The Formation of Our Digits Points to a Process with Foresight
Have you ever wondered how our fingers and toes form during embryonic development? Our digits are, in fact, sculpted from a paddle-like structure in the embryo through the process of apoptosis — that is, programmed cell death. During early development, the hands and feet begin as solid, webbed structures. Through carefully controlled apoptosis, the tissue between them is eliminated, facilitating the separation of the digits. As one paper put it, “the role of apoptosis can be compared with the work of a stone sculptor who shapes stone by progressively chipping off small fragments of material from a crude block, eventually creating a form.”1 Apoptosis, of course, serves other important biological functions as well — such as eliminating old, damaged, or infected cells.
When cells die as a consequence of acute injury, they tend to swell and burst, releasing their contents into the surrounding tissue. This is known as necrosis, and it can result in an inflammatory response that can be damaging to the cells around them. Death by apoptosis, by contrast, is much cleaner. During apoptosis, the cytoskeleton breaks down and the nuclear envelope disassembles, and the genetic material is broken down into smaller fragments. The surface of the cell is modified such that it attracts macrophages that phagocytose (engulf) the cell before its contents can spill out into the environment and cause damage.
The process of apoptosis is tightly regulated by genetic and biochemical signals, ensuring that the correct number of cells die in the right areas. But how could such a developmental process involving programmed cell death evolve in a gradual, incremental fashion without any awareness of where the target is? This presents a significant obstacle to unguided evolutionary mechanisms. Here, I will give a brief overview of how this remarkable process is regulated and controlled.
Initiation of Apoptosis
The zones of undifferentiated cells between what will become the digits are called interdigital mesenchyme. It is here that apoptosis is initiated by signaling molecules. For example, bone morphogenetic proteins (BMPs) are secreted signaling molecules that are critical for inducing apoptosis in the cells of the interdigital spaces.2 Indeed, knocking out BMP molecules has been shown to result in webbed feet in chickens.3 BMPs are upregulated in the regions between the forming digits, resulting in cellular death and tissue regression.
These BMPs bind to receptors on the surface of target cells in the developing limb bud.4 This, in turn, activates intracellular SMAD proteins, which translocate to the nucleus and regulate the expression of pro-apoptotic and anti-apoptotic genes.5 For instance, pro-apoptotic genes such as Bax and Bak (discussed later) are upregulated. Anti-apoptotic genes, such as Bcl-2, are also downregulated. This facilitates cell death in areas where tissue needs to be removed.
The activity of BMPs is regulated by antagonists, such as Noggin, which binds directly to BMPs, forming a complex that inhibits them from interacting with their receptors. This ensures that apoptosis only occurs in the interdigital spaces, while preserving the cells that will form the digits.6
Executioner Caspases
A family of proteases called caspases comprise the molecular machinery responsible for apoptosis.7,8 These proteases are initially produced as inactive precursors known as procaspases. In response to apoptosis-inducing signals, they are activated. Executioner caspases are responsible for dismantling essential cellular proteins — these are themselves cleaved (and thereby activated) by initiator caspases. One executioner caspase targets for destruction the lamin proteins that comprise the nuclear lamina, resulting in its disintegration.9 This facilitates the entry of the nucleases into the nucleus where they degrade the cell’s DNA. Other targets of executioner caspases include the cytoskeleton10 and other critical cellular proteins.
Execution of the Death Program: The Intrinsic Pathway
There are two ways in which the cell’s death program can be initiated — the extrinsic and intrinsic pathways. The extrinsic pathway is initiated by external signals through the binding of ligands to death receptors on the cell surface. The intrinsic pathway is triggered by signals from within the cell itself. Since the intrinsic pathway is associated with digit formation, it will be my focus here.
In nucleated animal cells, inactive procaspases roam, waiting for a signal to activate the death program and kill the cell. Unsurprisingly, then, the activity of caspases must be very carefully controlled. This presents another conundrum for their origins — how could they arise without a mechanism in hand for holding them in check until required?
The Bcl2 family of proteins is responsible for regulating caspase activation.11 Some of these proteins promote activation of caspases and apoptosis, while others negatively regulate these processes. Two essential proteins for promoting cell death are Bax and Bak.12 These proteins trigger the release of cytochrome c from the mitochondria. Other Bcl2-family proteins sequester apoptosis by inhibiting Bax and Bak from releasing cytochrome c.13 Critical to a cell’s survival is the balance between the activities of the pro-apoptosis and anti-apoptosis Bcl2-family members.
Image credit: David Goodsell, CC BY 3.0 https://creativecommons.org/licenses/by/3.0, via Wikimedia Commons
Upon release of cytochrome c from the mitochondria, the cytochrome c molecules bind to Apaf-1 (apoptotic protease activating factor 1).14 Apaf-1 has a specific region called the WD40 repeat domain that interacts with cytochrome c.15,16This binding induces a conformational change in Apaf-1, which allows it to oligomerize. The Apaf-1 monomers thus assemble into a large heptameric complex called the apoptosome (shown in the figure above). This wheel-like structure serves as a scaffold for further recruitment of procaspase-9 molecules.17 Within the apoptosome, the proximity of multiple procaspase-9 molecules results in their autocleavage and activation.18 This induces a caspase cascade (involving the activation of downstream effector caspases, such as caspase-3 and caspased-7), ultimately resulting in programmed cell death.19
The Need for Foresight
We began by comparing the role of apoptosis in digit formation to a stone sculptor, chipping off tiny fragments from a block with a view towards ultimately creating a form. Of course, an actual stone sculptor has a vision of the final form — the ability to visualize a distant outcome. Conversely, a feature of natural selection is that it lacks foresight, or any awareness of complex end goals. How can a mindless evolutionary process select for a process of carefully regulated programmed cell death during development, without knowledge of the target? It would seem that any process capable of producing this mechanism would have to possess intelligence and foresight — characteristics uniquely associated with a conscious mind.
Notes
Suzanne M, Steller H. Shaping organisms with apoptosis. Cell Death Differ. 2013 May;20(5):669-75.
Storm EE, Kingsley DM. GDF5 coordinates bone and joint formation during digit development. Dev Biol. 1999 May 1;209(1):11-27.
Zou H, Niswander L. Requirement for BMP signaling in interdigital apoptosis and scale formation. Science. 1996 May 3;272(5262):738-41. doi: 10.1126/science.272.5262.738. PMID: 8614838.
Ovchinnikov DA, Selever J, Wang Y, Chen YT, Mishina Y, Martin JF, Behringer RR. BMP receptor type IA in limb bud mesenchyme regulates distal outgrowth and patterning. Dev Biol. 2006 Jul 1;295(1):103-15.
Gomez-Puerto MC, Iyengar PV, García de Vinuesa A, Ten Dijke P, Sanchez-Duffhues G. Bone morphogenetic protein receptor signal transduction in human disease. J Pathol. 2019 Jan;247(1):9-20.
Guha U, Gomes WA, Kobayashi T, Pestell RG, Kessler JA. In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb. Dev Biol. 2002 Sep 1;249(1):108-20.
McIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol. 2013 Apr 1;5(4):a008656. Erratum in: Cold Spring Harb Perspect Biol. 2015 Apr 01;7(4):a026716..
Cohen GM. Caspases: the executioners of apoptosis. Biochem J. 1997 Aug 15;326 ( Pt 1)(Pt 1):1-16.
Gheyas R, Menko AS. The involvement of caspases in the process of nuclear removal during lens fiber cell differentiation. Cell Death Discov. 2023 Oct 21;9(1):386.
Vakifahmetoglu-Norberg H, Norberg E, Perdomo AB, Olsson M, Ciccosanti F, Orrenius S, Fimia GM, Piacentini M, Zhivotovsky B. Caspase-2 promotes cytoskeleton protein degradation during apoptotic cell death. Cell Death Dis. 2013 Dec 5;4(12):e940.Kale J, Osterlund EJ, Andrews DW. BCL-2 family proteins: changing partners in the dance towards death. Cell Death Differ.2018 Jan;25(1):65-80.
Westphal D, Kluck RM, Dewson G. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis. Cell Death Differ. 2014 Feb;21(2):196-205.
Dlugosz PJ, Billen LP, Annis MG, Zhu W, Zhang Z, Lin J, Leber B, Andrews DW. Bcl-2 changes conformation to inhibit Bax oligomerization. EMBO J. 2006 Jun 7;25(11):2287-96.
Kim HE, Du F, Fang M, Wang X. Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-1. Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17545-50.
Hu Y, Ding L, Spencer DM, Núñez G. WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation. J Biol Chem. 1998 Dec 11;273(50):33489-94.
Shalaeva DN, Dibrova DV, Galperin MY, Mulkidjanian AY. Modeling of interaction between cytochrome c and the WD domains of Apaf-1: bifurcated salt bridges underlying apoptosome assembly. Biol Direct. 2015 May 27;10:29.
Yuan S, Yu X, Topf M, Ludtke SJ, Wang X, Akey CW. Structure of an apoptosome-procaspase-9 CARD complex. Structure. 2010 May 12;18(5):571-83.
Li Y, Zhou M, Hu Q, Bai XC, Huang W, Scheres SH, Shi Y. Mechanistic insights into caspase-9 activation by the structure of the apoptosome holoenzyme. Proc Natl Acad Sci U S A. 2017 Feb 14;114(7):1542-1547.
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997 Nov 14;91(4):479-89.
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