bifid
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).
Leg axis formation in Drosophila is organized by Wingless (Wg) and Decapentaplegic (Dpp) that control a number of downstream factors to pattern the dorsoventral (DV) and proximodistal (PD) axis. The T-box genes are important downstream factors mainly involved in dorsoventral leg axis formation. The ventral side is specified by H15 and midline, whereas optomotor-blind (omb) and Dorsocross (Doc1) are factors to specify dorsal cell fates. This study shows that omb also organizes PD leg axis patterning in the beetle Tribolium castaneum. In the legs, Tc-omb is expressed along the dorsal side and represses ventral factors like wg and H15. Intriguingly, removing Tc-omb function leads to the activation of the Dpp pathway along the dorsal side of the legs, thus mimicking normal dpp expression in Drosophila. Dpp activity along the dorsal side leads to altered expression of proximal-distal patterning genes such as Distal-less (Dll) and dachshund (dac). These results indicate a cell-autonomous activation of Dll and repression of dac by dpp. These findings are compatible with the cross-regulatory "cascade model" of proximal-distal leg imaginal disc patterning of Drosophila (Pechmann, 2022).
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).
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). 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).
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).
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).
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 ).
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.
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
The progenitor cells in the cerebral cortex coordinate proliferation and mitotic exit to generate the correct number of neurons and glial cells during development. However, mechanisms for regulating the mitotic cycle of cortical progenitors are not fully understood. Otx1 (see Drosophila ocelliless) is one of the homeobox-containing transcription factors frequently implicated in the development of the central nervous system. Mice bearing a targeted deletion of Otx1 exhibit brain hypoplasia and a decrease in the number of cortical neurons. It was hypothesized that Otx1 might be crucial to the proliferation and differentiation of cortical progenitors. Otx1 knockdown by in utero electroporation in the mouse brain reduced the proportion of the G1 phase while increasing the S and M phases of progenitor cells. The knockdown diminished Tbr1+ neurons (see Drosophila bifid) but increased GFAP+ astrocytes in the early postnatal cortex as revealed by lineage tracing study. Tbr2+ basal progenitors lacking Otx1 were held at the transit-amplifying stage. In contrast, overexpression of wildtype Otx1 but not an astrocytoma-related mutant Y320C inhibited proliferation of the progenitor cells in embryonic cortex. This study demonstrates that Otx1 is one of the key elements regulating cortical neurogenesis, and a loss-of-function in Otx1 may contribute to the overproduction of astrocytes in vivo (Huang, 2018).
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