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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 Tbx5 and the 5'Hoxd genes in regulating several aspects of limb development, including early inductive events (Tbx5), 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 to unravel the regulatory networks controlling limb development. 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.
Tbx5 function in regulating limb bud initiation:
The T-box transcription factor Tbx5 has been implicated in initiation of limb development, activating mesodermal Fgf10 expression, which in turn induces apical ectodermal ridge (AER) formation in the overlying limb bud ectoderm. The AER is a specialized structure at the distal limb bud edge that secretes FGF signals essential for limb outgrowth. We have begun to profile Tbx5 targets, using chromatin-immunoprecipitation (ChIP) and analysis of expression changes in Tbx5 mutant limb buds, and identified additional early targets that may act in parallel with Fgf10 during AER induction.
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 been proposed that Shh acts in a temporal gradient, with digit precursors exposed to short-range Shh signals for the longest time becoming the most posterior digits, and that patterning and growth are highly integrated. However, other experiments mapping Shh-responsiveness in vivo indicate that the posterior cells exposed to the highest autocrine signals become refractory in responding to Shh over time.
Our lab is using genetic approaches in mice to test these 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 not compatible with predictions from either spatial or temporal morphogen gradient models of Shh function. The order of digit loss correlates 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), rather than with ‘posterior’ digit-type (requiring more Shh activity). These results indicate that Shh is required only very transiently for patterning but sustained activity is required to attain normal cell numbers and hence digit numbers. Current analyses are focused on understanding how this is achieved mechanistically. We are testing several aspects of this new model by altering cell survival to rescue of Shh mutant phenotype. Our results indicate a class of Shh targets that require only transient activity for stable expression, and a potential induction of downstream signaling centers by early Shh activity.
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-Seq approaches are being developed to identify direct targets of Hoxd proteins in embryo limb buds. In comparison with Gli3 direct targets, these analyses will illuminate the role of Gli3-Hoxd interactions in gene regulation.
Late Hoxd and Gli3 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. Prolonged 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. 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 the role of Hoxd genes in normal joint formation. Using mutant mouse models, we have found that 5’Hoxd genes may act indirectly, in a non-cell autonomous fashion, to promote digit joint formation. Our genetic data also indicate a role for 5’Hoxd-Gli3 balance in regulating the formation, and perhaps the positioning of digit joints.
This page was last updated on 11/20/2013.