early establishment of the limb field in the lateral plate mesoderm but is a primary and direct initiator of forelimb bud formation. These data suggest common pathways for the differentiation and growth of embryonic structures downstream of T-box genes (Agarwal, 2003).

Tbx3, a T-box gene family member related to the Drosophila gene optomotor blind (omb) and encoding a transcription factor, is expressed in anterior and posterior stripes in developing chick limb buds. Tbx3 haploinsufficiency has been linked with the human condition ulnar-mammary syndrome, in which predominantly posterior defects occur in the upper limb. Omb is expressed in Drosophila wing development in response to a signalling cascade involving Hedgehog and Dpp. Homologous vertebrate signals Sonic hedgehog (Shh) and Bone morphogenetic protein 2 (Bmp2) are associated in chick limbs with signalling of the polarizing region, which controls anteroposterior pattern. Tissue transplantations and grafting with beads soaked in Shh, Bmps, and Noggin have been carried out in chick limb buds, and Tbx3 expression has been analyzed. Tbx3 expression was also analyzed in limb buds of chicken and mouse mutants and retinoid-deficient quail in which anteroposterior patterning is abnormal. Tbx3 expression in anterior and posterior stripes is regulated differently. Posterior Tbx3 expression is stable and depends on the signalling cascade centered on the polarising region involving Shh and Bmps, while anterior Tbx3 expression is labile and depends on the balance between positive Bmp signals, produced anteriorly, and negative Shh signals, produced posteriorly. These results are consistent with the idea that posterior Tbx3 expression is involved in specifying digit pattern and thus provides an explanation for the posterior defects in human patients. Anterior Tbx3 expression appears to be related to the width of limb bud, which determines digit number (Tümpel, 2002).

Tbx4 is a member of the T-box family of transcription factor genes, which have been shown to play important roles in development. Tbx4 function has been ablated using targeted mutagenesis in the mouse. Embryos homozygous for the null allele fail to undergo chorioallantoic fusion and die by 10.5 days post coitus. The allantoises of Tbx4-mutant embryos are stunted, apoptotic and display abnormal differentiation. Endothelial cells within mutant allantoises do not undergo vascular remodeling. Heterozygous embryos show a mild, transient growth defect in the allantois. Induction of a hindlimb field occurs normally in Tbx4 mutants and initial patterning of the hindlimb bud appears normal. However, hindlimb buds from Tbx4 mutants fail to develop either in vivo or in vitro and do not maintain Fgf10 expression in the mesenchyme. The expression of another, closely-linked, T-box gene, Tbx2, is reduced in both the hindlimb and the allantois of Tbx4-mutant embryos prior to the development of overt morphological abnormalities, which suggests that Tbx4 regulates Tbx2 in these tissues (Naiche, 2003).

A tight loop between members of the fibroblast growth factor and the Wnt families plays a key role in the initiation of vertebrate limb development. Tbx5 and Tbx4 are directly involved in this process. When dominant-negative forms of these Tbx genes were misexpressed in the chick prospective limb fields, a limbless phenotype arises with repression of both Wnt and Fgf genes. By contrast, when Tbx5 and Tbx4 are misexpressed in the flank an additional wing-like and an additional leg-like limbs are induced, respectively. This additional limb formation is accompanied by the induction of both Wnt and Fgf genes. These results highlight the pivotal roles of Tbx5 and Tbx4 during limb initiation, specification of forelimb/hindlimb and evolution of tetrapod limbs, placing Tbx genes at the center of a highly conserved genetic program (Takeuchi, 2003a).

The data reveal that Tbx5 and Tbx4 specifically regulate Wnt2b and Wnt8c, respectively, to initiate limb outgrowth in the early stages of development. In the later stages, Tbx5 and Tbx4 exert different actions to form distinct forelimb and hindlimb structures, respectively. These indicate that these genes play distinct roles with distinct specificity. Nonetheless, Tbx5 and Tbx4 are derived from the same ancestral gene. During evolution, these genes have diversified their biological functions to regulate different Wnt genes and make different limb structures. This is related to the observation that EnTbx5 and EnTbx4 (dominant negative proteins) failed to repress Wnt8c in the leg and Wnt2b in the wing. As expected, misexpression of EnTbx5 in the leg and EnTbx4 in the wing does not affect limb development. This suggests that Tbx5 and Tbx4 have acquired different target specificities during evolution (Takeuchi, 2003a).

Tbx5 is a T-box transcription factor expressed exclusively in the developing forelimb but not in the developing hindlimb of vertebrates. Tbx5 is first detected in the prospective forelimb mesenchyme prior to overt limb bud outgrowth and its expression is maintained throughout later limb development stages. Direct evidence for a role of Tbx5 in forelimb development was provided by the discovery that mutations in human TBX5 cause Holt-Oram Syndrome (HOS), a dominant disorder characterized predominantly by upper(fore) limb defects and heart abnormalities. Misexpression studies in the chick have demonstrated a role for this gene in limb-type specification. Using a conditional knockout strategy in the mouse to delete Tbx5 gene function in the developing forelimb, it has been demonstrated that this gene is also required at early limb bud stages for forelimb bud development. In addition, by misexpressing dominant-negative and dominant-activated forms of Tbx5 in the chick wing evidence is provided that this gene is also required at later stages of limb bud development for continued limb outgrowth. These results provide a context to understand the defects observed in HOS caused by haploinsufficiency of TBX5 in human. Moreover, these results also demonstrate that limb bud outgrowth and specification of limb identity are linked by a requirement for Tbx5 (Rallis, 2003).

Despite extensive studies on the anterior-posterior (AP) axis formation of limb buds, mechanisms that specify digit identities along the AP axis remain obscure. Using the four-digit chick leg as a model, Tbx2 and Tbx3 are shown to specify the digit identities of digits IV and III, respectively. Misexpression of Tbx2 and Tbx3 induced posterior homeotic transformation of digit III to digit IV and digit II to digit III, respectively. Conversely, misexpression of their mutants VP16ΔTbx2 and VP16ΔTbx3 induced anterior transformation. In both cases, alterations in the expression of several markers (e.g., BMP2, Shh, and HoxD genes) were observed. In addition, Tbx2 and Tbx3 rescued Noggin-mediated inhibition of interdigital BMP signaling, signaling which is pivotal in establishing digit identities. Hence, it is concluded that Tbx3 specifies digit III, and the combination of Tbx2 and Tbx3 specifies digit IV, acting together with the interdigital BMP signaling cascade (Suzuki, 2004).

Thus chick Tbx3 and Tbx2 specify posterior digit identities by regulating interdigital BMP signaling. Misexpression of Tbx3 and Tbx2 induced posterior homeotic transformation of digit II to III and digit III to IV, respectively. In contrast, misexpression of VP16ΔTbx3 and VP16ΔTbx2 induced anterior transformation, thereby converting digit III to II and digit IV to I or II. In some cases, truncation of the posterior digits was observed, indicating that Tbx3 and Tbx2 also control the development of the posterior digits. Tbx2 and Tbx3 are known to have specific expression patterns in the interdigital autopod regions; namely, chick Tbx3 is expressed in ID3 and 4, and Tbx2 in ID4. Since the interdigit BMP level regulates its anterior digit identity, these expression patterns suggest that Tbx2 and Tbx3 might be direct regulators of the posterior digit identities. More specially, Tbx2 acts upstream of Shh and BMP2, and Tbx3 regulates BMP2. Conversely, Shh and BMP4 upregulate the posterior expression of Tbx2 and Tbx3. These lines of evidence suggest that the feedback and feedforward regulation between Tbx2/3 and the Shh and BMP signaling cascades is pivotal for the specification of posterior digit identities (Suzuki, 2004).

Small patella syndrome (SPS) is an autosomal-dominant skeletal dysplasia characterized by patellar aplasia or hypoplasia and by anomalies of the pelvis and feet, including disrupted ossification of the ischia and inferior pubic rami. An SPS critical region of 5.6 cM on chromosome 17q22 was identified by haplotype analysis. Putative loss-of-function mutations were found in a positional gene encoding T-box protein 4 (TBX4) in six families with SPS. TBX4 encodes a transcription factor with a strongly conserved DNA-binding T-box domain that is known to play a crucial role in lower limb development in chickens and mice. The present identification of heterozygous TBX4 mutations in SPS patients, together with the similar skeletal phenotype of animals lacking Tbx4, establish the importance of TBX4 in the developmental pathways of the lower limbs and the pelvis in humans (Bongers, 2004).

Tbx5 is essential for initiation of the forelimb, and its deletion in mice results in the failure of forelimb formation. Misexpression of dominant-negative forms of Tbx5 results in limb truncations, suggesting Tbx5 is also required for forelimb outgrowth. This study shows that Tbx5 is expressed throughout the limb mesenchyme in progenitors of cartilage, tendon and muscle. Using a tamoxifeninducible Cre transgenic line, the time frame during which Tbx5 is required for limb development was mapped. Deletion of Tbx5 subsequent to limb initiation does not impair limb outgrowth. Furthermore, two distinct phases of limb development are distinguished: a Tbx5-dependent limb initiation phase, followed by a Tbx5-independent limb outgrowth phase. In humans, mutations in the T-box transcription factor TBX5 are associated with the dominant disorder Holt-Oram syndrome (HOS), which is characterised by malformations in the forelimb and heart. These results demonstrate a short temporal requirement for Tbx5 during early limb development, and suggest that the defects found in HOS arise as a result of disrupted TBX5 function during this narrow time window (Hasson, 2007; full text of article).

Tbx4 is a crucial gene in the initiation of hindlimb development and has been reported as a determinant of hindlimb identity and a presumptive direct regulator of Fgf10 in the limb. Using a conditional allele of Tbx4, Tbx4 function was ablated before and after limb initiation. Ablation of Tbx4 before expression in the hindlimb field confirms its requirement for limb bud outgrowth. However, ablation of Tbx4 shortly after onset of expression in the hindlimb field, during limb bud formation, alters neither limb outgrowth nor expression of Fgf10. Instead, post-limb-initiation loss of Tbx4 results in reduction of limb core tissue and hypoplasia of proximal skeletal elements. Loss of Tbx4 during later limb outgrowth produces no limb defects, revealing a brief developmental requirement for Tbx4 function. Despite evidence from ectopic expression studies, this work establishes that loss of Tbx4 has no effect on hindlimb identity as assessed by morphology or molecular markers (Naiche, 2007; full text of article).

Vertebrate limb bud formation is initiated by localized epithelial-to-mesenchymal transition

Vertebrate limbs first emerge as small buds at specific locations along the trunk. Although a fair amount is known about the molecular regulation of limb initiation and outgrowth, the cellular events underlying these processes have remained less clear. This study shows that the mesenchymal limb progenitors arise through localized epithelial-to-mesenchymal transition (EMT) of the coelomic epithelium specifically within the presumptive limb fields. This EMT is regulated at least in part by Tbx5 and Fgf10, two genes known to control limb initiation. This work shows that limb buds initiate earlier than previously thought, as a result of localized EMT rather than differential proliferation rates (Gros, 2014).

Tbx2 and Tbx3 act downstream of Shh to maintain canonical Wnt signaling during branching morphogenesis of the murine lung

Numerous signals drive the proliferative expansion of the distal endoderm and the underlying mesenchyme during lung branching morphogenesis, but little is known about how these signals are integrated. By analysis of conditional double mutants, this study shows that the two T-box transcription factor genes Tbx2 and Tbx3 (see Drosophila bi) act together in the lung mesenchyme to maintain branching morphogenesis (see Drosophila trachea development). Expression of both genes depends on epithelially derived Shh (see Drosophila hh) signaling, with additional modulation by Bmp (see Drosophila BMP signaling), Wnt (see Drosophila Wg), and Tgfβ (see Drosophila EGF signaling) signaling. Genetic rescue experiments reveal that Tbx2 and Tbx3 function downstream of Shh to maintain pro-proliferative mesenchymal Wnt signaling, in part by direct repression of the Wnt antagonists Frzb and Shisa3. In combination with previous finding that Tbx2 and Tbx3 repress the cell-cycle inhibitors Cdkn1a and Cdkn1b (see Drosophila dap), this study concludes that Tbx2 and Tbx3 maintain proliferation of the lung mesenchyme by way of at least two molecular mechanisms: regulating cell-cycle regulation and integrating the activity of multiple signaling pathways (Lüdtke, 2006).

A combination of activation and repression by a colinear hox code controls forelimb-restricted expression of tbx5 and reveals hox protein specificity

Tight control over gene expression is essential for precision in embryonic development and acquisition of the regulatory elements responsible is the predominant driver for evolution of new structures. Tbx5 and Tbx4, two genes expressed in forelimb and hindlimb-forming regions respectively, play crucial roles in the initiation of limb outgrowth. Evolution of regulatory elements that activate Tbx5 in rostral lateral plate mesoderm (LPM) was essential for the acquisition of forelimbs in vertebrates. This study identified such a regulatory element for Tbx5 and demonstrated Hox genes are essential, direct regulators. While the importance of Hox genes in regulating embryonic development is clear, Hox targets and the ways in which each protein executes its specific function are not known. This study reveals how nested Hox expression along the rostro-caudal axis restricts Tbx5 expression to forelimb. Hoxc9, which is expressed in caudal LPM where Tbx5 is not expressed, can form a repressive complex on the Tbx5 forelimb regulatory element. This repressive capacity is limited to Hox proteins expressed in caudal LPM and carried out by two separate protein domains in Hoxc9. Forelimb-restricted expression of Tbx5 and ultimately forelimb formation is therefore achieved through co-option of two characteristics of Hox genes; their colinear expression along the body axis and the functional specificity of different paralogs. Active complexes can be formed by Hox PG proteins present throughout the rostral-caudal LPM while restriction of Tbx5 expression is achieved by superimposing a dominant repressive (Hoxc9) complex that determines the caudal boundary of Tbx5 expression. These results reveal the regulatory mechanism that ensures emergence of the forelimbs at the correct position along the body. Acquisition of this regulatory element would have been critical for the evolution of limbs in vertebrates and modulation of the factors that were identified can be molecular drivers of the diversity in limb morphology (Nishimoto, 2014).

T-box family and eye development

Dorsal and ventral aspects of the mammalian eye are distinct from the early stages of development. The developing eye cup grows dorsally, and the choroidal fissure is formed on its ventral side. Retinal axons from the dorsal and ventral retina project to the ventral and dorsal tectum, respectively. Expression of the Tbx5 gene, an optomotor blind homolog, in the chick eye is first detected at stage 11 throughout the retina, with the strongest signal in the dorsal retina. Expression is later confined to the dorsal eye cup. Tbx5 is expressed in both retinal pigment epithelium (RPE) and neural retina (NR) at these early stages. Although expression in RPE starts to fade out at embryonic day 10, robust expression is maintained in the dorsal NR (all layers except the nerve fiber layer at the inner surface of the retina). Misexpression of Tbx5 induces dorsalization of the ventral side of the eye and altered projections of retinal ganglion cell axons. Thus, Tbx5 is involved in eye morphogenesis and is a topographic determinant of the visual projections between retina and tectum (Koshiba-Takeuchi, 2000).

When mouse BMP4 is misexpressed in the ventral half, round eyes are formed with expansion of Tbx5 expression in the ventral half. In such eyes, expression of Vax and Pax2 was repressed. Misexpression of BMP4 induces profound effects on eye morphology. These observations indicate that BMP4 acts upstream of Tbx5. The Pax2, Vax, EphrinB1, and EphrinB2 genes act downstream of the Tbx5 gene. This genetic hierarchy seems in good accordance with the timing of expression of BMP4, Tbx5, Pax2, EphrinB1, and EphrinB2 (stage 10+, 11, 11, 13 to 15, and 13 to 15, respectively). Misexpression of Tbx5 in the ventral side of the eye induces marked changes of the retinotectum projection without obvious morphological alteration. This rules out the possibility that the effect of Tbx5 misexpression on the projection is secondary to the dorsalization, because the exit of retinal axons into the optic nerve and the optic nerves themselves are formed normally in virus-infected eyes. Hence, it is concluded that the signaling cascade mediated by Tbx5 plays a key role in both eye morphogenesis and the visual projection (Koshiba-Takeuchi, 2000).

Several eye-field transcription factors (EFTFs) are expressed in the anterior region of the vertebrate neural plate and are essential for eye formation. The Xenopus EFTFs ET, Rx1, Pax6, Six3, Lhx2, tll and Optx2 are expressed in a dynamic, overlapping pattern in the presumptive eye field. Expression of an EFTF cocktail with Otx2 is sufficient to induce ectopic eyes outside the nervous system at high frequency. Using both cocktail subsets and functional (inductive) analysis of individual EFTFs, a genetic network regulating vertebrate eye field specification has been revealed. The results support a model of progressive tissue specification in which neural induction then Otx2-driven neural patterning primes the anterior neural plate for eye field formation. Next, the EFTFs form a self-regulating feedback network that specifies the vertebrate eye field. Striking similarities and differences are found in the network of homologous Drosophila genes that specify the eye imaginal disc, a finding that is consistent with the idea of a partial evolutionary conservation of eye formation (Zuber, 2003).

These remarkable similarities in general developmental design are perhaps logically predicated based on the functional and structural homologies between the Drosophila eye genes and the vertebrate EFTFs. orthodenticle (otd), the Drosophila homolog of Otx genes, is required for development of the eye, antenna and anterior brain, and is normally expressed in a wide domain that spans the dorsal midline and encompasses the entire dorsal head ectoderm. Its expression is turned off in the head midline during development and in the part of the visual primordium that forms the posterior optic lobe and the larval eye. This is strikingly similar to the changes seen in the Xenopus Otx2 expression pattern. The optomotor-blind (omb) gene is a member of the Tbx2 T-box subfamily. ET shares more sequence homology with omb than any other gene in the fly genome. omb expression is first detected in the optic lob anlagen, later expanding to a larger part of the developing larval brain. In the eye imaginal disc, omb is detected in glial precursors, posterior to the morphogenetic furrow and in the optic stalk. Null omb mutants die in pupal stage and show severe optic lobe defects. The Drosophila Rx homolog is not expressed in the larval eye imaginal discs nor the embryonic eye primordia. However, it is expressed prior to ey in the procephalic region from which the eye primordia originates, suggesting a role for Drosophila Rx prior to ey during eye formation in the fly. It has therefore been suggested that Drosophila Rx may only be required for early brain development. Finally, the results showing Pax6 as the most critical component of the Xenopus EFTF cocktail with respect to the induction of ectopic eyes, meshes well with the general prominence given to Pax6 and its Drosophila homologs ey and toy as transcription factors centrally involved in early eye development (Zuber, 2003).

Canonical Wnt signaling is required for the maintenance of dorsal retinal identity

Accurate retinotectal axon pathfinding depends upon the correct establishment of dorsal-ventral retinal polarity. Dorsal retinal gene expression is regulated by Wnt signaling in the dorsal retinal pigment epithelium (RPE). A Wnt reporter transgene and Wnt pathway components are expressed in the dorsal RPE beginning at 14-16 hours post-fertilization. In the absence of Wnt signaling, tbx5 and Bmp genes initiate normal dorsal retinal expression but are not maintained. The expression of these genes is rescued by the downstream activation of Wnt signaling, and tbx5 is rescued by Bmp signaling. Furthermore, activation of Wnt signaling cannot rescue tbx5 in the absence of Bmp signaling, suggesting that Wnt signaling maintains dorsal retinal gene expression by regulating Bmp signaling. A model is presented in which dorsal RPE-derived Wnt activity maintains the expression of Bmp ligands in the dorsal retina, thus coordinating the patterning of these two ocular tissues (Veien, 2008).

This study has shown that Wnt signaling is required for the proper development of DV retinal polarity. Expression analysis suggests that Wnt signaling functions in the RPE, while Bmp ligands are expressed in both the RPE and retina. The results demonstrate that dorsal retinal genes initiate their expression normally at around 12 hpf in the absence of Wnt signaling, but soon thereafter require Wnt signaling for their maintained expression in the dorsal retinal domain. The expression of Bmp ligands in the dorsal retina is dependent on Wnt signaling, and following Wnt inhibition the loss of at least one Bmp ligand, gdf6a, can be rescued by activation of Wnt signaling. In addition, tbx5, an early marker of dorsal identity, is rescued by the activation of either Wnt or Bmp signaling following Wnt inhibition. By contrast, tbx5 cannot be rescued by the activation of Wnt signaling in the absence of Bmp signaling. These data together suggest a model for the maintenance of DV retinal identity in which Wnt signaling in the dorsal RPE transcriptionally maintains Bmp expression in the dorsal RPE and retina, which regulates the expression of downstream DV axis genes, including tbx5 and Ephrin B axon guidance molecules. This mechanism provides a conduit through which a Wnt signal in the RPE can exert its effects in the neural retina. It is likely that this mechanism functions to maintain the integrity of the dorsal retinal domain by coordinating its patterning with the dorsal RPE, but detailed fate-mapping in the developing retina and RPE is needed to show this conclusively (Veien, 2008).

Wnt signalling mediated by Tbx2b regulates cell migration during formation of the neural plate

During gastrulation, optimal adhesion and receptivity to signalling cues are essential for cells to acquire new positions and identities via coordinated cell movements. T-box transcription factors and the Wnt signalling pathways are known to play important roles in these processes. Zebrafish tbx2b, a member of the TBX2 family, is required for the specification of midline mesoderm. tbx2b transcripts are present during mid-gastrula before its expression is detected by whole-mount in situ hybridization. Isolated ectodermal cells deficient in Tbx2b have altered cell surface properties and the level of cadherins in these cells is lower. In chimaeric embryos generated by cell transplantation and single blastomere injections, Tbx2b-deficient cells are defective in cell movement in a cell-autonomous manner, resulting in their exclusion from the developing neural plate. Using this `exclusion' phenotype as a screen, it is shown that Tbx2b acts within the context of Fz7 signalling. The exclusion of cells lacking T-box proteins in chimeras during development was demonstrated with other T-box genes and may indicate a general functional mechanism for T-box proteins (Fong, 2005 ).

Emergence of dorsal-ventral polarity in embryonic stem cell-derived retinal tissue

Mouse embryonic stem cell-derived retinal epithelium self-forms an optic cup-like structure. In the developing retina, the dorsal and ventral sides differ in terms of local gene expression and morphological features. This aspect has not yet been shown in vitro. This study demonstrates that embryonic stem cell-derived retinal tissue spontaneously acquires polarity reminiscent of the dorsal-ventral (D-V) patterning of the embryonic retina. Tbx5 (see Drosophila Omb) and Vax2 (see Drosophila Emx) were expressed in a mutually exclusive manner, as seen in vivo. Three-dimensional morphometric analysis showed that the in vitro-formed optic cup often contains cleft structures resembling the embryonic optic fissure. To elucidate the mechanisms underlying the spontaneous D-V polarization of embryonic stem cell-derived retina, the effects of the patterning factors were examined, and endogenous BMP signaling was found to play a predominant role in the dorsal specification. Further analysis revealed that canonical Wnt signaling, which was spontaneously activated at the proximal region, acts upstream of BMP signaling for dorsal specification. These observations suggest that D-V polarity could be established within the self-formed retinal neuroepithelium by intrinsic mechanisms involving the spatiotemporal regulation of canonical Wnt and BMP signals.

T-box family and ear development

Inner ear sensory organs and VIIIth cranial ganglion neurons of the auditory/vestibular pathway derive from an ectodermal placode that invaginates to form an otocyst. In the mouse otocyst epithelium, Tbx1 suppresses neurogenin 1-mediated neural fate determination and is required for induction (Otx1) or proper patterning of gene expression (Bmp4) related to sensory organ morphogenesis. Tbx1 loss-of-function causes dysregulation of neural competence in otocyst regions linked to the formation of either mechanosensory or structural sensory organ epithelia. Subsequently, VIIIth ganglion rudiment form is duplicated posteriorly, while the inner ear is hypoplastic and shows neither a vestibular apparatus nor a coiled cochlear duct. It is proposed that Tbx1 acts in the manner of a selector gene to control neural and sensory organ fate specification in the otocyst (Raft, 2004).

Evidence of a common progenitor for VIIIth ganglion neurons and mechanosensory cells has been obtained by clonal analyses in chick. Furthermore, expression overlap of Lfng and neural fate markers in both chick and mouse had led to the suggestion that neural progenitors and utricular and saccular maculae derive from a common anterior otocyst region. In the wild-type anterior otocyst, overlapping Ngn1, NeuroD, and Lfng expression is found that is complementary to the Tbx1 domain. Neural bHLH gene expression persists in this region through E11.5, the latest stage assayed for these markers. Tbx1 loss-of-function has little to no effect on neurogenic activity in this region and does not preclude the subsequent development of anteroventral sensory epithelium. Thus, Tbx1-independent pathways probably control neural and sensory epithelial fate assignment at this otocyst region (Raft, 2004).

The Lfng-positive posteroventral otocyst is the presumptive anlage of the organ of Corti and initially, this region is Tbx1-negative. Transient wild-type expression of Ngn1 and NeuroD, together with delamination, precedes the local onset of Tbx1 expression in this region. Regression of posteroventral neurogenesis is delayed in Tbx1 heterozygotes, and neurogenesis persists in this region through E11.5 in Tbx1–/– otocysts. Conversely, TBX1 gain of function effectively eliminates posteroventral neurogenesis. Interestingly, Tbx1 heterozygotes at E11 show delayed regression of neurogenesis at the anterodorsolateral otocyst and loss of a definitive Bmp4 anterior stripe. These phenotypes are observed toward the end of a period (E9.75-E11) during which Tbx1 expression expands into the anterodorsolateral otocyst. Together these results suggest that at some otocyst regions, Tbx1 regulates the developmental timing by which neural and sensory epithelial competent states are expressed. Functionally, this differs from the effect of Tbx1 activity at the posterolateral otocyst, where neural competence is fully suppressed at all times and sensory organ structural epithelium is formed (Raft, 2004).

It is concluded that Tbx1 specifies regional identity in the otocyst and is required for the positioning of a fate boundary. The data support the hypothesis of a relationship between neural and sensory epithelial competence in the otocyst. Furthermore, absence of Tbx1 causes expression of neural competence in a portion of the otocyst associated with formation of sensory organ structural epithelia. Taken together, these results suggest that Tbx1 regulates otocyst gene expression locally but affects inner ear growth and morphogenesis in a global manner. Tbx1 may therefore function as an otocyst selector gene in its control of neurogenesis and sensory organ development. Studies aimed at dissecting the contributions of epithelial and mesenchymal Tbx1 activity to various aspects of inner ear development using tissue-specific gene inactivation strategies are currently in progress (Raft, 2004).

Humans TBX1 is implicated in the etiology of the DiGeorge syndrome. Inactivation of the Tbx1 gene in mice produces a variety of malformations including abnormal branching of the heart outflow tract, deficiencies in the branchial arch derivatives, agenesis of pharyngeal glands and abnormal development of the auditory system. This study analyzes the middle and inner ear phenotypes of the Tbx1 null mice. The middle ear is strongly affected. Its skeletal components are malformed to varying degrees, some being slightly hypoplastic and others completely absent. However, a seemingly normal-looking tympanic membrane can still be recognized. Middle ear anomalies are associated with other skeletal deficiencies in the branchial arch-derived skeleton. These phenotypes derive from a combination of the failure of the posterior branchial arches to develop and the misrouting of neural crest cells. The inner ears of Tbx1-/- animals are hypoplastic. No vestibular or cochlear structures are detectable, but the endolymphatic duct, the cochleovestibular ganglia and residual sensory patches are still identifiable. Molecular analyses reveal a seemingly normal spatial distribution of a variety of patterning markers in the otic vesicles of Tbx1 null mutants at E9.0. However, 1 day later, several of these markers present altered domains of expression in the otocysts of these mutant embryos, suggesting that Tbx1 is not required for the establishment of spatial patterns in the otocyst, but rather for their maintenance. The inability of the Tbx1-/- embryos to keep properly segregated functional domains in the otocyst is likely the cause of the strong inner ear phenotypes observed in these mutants (Moraes, 2005).

Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. This study demonstrates that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. A potential mechanism is discussed for the absence of neural precursors in the Eya1-/- inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant (Friedman, 2005 ).

Several lines of evidence support the existence of compartmental boundaries of gene expression within the otocyst governing the divergence of epithelial cell lineages. Examples include the expression of Dlx5 in the dorsal epithelium of the otocyst and its responsibility for development of the semicircular canals and the expression of Otx1 in the ventral otocyst and its essential role in cochlear morphogenesis. Specification of neural progenitors is the earliest identifiable fate determination event in the developing otocyst, beginning around E9. This subpopulation of ventral otic epithelial cells is identifiable by their expression of the Atonal-related basic helix-loop-helix genes, Neurogenin1 (Ngn1) and NeuroD. Ngn1 is necessary for neural progenitor determination and formation of the cochleovestibular ganglion (cvg). Supporting its role in inner ear development, studies in Ngn1 deficient mice show complete absence of the cvg. Ngn1 regulates another gene in this cascade, NeuroD. It is expressed in a spatially and temporally overlapping pattern with Ngn1 and promotes neuroblast delamination into the ventral mesenchyme and growth factor mediated neuronal survival. Tbx1 has recently been shown to act upstream of Ngn1 and NeuroD as a negative regulator of neural fate specification in the otocyst (Friedman, 2005).

Regional morphogenesis in the hypothalamus: A BMP-Tbx2 pathway coordinates fate and proliferation through Shh downregulation

A central challenge in embryonic development is to understand how growth and pattern are coordinated to direct emerging new territories during morphogenesis. This study reports on a signaling cascade that links cell proliferation and fate, promoting formation of a distinct progenitor domain within the developing chick hypothalamus. The downregulation of Shh in floor plate-like cells in the forebrain governs their progression to a distinctive, proliferating hypothalamic progenitor domain. Shh downregulation occurs via a local BMP-Tbx2 pathway, Tbx2 acting to repress Shh expression. Forced maintenance of Shh in hypothalamic progenitors prevents their normal morphogenesis, leading to maintenance of the Shh receptor, ptc, and preventing progression to an Emx2+-proliferative progenitor state. These data identify a molecular pathway for the downregulation of Shh via a BMP-Tbx2 pathway and provide a mechanism for expansion of a discrete progenitor domain within the developing forebrain (Manning, 2007).

Tbr2 directs conversion of radial glia into basal precursors and guides neuronal amplification by indirect neurogenesis in the developing neocortex

T-brain gene-2 (Tbr2) is specifically expressed in the intermediate (basal) progenitor cells (IPCs) of the developing cerebral cortex; however, its function in this biological context has so far been overlooked due to the early lethality of Tbr2 mutant embryos. Conditional ablation of Tbr2 in the developing forebrain resulted in the loss of IPCs and their differentiated progeny in mutant cortex. Intriguingly, early loss of IPCs led to a decrease in cortical surface expansion and thickness with a neuronal reduction observed in all cortical layers. These findings suggest that IPC progeny contribute to the correct morphogenesis of each cortical layer. These observations were confirmed by tracing Tbr2+ IPC cell fate using Tbr2::GFP transgenic mice. Finally, it was demonstrated that misexpression of Tbr2 is sufficient to induce IPC identity in ventricular radial glial cells (RGCs). Together, these findings identify Tbr2 as a critical factor for the specification of IPCs during corticogenesis (Sessa, 2008).

Tbr2-positive intermediate (basal) neuronal progenitors safeguard cerebral cortex expansion by controlling amplification of pallial glutamatergic neurons and attraction of subpallial GABAergic interneurons

Little is known about how, during its formidable expansion in development and evolution, the cerebral cortex is able to maintain the correct balance between excitatory and inhibitory neurons. In fact, while the former are born within the cortical primordium, the latter originate outward in the ventral pallium. Therefore, it remains to be addressed how these two neuronal populations might coordinate their relative amounts in order to build a functional cortical network. This study shows that Tbr2-positive cortical intermediate (basal) neuronal progenitors (INPs) dictate the migratory route and control the amount of subpallial GABAergic interneurons in the subventricular zone (SVZ) through a non-cell-autonomous mechanism. In fact, Tbr2 interneuron attractive activity is moderated by Cxcl12 chemokine signaling, whose forced expression in the Tbr2 mutants can rescue, to some extent, SVZ cell migration. It is thus proposed that INPs are able to control simultaneously the increase of glutamatergic and GABAergic neuronal pools, thereby creating a simple way to intrinsically balance their relative accumulation (Sessa, 2010).

Periventricular notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells

To understand the cellular and molecular mechanisms regulating cytogenesis within the neocortical ventricular zone, the spatiotemporal expression patterns of Ngn2 and Tbr2 were examined at high resolution. Individually DiI-labeled daughter cells were tracked from their birth in slice cultures and immunostained for Ngn2 and Tbr2. Both proteins were initially absent from daughter cells during the first 2 h. Ngn2 expression was then initiated asymmetrically in one of the daughter cells, with a bias towards the apical cell, followed by a similar pattern of expression for Tbr2, which was found to be a direct target of Ngn2. How this asymmetric Ngn2 expression is achieved is unclear, but gamma-secretase inhibition at the birth of daughter cells resulted in premature Ngn2 expression, suggesting that Notch signaling in nascent daughter cells suppresses a Ngn2-Tbr2 cascade. Many of the nascent cells exhibited pin-like morphology with a short ventricular process, suggesting periventricular presentation of Notch ligands to these cells (Ochiai, 2009).

Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex

The link between cortical precursors G1 duration (TG1) and their mode of division remains a major unresolved issue of potential importance for regulating corticogenesis. This study induced a 25% reduction in TG1 in mouse cortical precursors via forced expression of cyclin D1 and cyclin E1. In utero electroporation-mediated gene transfer transfects a cohort of synchronously cycling precursors, necessitating alternative methods of measuring cell-cycle phases to those classical used. TG1 reduction promotes cell-cycle reentry at the expense of differentiation and increases the self-renewal capacities of Pax6 precursors as well as of Tbr2 basal precursors (BPs). A population level analysis reveals sequential and lineage-specific effects, showing that TG1 reduction: (1) promotes Pax6 self-renewing proliferative divisions before promoting divisions wherein Pax6 precursors generate Tbr2 BPs and (2) promotes self-renewing proliferative divisions of Tbr2 precursors at the expense of neurogenesis, thus leading to an amplification of the BPs pool in the subventricular zone and the dispersed mitotic compartment of the intermediate zone. These results point to the G1 mode of division relationship as an essential control mechanism of corticogenesis. This is further supported by long-term studies showing that TG1 reduction results in cytoarchitectural modifications including supernumerary supragranular neuron production. Modeling confirms that the TG1-induced changes in neuron production and laminar fate are mediated via the changes in the mode of division. These findings also have implications for understanding the mechanisms that have contributed to brain enlargement and complexity during evolution (Pilaz, 2009).

Cyclin D2 is critical for intermediate progenitor cell proliferation in the embryonic cortex

Expression of cyclins D1 (cD1) and D2 (cD2) in ventricular zone and subventricular zone (SVZ), respectively, suggests that a switch to cD2 could be a requisite step in the generation of cortical intermediate progenitor cells (IPCs). However, direct evidence is lacking. In this study, cD1 or cD2 was seen to colabel subsets of Pax6-expressing radial glial cells (RGCs), whereas only cD2 colabeled with Tbr2. Loss of IPCs in cD2(-/-) embryonic cortex and analysis of expression patterns in mutant embryos lacking cD2 or Tbr2 indicate that cD2 is used as progenitors transition from RGCs to IPCs and is important for the expansion of the IPC pool. This was further supported by the laminar thinning, microcephaly, and selective reduction in the cortical SVZ population in the cD2(-/-)cortex. Cell cycle dynamics between embryonic day 14-16 in knock-out lines showed preserved parameters in cD1 mutants that induced cD2 expression, but absence of cD2 was not compensated by cD1. Loss of cD2 was associated with reduced proliferation and enhanced cell cycle exit in embryonic cortical progenitors, indicating a crucial role of cD2 for the support of cortical IPC divisions. In addition, knock-out of cD2, but not cD1, affected both G(1)-phase and also S-phase duration, implicating the importance of these phases for division cycles that expand the progenitor pool. That cD2 was the predominant D-cyclin expressed in the human SVZ at 19-20 weeks gestation indicated the evolutionary importance of cD2 in larger mammals for whom expansive intermediate progenitor divisions are thought to enable generation of larger, convoluted, cerebral cortices (Glickstein, 2009).

The level of the transcription factor Pax6 is essential for controlling the balance between neural stem cell self-renewal and neurogenesis

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. Pax6 was used as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. The genomic binding locations were identified of Pax6 in neocortical stem cells during normal development, and the functional significance of genes were ascertained that were found to be regulated by Pax6. Pax6 was found to positively and directly regulate cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, a core network regulating neocortical stem cell decision-making was identified in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. It was found that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal (Sansom, 2009).

Tbr1 regulates regional and laminar identity of postmitotic neurons in developing neocortex

Areas and layers of the cerebral cortex are specified by genetic programs that are initiated in progenitor cells and then, implemented in postmitotic neurons. This study reports that Tbr1, a transcription factor expressed in postmitotic projection neurons, exerts positive and negative control over both regional (areal) and laminar identity. Tbr1 null mice exhibited profound defects of frontal cortex and layer 6 differentiation, as indicated by down-regulation of gene-expression markers such as Bcl6 and Cdh9. Conversely, genes that implement caudal cortex and layer 5 identity, such as Bhlhb5 and Fezf2, were up-regulated in Tbr1 mutants. Tbr1 implements frontal identity in part by direct promoter binding and activation of Auts2, a frontal cortex gene implicated in autism. Tbr1 regulates laminar identity in part by downstream activation or maintenance of Sox5, an important transcription factor controlling neuronal migration and corticofugal axon projections. Similar to Sox5 mutants, Tbr1 mutants exhibit ectopic axon projections to the hypothalamus and cerebral peduncle. Together, these findings show that Tbr1 coordinately regulates regional and laminar identity of postmitotic cortical neurons (Bedogni, 2010).

The gene cascade Fezf1/Fezf2 -> Hes5 -> neurogenin 2 regulates the expression of Tbr2 and controls differentiation of the neural stem cells into the intermediate progenitors

Precise control of neuronal differentiation is necessary for generation of a variety of neurons in the forebrain. However, little is known about transcriptional cascades, which initiate forebrain neurogenesis. This study shows that zinc finger genes Fezf1 and Fezf2, homologs of Drosophila Earmuff, that encode transcriptional repressors, are expressed in the early neural stem (progenitor) cells and control neurogenesis in mouse dorsal telencephalon. Fezf1- and Fezf2-deficient forebrains display upregulation of Hes5 and downregulation of neurogenin 2, which is known to be negatively regulated by Hes5. FEZF1 and FEZF2 bind to and directly repress the promoter activity of Hes5. In Fezf1- and Fezf2-deficient telencephalon, the differentiation of neural stem cells into early-born cortical neurons and intermediate progenitors is impaired. Loss of Hes5 suppresses neurogenesis defects in Fezf1- and Fezf2-deficient telencephalon. These findings reveal that Fezf1 and Fezf2 control differentiation of neural stem cells by repressing Hes5 and, in turn, by derepressing neurogenin 2 in the forebrain (Shimizu, 2010).

An important question about neural development is how the differentiation of neural stem cells is precisely controlled in the forebrain. Asymmetric cell division of neural stem cells is thought to contribute to the differentiation of neural stem cells (radial glial cells) into either neurons or intermediate progenitors. Recent reports suggest that the orientation of stem cell division in the VZ might not directly control which of the two asymmetrically divided cells becomes a stem cell and which of the two becomes a differentiated cell. Although asymmetric centrosome inheritance during the asymmetric cell divisions was reported to play a role in the maintenance of the neural stem cells, it is not clear what factors determine cell fate. It is known that oscillation of Hes1 and neurogenin 2 expression in the telencephalic VZ plays an important role in maintenance of the neural stem cells and that stabilization of neurogenin 2 expression supports differentiation of the neural stem cells. However, it is still not understood what factor(s) control stabilization of neurogenin 2 expression and what factor(s) induce their differentiation. These reports imply that, besides asymmetric distribution of cell-fate determinants, extrinsic and intrinsic factors might bias the neural stem cells toward differentiation. Notch signaling plays an essential role in maintenance of the neural stem cells. Thus, regulators of Notch signaling and its downstream effectors might be involved in the decision as to whether to be a stem cell or a differentiated cell. This report demonstrates that Fezf1 and Fezf2, which are expressed in the neural stem cells at the beginning of mouse cortical development, inhibit the expression of the Notch effector Hes5 and promote differentiation of the neural stem cells. The findings suggest that Fezf1 and Fezf2 function as intrinsic factors to bias the neural stem cells toward differentiation (Shimizu, 2010).

Expression of fezf2 takes place in the radial glial cells of the telencephalic VZ of adult zebrafish (Berberoglu, 2009). fezf2 is also expressed in the neural progenitors and neurons in the pre-optic region and hypothalamus of the adult zebrafish brains (Berberoglu, 2009). In zebrafish, neurogenesis continuously takes place in adult brains. It is possible that fezf2 might control differentiation of the neural stem cells in the adult zebrafish forebrain as Fezf1 and Fezf2 do during early mouse cortical development (Shimizu, 2010).

Expression of Fezf1 or Fezf2 repressed both NOTCH1-dependent and NOTCH1-independent Hes5 promoter activity, but did not repress the Hes1 promoter or the artificial CBS-dependent promoter. Hes1 expression was not upregulated in the telencephalon of Fezf1-/-Fezf2-/- mice. Furthermore, FEZF1 and FEZF2 bound to the Hes5 promoter in vivo in the mouse forebrain. All of these data indicate that FEZF1 and FEZF2, rather than inhibit Notch cytoplasmic signaling, specifically bind to and directly repress the Hes5 promoter. FEZF1 and FEZF2 have an EH1 repressor motif. The data support the assertion that FEZF1 and FEZF2 function as transcriptional repressors and repress the Hes5 promoter at least during early cortical development. Hes5 deficiency suppressed neurogenesis defects in Fezf1-/-Fezf2-/- telencephalon, supporting the hypothesis that Fezf1 and Fezf2 suppress the expression of Hes5 and thereby control differentiation of the neural stem cells (Shimizu, 2010).

FEZF1 and FEZF2 repress only Hes5. Hes1 and Hes5 function redundantly in the maintenance of neural stem cells in the mouse central nervous system, whereas only Hes1 is reported to exhibit oscillatory expression in the neural stem cells, suggesting that Hes1 and Hes5 might have distinct roles in neurogenesis. Previous research has revealed that oscillation of Hes1 is involved in the maintenance of neural stem cells and, in the current study, it is speculated that Hes5 plays a different role in neurogenesis; specifically, it is proposed that Hes5, in contrast to Hes1, sets up the overall expression levels of Hes genes and neurogenin 2 in the forebrain. Once Fezf1 and Fezf2 expression exceeds a threshold, FEZF1 and FEZF2 might repress Hes5 expression, stabilize neurogenin 2 expression and thereby bias the neural stem cells toward differentiation (Shimizu, 2010).

The Drosophila homolog of Fezf1/2 (dFezf or Earmuff) has been shown to restrict the developmental potential of intermediate progenitors by negatively regulating Notch signaling. Although the mechanism by which dFezf represses Notch signaling is unknown, Fezf family genes function to negatively regulate Notch signaling in both vertebrates and invertebrates (Shimizu, 2010).

Fezf1 and Fezf2 function to repress the caudal diencephalon fate and their function is involved in proper rostro-caudal patterning of the forebrain (see Jeong, 2007). The prospective telencephalon domain is already smaller in Fezf1-/-Fezf2-/- mouse embryos than in the wild type at E9.5, before neurogenesis is initiated in the telencephalon. Therefore, the defect in rostro-caudal patterning is attributable to reduction of the telencephalon domain. In addition, Fezf2-/- or Fezf1-/-Fezf2-/- telencephalon lacks layer-V subcerebral projection neurons. Hes5 deficiency did not suppress the defects in rostro-caudal patterning of the forebrain or specification of layer-V neurons in Fezf1-/-Fezf2-/- forebrains. Therefore, Fezf1/2-mediated downregulation of Hes5 is not involved in the rostro-caudal patterning of the forebrain and the specification of layer-V neurons. Fezf1 and/or Fezf2 probably control genes other than Hes5 to elicit these functions (Shimizu, 2010).

Fezf1-/-Fezf2-/- telencephalon exhibited reduced formation of early-born neurons such as SP neurons and CR cells. A birthdate analysis revealed that the reduction of SP neurons and CR cells was not due to mis-specification of these neurons to other types of neurons. The data suggest that generation of the neural stem cells into SP neurons and CR cells is impaired in Fezf1-/-Fezf2-/- telencephalon. This finding is consistent with a reduction of differentiated (anti-neuron-specific βIII tubulin antibody TUJ1+) neurons in the Fezf1-/-Fezf2-/- telencephalon at E10.5, when subplate (SP) neurons and Cajal-Retzius (CR) cells were born in the VZ. Hes5 deficiency rescued neurogenin 2 expression at E10.5 and the generation of SP neurons and CR cells in Fezf1-/-Fezf2-/- telencephalon, indicating that Fezf1- and/or Fezf2-mediated repression of Hes5 plays an important role in the generation of these early-born cortical neurons. It is reported that formation of CR cells in the choroid plexus region, near the cortical hem, is controlled by a Hes-neurogenin cascade but that the Notch signal-mediated lateral inhibition is not involved in regulation of the Hes-neurogenin cascade in the CR cell development. Fezf1 and Fezf2 are expressed in the dorsomedial telencephalon. The current data suggest that Fezf1 and Fezf2 might control the development of CR cells by regulating Hes5 and neurogenin 2 expression in the choroid plexus domain (Shimizu, 2010). Fezf1-/-Fezf2-/- telencephalon had normal upper-layer (layer II, III) neurons but displayed a reduction of layer-IV neurons. There are two plausible explanations for this finding: Fezf1 and Fezf2 regulate the specification of layer-IV neurons or Fezf1 and Fezf2 control the generation of layer-IV neurons. Neither Fezf1 nor Fezf2 is expressed in differentiated layer-IV neurons, but both are expressed in their progenitors (neural stem cells or intermediate progenitors). Layer-IV neurons are normally born (differentiated) from E13.5 through E15.5. Birthdate analysis indicated that Fezf1-/-Fezf2-/- telencephalon contained a reduced number of Rorβ-positive neurons that were born at E13.5, suggesting that Fezf1 and Fezf2 control the generation of layer-IV neurons either from the neural stem cells or the intermediate progenitors. In Fezf1-/-Fezf2-/- telencephalon, differentiation of the neural stem cells into the TBR2+ intermediate progenitors was impaired. Tbr2 is an essential regulator of the intermediate progenitors and is directly regulated by neurogenin 2. These data suggest that the gene cascade Fezf1/Fezf2 -> Hes5 -> neurogenin 2 regulates the expression of Tbr2 and controls differentiation of the neural stem cells into the intermediate progenitors. The reduction of the TBR2+ intermediate progenitors in the Fezf1-/-Fezf2-/- telencephalon might contribute to a reduction of layer-IV neurons. Consistent with this idea, Hes5 deficiency rescued the development of TBR2+ intermediate progenitors as well as layer-IV neurons in Fezf1-/-Fezf2-/- telencephalon. It is reported that TBR1+ layer-VI neurons are increased in Fezf2-/- telencephalon, suggesting the transfate of layer-V to layer-VI neurons. However, they were not increased in Fezf1-/-Fezf2-/- telencephalon, implying that the gene cascade Fezf1/Fezf2 -> Hes5 ->neurogenin 2 controls the generation of layer-VI neurons. Future studies will clarify these issues (Shimizu, 2010).

In summary, FEZF1 and FEZF2 are transcriptional repressors that repress Hes5 expression and subsequently activate neurogenin expression. The Fezf1/Fezf2 -> Hes5 -> neurogenin 2 gene cascade controls differentiation of the neural stem cells into neurons or intermediate progenitors and contributes to the generation of a variety of neurons in the forebrain (Shimizu, 2010).

Af9/Mllt3 interferes with Tbr1 expression through epigenetic modification of histone H3K79 during development of the cerebral cortex

Mutations of leukemia-associated AF9/MLLT3 are implicated in neurodevelopmental diseases, such as epilepsy and ataxia, but little is known about how AF9 influences brain development and function. Analyses of mouse mutants revealed that during cortical development, AF9 is involved in the maintenance of TBR2-positive progenitors (intermediate precursor cells, IPCs) in the subventricular zone and prevents premature cell cycle exit of IPCs. Furthermore, in postmitotic neurons of the developing cortical plate, AF9 is implicated in the formation of the six-layered cerebral cortex by suppressing a TBR1-positive cell fate mainly in upper layer neurons. The molecular mechanism of TBR1 suppression is based on the interaction of AF9 with DOT1L, a protein that mediates transcriptional control through methylation of histone H3 lysine 79 (H3K79). AF9 associates with the transcriptional start site of Tbr1, mediates H3K79 dimethylation of the Tbr1 gene, and interferes with the presence of RNA polymerase II at the Tbr1 transcriptional start site. AF9 expression favors cytoplasmic localization of TBR1 and its association with mitochondria. Increased expression of TBR1 in Af9 mutants is associated with increased levels of TBR1-regulated expression of NMDAR subunit Nr1. Thus, this study identified AF9 as a developmental active epigenetic modifier during the generation of cortical projection neurons (Büttner, 2010).

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