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Susan Mackem, M.D., Ph.D.
Genes Regulating Pattern Formation During Embryonic Development
Developmental processes such as axis formation and organogenesis often re-employ regulatory components and interactions, which may even be used in the adult organism. Limb development is a particularly attractive model for unraveling the function and interaction of such regulatory components, because it is very accessible to study and is extremely well conserved between genetic (mouse) and embryologic (chick) model organisms and humans. Mouse genetics have spotlighted many mutations affecting this process, and a wealth of embryologic information from experimental manipulations in chick has uncovered a dense network of inductive tissue interactions, providing a rich conceptual framework for molecular analysis. My lab previously identified several transcription factors (homeobox genes and T-box genes) involved in formation and patterning of both the primary embryonic axis and the limb axis in vertebrates. Recently, we have focused on the role of the prototype T-box gene Brachyury (T), and the 5'Hoxd genes in regulating several aspects of limb development, including early inductive events (T), pattern formation (Hoxd), as well as condensation and differentiation of cartilage precursors for the limb skeleton (Hoxd). We are analyzing the normal developmental function of these transcription factors in the limb, with the long-term aim of linking regulatory cascades and patterned gene expression to the morphogenesis of specific structures.
A major goal of our research program is the identification of the direct transcriptional targets of these factors at the DNA level. Identifying the direct target promoters of developmental gene-regulators will be critical to understand how these regulators function and to link regulatory cascades in developmental genetic programs to the basic cellular processes that drive morphogenesis of anatomic structures. These factors operate in signaling pathways (eg. WNT, FGF, SHH) that are used extensively in a number of processes in both the embryo and adult. Thus, understanding how normal transcription programs are regulated during development may also help to decipher abnormal gene expression patterns in tumors and devise new strategies to intercept cellular targets driving tumor cell behavior. Developmental systems also afford an excellent avenue to study complex regulatory circuits designed to ensure the robust functioning of a normal process. The application of systems biology principles to developmental biology promises to reveal the quantitative interrelationships between signaling and regulatory systems necessary to support robust physiological processes, and thereby provide a framework for the interpretation of the pathology (especially cancer) resulting from gross or subtle disturbances in these interrelationships.
T gene (Brachyury) function in regulating early limb bud outgrowth:
The apical ectodermal ridge (AER) is a specialized structure that forms at the distal limb bud edge and secretes FGF signals essential for limb outgrowth. A cascade of FGF and WNT signals relayed from the midline of the embryo to the periphery regulates limb initiation and the formation of the AER. Finally, signals from the underlying mesoderm, including WNTs and FGFs, regulate early steps of AER induction. We have found that that the chick T gene (Brachyury), the prototype of the T-box transcription factor family, is expressed in the limb bud as well as the gastrulating embryo. T is expressed at some relay sites, in pre-limb field mesoderm at limb initiation, and later in mesoderm just beneath the AER. We showed that T plays a role in regulating AER formation, using retroviral T mis-expression in chick and analyzing early limb buds in T-/- mouse embryos. Our results suggest FGF and WNT signals operate both upstream and downstream of T and that T may be part of an epithelial-mesenchymal signaling loop that maintains the mature AER during limb outgrowth. We are developing strategies to further analyze the role of T in limb development, including conditional removal of T gene function in mouse to enable analysis of selective T gene loss in particular tissues.
Temporal requirements for Sonic hedgehog (Shh) signaling in digit pattern:
Shh acts as a mitogen and cell survival factor in many adult processes, and also appears to act as a morphogen in several developmental contexts. In the limb, Shh regulates both digit number and the identity of different AP digits (eg. A-to-P, thumb to pinky), and has long been thought to act as a morphogen forming a spatial gradient along the AP axis of the limb bud, with higher concentrations of signal specifying more posterior digit types. Since many of the posterior digits are descended from cells that previously expressed Shh before proliferating and moving out of the Shh-expressing zone, it has recently been proposed that Shh acts in a temporal rather than spatial gradient and digit precursors that are exposed to autocrine Shh signals for the longest time become the most posterior digits. However, other experiments mapping Shh-responsiveness in vivo indicate that the posterior cells exposed to highest autocrine signals become refractory in responding to Shh over time.
Our lab is using genetic approaches in mice to test these different models and evaluate the time-dependence of digit formation on Shh function in the early limb bud (using a limb-specific tamoxifen-regulated Cre recombinase to remove Shh at different times). We find the observed order of digit loss is invariant, but unexpected, and is not consistent with predictions from either current spatial or temporal morphogen gradient models. Rather, digit loss order appears to correlate with the order in which mesenchyme condenses to produce primordia for each of the digits (latest forming condensations most sensitive to loss of Shh signals) and analyses are underway to determine how this occurs. Our preliminary results suggest that Shh regulates digit pattern only very early and transiently and may even function more as a trigger than as a morphogen in the limb. During most of its expression course in the limb, Shh may be needed mainly to ensure cell survival and/or proliferation. If the limb bud has a reduced cell mass for whatever reason, fewer condensations and hence fewer digits can form but those that do form are normal. We are testing several aspects of this provocative new model by altering cell survival and/or proliferation rates to attempt rescue of Shh mutant phenotype.
Hoxd gene function in regulating digit pattern and role of Gli3-Hoxd interaction:
Digits arise as single chondrogenic condensations in the limb mesenchyme, that later segment and grow to acquire defining features such as the number, size and shape of their phalanges (segments). The AP pattern of different digits is controlled by posterior Sonic Hedgehog (SHH), in a dose-dependent fashion, but how different levels of Sonic hedgehog (SHH) specify the formation of different digit types (identities) in the developing limb remains unclear. SHH signaling protects the Gli3 transcription factor from cleavage to a repressor form. Without Shh, the Gli3 repressor predominates, SHH/Gli3 target genes are repressed, and digit formation fails. Eliminating Gli3 renders Shh dispensable for digit formation, but distinct, normal digit identities are lost and polydactyly occurs. Several 5' Hoxd genes function downstream of SHH/Gli3 to regulate digit pattern in an additive, semi-redundant manner, through as yet unknown targets. We have found that Gli3 interacts genetically and physically with 5'Hoxd members, converting Gli3 repressor into an activator of its target promoters. In vivo, this interaction promotes formation of digits with distinct, often posterior identities. Changes due to elevated Hoxd expression are greatly exacerbated by decreased Gli3, yet are reduced or lost in the total absence of Gli3, suggesting a physical interaction between Gli3-Hoxd. Varying [Gli3]:[total Hoxd] protein ratios in different parts of the limb bud may lead to differential activation of Gli3 target genes, serving to spatially and/or temporally extend the SHH activity gradient by altering the balance between 'effective' Gli3 activating and repressing functions. This model provides a mechanistic basis for the quantitative, dose-dependent nature of Hoxd gene function in regulating digit pattern and may also have implications for the mechanism by which certain human genetic diseases (PHS, PAP), due to Gli3 mutations producing a constitutive repressor form, cause polydactyly. We are currently using a combination of biochemical and genetic approaches to test the model, including site-directed mutagenesis of Gli3-Hoxd interaction domains and conditional modulation of Gli3 and Hoxd expression levels in mouse embryos. In parallel ChIP approaches are being developed to identify bona fide direct targets of endogenous Hoxd proteins in embryo limb buds. These analyses will also assist in comparing Gli3-Hoxd and Gli3-only target promoters.
Late Hoxd function in chondrogenesis and joint segmentation:
5'Hoxd gene expression persists long after chondrogenic condensations of the digits have formed in the mesenchyme. Late Hoxd expression has also been proposed to play a role in the establishment and growth of chondrogenic condensations. Hoxd expression normally shuts off within differentiating condensations, but persists at their periphery. It is not known whether Hoxd shut-off allows the transit of mesenchymal cells from proliferation to recruitment/differentiation. We are evaluating the role of Hoxd genes in balancing in this switch, using conditional transgenic approaches to prolong Hoxd gene expression through the early stages of chondrogenic differentiation. Prolonged Hoxd12 or Hoxd13 expression completely disrupts the normal differentiation program, both in vivo (particularly in long bone precursors) and in vitro in micromass cultures. Sustained Hoxd expression during this condensation-early differentiation phase actually reverses the differentiation program by repressing expression of the early master regulator for chondrogenic differentiation, Sox9, resulting in small condensations and yielding very small, attenuated limb cartilage models. ChIP-Chip approaches that are also being developed in the lab (see below) will assist in determining whether Sox9 is a direct Hoxd target, and what other targets implicated in the condensation-differentiation process are also targets. We have recently found that the 5'Hoxd13-d12-d11 knock-out has abnormal, incomplete segmentation of the digit joints. Segmentation to produce joints normally initiates by localized reversal of the cartilage differentiation program at the forming joint interzone region and we are currently investigating whether Hoxd genes play a role in normal joint formation by repressing chondrogenesis.
Global identification of direct target promoters regulated by limb transcription factors:
In parallel studies, we are developing chromatin immunoprecipitation (ChIP) assays in embryos to allow direct identification of target promoters in vivo for transcriptional regulators of limb development. ChIP techniques provide an exciting new tool to globally identify direct target promoters regulated by developmental control genes an exposition of the patterns of these targets for different factors is necessary to unravel the regulatory networks operating during pattern formation and morphogenesis. We are piloting this approach using Tbx5 to identify the endogenous target promoters bound by this factor in the limb buds of normal embryos with the aim of applying the analysis to promoter microarrays, which are now available covering the entire mouse genome. Tbx5 is an excellent factor as a prototype for this analysis because: 1) It is highly expressed in early forelimb bud (also heart), but not hindlimb bud and so a naturally paired positive and negative control may exist. (A related factor, Tbx4, is expressed only in hindlimb and may also be used reciprocally and serve as a control); 2) Tbx5 is essential for initiation of forelimb bud formation; the knock out does not form a forelimb bud and the mice have no forelimb. 3) At least one direct target gene, Fgf10, has been convincingly identified, which will assist in validating and troubleshooting the approach, yet leaving the vast majority of targets as low-hanging fruit; 4) Tbx5 is also a critical factor in heart development. We have already generated excellent high affinity polyclonal antibodies against Tbx5 and have verified that this antibody works efficiently and specifically on the Fgf10 promoter. We have optimized the non-selective amplification of ChIP'ed DNA and are currently hybridizing pilot selected-promoter microarrays.
We plan to ultimately then extend this type of analysis to several other developmental regulators important in digit specification and patterning. We have generated polyclonal antibodies for Hoxd12 and Gli3. Identifying direct binding targets for Hoxd and Gli3 proteins will be valuable in elucidating the role of Hoxd-Gli3 interaction in gene regulation. We are also generating a Hoxd13-epitope knock-in allele that will enable high affinity ChIP experiments with endogenous tagged-Hoxd13 in vivo to identify direct Hoxd target promoters using a ChIP-Chip approach with tiled genomic arrays.
This page was last updated on 3/21/2013.